Review pubs.acs.org/ac
Bioconjugated Nanoparticles for Biosensing, in Vivo Imaging, and Medical Diagnostics Brad A. Kairdolf, Ximei Qian, and Shuming Nie* Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, 1760 Haygood Drive, Atlanta, Georgia 30322, United States
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CONTENTS
Surface Coating and Bioconjugation Surface Coating Nonspecific Binding Bioconjugation Detection Modalities Fluorescence Raman Magnetic PET and CT Multimodality Cellular and Molecular Imaging Dynamic Cellular Imaging In Vivo Imaging and Spectroscopy Intraoperative Imaging and Spectroscopy In Vitro Diagnostics Concluding Remarks Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References
available from novel nanoparticles. We also discuss recent applications of nanoparticle probes for cellular and molecular imaging, intraoperative guidance, and in vitro diagnostics. Because the size of the nanomaterials typically falls within the same size regime as biomolecules or structures (e.g., globular proteins or small viruses), a large portion of the atoms within the materials are surface atoms and provide nanoparticle probes with an extremely large surface area to volume ratio. This high surface area can allow increased loading of functional agents (e.g., optical, radioisotopic, or magnetic) or the attachment of multiple bioaffinity molecules to the nanoparticle surface for multivalent binding effects.22,24 Of particular interest are the size tunable features of many nanomaterials (see Figure 1). Semiconductor quantum dots are one of the most widely known nanoparticles that exhibit this phenomenon. By reducing the radius of the quantum dots to a size below the exciton Bohr radius, a quantum confinement effect occurs and the electronic properties of the nanomaterial are altered.2 Exploiting this feature provides a method for tuning the fluorescence emission wavelength of the quantum dots over a wide range by simply changing the particle size25 and makes them a versatile tool for optical sensing and detection applications. Other nanomaterials exhibit tunable properties as well, including carbon nanotubes26 (structure tuning of emission wavelength), gold nanoparticles27,28 (shape/size tuning of surface plasmon resonance), and superparamagnetic iron oxide nanoparticles29 (size tuning of proton relaxivity). Here, we discuss recent developments and features of bioconjugated nanoparticles and their use in the sensing and measurement of biomolecules. We also discuss the use of nanoparticles for multimodal detection and in vivo molecular imaging. The recent developments in bioconjugated nanoparticle properties and preparation have highlighted the exciting potential of these agents for use in next generation biosensing, clinical diagnostics, and in vivo imaging.
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ioconjugated nanoparticles are of considerable current interest because materials on the nanometer scale have unique optical, electronic, and magnetic properties with broad applications in biosensing, molecular imaging, and medical diagnostics.1−10 In particular, recent advances have led to the development of nanomaterials for a variety of applications, including iron oxide nanocrystals for magnetic resonance imaging (MRI),11,12 fluorescent quantum dots (QDs) for multiplexed molecular diagnosis and in vivo imaging,13−17 and gold nanoparticles for surface-enhanced Raman scattering.18−20 A key factor is that materials in the nanoscale size range have unique structural and functional properties that are not available from bulk materials or small molecules, which make them ideal probes for many applications.1−3 In addition, coupling nanoparticles to bioaffinity ligands such as small organic compounds, proteins, or oligonucleotides enables their use in high sensitivity biomolecule assays or in vivo molecular imaging.21−23 Here, we discuss recent advances in the field of nanotechnology for applications in biosensing, molecular imaging, and biodiagnostics. We outline innovations made in surface coatings and coupling methodologies to prepare biocompatible nanoprobes capable of performing in complex biological environments and detail various detection modalities © 2016 American Chemical Society
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SURFACE COATING AND BIOCONJUGATION Surface Coating. Crystalline nanoparticles are typically incompatible with aqueous solutions and must be coated with a material that can stabilize the particles and render them watersoluble. Extensive research over the last few decades has led to many approaches to coating nanoparticles for use in biomedical applications. Initial successes were achieved with small molecules capable of coordinating the nanoparticle surface and presenting a polar functional group for water solubility. Historical work by Turkevich et al.30 and Frens31 developed methods for synthesizing and stabilizing gold nanoparticles Published: December 19, 2016 1015
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Figure 1. Tunable properties of nanomaterials. (a) Image of vials containing gold nanorods of increasing aspect ratio (left to right). (b) Absorbance spectra of gold nanorods showing the size-tunable nature of the surface plasmon resonance, with higher aspect ratios resulting in a shift in the absorbance peak into the near-infrared. (c) Fluorescence image showing vials containing CdTe semiconductor quantum dots with the particle sizes increasing from left to right. (d) Fluorescence spectra of quantum dot nanoparticles, showing a red shift of the emission peak as the size of the particles increases. Adapted from Kim, F.; Song, J. H.; Yang, P. J. Am. Chem. Soc. 2002, 124 (48), 14316−14317 (ref 27), Copyright 2002 American Chemical Society, and Kairdolf, B. A.; Smith, A. M.; Nie, S. J. Am. Chem. Soc. 2008, 130 (39), 12866−12867 (ref 25), Copyright 2008 American Chemical Society.
often bind nonspecifically to a variety of biomolecules and structures, including nucleic acids, phospholipids, proteins, cellular membranes, or extracellular matrix components.39−42 In complex biological environments, proteins in solution compete to bind the nanoparticle surface and lead to the formation of a protein corona that surrounds the nanoparticle.43−48 The nonspecific binding of biomolecules to the surface of a nanoparticle can significantly affect its properties (such as its hydrodynamic size) and can limit its targeting specificity and detection sensitivity. In diagnostic assays of tissues, cells, or fluids, this nonspecific binding can lead to false positive biomarker detection. Studies have also shown that the cellular association and uptake of nanoparticles is significantly affected by the properties and makeup of their protein corona.47,49,50 Recent work by Chan and co-workers has demonstrated that a quantitative model based on a nanoparticle’s protein corona fingerprint can be developed to predict its biological interactions more accurately than models using other properties of the nanoparticle.51 Many studies have shown that nanoparticles with highly positive or negative charges exhibit strong nonspecific binding to cells and tissues as well as soluble molecules in biological fluids.39−41 This is not surprising because biomolecules are charged or contain charged domains52 that can bind to oppositely charged nanoparticles through electrostatic attraction. This presents a major challenge because nanoparticles are often designed with highly charged surfaces to confer water solubility and provide stability against aggregation through electrostatic repulsion. A number of strategies have been employed to combat this problem and reduce nonspecific binding. One method that has been explored is to reduce the charge associated with the nanoparticle surface. Kairdolf et al. cross-linked carboxylic acid functional groups on the nanoparticle polymer coating using a hydroxyl-containing small molecule to yield nanoparticles with a relatively neutral hydroxyl-covered surface.42 These modified particles demonstrated extremely low nonspecific binding to proteins in solution and whole cells while maintaining high colloidal stability and a small size.
using reduction by citrate, which also binds the surface and stabilizes the particles in aqueous solution. Small molecules such as mercaptoacetic acid were also exploited by Chan and Nie to transfer semiconductor quantum dots to water for cell labeling applications.32 However, the stability of nanoparticles coated with small molecules is low over long time periods, and they are particularly susceptible to aggregation when exposed to complex biological solutions. Later methods using polymeric coatings or lipids proved more stable and provided superior performance and protection of the nanoparticles against degradation.14,29,33 However, the thickness of the polymer coating can dramatically increase the size of the nanoparticles,34 rendering them unsuitable for applications where small size is critical. In recent years, novel surface coatings have been developed to address the issues related to nanoparticle size and the reduced stability of small molecule coatings. Multidentate small molecule ligands which are capable of binding the surface of the nanoparticle at multiple points have been successfully used to increase the coating stability while maintaining a small size.35,36 Smith and co-workers have taken that concept further and developed multidentate polymer ligands to maintain a small hydrodynamic size while capitalizing on the benefits of polymeric nanoparticle coatings.37,38 These ligands consist of a linear polymer with chemical groups to bind the nanoparticle surface, provide aqueous solubility, and provide bioconjugation sites for coupling functional molecules such as targeting ligands. The polymers tightly bind the nanoparticle surface in a closed “loops-and-trains” conformation to eliminate any extraneous organic barrier between the nanoparticle and aqueous solvent, resulting in an extremely thin polymer shell. Recent advances in this concept have further optimized the preparation methods to reduce nanoparticle aggregation and polymer cross-linking between nanoparticles.37 These strategies have resulted in the synthesis of nanoparticles with a hydrodynamic size of 6 nm or less, ideal for a number of in vitro and in vivo biomedical applications. Nonspecific Binding. A major problem associated with the use of nanoparticles in biomedical applications is that they 1016
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Figure 2. Schematic illustration of stoichiometric nanoparticle probes for massively multiplexed molecular profiling in cells and tissues. (a) By relying on protein A as an intermediate coupling molecule, antibodies can be bound to the nanoparticle surface in a defined orientation without interfering with the binding site. This technology also allows destaining, washing, regeneration, and restaining to dramatically increase the number of unique biomarkers that can be analyzed (b−f). Reprinted with permission from Macmillan Publishers Ltd.: Nature Communications (ref 76) copyright 2013.
were used to fill in gaps between the biofunctionalized PEG molecules on the surface of 50 nm gold nanoparticles. Backfilling with additional PEG resulted in a reduction of the protein corona and restoration of targeted binding of the nanoparticles, which was lost in control samples without PEG backfilling. An important finding is that the polymer length of the PEG used for backfilling on the nanoparticle surface must be less than the length of the targeting molecule linker to prevent interference with specific receptor−ligand binding. Bioconjugation. Nanoparticles alone serve as excellent tools for producing high contrast signals but require the conjugation of a functional molecule for most sensing, diagnostic, or molecular imaging applications. Because of the highly versatile surface chemistries available for nanoparticles, many bioconjugation strategies have been utilized to couple them to functional molecules. Covalent bonding through the amine groups present on biomolecules using amide chemistry has been widely exploited to permanently attach proteins, oligonucleotides, and small molecules to the surface of nanoparticles.58 Malemide groups have also been used to couple nanoparticles to functional molecules containing a thiol group. This chemistry is particularly convenient for bioconjugation to antibodies, which have thiols outside of the affinity site so that coupling to nanoparticles does not disrupt specific binding to the targeted molecule.59,60 Coupling nanoparticles to biomolecules using an intermediary affinity group, such as biotin−streptavidin,14,61,62 or electrostatic interactions with highly charged biomolecules, such as oligonucleotides,63−69 can also be used to produce versatile nanomaterials. More recently for crystalline nanoparticles, techniques for coupling functional molecules directly to the crystalline surface have received attention. Thiol functional groups have been routinely used in the bioconjugation of gold nanoparticles due to their strong interaction with the surface atoms, and this method has resulted in a number of functionalized gold nanoparticle probes, particularly using thiol-modified oligonucleotides.70,71
Polyethylene glycol (PEG) has also been extensively used to coat the surface of nanoparticles to reduce charge and nonspecific binding and is the most common material used for nanoparticles in biomedical applications.14,39 PEGylated nanoparticles exhibit a nearly neutral surface charge and rely on the conformational flexibility and steric repulsion of the PEG chains on the particle surface to maintain colloidal stability in solution and reduce the adsorption of soluble proteins. Early work by Rosenthal and co-workers showed that coating the nanoparticle surface with PEG chains as short as 550 Da can significantly reduce the nonspecific binding to cells,39 and additional studies have proven the utility of PEGylation for nanoparticles in both in vitro14,39 and in vivo17,20,53,54 applications. Additional studies have closely examined the effects of PEG functionalization on protein adsorption, cellular uptake, and the conformation of the PEG molecules on the nanoparticle surface. Pelaz et al. recently found that PEGylation with PEG chains between 750 and 10 000 Da significantly reduced cellular uptake in comparison with bare nanoparticles, but some protein adsorption still occurred.55 Interestingly, the nanoparticle size increase upon PEGylation was much smaller than the theoretical size increase based on the length of a fully stretched PEG polymer, indicating that the PEG density was low enough for the polymers to take a mushroom-like conformation rather than a brush conformation and allowed proteins to become buried within the PEG shell. This corroborates work by Lai and co-workers who observed that PEG densities exceeding that required for the polymers to adopt a brush-like conformation had increased resistance to uptake by macrophages and increased blood circulation times compared with uncoated nanoparticles and less densely PEGylated particles.56 The importance of complete PEG coating was also demonstrated by Dai et al. with their investigations into the influence of PEG backfilling to mitigate the complications associated with the formation of a protein corona around nanoparticles.57 In this study, short PEG chains 1017
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The preparation of thin and stable coatings has also allowed the use of His-tags for directly coupling biomolecules to the surface of a quantum dot in a defined orientation for optimal target recognition.37,72−74 These short peptide tags can be fused onto recombinant proteins and have shown a strong affinity to charged metal atoms such as Ni2+ or Zn2+. Early studies showed the utility of this approach with nanoparticles coated using small molecule ligands where the nanocrystal surface is easily accessible.4,74,75 Surprisingly, this bioconjugation strategy has recently been shown to be effective with multidentate polymer coatings as well.37 A major challenge for preparing highly efficient nanoparticle probes is that many of the techniques described above involve random chemical processes, leading to the conjugation of biomolecules in orientations that are not ideal for binding a target. In addition, there is very little control over the number of bioconjugated molecules on the nanoparticle surface.6 This heterogeneity can have a significant impact on the avidity of individual nanoparticle probes and introduces variability for bioanalytical assays and molecular imaging. To address these issues, Gao and co-workers have developed stoichiometric fluorescent probes by linking target-specific antibodies to a universal quantum dot-based platform via protein A (see Figure 2) and have achieved great success in massively multiplexed profiling of single tumor cells.76,77 Also, Ting and co-workers have engineered monovalent avidin and bioaffinity tags for specific targeting of nanoparticle probes to cell surfaces78,79 Overall, more research is still needed to develop techniques to fully control both the loading density and orientation of biomolecules on the surface of nanoparticles.
Despite their many advantages, certain properties of quantum dots have restricted their use in some applications. A limiting factor in dynamic molecular imaging is that the fluorescence of a single quantum dot fluctuates in an intermittent on−off pattern called blinking, where the nanoparticle can be nonfluorescent for many seconds. This presents a major challenge for tracking molecules that are moving because the loss of signal means molecules cannot be tracked for long periods with complete certainty, particularly in a complex, heterogeneous environment with many moving particles. Efforts have been made to engineer the nanomaterials or surface chemistry in order to suppress or eliminate the blinking state of the quantum dots to enable their use in realtime single molecule detection and tracking. Surface passivation using an excess of small thiol moieties was shown to dramatically reduce quantum dot blinking.97 Other approaches, such as growing a “giant”, high bandgap shell around the quantum dot core, have also shown blinking suppression.98,99 Recently, Dubertret and co-workers devised a hybrid nanoparticle scheme to grow a large silica shell surrounding the quantum dot and deposit a gold nanoshell onto the silica.100 Despite the improvement in optical properties, these nanoparticles are not ideal for tracking dynamic cellular events at the molecular level because they can introduce toxic components or have an increased size which can hinder their mobility in a complex cellular environment. Lane et al. have addressed these issues by using a linearly graded alloy shell on small CdSe cores via a layer-by-layer process, which drastically suppressed the fluorescence blinking and allowed reduction of the nanoparticle size to approximately 10 nm (see Figure 3).101 Another challenge for quantitative, multiplexed biological assays using quantum dots is the disparity in fluorescence brightness observed between particles with different emission peak wavelengths. This is primarily due to the differences in extinction coefficients that arise from size-tuning, which scales with the volume of the particles and results in larger (redder) particles having significantly higher fluorescence than smaller (bluer) particles. This presents a problem for multiplexed molecular imaging because the signals from larger particles can overwhelm signals from smaller particles and the dynamic range of the imaging system cannot be fully exploited. Recent efforts to develop “brightness-equalized” quantum dots have resulted in nanoparticles with closely matched fluorescence intensity over a broad wavelength range. These next generation probes utilize a ternary or quaternary alloy core rather than the more conventional binary cores, which allows the bandgaps to be continuously tuned (from 500 to 800 nm) without changing the size of the nanocrystals and significantly reduces the differences in extinction coefficients.102 One of the major limitations of semiconductor quantum dots for in vivo applications is that they typically contain toxic heavy metals such as cadmium, mercury, or lead. Studies investigating the short- and long-term toxicology of quantum dots have produced mixed results,103 and there is not yet enough evidence to justify the in vivo use of these nanoparticles in human patients given their toxic components. Because of this challenge, another class of nanoparticles called carbon dots has seen considerable interest for fluorescence applications. These are carbon-based nanomaterials with a semispherical shape and exhibit quantum effects at sizes less than 10 nm.104,105 Like semiconductor quantum dots, these nanomaterials show sizedependent optical properties and have a strong resistance to photobleaching, making them useful for many biomedical
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DETECTION MODALITIES Fluorescence. Fluorescence emission is a mainstay in bioanalytical techniques as well as diagnostic assays and molecular imaging. Fluorescence detection and imaging has opened new avenues for the measurement and understanding of biological processes at the macroscopic and microscopic level and has dramatically expanded the toolkit for both clinicians and basic science researchers. Nanotechnology has shown tremendous promise to push the field even further due to the fluorescence properties of many types of nanoparticles. Most notably, semiconductor quantum dots have proven to be an important class of fluorescent probes that offers optical properties that are not available from conventional fluorescent dyes.58 By changing the size, chemical composition, or internal strain of the semiconductor nanocrystal, the band gap and fluorescence emission wavelength of the nanoparticle can be continuously tuned from the visible light range into the nearinfrared,2 allowing the synthesis of nearly identical probes for multiplexed assays using a variety of wavelengths. Quantum dots also offer optical properties that are superior to many organic dyes, including narrow and symmetric emission spectra as well as broad absorption profiles that allow them to be excited over a wide range of wavelengths. In addition, high quality quantum dots are extremely resistant to photobleaching and exhibit quantum yields approaching 100%, making them ideal probes for applications requiring ultrahigh sensitivity and stable fluorescent signals. Because of these properties, semiconductor quantum dots have seen use in a variety of bioassays and molecular imaging settings, including molecular profiling of cancer tissue specimens,15,16,80−82 detection of biomolecules or single virus particles in biological fluids,83−85 dynamic cellular imaging,86−92 and in vivo molecular imaging.17,93−96 1018
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amplification on the order of 1014 to 1015.116 Amplifications of this magnitude have allowed single molecule detection and the preparation of nanoparticle probes with ultrahigh sensitivity for many applications.20,117−120 SERS-based nanoparticle probes offer a number of advantages over other methods for bioassays and molecular bioimaging.115 The peak width of Raman signals is typically 1−2 nm (full width at half-maximum), which is 10 times more narrow than high quality quantum dot peaks and makes multiplexed imaging and detection much easier. In addition, SERS probes produce strong signals in the nearinfrared using 630 or 785 nm laser excitation, an ideal wavelength range for use in tissues where high light scattering and autofluorescence in the visible light range is a limiting factor for many optical probes. Another major advantage for in vivo bioimaging using SERS-active nanoparticles is that they are typically made from gold, which is chemically inert and expected to be nontoxic. Due to these properties, a number of groups have developed high quality SERS-active probes using spherical gold nanoparticles. While these spherical particles are easy to prepare and have proved to be capable probes for bioanalytical work, they typically have surface plasmon peaks between 500 and 600 nm, which is not optimized for enhancement using near-infrared laser excitation. A number of nonspherical nanoparticle shapes have been recently developed to shift the surface plasmon resonance into the near-infrared window to provide even greater field enhancement and yield higher Raman signal intensity, including nanorods121−124 and nanoprisms,125,126 among others.115 One challenge moving forward is that these unique nanoparticle shapes are often synthesized using surfactants that tightly bind their surface, which hinders the application of PEG coatings for biocompatibility and stability. Multiple strategies, such as using a citrate coating as an intermediate,127 have been proposed but further research and optimization is needed to take full advantage of the superior optical properties of these nanomaterials for biomedical applications. Magnetic. Magnetic resonance imaging (MRI) is a widely used technique for high resolution imaging in the clinic. A key advantage of MRI over optical techniques such as fluorescence or Raman is that the signal is not limited by tissue scattering, allowing the imaging of areas deep inside the body. In addition, a tremendous amount of information can be obtained using native signals without exogenous contrast agents. However, the sensitivity can be quite low, and targeted contrast agents are required to achieve molecular imaging. Magnetic nanoparticles have shown great promise for targeted molecular imaging using MRI.128,129 Most notably, superparamagnetic iron oxide (SPIO) nanoparticles have been shown to exhibit a reduction in the T2 signal of tissues and have received FDA approval for use in humans in some clinical applications. Early synthetic methods were performed using aqueous solutions with iron salts and dextran, resulting in water-soluble iron oxide nanoparticles with a polysaccharide coating.130−132 Later preparation methods using high temperature organic solvents produced highly crystalline and uniform nanoparticles with low polydispersity.133−136 However, these nanoparticles as prepared were stabilized with hydrophobic ligands and require polymer29,137 or lipid138,139 coatings for use in aqueous environments. Portions of these coatings intercalate between the hydrophobic domains of the nanoparticle’s surface ligands, leaving a thick, hydrophobic layer between the nanocrystal surface and the polar portion of the coating that confers
Figure 3. Traditional (blinking) and linking-suppressed quantum dots for single-particle tracking. (a) Tracking the trajectories of a nonblinking gradient shell quantum dot (GS-QD) compared with a commercially available quantum dot with an abrubt shell (AS-QD). Traditional quantum dots experience significant fluorescent intensity fluctuations and can have prolonged “off” times, leading to a series of blackout frames and disappearance of the signal. Conversely, gradient shell quantum dots emit uniform fluorescence signal without “off” times and allow real-time single tracking for long periods. Reproduced from Lane, L. A.; Smith, A. M.; Lian, T.; Nie, S. J. Phys. Chem. B 2014, 118 (49), 14140−14147 (ref 101). Copyright 2014 American Chemical Society.
applications.106,107 While the toxicological profile of carbon dots has not yet been fully studied, their chemical composition (primarily carbon with low amounts of oxygen, hydrogen, and nitrogen) is inert and initial toxicity studies have been promising.108−110 Recent efforts have focused on their adaptation for in vivo bioimaging and a variety of bioassays111−113 as well as fundamental material science studies to better understand the origin carbon dot fluorescence, which is still open for debate.114 The elucidation of this mechanism is a critical step toward optimizing the synthesis and properties of carbon dots and engineering them for in vivo bioimaging. Raman. With the recent developments of high quality, biocompatible nanoparticles, and an improved understanding of the fundamentals underlying Raman scattering and its enhancement, Raman-active nanoprobes have received considerable attention for their use in biomedical applications.115 Inelastic Raman scattering is a relatively rare optical event (approximately 1 in 10 million), with the majority of scattered photons undergoing elastic (Rayleigh) scattering. Due to this infrequent occurrence, the amplification of Raman scattering intensities is necessary for highly sensitive detection. Metallic nanoparticles (e.g., gold, silver) have been shown to dramatically increase the Raman scattering signal through a process referred to as surface-enhanced Raman scattering (SERS), with Raman signal 1019
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can be included to function as a core and drive self-assembly into nanoparticles. Biodegradable dendritic nanoparticles have also been developed for PET imaging using 76Br as the PETactive tracer.154 With the attachment of RGD targeting moieties, these nanoparticles showed a 6-fold increase in αvβ3 receptor-mediated endocytosis and increased accumulation in an in vivo mouse model at sites of angiogenesis compared with nontargeted nanoparticle probes. Other nanoparticle structures that have seen use in PET imaging include carbon nanotubes154,155 and liposomes.156−159 Often used together with PET, CT is a bioimaging technique which creates a 3-dimensional image using a series of X-ray images from multiple angles to reveal details regarding the anatomic features of tissues of interest. Imaging agents containing atoms with a high atomic number (such as iodine or barium) are often used to provide contrast in soft tissues due to their higher X-ray absorption.160 Bismuth sulfide nanoparticles with a polymer coating were shown to exhibit good contrast in CT imaging (5-fold higher than iodine), long circulation times, and a safety profile comparable to iodinated imaging agents in animal models.161 However, the shape and size of the nanoparticles were polydisperse, and improvements are needed in the coating for functionalization with targeting moieties. Subsequently, gold nanoparticles were explored for their efficacy in CT contrast.162−164 The X-ray absorption coefficient of gold is approximately 2.5-fold higher than iodine, and gold nanoparticle synthesis techniques have been extensively studied and refined. In addition, biocompatible coating methodologies have been developed to render gold nanoparticles extremely stable and provide multiple options for biofunctionalization to generate targeted probes for molecular imaging. By coupling gold nanoparticles with an antibody targeted to the A9 antigen, which is overexpressed in head and neck cancer cells, Popovtzer et al. were able to demonstrate cancer detection at the cellular and molecular level for the first time using a standard clinical CT instrument.165 More recently, polymeric micelles loaded with small gold nanoparticles have been shown to exhibit strong contrast for CT imaging.166 Using this scheme, large nanoparticles with long blood circulation times can be produced and engineered to degrade into small components that can then be cleared through renal filtration. While efforts have been made to develop both PET and CT nanoparticle agents for single modality use in vivo, the properties of the nanoparticles and the nature of PET and CT imaging make them naturally adaptable for multimodality imaging. Multimodality. Despite continued improvements in the technologies and instrumentation associated with various imaging modalities, no imaging method is perfect and all suffer from limitations. Some factors are likely to be alleviated through technological advancements, but other limitations are a function of the basic biology and physics, making it difficult or impossible in some cases to engineer a solution. In addition, each modality provides unique information about a patient. With recent innovations in nanoparticle technologies, many groups have worked to develop multifunctional or multimodal nanomaterials that enable imaging using more than one modality, allowing clinicians to exploit the advantages of each and obtain as much patient information as possible while minimizing limiting factors. Superparamagnetic iron oxide nanoparticles have been widely used as a basis for constructing multimodal nanoparticles due to the inherent magnetic properties of the
solubility in aqueous solutions. While this provides excellent protection against degradation, it has been shown that a thick hydrophobic intermediate layer significantly reduces the relaxivity of the iron oxide nanoparticles.29 Initial clinical work with liver cancer exploited the rapid phagocytosis of nontargeted SPIO particles by Kupffer cells for imaging in the liver parenchyma, which reduced T2 signal in normal liver and provided contrast for tumor regions that did not contain Kupffer cells.140,141 SPIOs were also used to detect metastatic lymph nodes in prostate cancer.142 In a clinical trial of 80 patients, contrast-enhanced MRI using SPIOs accurately identified all patients with nodal metastases, including a large percentage that did not meet the normal imaging criteria to identify malignancy. While these successes have excited the field about the future of nanomedicine for in vivo clinical applications, many of the SPIO nanoparticles which have been FDA approved are no longer commercially available, primarily due to business-related issues. Polymeric nanoparticles containing gadolinium(III) as an MRI contrast agent have also received attention recently.143,144 Small molecule gadolinium chelates have been extensively developed and tested for biomedical imaging over the last few decades. However, the low sensitivity of MRI makes molecular imaging with small molecule gadolinium contrast agents challenging. Formulating gadolinium into nanoparticles significantly increases the signal for molecular imaging due to the presence of multiple gadolinium ions in a single particle and an enhancement due to the geometric confinement of the gadolinium, which influences its paramagnetic behavior.145 In addition, the nanoparticle size and surface chemistry can be altered to tune the blood circulation time and biodistribution, as macromolecules and nanoparticles are known to passively accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect.146 Efforts have also been made to develop small molecule gadolinium agents that spontaneously bind to human serum albumin.147,148 These agents exhibit many of the same pharmacokinetic properties seen in more traditional nanoparticle agents because albumin is a globular protein with a size of approximately 5 nm. One such agent, gadofosveset trisodium, is FDA approved and has been used in a number of clinical imaging settings.149−151 PET and CT. Positron emission tomography (PET) and computed tomography (CT) are two imaging modalities that are commonly used in disease diagnoses and are often combined to provide clinicians with detailed information regarding abnormal biological activity and anatomical features. PET relies on radioactive tracers injected into the body, which can then be measured and imaged in three dimensions with a scanner. A number of small molecules are commonly used to provide contrast in PET imaging, including 18F-fluorodeoxyglucose (FDG), which is most common. FDG is a glucose analog which is rapidly taken up by tissues with high rates of metabolism and is used to image diseases where metabolic rates may be abnormal (e.g., cancer). Nanoparticles have the potential to greatly extend the utility of this technology by providing a highly engineered structure for the attachment of radionuclides and targeting moieties for molecular imaging.152 The physical properties of the nanoparticle can also be tuned to optimize the probes behavior in vivo.153 These nanoparticles often consist of linear block copolymers with a polyethylene glycol block for biocompatibility and reduction of nonspecific adhesion and a block with functional groups for chelating a PET-active tracer like 64Cu. In addition, a hydrophobic block 1020
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information for a better understanding of basic biological processes that is not currently possible using existing methods. The high sensitivity and unique properties of nanomaterials allows detection at the single molecule level, real-time tracking of single biomolecules, and in vivo molecular imaging. Already, novel nanoparticle probes have served as excellent tools for research into the understanding of cells and biomolecules. Dynamic Cellular Imaging. Extreme brightness, high photostability, and tunable fluorescence emission have made semiconductor quantum dots an ideal probe for dynamic imaging within live cells.58,177 In addition, the long-term in vivo toxicity concerns regarding these nanoparticles are not an issue for short-term experiments in vitro, where toxicity is not a major problem. Quantum dot nanoparticles have been used to elucidate a variety of biological phenomena in real time, including single receptor diffusion on the cellular membrane, tracking of individual kinesin motors, and receptor internalization and trafficking.87,89,91,178 For example, single nanoparticle tracking was used to study growth factor-activated Trk receptor dynamics and trafficking in neuronal soma.179 Neurons have complex cell membranes and exhibit dynamic receptor behavior for the proper functioning of synapses. Previous nanoparticle studies have shown that neurotransmitter receptor diffusion undergoes rapid fluctuations in different membrane regions.87,92 In this recent study, quantum dot conjugates showed that the trafficking of brain-derived neurotrophic factor (BDNF)-Trk receptor complexes exhibit diverse heterogeneity and little time-synchrony. In addition, the trajectories of individual complexes appeared to be random and meandering with no apparent end destination which stands in contrast to the reported linear nature of axonal transport data. Wang et al. used single nanoparticle tracking in live cells to study the membrane nanotube-mediated transport of biomolecules between individual cells.180 Quantum dots were coupled to wheat germ agglutinin and imaged in real time. The single particle tracking data shows their transport to be in a directed fashion, and it is hypothesized that the trafficking is driven by myosin molecular motors, rather than kinesin or dynein motors, based on the tracking data. Single nanoparticle tracking techniques have also been used in more complex environments. Biermann et al. demonstrated the feasibility of tracking individual nanoparticles in organotypic brain slice cultures to study the molecular dynamics of lipids and transmembrane proteins in correlation to synaptic membrane compartments.181 This technique enabled the verification of previous data obtained from primary neuronal cultures and allowed the monitoring of individual biomolecule diffusion within physiologically relevant time frames. Studies have shown that nanoparticle contrast agents can be used to detect and image larger constructs, such as individual virus particles. Agrawal et al. developed a method for the detection of virus particles based on fluorescent nanobeads or semiconductor nanoparticles.182 It has also been shown that fluorescent nanoparticles can be used to label and track individual viruses. Liu et al. tracked the entry of infectious hematopoietic necrosis virus (IHNV) into living cells.183 Biotin was introduced onto the surface of the virus particles, and they were labeled with streptavidin-conjugated quantum dots. Live cell fluorescence microscopy revealed that IHVN binds to the host cell membrane and is internalized through clathrin-coated pits. The results also showed that the viruses are transported within endocytic vesicles along the cellular cytoskeleton and that infectivity was dramatically reduced when this transport
nanocrystal core and the diverse chemistries available from organic coatings for functionalization. Functionalization of biocompatible iron oxide nanoparticles with small molecule organic dyes has led to the development of a class of nanoparticles with activity for both fluorescence and MRI.167−169 Near infrared fluorescent dyes, such as Cy5.5, are often used because of the increased optical penetration depth in tissues compared with dyes emitting fluorescence in the visible light range. These two imaging modalities provide an excellent complement to each other, as MRI provides detailed anatomic information and imaging contrast without depth penetration issues while targeted fluorescence imaging allows sensitive molecular detection and real-time visualization. This dual-modal imaging technique was recently used by Zhang et al. to perform fluorescence molecular tomography and MRI imaging in animal tumor models.170 Additional modifications have included the incorporation of therapeutic compounds such as taxol to create theranostic nanoparticles that are capable of imaging as well as treatment of disease.171 Other multimodal nanoparticles have incorporated fluorescent dyes and use gadolinium as the MRI contrast agent rather than iron oxide to obtain contrast in T1 imaging.172 Nam et al. developed self-assembled nanoparticles from biocompatible chitosan polymers that were functionalized with both nearinfrared fluorescent dyes (Cy5.5) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to chelate gadolinium(III).172 These nanoparticles showed excellent accumulation in the tumors of mice models and produced contrast under both fluorescence and MRI imaging. Nguyen et al. developed a similar strategy for fluorescence/MRI imaging using a generation 5 poly(amidoamine) dendrimer as the nanoparticle construct.173 The single nanoparticle agent enabled preopertative MRI for tumor detection and real-time intraoperative fluorescence guidance for detecting the tumor margins during surgical resection in animal models. Iron oxide nanoparticles have also been functionalized with radionuclides to produce PET/MRI-active dual modality nanoparticles. These nanoconstructs are similar in design to iron oxide nanoparticle-based MRI/Fluorescence probes with functional groups capable of chelating radioactive agents, such as 64Cu.174,175 More recently, other radiotracers have also been used. Thorek et al. have developed a multimodal PET/MRI nanoparticle probe using a clinical iron oxide nanoparticle formulation called ferumoxytol with desferrioxamine chelators and 89Zr as the radiotracer.176 These nanoparticles were successfully used to map lymph node drainage in preclinical animal models, including nodal drainage in deep tissue which is not possible with other imaging techniques. Taking this concept even further, Xie and co-workers recently added fluorescence capabilities to develop PET/NIRF/MRI trimodal nanoparticles using iron oxide as the core with a human serum albumin coating.175 These nanoparticles were dual labeled with 64 Cu and Cy5.5 to allow the use of three different imaging modalities. In this preliminary study, the multimodal approach was utilized to carefully investigate the in vivo fate of the albumin-coated nanoparticles in animal models. However, the versatility of this approach and the wealth of information provided could have future clinical implications for advanced disease imaging as well as theranostics.
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CELLULAR AND MOLECULAR IMAGING Nanoparticle imaging agents have the potential to revolutionize the cellular and molecular imaging fields and provide new 1021
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Figure 4. Surface-enhanced Raman scattering for in vivo molecular imaging. (a) Preparation of SERS nanoprobes consisting of a gold nanoparticle core, adsorbed Raman reporter molecules (green stars), biocompatible surface coating (pink layer), and ligands for molecular targeting. SERS spectra from the tumor and liver show specific accumulation of EGFR-targeted nanoparticles in cancerous tissue (b) while nontargeted nanoparticles accumulate primarily in nontargeted organs (c). (d) Photographs showing experimental setup for in vivo spectroscopic detection of SERS nanoparticles in mouse models. Adapted from Lane, L. A.; Qian, X.; Nie, S. Chem. Rev. 2015, 115 (19), 10489−10529 (ref 115), Copyright 2015 American Chemical Society, and adapted with permission from Macmillan Publishers Ltd.: Nat. Biotechnol. (ref 20) copyright 2008.
pathway was disrupted or when the acidification of the endosome was inhibited. More recent studies have further explored the microtubule-dependent behavior of viruses using nanoparticles to visualize virus movement within a live cell. The nanoparticles were again coupled with streptavidin and used to label biotinylated viruses.184 An interesting finding was that microtubule geometry within the cell has a significant influence on virus transport, where the intersection configuration of microtubules can interfere with intracellular transport and lead to long-time cessation of virus movement. The results also showed the rapid and directed transport of viruses from the cell periphery toward the microtubule organizing center (MTOC). This directed motion mirrors previous results from Ruan et al., who used peptide-conjugated QDs which were tethered to the inner surface of endocytic vesicles and exhibited similar directed motion toward the MTOC along microtubules.91 While the advancement of nanoparticle design for biomedical applications has led to a rapid increase in their adaptation for cellular imaging, a number of challenges remain for their widespread use. A major issue is the delivery of nanoparticles that are monodispersed and free to diffuse within the complex cellular environment. As described above, nanoparticles delivered to cells through endocytosis often remain trapped inside vesicles and are transported to the MTOC rather than freely diffusing within the cytoplasm.91 Manually injecting nanoparticles into cells using microneedles has proven to be effective but is extremely laborious.185 To increase throughput, biochemical or electrical methods have been used to temporarily permeabilize cell membranes of living cells in order for nanoparticle imaging agents to diffuse in. Nanoparticle surface coatings have also been engineered to induce transport across the cell membrane.91,186,187 These methods have been used successfully in many studies but can lead to aggregation, trapping within vesicles, or unwanted transport to specific domains within the cell. Hydrodynamic size is also a contributing factor to impeding the free diffusion of nanoparticles within cells and their use for labeling individual biomolecules. In addition to the size difference of large
nanoparticles compared with small biomolecules (where the significant size increase of a nanoparticle−molecule complex could alter the molecule’s native movement and function), an increase in hydrodynamic size leads to a corresponding decrease in free diffusion within the complex environment of a cell’s cytoplasm. Efforts have been made to address these issues through the development of ultrathin coatings, new synthetic procedures that allow tight control of particle size, and novel materials with optimized properties for ultrasmall nanoparticles. In Vivo Imaging and Spectroscopy. In vivo diagnostic techniques have revolutionized medicine by improving a physician’s ability to detect and diagnosis disease as well as revealing internal anatomic features to guide treatment decisions. Nanoparticle probes have an important role to play in a variety of different imaging modalities and can serve as contrast agents and molecular imaging probes for highly sensitive clinical imaging. To this end, nanoparticle probes have been used as imaging agents for MRI, PET, CT, and optical modalities to provide better contrast or target specific molecular markers that are not visible using standard techniques. Magnetic nanoparticles (such as Feridex and ferumoxytol) have been the most widely used for human clinical applications, and a number have been approved by the FDA for imaging contrast. Initial applications have relied on passive mechanisms for tissue contrast rather than molecular targeting with bioaffinity ligands. These nontargeted nanoparticles were shown to be phagocytosed by macrophages, and this phenomena was utilized to provide MRI contrast in liver cancer imaging.140,141 More recently, macrophage phagocytosis has also been exploited to image pancreatic islet inflammation in type 1A diabetes patients.188 Ferumoxtran-10 nanoparticles were infused into patients and allowed noninvasive MRI imaging of the pancreas and a distinction between recent-onset diabetes patients from nondiabetic control patients. Passive targeting can also be achieved in solid tumors using the enhanced permeability and retention (EPR) effect.189 This effect is a consequence of tumor biology, where rapidly growing 1022
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ultrahigh sensitivity could lead to contrast agents capable of identifying micrometastases that are not currently visible using existing clinical technologies. One major issue with typical SERS-active nanoparticles is their large size. These nanoparticle probes are generally over 60 nm in diameter and cannot be cleared from the body. Multiple studies have explored the effect of size on the renal clearance of macromolecules and nanoparticles.197,198 Choi et al. studied this effect in a mouse model using a size series of quantum dots, ranging in size from approximately 4 to 8 nm in diameter.198 They found that nanoparticles with a diameter of approximately 6 nm or less were able to undergo clearance through the urine. Because of their smaller size, tunable fluorescence signal, and the availability of fluorescence imaging systems, quantum dots have also received significant attention for in vivo imaging.3,94 Nie and co-workers first demonstrated in vivo cancer targeting and imaging with quantum dots in a mouse model.17 Polymercoated quantum dots functionalized with an antibody targeted to prostate specific membrane antigen (PSMA) were injected intravenously into a murine tumor model and successfully detected prostate cancer tissue under fluorescence imaging. Over the past ten years, quantum dots have been used in a variety of exciting imaging applications, including bioluminescence imaging with self-illuminating quantum dots,93 in vivo multiphoton fluorescence imaging,199 and tracking tumor cell extravasation.200 More recently, the use of quantum dots for intravital microscopy has also received attention. This technique enables the study of individual cells and their interactions in their native microenvironment rather than in culture on a Petri dish, which can dramatically alter a cell’s normal behavior. The multiplexing capabilities and bright, photostable signals make quantum dots an ideal probe for this application. Han et al. used quantum dot−antibody conjugates for single cell imaging and multiplexed cytometric analysis of rare cells in the bone marrow using intravital microscopy.201 These 15 nm particles were able to diffuse completely into the bone marrow and label rare populations of hematopoietic stem and progenitor cells (Sca1+c-Kit+ cells). Smith and co-workers were able to image the vasculature of breast cancer tumors via intravital multiphoton fluorescence microscopy and showed that green and red quantum dots fluorescence was capable of a depth penetration of 50 μm using an excitation wavelength of 780 nm.102 Despite these many advancements and the development of optically active nanoparticles with signal in the near-infrared, tissue scattering and absorption as well as native tissue autofluorescence remain a challenge and limit tissue penetration depth to between 1 and 2 cm. Previous studies suggested that it would be possible to further improve signal-tonoise in complex biological tissue by shifting the optical signals into a second window between 1000 and 1350 nm.202 Imaging in this second window has been hindered both by the lack of probes emitting signal in the appropriate wavelength range and the availability of cameras capable of sensitive detection at these wavelengths. With the cost of these cameras becoming more affordable and availability increasing, researchers have begun to explore the development of sensitive nanoparticle probes that emit fluorescence within the second near-infrared window. Dai and co-workers have prepared brightly fluorescent single-walled carbon nanotubes with emission between 1100 and 1700 nm.203 Using a phospholipid−polyethylene glycol coating for biocompatibility, the nanotubes demonstrated high signal-tonoise in a nude mouse model in the second near-infrared
tumors induce angiogenesis to meet the increased nutrient demands of the cells. This neovasculature is poorly formed and “leaky”, allowing nanoparticles in the blood to leak into the tumor tissue. In addition, tumors typically have poor lymphatic drainage, leading to an accumulation of nanoparticles in the tumor and providing a passive method for targeted imaging. This process has been widely used for the passive delivery of a variety of nanoparticles to many solid tumor types.190,191 Optically active nanoparticles have also been explored for in vivo spectroscopy and imaging applications but are still under development and are not yet FDA approved for use in the clinic. These contrast agents have shown excellent contrast and sensitivity for molecular imaging, and it is expected that they will significantly augment in vivo optical imaging in human patients. However, a major challenge for optical imaging in vivo is depth penetration because tissue strongly scatters and absorbs photons, particularly in the visible wavelength range. Tissue becomes more transparent as wavelengths shift to the near-infrared, with a “clear window” existing for wavelengths between 650 and 950 nm.192,193 As such, nanoparticles generating optical signals in this wavelength range have received increased focus and a number of nanoparticle probes have been developed for use in bioimaging applications. Surface-enhanced Raman scattering (SERS) nanoparticles are of particular interest because of their intense signal in the nearinfrared and their biocompatible and inert components. Qian et al. first demonstrated the potential of SERS-active gold nanoparticles in vivo using animal models and EGFR-targeted nanotags (see Figure 4).20 The SERS-active nanoparticles used highly stable PEG coatings to provide biocompatibility, reduce nonspecific binding, and trap the organic Raman reporter molecule on the gold surface. These nanoparticles were also significantly brighter than semiconductor quantum dots and showed targeted accumulation in xenograft tumor models after intravenous injection. Another advantage of SERS-nanoparticles is their adaptability for multiplexed imaging applications. Zavaleta et al. performed proof-of-concept studies to show the multiplexing capability of SERS-active nanoparticles in vivo.194 Ten different nanotags subcutaneously injected into a live mouse could be identified and spectrally separated. The five most intense and spectrally unique nanoparticles were then injected intravenously to image natural accumulation in the liver and were easily identified and spectrally separated using a noninvasive Raman imaging system. A later study by Dinish et al. confirmed the multiplexing potential of SERS nanoparticles by intratumoral injection of nanoprobes targeted to EGFR, CD44, and TGFβRII in a breast cancer mouse model.195 Interestingly, the targeted probes remained at the tumor site for 48 h compared with nontargeted nanoprobes which showed no signal after 6 h. More recently, surface-enhanced resonance Raman scattering (SERRS) nanoparticles were shown to have a sufficient signal enhancement to enable the detection of malignant lesions in genetically engineered mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma and in one human sarcoma xenograft model.196 The nanotags consisted of a 75 nm starshaped gold core with a resonant Raman reporter molecule (IR-780) and a silica coating and relied on passive accumulation from the EPR effect rather than specific molecular targeting. Moreover, the increased sensitivity of the SERRS nanoprobes (1.5 fM limit of detection) allowed the detection of premalignant lesions of pancreatic and prostatic neoplasias. These advancements in optical imaging nanoprobes and the 1023
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window, where tissue autofluorescence was nearly zero. More recently, a new generation of quantum dots with a Ag2S composition has been developed to further shift fluorescence emission between 1000 and 1350 nm.204−206 Hong et al. have shown that these biocompatible and heavy metal-free quantum dots can provide bright tumor contrast in vivo.207 The nontargeted Ag2S nanoparticles had an emission peak within the second near-infrared window (∼1200 nm) and accumulated in the tumors of a mouse model through the EPR effect. This new class of quantum dots has also shown utility for targeted cancer imaging in vivo. Achilefu and co-workers recently coupled Ag2S quantum dots with an integrin-targeting pentapeptide for specific tumor targeting.208 These targeted nanoparticles showed highly selective internalization in cancer cells compared with nontargeted controls. Interestingly, in vivo tumor models showed a biodistribution profile with exceptionally high accumulation in the tumor compared with the liver, which often captures nanoparticles nonspecifically. It is hypothesized that the small size of these quantum dots (hydrodynamic size
