Anal Bioanal Chem (2011) 399:3–27 DOI 10.1007/s00216-010-4207-5

REVIEW

Nanoparticles as contrast agents for in-vivo bioimaging: current status and future perspectives

Megan A. Hahn & Amit K. Singh & Parvesh Sharma & Scott C. Brown & Brij M. Moudgil

Received: 18 August 2010 /Accepted: 7 September 2010 /Published online: 6 October 2010 # Springer-Verlag 2010

Abstract Nanoparticle-based contrast agents are quickly mography (PET), computed tomography (CT), ultrasound becoming valuable and potentially transformative tools for (US), and photoacoustic imaging (PAI). Clinical and preclin- enhancing medical diagnostics for a wide range of in-vivo ical applications of NPs are identified for a broad spectrum of imaging modalities. Compared with conventional molecular- imaging applications, with commentaries on the future scale contrast agents, nanoparticles (NPs) promise improved promise of these materials. Emerging technologies, for abilities for in-vivo detection and potentially enhanced example multifunctional and theranostic NPs, and their targeting efficiencies through longer engineered circulation potential for clinical advances are also discussed. times, designed clearance pathways, and multimeric binding capacities. However, NP contrast agents are not without Keywords Nanoparticles . In-vivo imaging . Clinical . issues. Difficulties in minimizing batch-to-batch variations Characterization . Multifunctional . Theranostic and problems with identifying and characterizing key physicochemical properties that define the in-vivo fate and transport of NPs are significant barriers to the introduction of Introduction new NP materials as clinical contrast agents. This manuscript reviews the development and application of nanoparticles and Noninvasive imaging and minimally invasive in-vivo bio- their future potential to advance current and emerging clinical imaging techniques are valuable tools in the arsenal of clinical bioimaging techniques. A focus is placed on the application of diagnostics. Many types of bioimaging are available, span- solid, phase-separated materials, for example metals and ning from techniques that enable whole-organism anatomical metal oxides, and their specific application as contrast agents imaging (e.g., magnetic resonance imaging, MRI) to others for in-vivo near-infrared fluorescence (NIRF) imaging, that provide specific molecular imaging (e.g., optical fluores- magnetic resonance imaging (MRI), positron emission to- cence) at subcellular resolution. Such tools are expected to be pivotal for advancing early-stage cancer diagnosis, guided stem cell therapies, drug delivery, pathogen detection, gene Published in the special issue Nanomaterials for Improved Analytical therapy, image-guided surgery, and cancer staging [1], in Processes with Guest Editors Miguel Valcárcel and Bartolomé M. addition to many other clinically relevant procedures, Simonet. diagnostics, and therapies. : : : M. A. Hahn (*) P. Sharma S. C. Brown B. M. Moudgil Nanoparticles (NPs) are a class of materials generally Particle Engineering Research Center, University of Florida, ranging in size from 1 to 100 nm that are emerging as 205 Particle Science and Technology Building, potentially powerful probes for in-vivo imaging in medical P.O. Box 116135, Gainesville, FL 32611, USA e-mail: [email protected] and biological diagnostics. Several NP-based contrast : agents have been developed to overcome issues that plague A. K. Singh B. M. Moudgil conventional contrast agents; improvements in chemical Department of Materials Science and Engineering, and photostability of NP fluorophores, and contrast agent University of Florida, 205 Particle Science and Technology Building, detection limits, have been demonstrated in a broad array of P.O. Box 116135, Gainesville, FL 32611, USA imaging modalities. The ideal NP agent must fulfill a 4 M.A. Hahn et al. number of stringent requirements: it should be easily dispersible and stable (i.e., resist aggregation) in a variety of local in-vivo environments and not be affected by differences in solvent polarity, ionic strength, pH, or temperature when these conditions are not intended for follow many labels radionuclide, requires radioactivity tissues works poorly in air-containing organs and machines still being optimized measurement; it should exhibit limited nonspecific binding and be resistant to reticuloendothelial system (RES) uptake, and have programmed clearance mechanisms; and it should have high sensitivity and selectivity for the target (e.g., antigen, cell, tissue) with good contrast quality (high signal- to-noise ratio, SNR) and sufficiently long circulation times HighLow LowLow Poor depth penetration HighLow Low sensitivity, cannot HighLow Can detect only one High High High Requires radioactivity High Poor resolution Low of soft Poor image contrast, Low Information processing in the blood if administered intravenously. Ideally, these Throughput Cost Main limitation materials will be suitable for long-term quantitative imaging at low doses and be safely cleared from the body after imaging is complete. 6 Important areas in which NP-based contrast can prove − 10 – 12 9 15 14 6 8 12 advantageous include tumor imaging for guided surgery, − − − − − − − imaging of gene expression in vivo to elucidate disease of label detected) development, and efficacy of anti-cancer drugs. For example, a combination of in-vivo imaging can be used to image a tumor pre-operatively utilizing MRI and intra- operatively using optical imaging that can then aid image- guided tumor resection. In-vivo imaging could track tumor — m No limit 10 m Several cmm<5cm10 10 response to chemotherapy treatments in real time saving m No limit 10 μ μ μ μ 2 mm No limit 10 2 mm No limit 10 3mm <1cm 10 – – time and money while diminishing patient discomfort and – ]) side effects—which would be an immensely powerful role 226 in the drug development of cancer therapeutics. The performance of the bioimaging modality used, and ] and [ the contrast agents or probes, is dependent on the type of 225 information desired, the characteristics of the biological target, and the size and thickness of the subject. Different techniques are more powerful and safer on humans (e.g., MRI) in a clinical setting, rather than fluorescence imaging, which is better suited toward small animal imaging for cancer and other disease models. Each type of in-vivo -rays 1 X-rays 50 SoundSound 50 50 γ Positron from radionuclides 1 Alterations in magnetic fields 50 imaging technique has its own advantages and limitations, Light, particularly in the near-infrared 1 which include spatial and/or temporal resolution, sensitiv- ity, SNR, penetration depth in tissue, and quantitative In) 111 C, Xe) accuracy. Table 1 highlights the most commonly used in- 11 Tc, 129 F, vivo imaging modalities. There is a high desire for earlier 18 99m detection and characterization of disease development and determining treatment effectiveness, rather than the end effects of disrupted molecular processes (e.g., malignant cancers and metastases). One area of research which has led to significant advancement in achieving specific molecular targeting is the development of biomarkers based on NP constructs. I) Regardless of composition, surface functionalization of the 124 Cu, CEST and hyperpolarized probes (e.g., nanomaterials polystyrene NPs gold NPs, SWNTs, dye-doped NPs 64 nanomaterial is often required to enable targeting and SWNTs and other carbon-based nanomaterials stealth for long circulation times with minimal nonspecific binding. There is a plethora of entities that can be Comparison of commonly used bioimaging techniques (adapted from Refs. [ incorporated on to a NP’s surface, with covalent bonding MRI Iron oxide NPs, Gd(III)-doped NPs, NP-based CT Iodinated NPs, gold NPs, iron oxide-doped PETSPECT NPs incorporating radioisotopes (e.g., NPs incorporating radioisotopes (e.g., USPAI Microbubbles, nanoemulsions, silica NPs, Gold nanoshells, gold nanocages, gold nanorods, NIRF QDs, dye-doped NPs, upconverting NPs, preferred over electrostatic interactions: DNA, RNA, and Table 1 Technique Typical NP label Signal measured Resolution Depth Sensitivity (moles NP contrast agents for bioimaging 5 oligonucleotides (aptamers); peptides, proteins, peptidomi- thin tissue sections; the requirement for deeper penetra- metics, enzymes, antibodies, and antibody fragments; tion depths for most clinical applications is driving tumor cell receptors, for example folate and Her2, or fluorescence-based techniques into the NIR region ligands against particular antigens or epitopes; carbohy- (650–950 nm). In this NIR window, the absorption of drates; and agents to reduce the chance of an immunogenic water, hemoglobin, and lipids are at their minimum while response while increasing the circulation time in the blood, autofluorescence and tissue scatter are low, enabling and stealthily avoiding the RES and promoting dispersi- maximum light penetration; therefore, high SNRs and bility and solubility (e.g., poly(ethylene glycol) (PEG), sensitive detection limits result. Typically <1 cm, light polymers, phospholipids, dextran, latex). No matter what penetration depth depends on the type of tissue imaged: the surface moiety, its activity must not be altered once skin and muscle are more transparent than organs having anchored to the NP surface. lots of vasculature (e.g., liver and spleen) because of This review will focus on several principle types of absorption by hemoglobin. However, new advances in NPs currently affecting or with the ability to improve optical microscopy imaging techniques have increased upon powerful in-vivo imaging techniques, for example light penetration depths [4]. The fluorophores must be near-infrared fluorescence (NIRF) imaging, MRI, posi- bright with, preferably, large Stokes shifts and high tron emission tomography (PET), computed tomography fluorescence quantum yields in the NIR, photostable, and (CT), ultrasound (US), and photoacoustic imaging (PAI). resistant to degradation in biological systems. Reviews on Table 1 provides an overview of the various techniques NPs used in in-vivo fluorescence imaging are available [3, that are discussed, highlighting current NP-based contrast 5, 6]. A targeted approach is usually used for in-vivo labels for each technique. Multifunctional and theranostic imaging, with a moiety for a particular cellular target NPs, and emerging technologies and their potential for conjugated to the NP surface. clinical use, will also be discussed. Many types of NPs are under investigation as potential contrast agents for in- Quantum dots vivo imaging; however, the scope of this review will be limited mainly to rigid, sparingly soluble NPs, for With their broad absorption spectra, large absorption cross example those composed of metals, metal oxides, semi- sections, narrow and tunable emission spectra, high conductors, and ceramics, and will not discuss in detail fluorescence quantum yields, and high photostability, other “softer” NPs, for example those formed from inorganic semiconductor nanocrystals, or quantum dots liposomes, dendrimers, polymers, proteins, and viruses. (QDs), are a popular choice for fluorescence imaging In addition to the online resource http://www.mi-central. applications. Their optical properties enable multiplexing, org, information on numerous imaging techniques can be where different colors of QDs are used in a single assay found in reviews by Massoud et al. [2]andDebbageetal. with only one excitation source. Figure 1 demonstrates the [3], the latter concentrating on molecular imaging using power and versatility of use of multiple QDs in a single nanoparticles. assay to visualize several lymphatic drainages in a mouse. QDs that emit in the NIR include II–VI, IV–VI, and III–V compounds, for example CdSe, CdTe, HgTe, Near-infrared fluorescence (NIRF) imaging PbS, PbSe, PbTe, InAs, InP, and GaAs, alloys of these component materials, and even core/shell structures, Fluorescence imaging is a powerful molecular imaging which can tune the emission further and alter fluores- technique in which specific probes (i.e., fluorophores) are cence lifetimes. An interesting probe is self-illuminating excited by incident radiation, usually in the visible or NIR, QDs, using fluorescence resonance energy transfer and emit energy at a (usually) lower energy than that with (FRET) from bioluminescent proteins conjugated to the which they were excited. Despite its extremely high- QD [7]. However, potential toxicity from the heavy metal sensitivity detection and location of individual cells, ions may preclude their use in clinical bioimaging and mRNA, DNA, proteins, peptides, receptors, low- may limit their uses to in-vitro and diagnostic assays. expressing cellular markers, and epitope distributions, it However, they have been used successfully in a sentinel lacks the ability to provide anatomical resolution. In fact, its lymph node mapping procedure that utilized intraoperative resolution is limited to 2–3mm[2]. However, fluorescence NIRF imaging [8]. and autofluorescence thoroscopic and endoscopic techni- ques are emerging as powerful diagnostic tools for Dye-doped silica nanoparticles identifying disease and abnormal structural features on body cavity surfaces. Regarding noninvasive imaging, NIR dye-doped silica NPs are becoming popular choices of fluorescence in the visible region is acceptable only for contrast agent for a number of reasons: silica NPs are 6 M.A. Hahn et al.

Fig. 1 In-vivo five-color lymphatic drainage imaging was able to the schematic diagram. Five primary draining lymph nodes were visualize five distinct lymphatic drainages. In-vivo and intrasurgical simultaneously visualized with different colors through the skin in the spectral fluorescence imaging of a mouse injected with five carboxyl in-vivo image and are more clearly seen in the image taken at the Qdots (565, blue; 605, green; 655, yellow; 705, magenta; 800, red) surgery. (Reprinted, with permission, from Ref. [227], Copyright 2007 intracutaneously into the middle digits of the bilateral upper American Chemical Society) extremities, the bilateral ears, and at the median chin, as shown in optically transparent, water dispersible, biologically inert, of these NPs can be increased by making mesoporous nontoxic in the amorphous form, with well established silica NPs; this method enables loading an additional conjugation strategies to modify the surface to proteins, component into the resulting pores, for example a peptides, and other ligands for cellular receptors using therapeutic agent capable of photothermal ablation or a silane chemistry. Utilizing this matrix, numerous NIR drug able to be released at the appropriate time and fluorophores can be encapsulated, reducing the potential location. toxicity of these fluorescent probes and shielding the NIR emitter from the aqueous environment, where the Upconverting nanomaterials dye usually suffers from low fluorescence quantum yield, degradation, and insufficient photostability. Dyes emit- Upconverting NPs are a relatively new class of compounds ting in the NIR that can be incorporated into silica NPs being developed as agents for in-vivo fluorescence imaging. include polymethines (e.g., Cy5.5, Cy7), indocyanine Doped with rare-earth ions, these materials absorb NIR light green (ICG), Alexa Fluor 750, and IRDye78, among (usually 980 nm) and emit upconverted light at a higher others. Encapsulating thousands of dye molecules within energy, usually in the green or far-red/NIR, with long one silica NP provides a tremendous advantage: a single fluorescence lifetime (μstoms)[9]. An example of such a

NP loaded with dye molecules is much brighter and system is yttrium oxide (Y2O3) NPs doped with erbium and more stable than its single-molecule counterpart. Dye- yttrium, which have excellent photostability in the NIR and doped silica NPs are usually synthesized by a sol–gel low toxicity [10]. In another study, Zhang and coworkers process (i.e., Stöber) or a microemulsion system by used upconverting polyethyleneimine-coated NaYF4:Yb,Er simply adding the dye (or a modified form of the dye) to and NaYF4:Yb,Tm NPs excited with NIR laser light to the silica-forming solution. In addition, the surface area demonstrate the imaging of visible fluorescence through NP contrast agents for bioimaging 7 mouse skin and even thigh muscle up to 10 mm deep Other probes and NIRF techniques [11]. However, upconversion may not be an advantage if the fluorescence is in the visible region, which suffers Other NIR fluorescence-based contrast agents under devel- from the aforementioned tissue penetration issue, but opment for in-vivo imaging include ICG-doped calcium may be satisfactory for imaging thin tissue sections. A phosphate NPs that are cleared by a hepatobiliary mecha- 3+ 3+ recent study reports YF3:Yb /Er NPs having upcon- nism [21]; the appeal of degradation into innocuous version luminescence in the NIR rather than in the biological byproducts combined with an FDA-approved visible, which enables greater penetration of the light fluorophore is understandable. Luminescent porous silicon [12]. Some upconverting NPs also have NIR fluorescence NPs emitting at ~800 nm are also attractive candidates, at energies lower than their excitation wavelength, thus excitable by NIR or two-photon excitation, and they were affording them even more advantages for bioimaging found to be biodegradable [22]; however, their synthesis applications [13]. An overview of luminescent NPs using requires use of hydrofluoric acid, and it takes approximate- such doped host lattice systems can be found in ly two weeks to activate their NIR luminescence—potential

Bachmann et al. [14]. Other examples include NdF3/ drawbacks for commercial contrast agents. One area for SiO2 core/shell NPs having excitation and emission in the growth includes the use of “smart” probes, those that are NIR range, and efficient deep tissue imaging of small animals “off” until reaching the desired target and then turn “on” [9]. All of these materials can be dispersed in aqueous [23]; these molecular beacons are commonly used with solutions and conjugated to relevant biomolecules for fluorescent agents via FRET or quenching mechanisms. targeting purposes. In addition to the development of the actual fluorescent contrast agents, other fluorescence-based imaging techniques Carbon nanomaterials are being developed that minimize the effects of autofluor- escence that plague bioimaging. One method utilizes time- Carbon-based nanomaterials are also potential NIR gated fluorescence imaging, which separates short-lived contrast agents for in-vivo imaging. Single-walled carbon autofluorescence (fluorescence lifetime of a few ns) from the nanotubes (SWNTs) can have emission in the second IR emission of fluorescent probes like QDs, with their long window (1000–1350 nm), which would enable even fluorescence lifetimes of a few hundred ns to 1 μs. Similarly, deeper light penetration [15]. However, the toxicity of fluorescence lifetime imaging (FLIM) is another potential SWNTs is hotly debated, and reproducible synthesis and growth area based on separation of probes on the basis of their functionalization are lacking, as are the methods to obtain lifetimes [24]. Bioluminescence imaging is popular for small high-purity samples. Carbon dots were found to have animal imaging because no external excitation light source is emission in the visible region when passivated by polymer required, thus eliminating autofluorescence, but this method chains [16]. These materials are being investigated for is not likely to be used for humans because of the necessary optical imaging agents using both one and two-photon injection of a substrate such as luciferin. With high spatial excitation [17–19]; however, with their visible fluores- resolution and using a normalized Born approximation, cence, small animal or thin tissue imaging remains fluorescence molecular tomography (FMT) is emerging as possible but clinical applications will most likely remain a powerful imaging technique for resolution of tumors in elusive. Colloidal diamond NPs (i.e., nanodiamonds) are mice using NIR fluorophores; the subject is imaged over yet another nanomaterial being investigated as potential several projections (i.e., different angles of source/detector in-vivo fluorescent probes. With fluorescence originating positions rotated through 360°), and a three-dimensional from N–vacancy, Si–vacancy, and Ni–N complexes, they image is mathematically reconstructed from the ratio of are biocompatible, not cytotoxic, and have a highly fluorescence intensity (at the fluorophore’s emission wave- reactive surface that is easily functionalized with biolog- length) to intrinsic intensity (at the wavelength of excitation) ical entities. Fluorescence arising from Si–vacancy defects [25]. This technique has been used to visualize the is preferred over the N–vacancy defects, because the distribution and localization of GFP-expressing T cells deep former emits narrowly at 738 nm; however, generating within mice [26] and even to quantify the number of GFP+ these particular defects after synthesis (which involves T cells in lymphoid organs in mice [27]. detonation of carbon-containing precursors) has not yet Two-photon absorption (TPA) is emerging as a powerful been demonstrated. The N–vacancy defects can be tool in fluorescence imaging. It is an excellent option for produced, but this method usually involves proton-beam excitation, because it can discriminate fluorophores at irradiation from an accelerator, followed by high temper- different depths owing to its sensitive temporal and spatial ature annealing—ahighlycostlyprocedure.Acompre- resolution; excitation occurs only at the focal point [5]. As hensive review of use of nanodiamonds as biolabels was mentioned previously, TPA can be used with numerous recently compiled by Barnard [20]. types of fluorescent nanomaterials, for example quantum 8 M.A. Hahn et al. dots, carbon dots, and silicon NPs. In addition, gold (paramagnetic) contrast agents, which does not display nanoshells, consisting of a silica core with a thin layer of this “blooming effect,” can easily be detected with high gold shell surrounding it, can be excited by TPA and may spatial resolution, but the major challenge in developing be candidates for future biological imaging applications [5]. T1 probes is achieving the high sensitivity obtained with Using NIR fluorophores for image-guided surgery is T2 agents. currently underway. Clinical instrumentation includes the Spy system from Novadaq (Bonita Springs, FL, USA), the Iron oxide nanoparticles Photodynamic Eye from Hamamatsu, and the Fluorescence- Assisted Resection and Exploration (FLARE) system devel- As with other imaging modalities, NP-based probes have oped by Khullar and coworkers. The latter researchers have been developed for MRI to achieve high tissue contrast and actually used NIR probes for fluorescence-guided sentinel to improve imaging sensitivity. The most popular material lymph node (SLN) mapping and nodal treatment in preclinical studied for T2 (superparamagnetic) contrast agents is iron and clinical trials [28]. SLN mapping is a growing area in oxide NPs, which are generally coated with dextran, PEG, which NIR agents can make an impact by enabling easy or other polymers, and are used for clinical MRI [31, 32]. identification of the location of the sentinel lymph node to Based on their size, these NPs are classified as magnetic check for malignancies and to determine if removal of the iron oxide nanoparticles (MION, μm), superparamagnetic lymph nodes is necessary. In addition, drug delivery to iron oxide (SPIO, hundreds of nm), and ultra-small lymph nodes or metastases by use of NP devices can localize paramagnetic iron oxide (USPIO, <50 nm). SPIO contrast chemotherapy drugs, which may improve cancer prognoses agents have been used clinically for diagnosis of liver and outcomes [29]. Combining fluorescent probes with diseases [33], whereas USPIO probes are generally used for microendoscopy could lead to imaging deep within tumors lymph-node imaging, angiography, and blood-pool imaging [24]. [32]. Besides their clinical use, MRI contrast agents based on iron oxide nanoparticles have been developed for studying biological processes: Weissleder and coworkers Magnetic resonance imaging (MRI) have contributed significantly in this research area and demonstrated use of these particles for molecular and MRI is a noninvasive and nonionizing imaging method that cellular imaging applications [34–37]. provides physiological and pathological information about As shown in Fig. 2, the efficiency of iron oxide probes is living tissue, usually by measuring water proton relaxation size-dependent and increases with higher particle crystal- rates. MRI offers high soft tissue contrast and is capable of linity [31]. However, these nanoparticles are generally deep tissue imaging with high spatial resolution (~50 μm). Its synthesized at low temperatures, have poor crystallinity, inherent drawback is its low sensitivity—millimolar concen- and lack monodispersity in particle size, as is common with trations of protons are needed—so the technique often other nanomaterials [38]. Thus, further optimization and requires use of exogenous contrast agents. These probes can improvement in synthetic processes is needed to make these alter relaxation processes when used in small amounts (of the probes useful for molecular imaging. Yet another challenge order of nmolL−1 to μmolL−1 concentrations). MR contrast for this class of contrast agents is the development of robust agents can broadly be divided into two classes: those that methods for dispersion in aqueous media and surface increase the T1 signal in T1-weighted images (positive functionalization for biological targeting. The inherent contrast agent, bright contrast), and those that reduce the T2 negative contrast associated with iron oxide NPs limits signal in T2-weighted images (negative contrast agent, dark their use in low-signal regions of the body or in organs with contrast). The effectiveness of a particular probe is defined intrinsically high magnetic susceptibilities, for example the by its longitudinal (r1) and transverse (r2) relaxivities— lungs. To solve this problem, specific methods based on enhancement of water proton relaxation rates by 1 mmolL−1 either pulse sequences [39, 40] or design of nanoparticles solutions of contrast agent is measured and compared with [41] have been developed by researchers to generate bright the intrinsic tissue or other MRI contrast agents (e.g., contrast from iron oxide NPs. Feridex). Although T2 agents enable highly sensitive To fulfill the high resolution and high sensitivity tracking of labeled cells, they suffer from poor contrast that requirements for in-vivo imaging applications, metal is sometimes weaker than the dark contrast produced by alloy-based T2 contrast agents with improved magnetic hypointense areas developed from pathogenic conditions. and physicochemical properties have been developed. Also, the “blooming effect” associated with T2 contrast Some examples of the bimetallic ferrite NPs in this agents makes staging of lesions difficult, because the category are CoFe2O4, MnFe2O4, and NiFe2O4 NPs [42]. signal from abnormal areas blends in with the back- However, the biological fate and long term toxicity of these ground signal [30]. In contrast, the signal produced by T1 new nanomaterials have yet to be assessed. NP contrast agents for bioimaging 9

available on the surface of the NPs. To solve this problem,

various paramagnetic nanoparticles, for example Gd2O3, GdF3, and GdPO4 [31]havebeenproposedtoyieldhigh magnetic moments because of the abundance of paramag- netic ions on their surfaces. Transition metal oxide (e.g., MnO) NPs have recently been developed by various groups for T1-contrast imaging of brain tumors, in addition to the liver and kidney [30]. In another report, hollow MnO NPs were synthesized that can carry drug molecules in their cavities for simultaneous imaging and therapy applications [46]. With relaxivities that depend on the biological environ- ment, “smart” T1 MR probes that respond to their surround- ings have been pursued extensively [43]. This class of probe primarily consists of Gd(III)-based complexes; incorporation of these smart probes into NPs will further enhance their utility for molecular imaging applications.

Other probes for additional MR techniques

Chemical exchange saturation transfer (CEST), a method used in NMR, is now currently being used as a technique for generating contrast in MRI [47]. In fact, MRI utilizing contrast agents for CEST could potentially be used for simultaneous visualization of multiple biological events. Fig. 2 (b–e) Nanoscale size effects of Fe3O4 (MEIO) nanoparticles on magnetism and MR contrast effects. (b) Transmission electron Upon application of a suitable radiofrequency (RF) pulse, microscopic (TEM) images of 4, 6, 9, and 12 nm sized MEIO CEST agents reduce the intensity of the bulk water signal nanoparticles. (c) Mass magnetization values, (d) T2-weighted MR by saturation transfer through their chemical exchange sites images (top: black and white, bottom: color). (e) Relaxivity coefficient [47]. CEST contrast can originate endogenously from r2 of the nanoparticles. (Reprinted, with permission, from Ref. [228], Copyright 2005 American Chemical Society, and Ref. [229], sugars, amino acids, nucleosides, and other diamagnetic Copyright 2008 Wiley–VCH) molecules (DIACEST). Because CEST contrast depends on the frequency difference (Δω) between the protons associ- Gadolinium-based agents ated with the contrast agent and the protons of bulk water, a large Δω is desirable as it gives rise to greater flexibility in According to the Solomon–Bloembergen–Morgon theory, selecting an RF pulse and enhanced contrast [43]. Although there are three important requirements for the design of highly high Δω can be achieved, this approach is not attractive for sensitive paramagnetic NPs: large number of labile water clinical translation because of the high magnetic fields molecules coordinated to the metal; optimum residence required. Various lanthanide-based paramagnetic complexes lifetime at the metal site; and slow tumbling motion of the (PARACEST) with large Δω (~50 ppm for Eu(III) NP containing the contrast agent [43]. A well investigated complexes compared with <5 ppm for diamagnetic probe for T1 contrast, Gd(III), has been incorporated into molecules) are currently being developed for higher various nanomaterials, for example silica and perfluorocar- sensitivity and improved contrast in MRI [43]. Various bon nanoparticles, carbon nanotubes [30, 31], and nano- nanocarriers with efficient PARACEST contrast include diamonds [44], which all yield high MR contrast because of those based on paramagnetic liposomes and perfluorocarbon a high payload of gadolinium ions and a slow tumbling NPs [31]. motion of particles. For a Gd(III) complex attached to The recent development of hyperpolarization techniques nanodiamonds, a 10-fold relaxivity increase was observed offers novel opportunities for using NMR-active heteronuclei compared with the monomeric Gd(III) complex [44]. In such as 13Cand15N in MRI. Use of nuclei other than another example of a T1 contrast agent, gadolinium chelates protons drastically improves the SNR, because of the were grafted on to mesoporous silica NPs to yield particles inherently low natural abundance of these heteronuclei in capable of drug delivery, thus granting them a therapeutic biological tissue. A hyperpolarized state has a higher number function [45]. However, gadolinium loading on these of spins aligned with the magnetic field compared with the constructs is limited by the number of anchoring sites normal Boltzmann distribution, resulting in an increase in 10 M.A. Hahn et al. the NMR signal intensity. In contrast with standard MRI 82Rb, and 86Y. Oftentimes PET tracers have been incorporated contrast agents, the hyperpolarized molecules themselves are with another modality in NPs, most notably CT [54, 55]. the source of the NMR signal and, therefore, the signal Figure 3 illustrates the power of this dual technique using 18F- intensity and SNR is directly proportional to their concen- doped cross-linked iron oxide (CLIO) NPs to image the liver tration and polarization level [43]. 129Xe has been shown to and blood pool of a mouse. In fact, since the inception of the be a potential tissue-specific CEST contrast agent because of first PET–CT scanner [56], commercial instruments that its exquisitely sensitive chemical shift. The extremely low provide solely PET imaging have been rendered virtually sensitivity of the 129Xe nucleus was improved by five orders obsolete. A source of information from the Academy of of magnitude simply by hyperpolarizing it before imaging Molecular Imaging on PET and PET–CT technology can be [48, 49]. Ongoing efforts in this area explore the slow rate of found online at http://www.ami-imaging.org. exchange between caged and free xenon for further improving the sensitivity of these probes [50]. For in-vivo Other contrast agents MRI, hyperpolarized 13C has been used extensively because of its relatively higher sensitivity and the availability of Other modalities that have been combined with PET dedicated RF coils [43]. include NIRF agents within silica NPs [57, 58] or QDs In addition to technical challenges related to the design of [59] and MRI agents in conjunction with iron oxide improved pulse sequences, the need to design contrast agents with higher sensitivity and target specificity is critical. Particular attention should be given to the development of NP-based CEST and hyperpolarized probes, because of their promising potential in MR-based molecular imaging applications. However, incorporating the agents that are used with CEST and hyperpolarized techniques into nanomaterial carriers is not straightforward, and more research is needed to determine the best way to take advantage of these more advanced magnetic imaging techniques.

Positron emission tomography (PET)

PET is an imaging technique that relies on emission from radioisotopes in the form of positrons without the need for external excitation. Approved by the FDA as a clinical molecular imaging technique with a resolution of 1–2mm [2], it lacks the anatomical resolution of a technique such as MRI. However, having the highest sensitivity of all imaging modalities enables quantification of the local concentration of radionuclide tracer, with the possibility of detecting a single abnormal cell labeled with only a few trace isotopes [3]. Furthermore, the penetration depth of this technique is unlimited, so the probe can always be imaged, irrespective of the location of the target. Especially important in cancer imaging and research, PET is capable of detecting molecular changes that are occurring in the body before the macroscopic disease is observed [51, 52] and of monitoring disease progression after treatment (i.e., Fig. 3 Dynamic PET/CT imaging of BALB/C mouse injected with tumor response to therapy) [53]. 18F-CLIO. Fused PET/CT coronal images at 2 h (a),7h(b), and 16 h (c) postinjection of 18F-CLIO. PET only coronal images at 2 h (d), 7 h Radioisotope-based agents (e), and 16 h (f) postinjection of 18F-CLIO. CT only coronal image (g). Three-dimensional rendering of fused PET-CT images at 2 h (h) and 16 h (i) postinjection. The green arrow indicates blood pool Common isotopes that can be chelated on to or incorporat- region of interest (ROI) and the asterisk indicates liver ROI. ed within NPs (in an analogous way to the gadolinium ions (Reprinted, with permission, from Ref. [209], Copyright 2009 used for MRI) include 18F, 11C, 15O, 13N, 64Cu, 124I, 68Ga, American Chemical Society) NP contrast agents for bioimaging 11 nanomaterials [60, 61], with the latter being a powerful ter performance in CT imagery. Figure 4 provides the mass combination of sensitivity and anatomical resolution with attenuation coefficients as a function of incident X-ray energy dual instrumentation unveiled in 2007 and 2008 [62, 63]. for several relevant elements, illustrating this effect. Although However, the amount of PET tracer must be carefully mass attenuation coefficients are important tools for rational- controlled compared with the amount of MRI contrast izing the composition of radio-opaque nanomaterials, mass agent, because of PET’s extreme sensitivity and MRI’s lack energy absorption coefficients rely on several assumptions of it. Single-photon emission computed tomography that are not necessarily valid for NP systems; as with any data, (SPECT), a similar technique, relies on the detection of due care is warranted before extrapolating information derived gamma rays from radioisotopes such as 99mTc, 111In, 123I, and from the bulk material [66]. 131I; however, SPECT is an order of magnitude less sensitive Although X-ray imaging has been a clinical workhorse than PET [2], but it has been used in combination with CT for more than half a century, little activity was devoted to by loading SWNTs with 125I[64]. One advantage of SPECT the development of new engineered nanomaterials as over PET is that numerous radionuclides can be multiplexed X-ray contrast agents until the last decade, when the for use in the former because different isotopes emit different number of publications involving nanomaterials as X-ray energies of gamma rays, whereas the various isotopes for the contrast agents has more than quadrupled. The first latter all emit the same energy in the form of positrons [2]. widespread clinical use of nanoparticulates as X-ray The major advantages of PET are its extreme sensitivity and contrast agents in humans goes back to the 1930s when FDA approval; therefore, the transition from cell culture to Thorotrast [67], a suspension of 3 to 10-nm thorium small animal to clinical setting is quite feasible. These dioxide nanoparticles, was applied as an IV radiographic advantages come at a high price, quite literally. A cyclotron contrast agent; because of long-term radiation effects source is the only way to generate the positron-emitting and significant carcinogenicity of the 232Th, however, isotopes; therefore, the setting in which the PET imaging takes the clinical application of Thorotrast was abandoned place must be located near such a source or at least have a rapid within 20 years. Because of the questionable biocom- pathway to get the radionuclides to it. In addition, hospital or patibility of many high atomic number (Z)elements,the clinical settings must have appropriate areas for storage and number of materials explored for in-vivo applications handling of these radioactive materials [3]. Exposure to this has been limited; today safer hydrophilic iodinated ionizing radiation, for the patient and the clinicians adminis- molecules are universally used as radiographic contrast tering the agents, should be limited and may be a severe agents. However, a renewed interest in NP-based agents drawback to the widespread use of PET. Nevertheless, it has emerged with the promise of more detailed and remains a technique that will continue to be developed and quantitative imaging, and potential for therapeutic potentially improved upon with the help of NP carriers. applications.

Iodinated nanoparticles X-ray imaging and computed tomography (CT) The widespread clinical use of iodinated compounds has Clinical X-ray imaging works by the principle of photon spurred the development of iodinated nanomaterials. There attenuation differences between materials according to the well established Bourguer–Lambert–Beer exponential absorption law. For clinical applications, both X-ray energy specific mass attenuation coefficients, and mass energy absorption coefficients of contrast materials are important to evaluate the relative penetration attenuation and energy deposition (e.g., ionization) of imaging X- rays caused by these materials. The mass attenuation coefficient is a measure of the X-ray opacity of a material, whereas the mass energy absorption coefficient provides an indication of the fractional amount of ionization that occurs in the sample caused by incident X-rays [65]. When designing X-ray probes for clinical applications, the resulting contrast is highly dependent on the energy of the incident X-rays, which varies with the Fig. 4 Calculated values of the mass attenuation coefficient for different imaging purpose. Hence, contrast agents that perform well materials from NIST (database: http://physics.nist.gov/PhysRefData/ in projectional radiographic imaging may exhibit lacklus- XrayMassCoef/tab3.html) 12 M.A. Hahn et al. is much research on, essentially, incorporation of iodinated NPs consisting of bismuth sulfide [95] and composite organic compounds into a NP, with designs ranging from ceramics containing iron oxide [96] and lanthanide materi- emulsions [68, 69], liposomes [70], and lipoproteins [71]to als [97] have been reported. Bismuth sulfide NPs have nano-milled insoluble compounds [72–75] and polymeric recently been shown to have superior performance to iodine NPs [76, 77], many of which have been successfully on a molar basis [95]. Although the initial report suggests applied in vivo [70, 71, 73–77]. The design principle for limited toxicity, the overall similar mass attenuation many of these nanomaterials has simply been to enhance coefficients for bismuth and gold, in addition to bismuth’s localized iodine concentrations, resulting in higher local higher k-edge transition, indicate that bismuth-based nano- contrast compared with conventional water-soluble com- materials may not perform significantly better than gold- pounds. In addition to modifying particles to alter physio- based materials. Hence, the toxicological risks from the logical fate and transport, doping with an iodinated presence of bismuth may preclude clinical use of these compound is used to enhance X-ray contrast for the materials; however, more research describing any biological purpose of creating multifunctional particles [69, 71, 78]. side effects is still needed. Other forms of contrast agents, Despite iodine having a lower atomic number than both for example those used in MRI, in particular iron oxide and gold and bismuth, it has a superior elemental mass attenuation gadolinium-doped materials, have also been shown to have coefficient and incident X-ray energies that are relevant for X-ray contrast properties. Toxicologically and practically, projectional radiographic imaging. A recent comparison of the the biodegradability and known absorption, distribution, performance of gold NPs with that of iodinated contrast metabolism, and excretion (ADME) profiles of iron oxide agents demonstrated that under conditions used for coronary NPs in humans make them attractive alternatives to angiography, both materials performed equivalently [79]. iodinated and gold-based materials for non-critical, low- Although these nanomaterials may be viewed as a simple energy X-ray imaging applications. Although the clinical evolution of conventional iodinated compounds, the strategy applications of X-ray contrast materials based on gadolin- of adding iodine to NPs with fairly well understood fate and ium and other lanthanides are limited, primarily because of transport properties is promising. the associated toxicological risk, gold-based X-ray poten- tiated therapy may realistically complement established Gold nanoparticles image-guided X-ray therapy in the near future. Use of these materials in the clinic will depend strongly on added value, Within the last decade, there has been substantial interest in with efforts being made to combine therapeutic components gold NP-based contrast agents for in vivo X-ray imaging. with these contrast agents to broaden their potential market Various gold [42, 80–87], gold–dielectric hybrid [88–90], value. Design criteria for such materials are currently being and multimodal materials [83, 91], have been fabricated established, and the promise of X-ray potentiated therapy and tested for X-ray contrast performance. These particles seems to be real. have been shown to have in-vivo functionality as CT contrast agents for cancer [82, 84, 91], tissue-specific [87], and blood-pool imaging [86]. In fact, gold NPs have been Ultrasound (US) shown to match or exceed the performance of conventional iodinated contrast agents under conditions relevant for As with X-ray imaging, US is a well established clinical mammography and relevant for CT and trans-torso imaging imaging modality. A relatively inexpensive and versatile [79], as expected from gold’s higher k-edge energy shown method, it is based on the pulse–echo principle, whereby in Fig. 4. Recently, it was found that gold NP-induced X- sound waves having frequencies greater than 20 kHz are ray contrast in CT imaging is further enhanced as the gold emitted and received. Ultrasound has been used for NP size is reduced [92]. Gold nanomaterials are currently molecular imaging and is capable of resolving nanoscale being explored in multiple clinical trials. The depth of features, albeit at limited penetration depths using non- research on the fate, transport, and toxicology of gold-based clinical wave frequencies. The clinical application of US NPs make them a promising next generation candidate for involves sound waves in the range of 2–3 MHz for X-ray contrast materials, and their use to potentiate pediatric imaging and 5–12 MHz for adult imaging, combined radiotherapy [93, 94] adds yet another dimension providing spatial resolution in the range 0.2 to 1 mm. to the application of these materials. Traditionally, contrast in US is provided by the variable ability of sound to propagate through media, resulting in Other contrast materials reflection and refraction of the sound waves. The extent of this reflection and refraction is based on the mismatch of Although the bulk of the current literature involves acoustic impedance between materials, which is defined as iodinated and gold-based NPs as contrast agents, other the speed of sound through the material multiplied by its NP contrast agents for bioimaging 13 density. Because sound travels poorly through gaseous Photoacoustic imaging (PAI) phases, several microbubble-based contrast agents have been developed and are applied clinically to enhance the PAI, also known as laser optoacoustic imaging, is an echogenicity of vasculature and organ-specific regions [98, emerging noninvasive, nonionizing, imaging modality that 99]. These microbubbles are composed of surfactant, combines the high sensitivity of optical methods with the protein, and/or polymer shells containing gas cores, for excellent resolution of acoustic methods [110, 111]. example air, perfluorocarbons, or nitrogen. The last two are Illuminated by a short-pulsed laser, the biological sample preferred because their minimal plasma solubility leads to absorbs this light on the basis of the characteristics of its improved longer-term imaging performance. In addition to composition. This excitation is followed by a transient microbubbles, perfluorocarbon emulsions have been used increase in temperature (~10 mK) and subsequent thermo- as US contrast agents [100]. Whereas microbubbles suffer elastic expansion of the absorbent, generating an ultrasonic from instability to insonification pressures, marked attenu- acoustic signal which is detected by wideband transducers ation, “blooming” effects, and short circulation times, surrounding the object and used to determine its geometry. emulsion-based US contrast agents are mechanically more PAI is generally performed using two main techniques, resilient to these effects, despite their reduced echogenicity. photoacoustic microscopy (PAM) and photoacoustic com- Although reports of use of nanobubbles [101–105] and puted tomography (PAT) [112]. PAM employs a coupled, nanoemulsions [106] as US contrast agents have been focused ultrasonic detector–confocal optical illumination frequent in recent years, these contrast agents rarely exist as system to generate multidimensional tomographic images true nanoparticles, because they are typically between 150 without the need for reconstruction algorithms, whereas the and 1000 nm in diameter. As with microbubbles, these detectors in PAT scan the laser-illuminated object in a materials are typically composed of a perfluorocarbon gas circular path and use inverse algorithms to construct three- (or liquid in the case of nanoemulsions) encapsulated by a dimensional images. surfactant, protein, and/or polymer shell. Because of their In-vivo imaging, especially with optical techniques, lower scattering cross sections and the often less-than- suffers from the issues of hemoglobin absorption and tissue optimum mechanical properties of the shell, the perfor- scatter, which limit overall light penetration depth. PAI can mance of these materials is often inferior to that of overcome this primary challenge because of the lower comparable microbubbles. A recent theoretical evalua- ultrasonic scattering coefficients (by 2–3 orders of magni- tion of US contrast agents indicates that the optimum tude) of absorbents compared with their optical equivalents; bubble size for current imaging practice is in the therefore, the propagation of the photons in the diffuse vicinity of 2–3 μm[107]. However, because of the regime [113] enables PAI up to ~50 mm deep with a clinical significance of US and potential labeling advan- resolutionof<1mm[114]. Further, because PAI’s tages of nanomaterials, there is a continued interest in sensitivity is primarily based on the optical absorption developing smaller ultrasound contrast agents. Solid properties of the specimen, all absorbed photons produce particles composed of silica and polystyrene have also photoacoustic signals, whether or not they were scattered. been used as US contrast agents to visualize mice livers, Selection of the appropriate central frequency and band- although the size of the particles ranged from 500 to width of the ultrasound enables variation of the penetration 3000 nm, which may not necessarily classify them as depth and spatial resolution, so samples of various thick- nanoparticles [108]. nesses and sizes can be studied using the same technique. As with other imaging modalities, there is an emerging In fact, the depth issue is completely eliminated when trend to combine both imaging and therapy. Rapoport et al. microwaves or radio waves (referred to as thermoacoustic have used a polymeric micelle and perfluorocarbon nano/ tomography (TAT)) are used as illumination sources [115]. microbubble system that encapsulated a drug, which was Using only endogenous contrast, PAI has been used to released locally within tumor cells; US was also used to image blood vessels [116], tumors [117, 118], tumor determine the efficacy of this drug therapy [105]. However, angiogenesis [119], and hemoglobin oxygenation [120]. the imaging performance of nanoscale US contrast agents UsinglaserPAT,lesions18mminsizehavebeendetected may become less significant when compared with their in a human breast 23 mm below the laser source [114]. potential therapeutic potency. Sonodynamic therapy, Exogenousagentssuchasdyes[121, 122], which absorb ultrasound-induced apoptosis, sonoporation/sonotransfec- and fluoresce at desirable wavelengths, have also been tion, ultrasound-induced drug/gas delivery, and focused used for cancer staging by PAM. For example, methylene ultrasound-induced thermal ablation are currently being blue was injected into the lymphatic system and accumu- explored for therapeutic clinical applications [109]. Reports lated in the sentinel lymph node, which was then identified of US contrast agent-based theranostics are emerging, and and imaged with an axial resolution of 144 μmanda their incorporation into multimodal probes. penetration limit of 30 mm [121]. PAI using multi- 14 M.A. Hahn et al. wavelength illumination further increases the potential to they were found mostly within the tumor cortex and were image multiple chromophores such as biomarkers, intrin- almost absent from the tumor core [131]. In a comparison sic, and exogenous agents [123, 124]. Functional imaging of gold plasmonic nanostructures (surface plasmon reso- is routinely performed on biological specimens on the nance (SPR) tuned to 800 nm), Hu et al. noted that gold basis of endogenous contrast, and engineered nanomate- nanorods and nanocages have much larger absorption and rials are also being used to perform molecular imaging in scattering cross sections than gold nanoshells [161]; future vivo. development of these materials may therefore increase. Currently, gold nanorods have similarly been used as NIR Gold-based nanomaterials photoacoustic contrast agents with high sensitivity [162]. Manipulation of the aspect ratio enables tuning of the SPR Gold-based nanomaterials stand out as the most significant of the resulting nanorods, which has led to multiplexing class of materials being explored for PAT applications. applications [163, 164]; for example, Li et al. used antibody- Some of those commonly used for PAT include spherical conjugated gold nanorods with two different aspect ratios gold NPs [125], nanorods [126, 127], nanocages [128], (peak absorptions at 785 and 1000 nm) to detect Her2 and agglomerates [129], and even hollow nanoshells [130], and CXCR4 target molecules by PAT [164]. A combination of composite materials such as gold nanoshells having silica PAT and ultrasound has also been used to target and detect cores [131, 132], cobalt/gold core/shell NPs [133], gold- prostate cancer, using functionalized gold nanorods to speckled silica [134], polymer–gold hybrids, and gold provide high photoacoustic contrast and anatomical details nanobeacons [135]. The reasons for the increasing of the targeted tissue [126]. Gold nanorods have recently attention to gold nanoconstructs are many: their size- been shown to be effective as tracers for noninvasive in-vivo dependent and shape-dependent plasmonic properties spectroscopic photoacoustic SLN mapping in a rat model [136, 137] enable them to absorb and scatter light in the [165]. In addition to the aforementioned studies, gold visible to NIR region, which may render them suitable for nanorods have been used in other bioimaging applications, image-guided therapy [138] and photothermal ablation of for example detecting inflammatory response from cells tumors [139–141]. Because of the prevalent use of gold- [166], measuring quantitative flow in biological samples based compounds in medicine (e.g., chrysotherapy [167],andmonitoringdrugdelivery[168]. Gold nanoc- [142]), the benign toxicity profile of NPs [143–145], ages [128, 169, 170] and hollow gold spheres [130]with and ongoing clinical trials [146], the possibility of NIR absorption profiles are also being explored as contrast approval of these gold-based materials for clinical and agents for PAI. medicinal applications is greatly enhanced. Conjugation of biologically relevant entities to gold surfaces is well Carbon nanomaterials established [147–149], and most of these nanomaterials have multimodality capabilities incorporating the other Carbon nanomaterials are being extensively used for techniques outlined here, for example PET [150], CT pharmaceutical, biomedical [171, 172], and bioimaging [141], MRI [151], conventional microscopic optical applications [173], including PAI and thermoacoustic techniques [152], reflective confocal microscopy [153], imaging [174]. The characteristic optical properties of multi-photon plasmon resonance microscopy [154], opti- SWNTs, particularly those with optical properties in the cal coherence tomography (OCT) [155] (including phase NIR region [175], play an important role in photoacoustic sensitive OCT) [156], scattering [157], surface enhanced imaging [176]. In an in-vivo tumor-targeting study, Raman spectroscopy (SERS) [158], and diffuse optical SWNTs~2nmindiameterand50–300nminlengthwere spectroscopy [159]. coupled to RGD peptides and were shown to bind to tumor Gold nanoshells, nanorods, and nanocages have vasculature, producing an ~8× higher photoacoustic signal attracted the most attention for photoacoustic applications, and an ~4× higher Raman signal (ex vivo) than unconju- because of the tunability of their peak absorption in the gated SWNTs [177]. Antibody-conjugated SWNTs tar- NIR region; gold nanoshells with a silica core, and geted to integrins αvβ3-positive U87 human glioblastoma nanorods, are the systems most investigated to date. An tumors in mice similarly resulted in high photoacoustic in-vivo study imaging rat brain cortical blood vessels contrast in vivo [178]. As an alternative and noninvasive reported a marked increase in blood vessel absorption approach to detection of the SLN, SWNTs have been with nanoshells, thus demonstrating successful application shown to result in significant signal enhancement for as a NIR PA contrast agent [160]. Gold nanoshells and the detection by PAI [179]. Despite various in-vivo applica- enhanced permeability and retention (EPR) effect were tions of SWNTs, their absorption coefficients are relatively recently used to image subcutaneous tumors by employing low compared with those of gold nanoparticulates [180]. high-resolution PAM, with heterogeneous localization— To overcome this limitation, nanotubes are being modified NP contrast agents for bioimaging 15 to enhance their NIR absorption and, thus, photoacoustic are reported to provide an ~300× improvement in photo- contrast. In one modification, nanotubes have been plated acoustic sensitivity compared with unmodified SWNTs in with a thin layer (4–8 nm) of gold; use of these golden vivo. carbon nanotubes (GNTs) resulted in a 100-fold increase in photoacoustic signal enhancement [180]. In a compar- Dye-doped nanoparticles ison of photoacoustic signals of GNTs with those of other NIR contrast agents they were shown to exhibit higher PA Although dyes alone are sufficient to increase SNR, thus signals and correspondingly lower bubble-formation improving image contrast, their encapsulation within NPs thresholds than those of pristine carbon nanotubes and provides additional advantages as mentioned previously gold nanospheres, with comparable properties to gold [183]: loading numerous dye molecules into a protective nanorods and nanoshells. The high photoacoustic sensi- NP matrix enables signal amplification, reduced chemical tivity of GNTs has been demonstrated by employing and photodegradation, improved contrast, and the ability to folate-conjugated GNTs as a secondary contrast agent to target specific biologically relevant sites. Besides the desired enable photoacoustic detection of magnetically captured absorption profile, selection of the dye can be based on other circulating tumor cells, thus potentially enabling early properties, for example fluorescence imaging in addition to diagnosis of cancer [181]. Antibody-conjugated GNTs PAI or incorporating a therapeutic function, for example have been used to target lymphatic vessels in vivo by PAI photodynamic therapy (PDT). ICG is the most commonly [180]. In another modification of SWNTs, ICG dye used dye for photoacoustic imaging in molecular and molecules attached to the surface of the nanotubes by nanoparticulate formulations. Currently the only FDA- π–π stacking interactions had 20-fold higher absorbance approved dye for human applications, ICG’s absorption peak than bare SWNTs (Fig. 5)[182].AidedbytheNIR at ~780 nm lies within the biologically relevant NIR window absorption property of the dye, SWNT–ICG nanomaterials and enables deep tissue imaging. PAT has been used to image objects containing blood and ICG that were embedded at depths greater than 5 cm in chicken breast tissue [184]. However, the in-vivo applications of ICG are limited, primarily because of its rapid degradation in aqueous media and its short plasma half life of up to 4min[185, 186]. Encapsulation of ICG within different NP matrices (e.g., organically modified silica (ORMO- SIL), poly(lactic-co-glycolic acid) (PLGA), and calcium phosphate) has been shown to improve its stability and blood circulation time [160], thereby enabling in vivo applications.

Current challenges and opportunities

Characterization and toxicology

One obstacle to overcome when using NPs is the lack of reproducibility in both synthesis of the actual material and functionalization to render it biologically active. Batch-to- Fig. 5 Photoacoustic detection of SWNT-ICG in living mice. (a) batch variations within the same laboratory, between Mice were injected subcutaneously with SWNT-ICG at concentrations of 0.82–200 nmolL−1. The images represent ultrasound (gray) and laboratories, and even between different techniques photoacoustic (green) vertical slices through the subcutaneous employed in synthetic and modification procedures are injections (dotted black line). The skin is visualized in the ultrasound common, often yielding the same NPs but with slightly images, and the photoacoustic images show the SWNT-ICG distribu- different characteristics (e.g., purity of size distribution, tion. The white dotted lines on the images illustrate the approximate edges of each inclusion. (b) The photoacoustic signal from each number of conjugated biomolecules). In part, these diffi- inclusion was calculated using 3D regions of interest and the culties arise from complex characterization of these “background” represents the endogenous signal measured from materials off-line, on-line, and in-situ. There is a need for tissues. Linear regression (R2=0.97) of the photoacoustic signal curve − consistency and scale-up in NP production and functional- indicates that 170 pmolL 1 SWNT-ICG will give the equivalent background signal of tissues. (Reprinted, with permission, from Ref. ization, especially to produce high yields at low cost if [182], Copyright 2010 American Chemical Society) commercialization is desired. This area is one that is both 16 M.A. Hahn et al. challenging and has great potential for growth in the future, predict the same behavior in humans. More complex issues before extensive use of NPs can be realized. (e.g., penetration depth, toxicity) must be explored before As mentioned previously, the biggest knowledge gap use in the clinic. Repeated ad nauseam, toxicity studies of existing in this research area is the lack of comprehensive nanomaterials are either lacking, especially long-term characterization of NPs that are subsequently used in effects, or inconsistent. Gadolinium chelates, ICG, biological research and bioimaging. Currently most studies, polymer-coated iron oxides, and gold nanocomposites are whether in-vitro or in-vivo, involving the use of NPs in either approved or in clinical trials (Table 2). However, for biological systems and in bioimaging report only minimal the remaining NP imaging agents under development for particle characterization. An in-depth understanding of the possible clinical use, information on short-term and long- structure–activity relationship of NP behavior in biological term toxicity, biodistribution, nonspecific uptake, and systems is imperative if better contrast agents are to be routes of elimination must be determined. designed; knowing what properties affect behavior will lead The need for benchmarking characterization techniques to more advances and improvements in contrast agents. A and toxicity tests is imperative [188–194]. Deciding on a base set of, admittedly extensive, characterized properties is set of standard toxicity assays is difficult, especially when required: size (actual diameter and hydrodynamic diameter) researchers from different laboratories use different tests to and size distribution; shape and shape distribution; surface confirm or deny toxicity. Toxicological characteristics may area; surface charge; surface chemistry and reactivity; include various in-vitro assays (often yielding different quantification of surface components; thickness and com- conclusions), reactive oxygen species (ROS) and singlet position of coating, elemental/chemical composition; crys- oxygen production, safe working concentrations (LD50 tal structure; porosity; and identification and levels of any values), in-vivo studies, and biodistribution profiles [195]. impurities. Depending on the material composition and It must be remembered that the various toxicological tests application, other techniques may be required, for example study different aspects of toxicity (e.g., membrane perme- UV–visible–NIR absorption spectroscopy, fluorimetry, de- ability, apoptosis) and that commercial testing kits designed termination of absorption cross sections, fluorescence for molecular toxins may not be the optimum tests for NPs, quantum yields, and fluorescence lifetimes for optically because the materials themselves may interfere with these active NPs. Magnetic NPs would require characterization of commercial assays. In addition, results of in-vitro analyses their magnetic properties, for example susceptibility and may not necessarily be valid in vivo. relaxivity, whereas NPs for PAI would require tests of their A standard is needed against which all nanomate- thermoelastic properties. Zhang and Yan have reviewed rials can be tested, and an established set of tests that some advances in the study of surface chemistry, including should be administered. The International Standards NMR, Fourier transform infrared (FTIR) spectroscopy, Organization (ISO) has outlined the work necessary, liquid chromatography–mass spectrometry (LC–MS), and and the MINChar initiative has been formed to combustion elemental analysis for characterizing molecules improve toxicological studies of nanomaterials [188]. on the NP surface [187]. However, filling this knowledge NP characterization remains an almost overwhelming gap remains a daunting task. challenge; however, this area is a major opportunity for Complicating matters further, different bioimaging techni- further development. The Nanotechnology Characteriza- ques require different administered doses, based on the tion Laboratory (http://ncl.cancer.gov), under the Na- technique’s sensitivity, host biology, route of delivery, and tional Cancer Institute (NCI), is working with the FDA the targeting strategy used. When used in bioimaging, even to develop standard tests to determine a NP’ssafetyand more characterization is needed to fully understand and exploit efficacy, and the properties of NPs which must be the structure–function relationship of engineered NPs, for characterized if they are to be used in conjunction with example particle number and dose administered (i.e., balanc- drugs or therapeutics; this task is quite complex, ing safety against good SNRs), behavior in the biological considering that the tests should work for multiple environment (e.g., dispersibility or aggregation), and inter- particle types, despite the fact that different NPs have actions between the moiety on the surface of the functional- different characteristics and behavior (e.g., fluorescent, ized NP and the target of interest (i.e., binding kinetics and magnetic, metallic). Their current database groups NPs thermodynamics). Reproducibility of bioconjugation and based on size, surface charge, and solubility (Fig. 6) study of the activities of those biomolecules once on the NP [196]. surface are immense challenges but also research opportuni- ties. One of the most difficult properties to study is the number Biological considerations of NP treatment modalities and activity of the conjugated molecules on a NP’ssurface. Small-animal imaging studies are a valuable tool when Important considerations when developing an imaging studying disease models, but they may not necessarily probe are routes of delivery and bioavailability. Most NP contrast agents for bioimaging 17

Table 2 Selected NP-based therapeutics approved or in clinical trials (adapted from Refs. [196] and [223])

Product Nanoparticle drug component Delivery route Indication FDA status Company

Doxil PEGylated liposome/doxorubicin IV Ovarian cancer Approved 11/17/1995 Ortho Biotech hydrochloride FDA050718 Amphotec Colloidal suspension of lipid-based Subcutaneous Invasive aspergillosis Approved 11/ 22/1996 Sequus amphotericin B FDA050729 Estrasorb Micellar NPs of estradiol Topical emulsion Reduction of vasomotor Approved 10/9/2003 Novavax hemihydrate symptoms FDA021371 Abraxane Nanoparticulate albumin/paclitaxel IV Various cancers Approved 1/7/2005 American Pharmaceutical FDA021660 Partners Triglide Nanocrystalline fenofibrate Oral tablets Lipid disorders Approved 5/7/2005 SkyePharma PLC FDA021350 Megace ES Nanocrystal/megestrol acetate Oral suspension Breast cancer Approved 7/5/2005 Par Pharmaceutical FDA021778 Companies Combidex Iron oxide IV Tumor imaging Phase III Advanced Magnetics Aurimune Colloidal gold/TNF IV Solid tumors Phase II CytImmune Sciences NB-00X Nanoemulsion droplets Topical Herpes labialis caused by Phase II NanoBio herpes simplex I virus AuroShell Gold-coated silica NPs IV Refractory head and neck Phase I Nanospectra Biosciences cancer CALAA-01 Cyclodextran-containing siRNA IV Various cancers Phase I Calando Pharmaceuticals delivery NPs Cyclosert Cyclodextran NP IV Solid tumors Phase I Insert Therapeutics INGN-401 Liposomal/FUS1 IV Lung cancer Phase I Introgen SGT-53 Liposome Tf antibody/p53 gene IV Solid tumors Phase I SynerGene Therapeutics delivery routes now consist of intravenous injection; it is, functionalization of their surfaces may result in NPs that are therefore, crucial to optimize circulation time. The func- larger than their “core” components, especially if very thick tionalized NPs must be able to pass through the blood- shells of silica, PEG, or polymer are used to render them stream and reach their desired target intact. The necessary well dispersed in aqueous solutions. This size increase may

Fig. 6 The physicochemical characteristics of a nanoparticle affect biocompatibility. Here we qualitatively show trends in relationships between the inde- pendent variables particle size (neglecting contributions from attached coatings and biologics), particle zeta potential (surface charge), and solubility and the dependent variable biocompati- bility—which includes the route of uptake and clearance (green), cytotoxicity (red), and RES rec- ognition (blue). (Reprinted, with permission, from Ref. [196], Copyright 2009 John Wiley and Sons) 18 M.A. Hahn et al. be crucial depending on the type of cancer, stage of cancer, performance (i.e., signal detection) must be optimized in location of tumor, and vasculature permeability. Steric order to take advantage of newer imaging modalities, and interactions of these larger NPs may enable only a small enhancing the sensitivity of emerging imaging techniques is fraction to bind to the target and/or affect bloodstream imperative. For fluorescence imaging, for example, detec- circulation: passing through thicker veins or arteries is tors in the NIR range are often less sensitive than those in much different from passing through thinner capillaries. the visible range; however, the use of CCDs and similar In addition to IV administration, intramuscular injec- equipment has helped in this regard. Silicon detectors can tions, oral, transdermal, and inhalation routes are also cover the first NIR window, and InGaAs detectors are more possible, depending on the desired target (Table 2). The efficient in the second NIR window. The major problem in NPs being used must be able to survive their particular all optical imaging is tissue scattering; a method must be route of delivery; for example, oral routes require particles developed that can deconvolute the scattering effects that can withstand the highly acidic environment of the propagated by imaged tissue [5, 92, 199] or otherwise stomach. If a region of the brain is the desired target, the counteract this scattering by modification of imaging NP agent must be able to cross the blood–brain barrier if modalities [4]. A cost/benefit analysis should be performed administered intravenously. Any loss of biological activity to balance the cost of the agents and detection systems of the component conjugated to the NP surface must be versus the improvement that those imaging techniques can determined. The probe will not be effective if it cannot do provide in terms of understanding disease progression, the desired function or bind properly to the desired target. early detection, better prognoses, and improved patient quality of life and/or survival. Developments in imaging techniques and instrumentation Design of improved NP probes Other types of bioimaging are becoming important. In-vivo Raman imaging has been developed using SERS probes Additional types of NP contrast agents may see develop- composed of Texas red and cresyl fast violet adsorbed on ment in the future. Currently, “smart” probes that turn “on” metallic gold, silver, or platinum NPs [197]. OCT and when exposed to the target are being developed; for spectroscopic optical coherence tomography (SPOCT) are example, superparamagnetic iron oxide NPs that contain being developed, as are probes for their use. Two-photon an optical probe which can be cleaved by proteases and excitation for optical imaging is also envisaged, with the then fluoresce combine an MRI technique with a selective advantages of reduced tissue scattering and absorption, and fluorescent optical probe that turns “on” only when in the increased resolution. MRI/PET probes are also powerful presence of the desired target [23]. emerging tools, combining the sensitive, metabolically Most in-vivo imaging is focused on the detection and functional PET with the high-resolution, anatomical detail location of abnormal lesions (e.g., cancer), differentiating a provided by MRI. Their growth had been slow until the cluster of cells that are drastically different from surround- successful development of MRI/PET scanners, because ing cells. If administered intravenously, the imaging agent PET detectors could not operate in the high magnetic fields must selectively find and bind the tumor, which usually required by MRI, so alternative instrument setups were involves taking advantage of the EPR effect and targeted required. However, the first MRI/PET scanners were agents. Relying on the EPR effect alone is often time- unveiled in 2007 and 2008 [62, 63]. Weissleder’s group consuming and may not provide enough contrast or signal has previously demonstrated the multimodal use of NPs intensity. Currently, fewer than 10% of engineered NPs that were fluorescent, magnetic, and contained a radiotracer reach tumor sites when both active targeting and EPR effect for nuclear imaging [198]. This area will most likely see are combined; improvement in targeted delivery is therefore growth in the future. In addition to optical projection needed. In the future, NPs will hopefully be used as tomography (OPT) and selective plane illumination micros- contrast agents not simply to find tumors, but to elucidate copy (SPIM), other imaging techniques are in development, biological processes and cellular mechanisms, which may including macroscopic and mesoscopic methods [4]. These be acute or transient processes, that are vital to understand- systems can increase light penetration depths compared ing and perhaps even curing debilitating diseases such as with typical optical microscopy, thus enabling deep tissue cancer, Alzheimer’s, Parkinson’s, multiple sclerosis, rheu- imaging, but may come at the cost of spatial resolution. matoid arthritis, and diabetes. Not only improvements in imaging probes, but also improvements in the instrumentation and detection systems Multimodal bioimaging that make the imaging possible are required. Improved image-analysis software and expanded data storage can A current popular approach to overcoming the limitations make existing technologies even more powerful. System imposed by a single imaging technique is to combine two NP contrast agents for bioimaging 19 or more contrast agents into a single NP entity which can combined MRI and PTA, and PTA has been used with then be imaged by those multiple techniques [1, 54, 200– SWNTs [213]. Imaging strategies utilizing PTA will most 202]. Combining the anatomical resolution of MRI with the likely see tremendous growth in the future, as gold sensitivity of optical imaging is common and could prove nanoshells are already in clinical trials for cancer therapy. to be a powerful technique for finding and quantifying the PDT uses photosensitizers that, when excited by light, size of tumors, especially tumors or metastases that are too react with molecular oxygen in the biological environment small for MRI detection alone. These MR/optical imaging to produce ROS, which are cytotoxic to cells. Weissleder et agents have been used to monitor enzyme activity, in brain al. have developed a multifunctional NP combining MRI, tumor imaging, and to detect and monitor apoptosis and NIRF, and PDT [214]. If the photosensitizer requires visible atherosclerosis [203]. Other types of multimodal contrast excitation, two-photon absorption is an option to increase agents are also in development: PET/NIRF used with ICG the penetration depth of the excitation source. Even X-rays [57] and QDs [59], SPECT/fluorescence [203], PET/MRI can be used as the energy required for PDT: researchers [198, 204–207], MRI/PAT [200], TAT/PAT used with have applied lanthanide-based materials and porphyrins SWNTs [123], and US/MRI [208], among others. Probes with PDT derived from X-ray bombardment, which with three modes of imaging are being considered, for releases UV photons. Gold NPs can also be applied as example MRI/NIRF/PET [55, 203, 209], PET–CT/MRI/ radiosensitizers at realistic doses with low energy X-ray NIRF [55], and even four modes incorporating MRI/PET/ sources [169]; originating from these ionizing X-rays, BRET/fluorescence [210]. Auger electrons directly cause single and double-stranded An important concept to consider is that with any breaks in DNA or produce free radicals when interacting multimodal system employed, the enhancement of one with water molecules, both resulting in local inactivation of modality must not be at the expense of another. For instance, tissue and cells. Theranostic NP probes can be used as the concentrations of MRI agents must usually be higher than actual therapeutic itself (e.g., gold nanoshells) or simply as agents for fluorescence imaging, because of the different the drug-delivery vehicle (e.g., liposomes). One material sensitivities of the two methods. Therefore, controlling the that may prove useful in combining a dual imaging and ratios of the two types of agents is necessary: high MRI agent therapy is mesoporous silica NPs; with their large surface loading must be combined with a low fluorescent agent areas and pore volumes, one modality can be incorporated loading. Additionally, incorporating more than one modality into the silica matrix while loading the other modality into may cause interferences between the two (e.g., iron-based its pores. This approach has already been done with an MRI agents quenching fluorescence agents) or complicate optical imaging agent and an anticancer drug [215]. fabrication, resulting in higher production costs or commer- NP therapeutics can revive drugs that were previously cial infeasibility. However, multimodal NPs would require discontinued because of toxicity or solubility issues and only one dose of multiple agents to the patient, hopefully have the potential to mitigate side effects of the free drug reducing side effects from having to use multiple doses of by delivering it directly to the site of interest. By different agents. This approach is likely to remain an area of encapsulating a concentrated drug payload, NPs prevent interest in the future, but unambiguous data showing exposure of healthy cells to the cytotoxic drug and may enhanced imaging with each technique incorporated in such prove more beneficial (e.g., lower toxicity and fewer side multimodal NP agents remains to be seen. effects) at lower doses than the free drug. However, NPs are much more complex than the simple small-molecule drugs that Theranostic NPs are easily characterized. In addition, the NP must remain intact until reaching the tumor site and then release the drug In addition to merely dual imaging, theranostic NPs are controllably through its desired mechanism—issues that will prominently finding their way into cancer research; they require further research and development. Kim et al. [201]and provide the diagnostic capability using an imaging modality Jarzyna et al. [54] have reviewed nanomaterials used for to detect a tumor, while supplying the component for multimodal imaging and theranostic applications. Summarized therapy against that disease, commonly utilizing photo- in Table 2, information on NP-based imaging agents and thermal ablation (PTA) or PDT. PTA works by exciting a therapeutics either approved or in clinical trials can be found NP with a large absorption cross section (e.g., gold), which in articles by Adiseshaiah et al. [216]andMcNeil[196]. causes localized heating that then kills the tumor cells into which the NPs have been injected. Gold nanoshells surrounding a silica core have been used in photoablative Toward the future: cancer, therapy, and the public therapies, and an approach using OCT in combination with PTA has been used with gold nanoshells [211]and In-vivo cancer imaging is likely to be the most prominent nanocages [212]. Iron oxide NPs with a gold shell enable area where nanomaterial research will be focused. The 20 M.A. Hahn et al. statistics are staggering: the NCI has estimated that cancer toward the development of new NP contrast agents for expenditure was approximately $104.1 billion in 2006, with bioimaging are government regulations and pure econom- breast, colorectal, and lung being the most costly [217]. ics. Getting an agent into clinical trials and approved by the According to the NCI over $3 billion was invested in FDA is a time-consuming and costly process, lasting up to cancer research in 2009, when the top four cancers 18 years and costing up to $2 million [222]; procedures that researched were breast, prostate, colon/rectum, and lung can shorten the development time and/or reduce the cost of [218]. According to estimates from the NIH, cancer will be bringing NP agents to market will therefore be most the third most heavily funded area in 2010–2011, preceded lucrative. Examples include incorporating a NP on to an only by grants for clinical research and genetics [219]. already-approved marker or combining the NP agent with a Therefore, early cancer and disease detection, and combin- therapeutic. Bawa provides an overview of getting NP- ing detection and therapy, are potential areas for growth and based therapeutics through the drug development process, development of NP contrast agents. highlighting the importance of federal agencies such as the Considering the amount of money spent on the second FDA and the USPTO [223]. A Nanoparticle Task Force has leading cause of death in the United States, this important been set up by the FDA to handle such regulatory issues area is a major opportunity for NP-based agents. Oncolo- [224]. gists are interested in using NPs for SLN mapping; this Investment in a new NP for clinical imaging is highly technique involves using an agent to locate the sentinel dependent on a number of factors. Newer agents must be lymph node before surgery and doing a biopsy to see if any proved to be vastly superior to any currently inexpensive malignancies are present to warrant removal of all lymph version that provides the same function; otherwise, there nodes, which may or may not have cancerous lesions. will be no incentive to continue its development. Therefore, Having been especially useful in breast cancer cases and agents that incorporate additional functionality (i.e., thera- significantly improving the detection of small metastases peutic agent, measure of disease progression, or evaluation [220], this technique has been proposed as a standard of treatment effectiveness) with the in-vivo imaging procedure for cutaneous melanoma, in which trials have modality will most likely see growth, especially if been successful [221]. Tracers currently used in the United incorporated into an already-approved material or device. States are isosulfan or “blue dye” (Lymphazurin 1%, US Also, investors in the development of new imaging and Surgical, North Haven, CT, USA), which requires visual therapies are generally hesitant unless the novel agent is inspection, and 99mTc–sulfur colloid (approved by the FDA potentially lucrative with widespread clinical use: probes for imaging liver and spleen), which requires a gamma ray that will benefit a large population of patients are more counter; other radiotracers based on 99mTc are available in likely to see development than those that target a rare Australia, Canada, and Europe. The technique is compli- disease or small subset of patients. Ultimately, it will take cated because different procedures must be used for an interdisciplinary team, consisting of a variety of different cancers and tumor locations, and the success of researchers with diverse backgrounds, for example materi- mapping is linked very heavily to the experience and skills als scientists, chemists, biologists, electrical engineers, of the surgeon performing the procedure. NP agents with surgeons, clinicians, and regulators, to make the prospect better contrast or ease of detection would prove beneficial of widespread NP use for clinical in-vivo imaging a reality. for increasing the usefulness and success of SLN mapping, leaving it less dependent on the individual clinician. Acknowledgments This work was supported by the National Besides the agents, other types of imaging, for example Science Foundation’s Division of Chemical, Bioengineering, Envi- SPECT–CT, may help elucidate SLN location with tumors ronmental, and Transport Systems (CBET) grant 0853707, the displaying ambiguous lymph node drainage. Researchers National Science Foundation’s Nanoscale Interdisciplinary Research are delving into other nanomaterials and imaging proce- Team (NIRT) Engineering Education and Center (EEC) grant 0506560, the Center for Nano-Bio Sensors (CNBS) at the University dures for SLN mapping: Ravizzini et al. provide an of Florida, and the Bankhead Coley Florida Biomedical Research overview of various methods using CT, US, MRI, and Program. fluorescence imaging for this procedure, with optical and MRI proposed as the future path for SLN mapping [29]. 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