Nanochemistry and Nanomedicine for Nanoparticle-based

Review pubs.acs.org/CR

Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy Guanying Chen,‡,† Indrajit Roy,†,§ Chunhui Yang,*,‡ and Paras N. Prasad*,† †

Institute for Lasers, Photonics, and Biophotonics and Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York 14260, United States ‡ School of Chemical Engineering and Technology, Harbin Institute of Technology, Harbin, Heilongjiang 150001, China § Department of Chemistry, University of Delhi, Delhi 110007, India 5.1.2. Nonlinear Optical Bioimaging 5.2. Magnetic Resonance Imaging 5.3. Radioisotope Imaging 5.4. CT Bioimaging 5.5. Multimodal Bioimaging 6. Nanoparticle-Based Biosensing 6.1. Fluorescence Sensors 6.1.1. Dye-Doped Nanoparticles 6.1.2. Quantum Dots 6.1.3. Molecular Beacon 6.1.4. Upconversion Nanoparticles 6.2. Plasmonic Sensors 6.2.1. Localized Surface Plasmon Resonance (LSPR) Colorimetric Sensors 6.2.2. Localized Surface Plasmon Resonance (LSPR) Fluorescence Sensors 6.2.3. Surface Enhanced Raman Scattering (SERS) Sensors 6.2.4. Nanomaterial-Enhanced Surface Plasmon Resonance (SPR) Sensors 7. Nanoparticle-Based Therapy and Controlled Release 7.1. Nanopharmacotherapy 7.1.1. Dispersibility and Protection in Vivo 7.1.2. Targeted Delivery 7.1.3. Controlled Release 7.2. Nanotherapies 7.2.1. Drug Delivery 7.2.2. Photodynamic Therapy (PDT) 7.3. Photothermal Therapy (PTT) 7.4. Magnetocytolytic Therapy 7.5. Antimicrobial Therapies 8. Nanoparticle Based Gene Therapy 8.1. Introduction to Gene Therapy 8.2. Challenges in Gene Therapy 8.3. Various Nanoparticles in Gene Therapy 9. Nanotoxicity and Toxicokinetics 9.1. In Vitro Toxicity 9.1.1. Oxidative Stress 9.1.2. Genotoxicity 9.1.3. Immunogenicity 9.1.4. Apoptosis and Necrosis 9.1.5. Biofouling 9.2. In Vivo Toxicity

CONTENTS 1. Introduction 2. Nanoparticle Formulations for Biomedical Applications 2.1. Advantages of Nanoparticles in Medicine 2.2. Principal Types of Nanoparticles 2.2.1. Polymeric Nanocarriers 2.2.2. Lipid-Based Nanocarriers 2.2.3. Carbon Nanostructures 2.2.4. Inorganic Nanocarriers 2.2.5. New Generation Nanocarriers 3. Nanochemistry Approaches for Preparation of Nanocarriers 3.1. Hot Colloidal Chemistry 3.2. Microemulsion Chemistry 3.3. Organic Chemical Synthesis of Dendrimers 3.3.1. Divergent Approach 3.3.2. Convergent Approach 3.4. Reprecipitation Method 3.5. Self-Assembly 3.6. Seed-Mediated Synthesis 3.7. Biosynthesis 4. Chemistry for Interface Engineering 4.1. Surface Engineering for Biocompatibility 4.1.1. Chemical Makeup 4.1.2. Micelle Encapsulation 4.1.3. Hydrophilic Surface Reactive Molecules (SRMs) 4.2. Bioconjugation Chemistry 5. Nanoparticle-Enabled Bioimaging 5.1. Optical Bioimaging 5.1.1. Linear Optical Bioimaging

© 2016 American Chemical Society

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Received: March 12, 2015 Published: January 22, 2016 2826

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Chemical Reviews 9.3. Physicochemical Control of Nanoparticle Toxicity 9.3.1. Nanoparticle Surface 9.3.2. Nanoparticle Dimensions 9.3.3. Composition 9.4. Nanotoxicity Studies on Selected Nanoparticles 9.4.1. Quantum Dots 9.4.2. Gold Nanoparticles and Nanorods 9.4.3. ORMOSILS 9.4.4. Carbon Nanostructures 10. Current Status and Future Outlook Author Information Corresponding Authors Notes Biographies Acknowledgments References

Review

biosensing,31 stem-cell modulation,32 and tissue engineering,33 to name a few. This review comprehensively covers nanomedicine, defining its scope and providing an account of the use of nanostructures for imaging and sensing, as well as their function as nanocarriers to effect therapy. A special emphasis has been placed on highlighting the role of nanochemistry in advancing nanomedicine, with the objective of increasing the participation of the chemical community in this high societal impact field.

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2. NANOPARTICLE FORMULATIONS FOR BIOMEDICAL APPLICATIONS Nanoparticles play a key role in nanomedicine, as they can efficiently carry and deliver imaging probes, therapeutic agents, or biological materials to targeted sites such as a specific organ, tissue, or even underlying cell. Also, some of them possess active functions that facilitate their use as nanoprobes for imaging/sensing or agents for novel therapies. This section introduces the benefits of using nanoparticles in medicine. It also presents some principal nanoparticle formulations that have been amply utilized for biomedical applications.

1. INTRODUCTION Nanoparticles are generally defined as any particulate material for which at least one dimension lies in the range of 1−100 nm.1−3 They can exist in various shapes, such as spheres,4,5 rods,6,7 wires,8 planes,9,10 stars,11 cages,12 multipods,13,14 etc. The various chemical processes that guide the synthesis of materials in the nanometer scale can be defined as nanochemistry, which plays a critical role in tailoring the physical and chemical properties of nanoparticles.15,1 Nanoparticles have several unique properties not found in their bulk counterparts, which include high surface-to-volume ratio, high surface energy, unique mechanical, thermal, electrical, magnetic, and optical behaviors, etc.3,16 These properties make them suitable for a wide range of applications, ranging from electronics, to energy harvesting and storage, to communications, to biology, and to medicine.1−3 Their chemical reactivity and dispersibility in various solvents can be regulated by modifying their surface with desired functional groups to suit a particular application.17,18 Nanomedicine is the application of nanoparticles in medicine.1,19 This field of advanced medicine seeks to address various medical challenges and shortcomings faced by conventional medicine, which include poor bioavailability, impaired target specificity, systemic and organ toxicity, etc.20−22 Nanoparticles intended for medical use have drawn inspiration from the various “natural” nanoparticles discovered in the body. These include various nanosized vesicles, lipids, proteins, and complex biomacromolecules that regulate the natural functioning in the body, and may act as carriers of active molecules. Most earlier examples of nanomedicine involved lipid- and polymer-based nanocarriers with encapsulated drugs for targeted and sustained drug delivery.23−25 Alongside, an increasing awareness about novel medical applications of smaller, inorganic-based nanoparticles, possessing unique properties at the nanoscale, has led to a burst of research activities in the development of “nanoprobes” for diagnostic medicine and agents for novel, externally activated therapies.26−30 Nowadays, nanodiagnostics and nanoparticle-mediated drug delivery are being recognized as complementary technologies, with the popular term “theranostic” being used to describe their combination, that can bring about unprecedented advances in medicine.1 Other modern medical techniques which critically hinge on the advances in nanomedicine include

2.1. Advantages of Nanoparticles in Medicine

As stated earlier, many nanoparticles possess unique properties at the nanoscale, which give rise to several novel diagnostic and/or therapeutic applications.34,35 These applications will be discussed in detail later. In addition, nanoparticles serve as excellent carriers of other active molecular or macromolecular agents, which can be incorporated within their bulk and/or pore network within them, and can be attached to the surface. Nanoparticulate carriers or nanocarriers have several key advantages over conventional, molecular agents in medicine.36,37 First, they enable stable aqueous dispersions of active, but poorly water soluble molecular agents, for delivery in the biological milieu. Their composition, size, shape, and surface properties can be exquisitely tailored so that, when introduced in the biological milieu, they can protect the encapsulated agents from degradation by various endogeneous defense mechanisms. These mechanisms include enzymatic degradation, immunodegradation, sequestration by the reticuloendothelial system (RES) in the bloodstream, acid hydrolysis in the stomach, mucociliary clearance in the lungs, etc.1 Control of their size, shape, and surface properties also allows them to be targeted to not only specific organs/tissues in the body, but even with a cellular and subcellular specificity.38 Another major benefit is that a nanocarrier matrix can be designed for controlled release of drugs at target areas for optimal and sustained drug action.39−41 A unique aspect of nanomedicine is multimodality, i.e. to perform several diagnostic and/or therapeutic functions in tandem.42−45 Finally, the nanocarrier should exhibit no toxicity and be safely excreted from the body. These advantages are listed in Figure 1. 2.2. Principal Types of Nanoparticles

Figure 2 lists some principal nanoparticles which are discussed here. In addition to a simple nanostructure, a core−shell or a core−shell−shell (multiple shells) type structure built hierarchically provides an opportunity to incorporate different therapeutic or diagnostic payloads in the core or different shells, thus making them suitable for multimodal applications. Table 1 summarizes the main biomedical applications of several selected nanoparticles as well as their future perspectives. 2.2.1. Polymeric Nanocarriers. Polymeric nanocarriers offer a great deal of flexibility in tailoring of their chemical 2827

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extracted from crab shell, is another biodegradable material. It is hydrophilic and cationic, and can electrostatically bind to anionic genetic material such as DNA and siRNA, for applications in gene therapy.59,60 Recently, we have produced water dispersible nanoparticles with a PLGA core and a chitosan shell.61 The inner PLGA core can carry a hydrophobic payload, while the chitosan shell can incorporate hydrophilic components (e.g., DNA, RNA, biorecognition agents). The natural biocompatibility and biodegradability of biopolymers, such as proteins and sugars, make them extremely promising in drug delivery applications. In this respect, protein nanoparticles have gained increasing attention as their amphiphilicity promotes their interaction with both waterinsoluble drugs and physiological fluids.62 Nanoparticles can be prepared from natural proteins via simple techniques, such as spontaneous self-assembly, emulsification, salt-induced coacervation, chemical cross-linking, etc.63,64 Moreover, their rich chemistry, via free availability of functional groups such as amino, carboxyl, and thiol, facilitates chemical linkages with drugs, imaging agents, bioaffinity ligands, etc.65 Several natural proteins have been tested for drug delivery, which includes albumin,66 gelatin,67 casein,68 ferritin,69 elastin,70 gliadin,71 etc. Among them, albumin has attracted special attention as it is known to naturally bind to several drugs and endogenous molecules, as well as function as a physiological transporter.66 Another attractive protein is gliadin, which has mucoadhesive properties owing to its tendency to form hydrogen bonding and provide other interactions with mucosal surfaces. Nanoparticles of gliadin can be used for oral and topical drug delivery.72 Block copolymers comprising at least a hydrophilic and a lipophilic block have also attracted great attention in drug delivery.73 In aqueous media, they spontaneously self-assemble into a core−shell structure with a hydrophobic inner core. These “polymeric nanomicelles” remain intact in very dilute solutions, as they have much lower critical micelle concentrations (cmc’s) than surfactant micelles. Especially attractive are those where the hydrophilic block contains a pH or thermosensitive group, such as acrylamide. These groups impart “hydrogel” nature to the nanoparticles that undergo swelling and drug release upon change in pH or temperature.74 Another class of block copolymers, which contain phospholipids as the lipophilic unit, has found versatile applications in drug delivery, either as phospholipid nanomicelles or as coating agents on other nanoparticles.75 Other examples include a

Figure 1. Advantages of nanocarrier formulations in medicine.

composition, size, biodegradability, morphology, and surface functionality.46 As a result, they serve as excellent drug carriers for a range of applications in sensing, imaging, and therapeutics. The drug release pattern can be exquisitely tailored via controlled polymer biodegradation or appropriate stimulus activation. Stimulus activation mechanisms of drug release range from passive (e.g., a pH change in a hydrogel, leading to swelling)47,48 to active, externally activated ones (e.g., photodegradation via light activated cleavage of photolabile bonds in a polymer backbone).49−51 Finally, it must be mentioned that several hydrophilic polymers, such as polyethylene glycol (PEG), chitosan, and dextran, are widely used as coating agents on other nanoparticles to enhance their aqueous dispersibility, bioavailability, and targeting efficacy.52−54 Examples of biodegradable polymeric nanoparticles include those made from polyesters, poly(amino esters), polyanhydrides, polyamides, chitosan, etc.55,56 Mostly, biodegradable polymers are characterized by having a heteroatom (−C−O−, − C−N−, C−S−, etc.) backbone, which facilitates hydrolysis and bond cleavage.57 These nanocarriers can be degraded by hydrolysis in vitro and in vivo under specific conditions, thus providing control in drug release and facilitating excretion from the body. Poly lactide-co-glycolide (PLGA) is a U.S. Food and Drug Administration (FDA) approved polyester whose microand nanoparticles have been successfully used in nanoformulations of a number of drugs. However, owing to their lipophilicity, PLGA nanoparticles have to be coated with a hydrophilic biocompatible polymer, such as PEG, for optimal in vivo use.58 Chitosan, a natural polysaccharide, primarily

Figure 2. Various principal nanoparticle formulations. 2828

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scaled-up synthesis, controlled drug release, detailed toxicological analysis, theranostics

multimodal bioimaging, theranostics

magnetic-resonance imaging (MRI), magnetically guided drug delivery, magnetic biosensing and cell separation, gene therapy multimodal bioimaging, drug release, gene therapy, tissue engineering

scaled-up synthesis, externally activated drug release, theranostics

detailed toxicological analysis, externally activated therapies, theranostics

polymer core decorated with polymeric brushes, which can act as biosensors or contain imaging or diagnostic agents.76 A special class of organic nanoparticles is dendrimers, which are supramolecular architectures consisting of well-defined hyperbranched organic units. Owing to their ultrasmall and highly monodispersed diameters (below 20 nm), they have been widely investigated for several biomedical applications, ranging from sensing to drug delivery.77 A dendrimer can encapsulate a payload in the core conjugated to a branch point, with the drug loading controlled by the generation number. By attaching the payload to a branch through a biodegradable chemical linker, controlled release of a payload can be achieved. The chemistry of the external corona in a dendrimer can also be exploited for conjugation to PEG or a targeting group. 2.2.2. Lipid-Based Nanocarriers. Three major lipid-based nanocarriers are (a) phospholipid-polymer nanomicelles (already discussed), (b) lipid-bilayer vesicular nanostructures (liposomes), and (c) solid-lipid nanoparticles (SLNs). While the first two classes use diglycerides as components, the third class is made up of high-melting triglycerides. Liposomal nanocarriers generally utilizing phospholipids consist of one or several lipid bilayers, with a polar aqueous “core”, a lipophilic bilayer compartment, and a hydrophilic exterior.78 The aqueous interior can incorporate water-soluble drugs, while lipophilic payloads can be included in the bilayer region. Cationic liposomes have been used as gene carriers for gene therapy. PEG chains are usually incorporated on their surface for improved circulation and bioavailability. SLNs are produced in the size range 10−1000 nm using high-melting lipids. They are solid at room temperature and at body temperature.79,80 They are biocompatible and biodegradable, with considerably reduced side effects compared to other lipid/micellar nanostructures. They are more rigid, thus structurally more stable, and offer better protection to encapsulated drugs against chemical degradation. 2.2.3. Carbon Nanostructures. Carbon nanostructures include fullerenes, carbon dots (CDs), carbon nanotubes (CNTs), and graphene dots (GDs), and possess unique electrical and tensile properties. CNTs are particularly useful in biomedical applications, such as sensing, imaging, and drug delivery. For example, high-resolution intravital microscopic imaging of tumor vessels beneath thick skin and whole-animal in vivo imaging have been successfully demonstrated using the intrinsic photoluminescence of single-walled carbon nanotubes, lying within the “second optical imaging window” of 1000− 1350 nm.81 Indeed, simulations and modeling of optical imaging in turbid media (such as tissue or blood) suggested that the signal-to-noise ratio can be improved over 100-fold by using fluorophores that emit light at 1320 nm, instead of 850 nm.82 Moreover, through-scalp and through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy has also been demonstrated using the intrinsic photoluminescence of single-walled carbon nanotubes at ∼1.3−1.4 μm due to the significantly reduced photon scattering.83 The unique electrical properties of CNTs have been exploited in the fabrication of amperometric biosensors. CNTs may contain functional groups on their surface, which facilitates their conjugation with diagnostic/therapeutic/biotargeting agents. Moreover, CNTs are also known to encapsulate and release drugs using their interior volume. Carbon dots (CDs) represent an important class of fluorescent materials and provide potential applications in biomedicine.84 They possess a typical size under 10 nm in all

carbon nanostructures (carbon dots, nanotube, graphene) polymeric and lipid nanocarriers (polymeric micelles, hydrogels, dendrimers, liposomes, solid−lipid nanoparticles) magnetic nanoparticles (iron oxide nanoparticles, cobalt ferrites) nanoscale metal−organic frameworks (NMOFs)

dark field optical imaging, multiphoton bioimaging, photoaccoustic (PA) bioimaging, photothermal therapy (PTT), plasmonic sensors, gene therapy fluorescence bioimaging, multiphoton bioimaging, multimodal bioimaging, drug release, PDT, gene therapy fluorescence bioimaging, multiphoton bioimaging, amperometric biosensing, drug release, gene therapy, tissue engineering sustained and stimuli-responsive drug release, gene delivery, PDT, photothermal therapy (PTT), gene therapy, tissue engineering plasmonic nanoparticles (metallic nanostructures, selfdoped semiconductor)

detailed toxicological analysis, theranostics

deep tissue bioimaging, sensitive biosensing (oxygen, metal ions, DNA, etc.), multimodal bioimaging (MRI, PET, CT), PDT rare-earth-doped upconversion nanoparticles

silica-based nanoparticles (ORMOSIL, doped silica)

fluorescence bioimaging, multiphoton bioimaging, fluorescence and ratiometric biosensing, in vitro diagnostics

development of heavy-metal free quantum dots/rods, surface modification to prevent degradation/erosion, detailed toxicological analysis, two-photon photodynamic therapy (PDT) scaling-up sythesis, surface modification to prevent of leaching of rare-earth ions, detailed toxicological analysis, light-regulated drug release, intraoperative light guidance, theranostics development of core−shell nanoparticles, multimodal bioimaging, light-regulated drug release, theranostics

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semiconductor nanoparticles (quantum dots, rods)

main biomedical applications nanomaterials

Table 1. Several Selected Nanomaterials, Their Main Biomedical Applications, and Future Perspectives

challenges and future perspectives

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Figure 3. (a) Energy diagrams for bulk semiconductor and QDs. (b) Example of cysteine-capped CdTe/ZnTe core/shell QDs synthesized in aqueous media, with a size-dependent luminescence. Upper panel: photographic images of CdTe/ZnTe QDs dispersed in water under UV irradiation. The emitted peak wavelength is marked in white numbers on the top of the image. Lower panel provides an illustration of the corresponding core/shell nanoparticle size from 2 to 8 nm. These core/shell QDs were prepared at the Institute for Lasers, Photonics, and Biophotonics, University at Buffalo. Reproduced with permission from ref 101. Copyright 2009 John Wiley & Sons.

luminescence quantum yield of as high as 11.4%.95 Moreover, recent advances of solution-based chemistry enable production of large, stable colloidal graphenes with uniform size and shape.96,97 This elicits an implication for them to be epidemiologically utilized in cancer phototherapy and imaging.98,99 2.2.4. Inorganic Nanocarriers. Inorganic nanoparticles as carriers offer the advantage of being extremely robust, and thus very stable and highly resistant to enzymatic degradation. They can be prepared in ultrasmall sizes (40 h) tumor accumulation time was observed, without any sign of toxicity. Sailor et al. have shown luminescent porous silicon nanoparticles (LPSiNPs) can carry a drug payload, while their intrinsic near-infrared photoluminescence enables monitoring of both accumulation and degradation in vivo.246 Furthermore, in contrast to most optically active inorganic nanomaterials (carbon nanotubes, gold nanoparticles, and quantum dots), LPSiNPs self-destruct in a mouse model into renally cleared components, in a relatively short period of time, with no evidence of toxicity. Other heavy-metal-free QDs, encompassing CuInS2,247 InP,226 and CuInSe/ZnS core/shell nanostructures,248 are also being utilized for fluorescence bioimaging. 5.1.1.2. Phosphorescence Bioimaging. Phosphorescence has also been explored for bioimaging. Heavy-metal complexes that possess unique d6, d8, and d10 electron configurations are found to have strong spin−orbit coupling, resulting in efficient intersystem crossing from the singlet excited state to the triplet manifold, and the spin-forbidden nature of the T1−S0 radiative transition can be overcome to a large extent.249 As a result, highly intense room temperature phosphorescence emission with useful radiative decay times can be achieved for steadystate or time-gated optical imaging. For example, phosphorescent iridium(III) complexes [Ir(C−N)2(PhenSe)]+ with tunable emission colors were developed to image mitochondria and track the dynamics of the mitochondrial morphology.250 High contrast time-gated cellular phosphorescence imaging of live CHO-K1 cells in the presence of solution of fluorescein have been demonstrated, which explored the longer phosphorescence lifetimehundreds of nanoseconds to microseconds from the PtLCl complex. Long time decay offers improved discrimination by allowing time-gated experiments to distinguish from short-lived autofluorescence.251 In addition, the red phosphorescence from Ir(btp)2(acac) (BTP) is dependent on the content of oxygen in the surrounding environment. This oxygen-quenching feature has been utilized for imaging tumor hypoxia.252 Recently, we reported the use of polymeric nanomicelles encapsulating NIR phosphorescent dye [Pt(II)tetraphenyltetranaphthoporphyrin, Pt(TPNP)] as efficient probes for the high contrast optical imaging and diagnosis of tumors in small animals.210 The main advantage of the NIR phosphorescent optical probes over conventional NIRfluorescent probes is a large spectral separation between absorption and phosphorescence emission, which ensures a dramatic decrease in the level of background autofluorescence and scattered excitation light in the spectral range where the signal from phosphorescent probe is observed (Figure 11). However, the use of NIR phosphorescence for bioimaging still remains rather limited. 5.1.1.3. Persistent Luminescence Bioimaging. Persistence luminescence from metal-ion-doped inorganic phosphors is analogous to long lifetime phosphoresce from organic dyes, but

Figure 10. Multiplexed in vivo fluorescence imaging of separate lymphatic networks with QD accumulation in sentinel lymphatic nodes (SLNs). Reproduced from ref 245. Copyright 2012 American Chemical Society. 2842

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Figure 11. Phosphorescence bioimaging using polymeric micelles containing Pt(TPNP) (Pt(II)-tetraphenyltetranaphthoporphyrin). (a) Structure of Pt(TPNP). (b) Room temperature luminescence spectra of Pt(TPNP) in DSPE−PEG nanomicelles at different concentrations of 2.5 (1, 2), 12.5 (3), and 25 μM (4). (c) Photoluminescent images of a tumored nude mouse at various time points (2, 24, 96 h) postinjection with DSPE−PEG polymeric nanomicelles containg Pt(TPNP). Reproduced from ref 210. Copyright 2009 American Chemical Society.

Figure 12. Persistent luminescence bioimaging. (a) Excitation (blue curve, for emission at 700 nm) and emission (red curve, when excited at 254 nm) spectra of an aqueous dispersion of PEG−LPLNPs of Zn2.94Ga1.96Ge2O10:Cr3+,Pr3+. The inset shows the digital photo of the aqueous dispersion of LPLNPs under 254 nm UV excitation. (b) NIR afterglow decay images recorded by a CCD camera at different times after stopping UV irradiation. (c) In vivo NIR luminescence images of a normal mouse after intravenous injection of PEG−LPLNPs (0.6 mg, 10 min irradiation with a 254 nm UV lamp before injection). Reproduced from ref 255. Copyright 2009 American Chemical Society.

breakthrough on deep-tissue-penetrating NIR persistent phosphors elicits a great deal of attention for their use in optical bioimaging.254 For example, Yan et al. prepared NIRemitting long-persistent luminescencent nanoparticles (LPLNPs) Zn2.94Ga1.96Ge2O10:Cr3+,Pr3+ with suitable Zn deficiency in the zinc gallogermanate, which exhibits bright NIR luminescence at ∼720 nm in the biological transparency window, with a superlong afterglow time of over 15 days (Figure 12). PEGylation greatly improves the biocompatibility and water solubility of LPLNPs, while bioconjugation with c(RGDyK) peptide makes LPLNPs promising for long-term in

with lifetimes from hours to days. Persistent luminescence nanophosphors utilize electron traps located within the band gap of the host lattice to store the excitation energy, and then slowly produce photonic emissions by detrapping of electrons over a certain period. As a consequence, these persistent luminescence nanoparticles can be excited before injection, and then be followed in vivo in real time for more than hours and even days, without the need for any external illumination source.253 This will create high contrast autofluorescence-free optical imaging. Though significant achievements have been made in producing visible persistent phosphors, only a recent 2843

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vivo targeted tumor imaging with low toxicity.255 Similarly, LiGa5O8:Cr3+ LPNPs with diameters of 50−150 nm, with polyethylenimine (PEI) coating, were prepared and then used to label 4T1 murine breast cancer cells. After illuminating with a 254 nm UV lamp, these PEI-coated LPNPs were subcutaneously injected and could be clearly imaged even 4 h after the injection.256 However, actual generation of NIR persistent luminescence nanoparticles necessitates ex vivo activation before systemic administration, which prevents long-term imaging in living animals. A recent work showed that LPNPs of chromium doped zinc gallate can be activated in vivo through living tissues, using high-penetrating low energy red photons. Notably, after phagocytosis of these LPNPs in RAW cells, these labeled cells can be tracked using a simple whole animal optical detection system, opening new perspectives for cell therapy research and for a variety of diagnosis applications.257 5.1.1.4. Photoacoustic Bioimaging. Photoacoustic (PA) imaging is a hybrid biomedical imaging method which explores the merit of the photoacoustic effect that is the generation of an acoustic wave by the heat produced by light absorption. This imaging modality, which utilizes acoustic (ultrasonic) detection, is presently receiving increasing attention. The image contrast for PA modality derives from the difference in the optical absorption between the target and its surrounding area, while the spatial resolution of PA is determined by ultrasonic detection. The advantage gained by PA is that one combines the merits of both optical imaging (higher contrast compared to ultrasound imaging) and ultrasound imaging (longer penetration depth compared to optical imaging).258 Moreover, the spatial image resolution is scalable with the ultrasonic frequency, while the imaging depth is limited by the penetration depth of excitation photons. Endogenous chromophores such as hemoglobin or melanin serve as contrast agents for label-free morphological and functional PA imaging of blood vessel and tumor angiogenesis.259,260 However, endogeneous agents provide inadequate contrast and spatial specificity, and consequently poor signal-to-noise ratio in PA imaging. The use of strongly light-absorbing nanoparticles, or chromophoreencapsulated nanoparticles, targeted to biological sites of interest can lead to significant contrast enhancement in PA. For example, tumor-targeting polyacrylamide (PAA) hydrogel nanoparticles containing Coomassie Blue (CB) dye enable the PA technique to delineate brain tumor for an intraoperative surgerical guidance.261 Furthermore, these nanoparticles can be designed to absorb in the NIR region, thus allowing higher tissue penetration of light. Kopelman and co-workers have demonstrated the use of PEBBLE nanoparticles, embedded with the NIR fluorophore indocyanine green (ICG), to provide significant PA contrast between targeted and untargeted prostate cancer cells in a single layer (LNCaP cell line).262 Their group further demonstrated the use of ICG-doped PEBBLEs and plasmonic gold nanorods in enhanced PA imaging of the mouse brain ex vivo and in vivo.263 Gold nanocages have been used by Song et al. for photoacoustic sentinel lymph node mapping on a rat model.114 The ability to produce gold nanocages in a broad size range from 35 to 100 nm provides the opportunity to tune their LSPR absorption to cover the NIR region, which allows deeper tissue penetration. Recently CuS nanoparticles have been used to enhance the contrast as well as to provide target imaging.264 Our group has used phospholipid−PEG encapsulated Cu2−xSe semiconductor plasmonic nanoparticles as an in vivo photoacoustic contrast

agent achieving an imaging depth of about 3.5 mm.265 We further improved the depth profile of photoacoustic imaging by synthesizing Au−Cu2−xSe plasmonic heterodimers, with broad LSPR across the visible and NIR wavelengths. In vivo NIR photoacoustic imaging in rats revealed a high imaging depth, up to 17 mm.119 In addition, organic nanomaterials of carbon nanotubes and porphysome nanovesicles (generated by porphyrin bilayers) have been utlized as photoacoustic molecular imaging agents in live mice.266,267 Pu et al. recently reported the use of NIR light absorbing semiconducting polymer nanoparticles as a new class of contrast agents for PA imaging, which exhibits a stronger signal than the commonly used single-walled carbon nanotubes and gold nanorods. Moreover, they are able to be engineered to allow real time NIR ratiometric photoacoustic in vivo imaging of reactive oxygen species (ROS) by exploiting their ROS-inert PA signal from the nanoparticles, as well as the ROS-dependent PA sensing ability of IR775S being incorporated into them.268 Zhang et al. recently developed ∼20 nm frozen micelles containing a high content of naphthalocyanine dyes that are known to be used as PDT drugs. Unlike conventional chromophores, these micelles, named as “nanonaps”, exhibit nonshifting spectra at ultrahigh optical densities and, following oral administration in mice, passed safely through the gastrointestinal tract. Noninvasive, nonionizing photoacoustic techniques were used to visualize nanonap intestinal distribution with low background and remarkable resolution, and enabled real-time intestinal functional imaging with ultrasound coregistration (see Figure 13).269 The high extinction coefficient in the NIR range and typically none or low luminescence ability are two common optical features shared by these exogenous PA imaging contrast agents. Nonlinear (excited state) absorption is being investigated in the curcumin BF2 and bis-styryl(MeOPh)2BODIPY dyes to enhance the photoacoustic effect.270

Figure 13. Photoacoustic imaging using frozen micelles containing naphthalocyanine. (a) Normalized absorbance of micelles formed from BPc (blue), ZnBNc (dark green), BNc (light green), or ONc (bronze). (b) Photograph of nanonaps in water. From left to right: BPc, ZnBNc, BNc, and ONc. (c) Depth-encoded PA image of the intestine visualizing frozen micelles containing ZnBNc. Reproduced with permission from ref 269. Copyright 2014 Nature Publishing Group. 2844

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5.1.2. Nonlinear Optical Bioimaging. Multiphoton processes which involve simultaneous nonlinear optical interactions of several photons or a series of coupled sequential absorptions to generate an excitation are playing an increasingly important role for biophotonics and nanomedicine. In addition to these resonant processes, there are also nonresonant nonlinear optical processes such as second harmonic generation that do not involve any energy deposition in the medium. The mulitphoton processes shown in Figure 14 are2 the following:

depositing any energy into the medium (Figure 14c). This process is thus different from the two-photon excited fluorescence in which the two photons are actually absorbed. Also, in SHG, the generated photon is always twice the frequency of the incident light, whereas in two-photon excited fluorescence, the emission frequency is determined by the energy gap between the excited emitting level and the ground level of the emitter. (d) Coherent anti-Stokes Raman scattering (CARS), generally used to induce a vibrational transition, involves nonlinear interaction of three photons, two of frequency νI and the third of frequency νS, to generate a photon of a new frequency 2νI − νS (Figure 14d). The difference νI − νS matches the frequency of a Raman-active vibration of a constituent chemical group of the medium (a biological specimen) or that of an external molecular probe (Raman probe). This process again has been used for high resolution three-dimensional vibrational (Raman) imaging. 5.1.2.1. Multiphoton Optical Bioimaging. Among this type of imaging, two-photon excited fluorescence imaging, commonly known as two-photon microscopy, has been widely used in biology. This imaging modality provides the following merits: (i) significantly reduced fluorescence background due to the relatively low two-photon cross section of most biomolecules responsible for autofluorescence in biological systems, (ii) reduced photobleaching by selective excitation of the focal volume, (iii) improved depth penetration in scattering samples due to the use of excitation light in a near-infrared (NIR) spectral range, and (iv) increased spatial resolution due to the nonlinear dependence of the luminescence intensity on the excitation power density. A set of nanoemitters with appreciable multiphoton absorption cross section have been developed to reach this purpose, which includes fluorphoredoped ORMOSIL nanoparticles,271 gold nanorods,272,273 QDs,274 and QRs,275 to name a few. Fluorophore-doped nanoplatforms can be very attractive in this regard, as a number of existing multiphoton dyes can be selected for doping. However, one major disadvantage for them is the phenomenon of aggregation-induced fluorescence quenching of the doped dyes upon high loading, thus limiting the two-photon luminescence intensity from one single nanoparticle. This problem can be overcome by using a class of dyes which exhibit aggregation induced enhancement of emission rather than quenching. We have demonstrated two-photon imaging using ORMOSIL nanoparticles doped with organic dye nanoaggregates of 9,10-bis[4′-(4″-aminostyryl)styryl]anthracene (BDSA). Two-photon excitation of this nanosystem resulted in aggregation enhanced fluorescence (AEF),271 leading to very high quality in vitro bioimaging. The ORMOSIL could be loaded with a high concentration of BDSA (about 30−40% BDSA with respect to the nanoparticle matrix) without any quenching. With 25% loading of the BDSA dye (∼2500 molecules per nanoparticle), they have a fluorescence quantum yield (QY) of 0.2 per dye molecule. The BDSA−ORMOSIL nanoprobe provides a 500-fold enhancement over a single fluorescent molecule, with a fluorescence QY of unity. With respect to gold nanomaterials, their emission quantum efficiency is typically not high. However, characteristic values for their two-photon action cross section are reported to be high, on the order of a few thousand Goeppert Mayer units, thus being able to yield a considerable amount of two-photon luminescence.272,273 Wang et al. demonstrated that the twophoton luminescence signal from a single nanorod is 58 times

Figure 14. Energy levels diagrams representing (a) two-photon absorption and subsequent upconverted emission, (b) sequential upconversion and subsequent emission (ESA is excited state absorption, ETU is energy transfer upconversion), (c) second harmonic generation (SHG), and (d) coherent anti-Stokes Raman scattering (CARS).

(a) Optically nonlinear two-photon excitation is a process involving simultaneous absorption of two photons through a virtual intermediate state, which is quadratically dependent on the intensity of light being absorbed, and is thus very localized near the focus of the light beam (Figure 14a). The advantage is that it enables point-by-point excitation by scanning the focal point of the beam in three dimensions. Thus, two-photon microscopy offers the merit of high resolution and threedimensional imaging. (b) Upconversion emission, as a result of sequential linear absorption through multiple real (not virtual) intermediate states, is another important multiphoton process, different from the above nonlinear processes (Figure 14b). Rare-earth-doped inorganic nanoparticles, as discussed before, represent upconversion nanoemitters. The advantage of sequential multiphoton absorption over the nonlinear simultaneous twophoton absorption is that it does not require high intensity and can be effected by using an inexpensive low power CW light source, whereas expensive pulsed laser systems are necessary for simultaneous two-photon absorption. (c) Second-harmonic generation (SHG) is a nonlinear optical process where two photons of a given frequency interact in a noncentrosymmetrical medium (i.e., material lacking a generalized mirror symmetry) to generate a new photon of twice the frequency (frequency doubling), without 2845

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nanoparticles, inducing strong SHG dipole sources within the nanoparticles. SHG imaging has been employed to examine the endogenous molecular structures of label-free biological samples, such as collagen fibrils in connective tissues or the actomyosin lattice of muscle cells.282,283 However, the use of label-free SHG remains rather limited for the centrally symmetrical structure of cell component materials, whereby only a small output of SHG at the interface layer is produced.284 As a result, SHG nanoprobes with noncentrosymmetric crystal structures have been developed, which include BaTiO3,280,285 ZnO,286 KNbO3,287 KTiOPO4 (KTP),288 Fe(IO3)3, etc.289 Moreover, SHG has also been observed from individual 150 nm diameter gold nanoparticles dispersed in gelatin.290 The SHG from single core/shell CdTe/CdS QDs with a diameter of 10−15 nm has been reported.291 Moreover, synergistic SHG effects from metallic and semiconductor nanostructures have also been manifested in the hybrid CdSe−Au nanoparticle; the strength of SHG is dependent not only on the size of the hybrid nanomaterials, but also on the interaction between two components.292 Some of these above-mentioned SHG nanoprobes have already been utilized for in vitro and in vivo imaging. For example, we have utilized phophospholid-coated ZnO nanoparticles carrying the ligand of folic acid for targeted imaging of KB cells.286 Pantazis et al. demonstrated the use of BaTiO3 as SHG nanoprobes for in vivo imaging of one-cell stage zebrafish embryos, circumventing many of the limitations of classical fluorescence probes such as photobleaching, photoblinking, and autofluorescence.280 It is worth mentioning that third harmonic generation (THG) from ∼40 nm gold nanoparticles and individual Si nanodisks has also been reported,293,294 which provides a potential use for bioimaging. 5.1.2.3. CARS Bioimaging. CARS microscopy utilizes a nonlinear Raman imaging technique that has received considerable attention for label-free biomedical imaging due to its ability of real-time, nonperturbative chemical mapping of live unstained cells and tissues utilizing molecular vibrations of the consituent biomolecules (proteins, lipids).232 The mechanism for CARS is depicted in Figure 14d. Like multiphoton and SHG microscopies, CARS microscopy is also able to implement three-dimensional (3D) optical sectioning, since the signal only can be generated in the focal volume of two laser beams.295 However, CARS microscopy differs from multiphoton and SHG microscopies in that it utilizes molecular vibrations to produce an imaging signal, rather than the electrons or polarization of molecules. As a consequence, CARS is able to image chemical species or cellular components that either do not fluoresce or cannot tolerate labeling. Though the first CARS microscope was reported in 1982,296 the collinear beam geometry with NIR excitation has resulted in the popularity of CARS microscopy in biology due to the achievement of high resolution three-dimensional sectioning with low imaging background. 297 The phase-matching conditions in this geometry are relaxed because of the large cone of wave vectors and the short interaction length producing CARS signals several orders higher than the corresponding spontaneous Raman intensity.297 CARS microscopy has been utilized for vibrational imaging of phospholipid bilayers, single lipid membrane of supported bilayers, giant unilamellar vesicles, and intact erythrocyte membrane using the strong resonant signal of the C−H stretching vibration in the lipid.298 The use of CARS microscopy to image brain structure and pathology ex vivo has also been reported through chemically selective

more than that from a single rhodamine molecule. The twophoton luminescence excitation spectrum coincides with the longitudinal plasmon band of gold nanorods, revealing a plasmon-enhanced two-photon absorption cross section. Visualization of two-photon luminescence from gold nanorods reveals the flow of single nanorods through mouse ear blood vessels.272 However, as the excitation is within the longitudinal plasmonic band, this increased the risk of causing strong local heating which is widely used in photothermal therapy to kill cancer cells. Tong et al. observed bright three-photon luminescence from Au/Ag alloyed nanostructures by excitation from a femtosecond laser at 1290 nm, manifested with an intensity level 1 order of magnitude higher than that from pure Au or Ag nanoparticles. As the excitation wavelength is outside the range of plasmon resonance, three-photon luminescence enables cellular bioimaging with negligible photothermal toxicity.276 In addition, we have successfully demonstrated the use of two-photon excited luminescence from CdSe/CdS/ZnS QDs to image live pancreatic cancer cells and CdSe/CdS/ZnS QRs to image HeLa cells. Receptor-mediated uptake of these bioconjugates has been verified by confocal and two-photon microscopy.274,275 More recently, Hyeon et al. demonstrated high-resolution in vitro and in vivo optical imaging by combining three-photon excitation of ZnS QDs and visible emission from the doped Mn2+ ions. The large three-photon cross section of the ZnS:Mn2+ QDs enabled targeted cellular imaging under high spatial resolution, approaching the theoretical limit of three-photon excitation. Moreover, this three-photon process was further successfully applied in highresolution in vivo tumor-targeted imaging.277 Despite these advances, the use of higher order multiphoton processes for in vivo bioimaging remains still limited. This is because multiphoton absorption cross sections of most of the commonly used luminophores are typically low, which necessitates the use of an expensive high power pulsed laser to produce the high local intensity needed to render observable signals. 5.1.2.2. SHG Bioimaging. SHG imaging is a promising nonlinear optical technique being explored in biology.278,279 The SHG mechanism is described in Figure 14c. As in twophoton microscopy, the quadratic dependency of the SHG signal on the excitation power also provides the optical sectioning capability in the axial direction, allowing threedimensional (3D) optical imaging.278 The problems of photobleaching and blinking in some popular imaging probes are circumvented in this technique, as SHG does not involve any real-state energy levels, and thus no absorption. Moreover, SHG nanoprobes are able to produce coherent and stable signals, with a broad flexibility in the choice of excitation wavelength for a specific imaging purpose.280 Unlike SHG in bulk materials, which is governed by phase matching condition, the phase matching requirement is relaxed for SHG from nanoparticles. A hyper-Rayleigh scattering (HRS) process is considered to occur in nanoparticles where the SHG signal produced by optical hyperpolarizability of the molecules scatters in all directions.280,281 The SHG scattered from nanoparticles encompass two parts: (i) the surface contribution, produced at the interface between the nanoparticle and the surrounding medium; (ii) the bulk contribution, produced by the constituents inside the nanoparticle, if its crystal lattice is noncentrosymmetric. Nanoparticles comprised of noncentrosymmetric and highly polarizable crystal structures are capable of efficient SHG due to the strong second-order susceptibility within the volume of 2846

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Figure 15. Whole body images of mouse injected with NaYF4:Yb3+/Tm3+UCNPs: intact mouse (a); same mouse after dissection (b). The red color indicates emission from UCNPs, while the green and black show background, as indicated by the arrows. The inset presents the PL spectra corresponding to the spectrally unmixed components of the multispectral image obtained with the Maestro system. (c) Whole body images of a BALB/c mouse injected via tail vein with hyaluronic acid coated α-(NaYbF4:0.5% Tm3+)/CaF2 core/shell UCNPs. (d) Bright-field image of a pork tissue (side view), displaying the imaging depth; (e) UC PL image of a cuvette containing α-(NaYbF4:0.5% Tm3+)/CaF2 core/shell UCNPs covered with a pork tissue. The insets in (c) and (e) show the spectra of the NIR UC PL and background taken from the circled area. Reprinted from ref 144. Copyright 2012 American Chemical Society. Reprinted from ref 304. Copyright 2008 American Chemical Society.

be reduced by using short-pass filters with the imaging camera, since the emitted light is considerably shifted in wavelengths relative to excitation.139 A highly suitable upconversion nanophosphor (UCNP) formulation for deep tissue in vivo imaging is nanocrystals of NaYF4 codoped with Yb3+ and Tm3+ ions, which are 10−20 nm in size, and exhibit upconverted emission at 802 nm when excited at 975 nm. In this case, both the excitation and the emission wavelengths are in the NIR range, which is the optical window of maximum transparency in biological media, thus allowing deep tissue access.2 Both light attenuation and scattering in vivo are significantly reduced in this spectral range and the autofluorescence of tissues is absent under the conditions of UC excitation and emission. We reported high contrast in vivo bioimaging using NIRin−NIRout NaYF4:Yb3+/Tm3+ UCNPs (Figure 15a,b).304 More recently, we reported on the development of novel efficient core/shell NIRin−NIRout (NaYbF4:Tm3+)/CaF2 UCNPs, which were intravenously injected in BALB/c mice to perform wholebody imaging; a high signal-to-background ratio of 310 was achieved (Figure 15c). It is shown that the upconversion luminescence signal can be readily detected and imaged, with a low background, through a 3.2 cm thick pork tissue (Figure 15d,e)).144 In analogy, Li et al. demonstrated the use of sub-10nm hexagonal NaLuF4:Gd3+/Yb3+/Tm3+ to reach an optical imaging depth of about 2 cm.305 Current research on upconversion for bioimaging is engaged in four directions: (i) synthesis of ultrasmall upconversion nanoparticles that can be cleared from the body and still have high luminescence efficiency,141 (ii) nanophotonic control of their excitation dynamics to improve their upconversion quantum yield,140 (iii) shift of the current excitation wavelength at ∼980 nm to a more effective excitaion wavelength to produce improved upconversion imaging,306−309 and (iv) development of multifunctional

imaging of different structures using their inherent vibrations. This holds promises for its potential use in definitive diagnosis of neoplasia prior to tissue biopsy or resection.299 Moreover, CARS microscopic imaging has also been demonstrated for visualization of the thermodynamic state (either liquid crystalline or gel phase) of lipid membranes.300 Combining CARS microscopy and two-photon excited fluorescence, we examined the spatial distribution of major types of biomoleculesproteins, lipids, and nucleic acids in the cellswhile monitoring their changes during apoptosis as well as during the regular cell cycle of replication. Our results indicate that multiplex nonlinear optical imaging can be used as a powerful tool for studying the molecular organization and its transformation in cellular processes.301 5.1.2.4. Upconversion Bioimaging. Rare-earth-doped upconversion nanoparticles have been considered to be excellent optical probes for photoluminescence bioimaging, as they eliminate many drawbacks associated with other photoluminescent probes, namely broad emission and susceptibility to bleaching for organic luminophores, blinking and toxicity for semiconductor QDs, etc.30 Moreover, the narrow and tunable emission bands produced by a single wavelength in NIR entails their uses in multiplexed bioimaging.302 In vitro cellular imaging has been reported,303 but their exquisite advantage resides in their ability to allow high contrast in vivo imaging. Unlike multiphoton imaging employing NIR excitation from expensive ultrashort pulsed lasers, a low-cost CW laser diode can be utilized as the NIR excitation source for upconversion photoluminescence (UCPL) imaging, with an excitation density of ∼10−1 W/cm2.140 Moreover, the anti-Stokes character of the upconversion excludes manifestation of the autofluorescence from the biological tissues, resulting in a decrease in the imaging background. In addition, the imaging background produced by the scattering of excitation light can 2847

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ratio of the particle. This size-depedent effect confirms that GdIII near the nanoparticle surface plays a vital role in defining the T1 relaxation times. Gd3+-containing nanostructures with a morphology-defined high surface-to-volume ratio will become new generation T1 MRI contrast agents. Superparamagnetic iron oxide (Fe3O4) nanoparticles, or SPIONs, serve as excellent T2 contrast agents in MRI. Iron oxide nanoparticles reduce the spin−spin relaxation time of proton, thus reducing the MRI signal and leading to a dark (or negative) contrast.320 Owing to their nontoxicity in biological systems, a number of iron oxide nanoformulations have been used in preclinical and clinical MRI. These nanoparticles can be co-incorporated with other imaging probes, such as an optical nanoemitter (e.g., quantum dots) for combined MRI and optical bimodal imaging.321−324 However, the relaxivity of current T2 contrast agents remains rather low, including commercially available ones prepared in aqueous media. This could lead to false positive diagnosis in the hypointense areas, such as blood pooling, calcification, and metal deposition.325 According to the quantum mechanical outer sphere theory, the T2 relaxivity is highly dependent on both the saturation magnetization (Ms value) and the effective radius of typically superparamagnetic core.326 The larger spherical iron oxide nanoparticles could have stronger saturation magnetization (Ms) and higher T2 relaxivity (r2). Yet, this could lead to ferri/ ferromagnetic properties at room temperature, resulting in interparticle agglomeration even in the absence of an external magnetic field. Size control and metal doping have been developed to produce iron oxide nanoparticles with a large Ms value,327,328 while morphology control of iron oxide nanoparticles can tune the effective magnetic core radius, leading to a significant increase of T2 relaxivity. For example, iron oxide nanoparticles with octapod shape have ultrahigh r2 relaxivity (679.3 ± 30 mM−1 s−1), which can be 5 times higher than the one with spherical shape and of similar volume (125.86 mM−1 s−1).329 Nanoparticle platforms also allow for a new generation MRI utilizing the spin transitions of another nucleus. For example, 19 F can easily be incorporated in a nanoparticle formulation to enhance the local concentration of 19F at a desired biological site, and thus increase the sensitivity.330 Moreover, the use of a 19 F-containing nanoformulation to label cells or to deliver drugs allows real-time tracking of their in vivo distribution, providing opportunities to probe their interactions with the body. Srinivas et al. intracellularly labeled T cells with a 19F-enriched perfluoropolyether (PFPE) nanoemulsion and transferred these cells to a host that received localized inoculation of antigen. Utilizing the MRI signal provided by 19F, they were able to track and quantify antigen-specific T cells, revealing their dynamic accumulation and clearance in lymph nodes.331 A strong merit of 19F MRI is that this nucleus is not intrinsic to the body, thus enabling MRI images with great spatial selectivity against a zero background. It is to be noted that incorporation of the 19F imaging capability requires only a minor modification of the instruments currently used for MRI.

upconversion nanoparticles for multimodal imaging and lightactivated therapy.146,310 5.2. Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) typically relies on the spin−lattice relaxation (T1 contrast) or the spin−spin relaxation (T2 contrast) times of protons contained in different microstructures of the organs to create imaging contrast. It provides good spatial resolution (25−100 μm) that can be sufficient for both morphological and functional imaging, such as neuroimaging, cardiovascular imaging, and liver and gastrointestinal imaging, to diagnose and evaluate different stages of a diease. Yet, approximately 35% of clinical MR scans need contrast agents to improve their sensitivity and diagnostic accuracy.311 Small or tiny tumor diagnosis or detection of lesions in normal tissues significantly relies on the use of contrast agents. Nanoprobes with magnetic functionality (i.e., magnetic nanoprobes) are intended for such a purpose, labeled as either T1 or T2 MRI contrast agents, depending on whether the relative change in relaxation times of proton is greater for T1 or T2. The contrast is quantitatively characterized by relaxivity (r1 or r2) which is the change in 1/T1 or 1/T2 of protons per concentration of the contrast agent. Magnetic functionality can be provided by paramagnetic ions, such as manganese (MnII),312 iron (FeIII),313 and gadolinium (GdIII).314 These ions can be intrinsic to the nanoparticles as in the case of iron oxide, or they can be incorporated as a dopant within a nonmagnetic matrix, or attached to the surface of a nanoparticle via chelation.313 The most popular T1 contrast agent used in MRI is GdIIIcontaining nanoprobes, because of their large magnetic moment. Chelates of Gd with diethyltriamine pentaacetic acid (DTPA) or tetraazacyclododecane tetraacetic acid (DOTA) are the most clinically used contrast agents,315,316 albeit with related major concern of toxicity of the released Gd ions. Placing GdIII−DTPA, GdIII−DOTA, as well as other GdIIIchelated ligands on the surface of a nanocarrier, such as polymeric nanomicelles, is a common yet effective way to provide additional MRI modality for real-time tracking the functionalities of the nanocarrier.317 A nanoparticle formulation utilizing lattice-bound Gd can overcome the toxicity problem caused by the possible Gd release from the contrast agent.149,150 Here, the GdIII ions can be purposely doped into an inorganic host lattice. Alternatively, a GdIII-based host lattice can be used to form the nanoparticle to be utilized as an MRI contrast agent.149,318,319 The GdIII units near the surface of the nanoparticles can affect the T1 relaxation times of protons more effectively than the GdIII inside the nanoparticle, as they interact more directly with the aqueous environment. We used GdIII in a NaGdF4 shell on the Tm doped NaYbF4 upconversion nanoparticle core for dual-modal MRI and upconversion optical imaging;150 the measured ionic T1 relaxivity (∼2.6 mM−1 s−1, unit Gd3+ concentration) can be about 18 times higher than the one for 20−30 nm sized GdIII-doped NaYF4 nanoparticles (0.14 mM−1 s−1).149 Moreover, the size of Gd3+-containing nanoparticles has a signifianct effect on T1 relaxivity. The ionic relaxivity values of NaGdF4 nanoparticles increased from 3.0 to 7.2 mM−1 s−1 when decreasing the particle size from 8 to 2.5 nm, and the relaxivity of the 2.5 nm sized particle is almost twice that of clinically used Gd−DTPA (Magnevist) relaxivity.318 The rate of increase in relaxivity with decreasing particle size was proportional to the rate of increase in the surface-to-volume

5.3. Radioisotope Imaging

Nanoparticles containing radioactive functionality have been used in biomedical imaging.The radioactive functionality is introduced via incorporation of a radioactive nucleus (radionucleotide) in a nonradioactive nanomatrix. The nature and dosage of the radionuclide is chosen in such a way that the emitted species should be of sufficiently low energy, so as to 2848

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CdTe/ZnS core−shell nanoparticles for in vivo SPECT/CT imaging.340

provide a reliable radioimaging signal, without any concurrent radiotoxicity. Examples include position emitters (e.g., 124I) used in positron emission tomography (PET),332 and lowenergy gamma emitters (e.g., 125I) used in single photon emission computed tomography (SPECT).333 The PET imaging technique utilizes pairs of γ rays emitted by a positron-emitting radionuclide in the imaging contrast agents to produce a three-dimensional image in the body.334 Compared with biologically relevant molecules (antibodies, proteins, and peptides), nanoparticles represent a new approach for PET molecular imaging probe design. The advantage of using a nanoparticle approach is the ability to attach an increased number of radionuclides to each nanoparticle, so that PET imaging can be performed at a much lower dose of nanoparticles. Methods for PET radiolabeling of nanoparticles can be classified into four categories:335 (1) surface chelation of radiometal ions through coordination chemistry; (2) bombardment of nanoparticles via hadronic projectiles; (3) nanochemical preparation of nanoparticles using mixed radioactive and nonradioactive precursors; (4) chelator-free postsynthetic radiolabeling (ion exchange). Among them, method 1 and method 4 are the most frequently used approaches. Polyaspartic acid coated iron oxide nanoparticles with a size of 5 nm were synthesized utilizing a coprecipitation method.336 The amino groups on the surface were activated to couple macrocyclic 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) for chelation of the radionuclide, 64Cu, for PET imaging, while surface functionalization with NHS− poly(ethylene glycol) (PEG)−maleimide (MAL) enabled introduction of thiolated arginine-glycine-aspartic (RGD) peptides for integrin αvβ3 targeting. This provides a bifunctional iron oxide nanoparticle probe for PET and MRI scans of tumor integrin αvβ3 expression. In addition, click chemistry has also been used to attach a large number of 18F atoms, another commonly used nucleotide for PET imaging.337 Owing to the short half-life of 18F (110 min), a quick conjugation of 18F into the probe with a high reaction yield is of vital importance. The exchange of radionuclide 18F with the fluoride element in lanthanide-doped NaYF4 upconversion nanoparticles (less than 5 min) can impart them for use in PET imaging. 18F-labeled upconversion nanoparticles can become a useful contrast agent to provide both upconversion and PET imaging in cells and lymph monitoring.338 We have synthesized ORMOSIL nanoparticles radiolabeled with 124I (with half-life of 4.2 days), for application in PET imaging.138 Radiolabeling of ORMOSIL was carried out using the Bolton−Hunter method. The free amino groups on the nanoparticle surface form a covalent linkage with the N-hydroxysuccinimide (NSH) ester group linked with 124I. Radiolabeling allowed tracking the nanoparticle biodistribution in the whole body, which was mainly found to be localized in the liver and spleen of the mice. With regard to SPECT imaging, a number of nanoparticle formulations incorporating suitable radionuclides have been reported. Commonly used radionuclides for SPECT are 123I, 125 I, 99mTc, 133Xe, 201Th, and 18F. Among them, 125I has received a great deal of attention for incorporating into a nanoparticle. An example is a silver nanoparticle which is capped with poly(N-vinyl-2-pyrrolidone) (PVP) with an average size of 12 nm, which was radiolabeled by chemisorption of 125I, with a >80% yield.339 In this work, these nanoparticles were intravenously injected in a BALB/c mouse and the in vivo distribution pattern by noninvasive whole-body SPECT imaging was investigated. Another example is 125Cd labeled

5.4. CT Bioimaging

X-ray computed tomography (CT) is an imaging procedure that uses computer-processed X-ray scans to produce tomographic images of a specific area of the body.341 These crosssectional images are used for diagnostic and therapeutic purposes in various medical disciplines. Utilizing differences in X-ray absorption and attenuation by different components in the body, one can achieve anatomical visualization of body structures and obtain high contrast images of blood vessels, stomach, and gastrointestinal organs.1 As inherent CT contrast within soft tissues is too small to be effectively detected, contrast agents need to be delivered to target tissues for their effective detection with respect to other tissues using CT. Usually large atomic weight (high Z) elements serve as good CT contrast agents owing to their high X-ray absorption. Also, it is desirable that the K-shell electron binding energy (K-edge) of the agents be located within the higher-intensity region of the X-ray spectrum (roughly within 50−70 KeV). Other factors that contribute to the choice of CT contrast agents are biocompatibility, nontoxicity, and low cost. Some of the common contrast agents are iodine (nonradioactive), gold, platinum, bismuth, tantalum, and ytterbium. These contrast agents have been used in the past in molecular or salt forms. However, these molecular contrast agents have a relatively short circulation half-life in the body, and are devoid of targeting ability to sites of interest. These factors impose serious limitations on imaging outcome. Since nanoparticles have enhanced circulation lifetime and can be targeted to sites of interest, high Z element containing nanoparticulate contrast agents for CT have been extensively explored in the past decade. Among them, gold nanostructures have received the maximum attention owing to their high X-ray attenuation coefficient, ease of synthesis, biocompatibility, and other complementary properties (such as SPR and NIR tunability). The use of gold nanoparticles as efficient contrast agents for X-ray CT in vivo was first reported by Hainfeld et al.342 These nanoparticles were ∼1.9 nm in diameter, and thus can clear through renal filtration after in vivo administration. They were shown to absorb X-rays 3 times more than equal weight of an iodine-based agent (Omnipaque), thus providing a higher contrast than the standard iodine agents. Using gold nanorods conjugated with an antibody specific to head-andneck cancer, targeted molecular CT imaging of cancer cells in vitro was shown by Popovtzer et al.343 The X-ray attenuation coefficient for the targeted cells was shown to be over 5 times higher than that for untargeted cancer cells or normal cells. Reuveni et al. reported the use of intravenously injected antibody-conjugated gold nanoparticles (30 nm) for in vivo tumor targeting and molecular CT imaging.344 More recently, Zhou et al. have applied PEGylated gold nanoparticles for blood pool and tumor imaging using X-ray CT. A prolonged half-decay circulation time of 11.2 h in rats was observed.345 A commercial formulation of these gold nanoparticles as X-ray contrast agents for in vivo CT imaging is available from Nanoprobes (Yaphank, NY) under the trade name AuroVist. They can be applied for imaging using standard micro CT, clinical CT, planar X-rays, or mammography. Another advantage of gold nanostructures is that their unique optical (SPR) absorption, NIR tunability, and photothermal effects can 2849

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Figure 16. Hexamodal bioimaging using porphyrin−phospholipid-coated upconversion nanoparticles. The nanoparticles were characterized in vitro and in vivo for imaging fluorescence (FL), upconversion (UC), positron emission tomography (PET), computed tomography (CT), Cerenkov luminescence (CL), and photoacoustic tomography (PA). Reproduced with permission from ref 154. Copyright 2015 John Wiley & Sons.

Figure 17. Various optical biosensing methods utilizing nanostructures. “LSPR” is the acronym for localized surface plasmon resonance discussed earlier.

and PEGylated NaYbF4, when both 80 and 140 kVp of input Xray voltages were used.

be exploited to combine CT imaging with other optical modalities. Several other nanoparticles for CT contrast enhancement have also been explored. A prominent example is the bismuth sulfide (Bi2S3) nanoparticle.346,347 As an injectable CT imaging agent, these nanoparticles exhibit excellent stability at high Bi concentration, with 5-fold more X-ray absorption than iodine, and more than 2 h of circulation time in vivo (compared to only minutes for conventional contrast agents). To make these nanocrystals more stable with long resistance time (avoid fast RES clearance), their surface is coated with a biocompatible polymer, polyvinylpyrrolidone (PVP). Size- and shape-controlled Yb containing (NaYbF4:Er) nanoparticles have been synthesized and used by Liu et al.151 Following PEGylation and intravenous delivery in rats, a clear enhancement of the CT signal of the heart, when compared to that of PVP-conjugated Bi2S3 nanoparticles, could be observed. Similarly, long-lasting liver and spleen CT signal enhancement could be seen using these Yb-containing nanoparticles, which did not diminish even upon reducing the dosage to half. In addition to CT imaging, these nanoparticles were also useful as upconverting nanophosphors with provision for NIR optical imaging. Zhang et al. have prepared silica-encased upconverting nanophosphors (UCNPs) with conjugated 5-amino-2,4,6-triiodoisophthalic acid as dual optical-CT nanoprobes. Following PEGylation and subsequent intravenous administration in rats, a prolonged circulation time in vivo was observed, with liver CT contrast visualized even after 30 min.348 Nanoparticles containing two or more CT-contrast elements have also been formulated, which can provide sufficient contrast enhancement at different X-ray input voltages. For example, BaYbF5 nanoparticles containing two contrast elements (Ba and Yb) have been developed.349 Following silica coating and PEGylation, remarkable enhancement in X-ray attenuation was observed in these BaYbF5 nanoparticles, when compared to Iobitridol

5.5. Multimodal Bioimaging

In recent years, a growing interest in the development of “fusion technologies”, combining multiple medical imaging techniques to provide complementary information, has fueled interest in developing multifunctional nanoparticles. Thus, we, as well as other laboratories, are developing nanoparticle probes containing multiple contrast agents for simultaneous use in different imaging modalities (e.g., optical, MRI, PET, SPECT, and CT imaging). Furthermore, these nanoparticle-based contrast agents can carry various biotargeting agents (e.g., antibodies) for targeting specific disease markers/sites. These targeted multimodal nanoparticle platforms will advance the sensitivity and selectivity of medical imaging technology, thus allowing for early detection of various diseases. Some representative examples include functionalized gold for CT/ MR/US (ultrasound), 350 and photoacoustic (PA)/MR/ Raman;351 functionalized iron oxide for FL/MR/PET,352 FL/ MR/CT,353 and FL/MR/PET/bioluminescence;354 polymeric porphyrins for FL/MR/PET;355 melanin nanoparticles for PA/ MR/PET;356 and upconversion nanoparticles for UC/MR/ CT357and UC/MR/CT/SPECT.310 Recently, we have demonstrated hexamodal bioimaging using porphyrin−phospholipidcoated upconversion nanoparticles (Figure 16).154 The NIR fluorescence, NIR-to-NIR upconversion luminescence, PA, Cerenkov luminescence, CT, and PET were simultaneously used for lymphatic mapping in mice. The porphyrin− phospholipid coating directly confers NIR biophotonic properties of FL and PA to the UCNP, while PET and CL imaging can be entailed through the exquisite affinity of copper for porphyrin. The UCNP (core−shell of NaYbF4:Tm−NaYF4) used was rationally designed for NIR-to-NIR upconversion luminescence imaging, and dense electron content from Yb3+ is 2850

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6.1. Fluorescence Sensors

also suitable for contrast enhancement in CT. An important advantage of having six distinct imaging functionalities on a simple nanoparticle formed from just two active components (porphyrin and upconversion nanoparticles) is based on its versatility without further modification. For example, in terms of trimodal imaging alone, this particle could be used in 20 (choose three modalities among the six modalities) unique combinations of modalities, spanning many potential applications.

Fluorescence-based biosensing has been used to monitor the physiological activities of cells that lead to a variety of intracellular diagnostics for disease profiling on a cellular level. We will discuss fluorescence sensors based on four types of nanostrctures, i.e., dye-doped nanoparticles, QDs, molecular beacon, and upconversion nanoparticles. 6.1.1. Dye-Doped Nanoparticles. Kopelman’s group has demonstrated fluorescence sensing of H+, Ca2+, Na+, Mg2+, Zn2+, Cl−, NO2−, O2, NO, and glucose, using their PEBBLE nanosensors (Figure 18); the sensing principle involves a

6. NANOPARTICLE-BASED BIOSENSING Biosensing forms a major thrust of medical diagnostics. In vitro biosensing utilizes biopsied cells, blood, urine, sweat, sputum, bronchio-alveolar lavage (BAL), and other body fluid samples collected from humans, which are potent indicators of any malfunctioning of the body, and can play a critical role in diagnosis and staging human diseases.2 In addition, biosensing can detect new strands of microbial organisms and the spread of infectious diseases. Nanotechnology has also provided major advancements in biosensing. This section describes some major optical biosensing techniques involving the use of a nanoplatform. Figure 17 shows the various optical biosensing methods, along with different types of nanostructures utilized. Electrical, electrochemical, and magnetic sensing will not be discussed in this review.358,359 A nanoplatform offers a large surface-tovolume ratio to enable surface binding of a large number of biorecognition elements to capture the analyte(s), thus entailing highly sensitive detection. A broad spectrum of spectroscopic techniques such as absorption, fluorescence, Raman, and plasmonics can be used to quantify molecular information through this nanoplatform. Among these, fluorescence sensors and plasmonic sensors have been widely used, and thus are the focus of this section. (i) Fluorescence biosensors utilize a range of fluorescence parameters to detect or obtain analytes, such as a change in the fluorescence intensity, a shift in the peak wavelength of fluorescence, a change of the fluorescence lifetime, or a ratiometric variation of the intensities of two emission bands. Biorecognition elements for selective capture of chemical entities or biospecies play a vital role to decipher the information (type and amount) on analytes into a discernible parameter change of fluorescence. Fluorescence may be an intrinsic feature of the biorecognition element, but more commonly, it is from a fluorophore which is conjugated with the biorecognition element. (ii) Raman biosensors utilize a change in the vibrational frequency or a variation in the intensity of a Raman active vibrational mode. The typically low cross section of Raman scattering results in insufficient Raman signal for high sensitivity detection. However, a metallic nanoparticle platform can produce a significant gain in sensitivity by the use of surface enhanced Raman scattering (SERS), when a Raman reporter is attached to the surface of a metallic nanoparticle or a metallic nanostructure. The increased local electric field due to the localized surface plasmon resonance (LSPR) in the metallic nanoparticles causes this enhancement. In addition, an LSPR sensor, where the oscillation of surface plasmon is very sensitive to the surrounding dielectric environment, can also be used as a sensitive technique for biodetection by monitoring the shift of its LSPR peak. Surface plasmon resonance (SPR) sensors, the other type of plasmonic biosensors, are already commercially available; they utilize the delocalization of surface plasmon resonance at the interface of a thin (∼50 nm) metallic film.

Figure 18. Schematics of a PEBBLE nanosensor. Reproduced with permission from R. Kopelman, http://www.umich.edu/~koplab/ research2/analytical/EnterPEBBLEs.html.

change in the fluorescence intensity of an appropriately encapsulated dye in the presence of an analyte to be detected.360,361 PEBBLEs is an abbreviation of “probes encapsulated by biologically localized embedding”, which are nanoscale devices consisting of sensor molecules entrapped in a chemically inert matrix, typically in a size range of 20−200 nm. The PEBBLE matrix is able to protect the fluorescent sensing molecules from interference by proteins, allowing reliable in vivo calibrations of dyes. For example, Ca ion activities have been monitored by encapsulating calcium green-1 and sulforhodamine dyes in hydrophilic polyacrylamide (PAA) nanoparticles. The calcium green fluorescence intensity increases with increasing calcium concentrations, while the sulforhodamine fluorescence intensity remains unaffected; the intensity ratio of fluorescence from the calcium green to that from the sulforhodamine was used to quantify the cellular calcium level. 6.1.1.1. pH Sensing. The PEBBLE technology was also used by chemically linking the pH sensitive dyes to a PAA nanoparticle for intracellular pH sensing.361 We have used a new design of nanoparticle-based fluorescence pH indicator for near-physiological use by matching the pKa to the pH of the experimental system.362 This design consisted of organically modified silica (ORMOSIL) nanoparticles concentrated with a ratiometric pH-responsive hydrophobic organic dye: naphthalenylvinylpyridine derivative (NVP) with the pyridyl unit as the proton receptor. NVP responds to protons by a red shift of fluorescence from high energy (blue) to low energy (yellow) upon protonation. The proton-sensing event takes place mainly near the surface of the NVP-concentrated ORMOSIL nanoparticle. Thus, the high-energy neutral and the low-energy protonated states closely coexist in the inner and surface parts 2851

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Stokes process involves optical excitation from thermally excited vibrational energy states of the ground state, followed by emission. 6.1.2. Quantum Dots. Quantum dots, because of their narrow emission band, tunability of emission, photostability against photobleaching, and ease of surface functionalization, have also been extensively investigated for optical biosensing.369−371 CdSe/ZnSe/ZnS core−shell−shell QDs capped with mercaptoacetic acid were used for intracellular pH sensing in human ovarian cancer cells.372 In this case, the emission intensity of QDs increased monotonically with increasing pH. Leblanc et al. reported the use of CdSe/ZnS core/shell QDs and organophosphorus hydrolase (OPH, the recognition element) to detect paraoxon.373 The negatively charged QDs and the positively charged organophosphorus hydrolase were brought together through electrostatic attraction. The luminescence intensity of the OPH/QD bioconjugate was quenched in the presence of paraoxon. Moreover, the use of CdSe QDs as fluorescent nanothermometers has been reported using twophoton fluorescence microscopy.374 Owing to temperaturedependent two-photon luminescence, a temperature gradient artificially created in a biocompatible fluid (phosphate-buffered saline) as well as in a single living cell at high resolution can be successfully imaged. Mauro et al. reported on a hybrid inorganic-bioreceptor QD−protein sensor for chemical sensing of maltose.375 In this design, each QD is linked to multiple copies of Escherichia coli maltose-binding protein (MBP) through the C-terminal oligohistidine segment, while the βcyclodextrin-QSY9 dark quencher is attached to the MBP saccharide binding site, resulting in fluorescence resonance energy-transfer (FRET) quenching of QD fluorescence. Added maltose analytes detach the β-cyclodextrin-QSY9 from the MBP−QD complex, thus leading to an increased photoluminescence. Following a similar strategy, Rosenzweig et al. reported on FRET-based QDs for protease sensing, in which an RGDC peptide labeled with a rhodamine dye molecule is bound to the surface of CdSe/ZnS QDs to form the sensing nanoplatform.376 Selective cleavage of the RGDC peptide by proteases removed the effect of the FRET process on the luminescence color of QDs, thus producing recovery of the authentic color of QDs. Nie and co-workers used QDs as tags for multiplexed detection of DNA by labeling a DNA target with a fluorescent dye and an oligonucleotide microbead with quantum dots emitting at different wavelengths.377 The capture of a DNA target on the microbead exhibited fluorescence from both emitters. A unique fluorescence spectral signature (emission at different wavelengths with variable intensities) for each individual target is obtained by varying the number and ratios of the multiple color quantum dots. Alivisatos and coworkers used CdSe/ZnS core−shell QDs in a chip-based assay to detect single base pair mutations in DNA.378 6.1.3. Molecular Beacon. A molecular beacon utilizing nanoparticle technology has been used by involving energy transfers between a fluorescent unit, a dye or a QD (F1), and a fluorescent quencher (Q).379,380 A molecular beacon consists of a loop, involving a single-stranded oligonucleotide in a specific sequence, and a stem, usually consisting of five to seven complementary base pairs. The two ends of the stem consist of an emitter (F1) and a quencher (Q). In the absence of a target analyte, the stem is intact, keeping the emitter and the quencher in close proximity which quenches the emission. In the presence of the analyte, the binding or the biorecognition process forces them apart, thus increasing the distance between

of the nanoparticle, respectively. By virtue of an inner-to-surface energy transfer from the absorbing neutral form in the inner part to the energy-accepting protonated surface molecules, the fluorescence signal from the protonated form is amplified, thus significantly improving the pH sensitivity. This approach also offers an easy way to increase the pKa value of the nanoparticles to a physiological level. However, due to the strong scattering of light in most biological tissues, the fluoresence technique for pH sensing remains rather limited, when working in a light diffusion regime. To overcome this problem, Kopelman et al. demonstrated the use of the PAA nanoparticle containing pHsensitive dye seminaphthorhodauor (SNARF-5F) for ratiometric photoacoustic determination of the pH level in a rat joint model, with a precision better than 0.1 pH unit.363 The pHinduced change of the absorption of the SNARF-5F dye was exploited, providing a variation of PA signal at 580 and 565 nm. 6.1.1.2. Oxygen Sensing. PEBBLE has also been used for oxygen sensing where a hydrophobic matrix such as ORMOSIL or polydecyl methacrylate (PDMA) encapsulating an oxygensensitive platinum based dye (platinum(II) octaethylporphyrin ketone) as well as a reference dye for ratiometric determination of oxygen was used. The use of an infrared dye has allowed the PEBBLE sensors to be operated in human plasma samples.364 In vivo quantification of blood oxygenation has been demonstrated by measuring the lifetime of the triplet state of Pd-tetra(4-carboxyphenyl) tetrabenzoporphyrin dendrimer using the photoacoustic lifetime (PALT) technique.365 An interaction of the intermediate triplet state of the dye with the surrounding oxygen shortened its relatively long lifetime (∼250 μs), producing a linear relation between the oxygen concentration and the lifetime. The essence of PALT lies in the use of a pump−probe technique, whereby a pump laser is used to create population of the triplet state, and the second probe laser can produce a population-dependent PA signal. As no optical absorption of hemoglobin for the oxygen level measurement is involved, PALT becomes a potentially powerful tool to evaluate oxygenation levels in tissues, such as in tumor hypoxia. 6.1.1.3. Intracellular Electric Field Sensing. Kopelman and co-workers developed E-PEBBLES to measure intracellular electric fields, where they utilized a fast responding voltagesensitive dye (di-4-ANEPPS) inside silane-capped (polymerized) micelles.366 Electrochromic effect of the dye produces a change in the fluorescence spectrum, providing the measurement of the electric field. PEBBLE also was used to detect glucose in which the enzyme glucose oxidase (GOx), an oxygen-sensitive ruthenium-based dye, and a reference dye were coencapsulated within the PAA nanoparticles. The principle utilized was that enzymatic oxidation of glucose to gluconic acid depletes the local oxygen concentration, leading to an increase in the fluorescence of the oxygen-sensitive dye.364 6.1.1.4. Temperature Sensing. The temperature of a subcellular unit can also be measured by the fluorescence method. Malignant cells have a higher temperature than the corresponding normal cells owing to the enhanced enzymatic activities in mitochondria of a malignant cell.367 This sensing method utilizes the ratio of the fluorescence intensities of the temperature dependent anti-Stokes process to the Stokes process to derive the local temperature.368 As opposed to Stokes fluorescence occurring from optical excitation of molecules in the zero point vibrational level of the ground electronic state to the first excited electronic state, an anti2852

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the linked 1O2 quencher until the interaction of the linker with tumor-associated protease. A precise selective control of PDTinduced cell death as well as tumor inhibition was demonstrated. 6.1.4. Upconversion Nanoparticles. Upconversion nanoparticles are promising for biosensing owing to the use of biocompatible NIR light that is silent to biological tissue constituents and common assay interferants in analytecontaining medium, to perform the excitation, while emitting anti-Stokes visible luminescence for detection or visualization. The ability to prepare small-size, efficient, and multicolor upconversion nanoparticles in colloidal forms enables them to be utilized as nanosensors for mutiplexed sensing.391−394 Upconversion biosensors have the potential to detect biochemical entities as well as to precisely monitor specific fundamental physiological processes with high sensitivity, which are deeply located in the living body systems.30 In addition, no background luminescence from biochemical assays is elicited, thus producing a detection limit unavailable from conventional fluorescence assays. In addition, the optical attributes of upconversion nanoparticles are inert to the environment (e.g., acid, base, and temperature), thus providing unaffected detection results, despite a large variety of environmental sampling conditions. Vetrone et al. developed a nanothermometer using water-dispersed PEI-coated NaYF4:Er3+/ Yb3+ upconversion nanoparticles.148 The strategy of this nanothermometry is based on measuring the temperaturedependent variation of the intensity ratio between the upconverted emissions of Er3+ ions at 520 nm from the 2 H11/2 → 4I15/2 transition and at 550 nm from the 4S3/2 → 4I15/2 transition. This nanothermometer is very sensitive, being able to measure the internal temperature of the living HeLa cervical cancer cell from 25 °C to the thermally induced death at 45 °C. Ratiometric upconversion biosensors were also utilized to detect mercuric ions in aqueous media.395,396 For example, Li et al. developed an upconverting sensor nanoplatform whereby the chromophoric ruthenium complex (N719) was labeled on the surface of NaYF4:20 mol % Yb3+,1.6 mol % Er3+,0.4 mol % Tm3+ nanoparticles.396 As the absorption peak of the N719 dye is varied with the mercuric ion concentration in the visible range, this causes a change of upconverted visible luminescence (at ∼541 nm), while leaving the NIR upconversion luminescence (at ∼800 nm) unaffected. Indeed, the ratiometric upconversion luminescence enables detection of Hg2+ down to 1.95 ppb (parts per billion), which is even lower than the maximum level (2 ppb) of Hg2+ in drinking water set by the U.S. Environmental Protection Agency. An upconverting oxygen sensor was presented by Wolfbeis’s group, in which both the NaYF4:Yb3+/Tm3+ nanoparticles as nanolamps and iridium(III) complexes as the oxygen indicators were incorporated in an ethyl cellulose thin film.397 Upon excitation at ∼980 nm, the FRET process from the upconversion nanoparticles will induce visible fluorescence from the iridium(III) complex, which is sensitive to the oxygen concentration. Determination of fluorescence intensity change quantified the content of molecular oxygen. This holds promise for clinical medical diagnosis as the developed tumor exhibits the phenomenon of hypoxia. Upconversion biosensing has also been utilized for detection of a wide range of analytes such as prostate-specific antigen in human prostate tissue,398 human chorionic gonadotropin,399 a drug of abuse panel,400 DNA detection,401 bacterial pathogen,402 and nuleic acids.403 Elimination of autofluoresence has made upconversion bio-

the emitter and the quencher sufficiently to inhibit quenching. The result is restoration of the fluorescence of the emitter. This process is illustrated in Figure 19.

Figure 19. Molecular beacon approach for biosensing. Hybridization with the DNA target molecules of a complementary sequence or unwinding of the DNA with an increase in temperature, change of pH, or presence of a denaturing agent produces an increase in emission.

Molecular beacons are ultrasensitive for DNA detection, as well as for probing of protein−DNA, protein−RNA, and RNA/ DNA/protein interactions.379,381 Krauss et al. have developed label-free biological sensor arrays based on fluorescence unquenching of DNA hairpins immobilized on metal surfaces, benefiting from a combined use of microarray patterning of DNA oligonucleotides on planar metal surface and oligonucleotide identification in solution.382 This produces numerous opportunities for high throughout sensing. Poddar reported on the detection of adenovirus through observation of recovered fluorescence from a molecular beacon post polymerase chain reaction (PCR) amplification.383 Recent efforts in the molecular beacon approach have focused on the utilization of quantum dots rather than organic dyes as the emitters. This substitution provides the added photostability of the QDs to avoid the photobleaching problem of dyes. Gold nanoparticles have been similarly utilized as the emission quencher.384,385 This process involves the dipolar quenching mechanism, when an emitter is in contact with a gold surface. Cady et al. used a QD and a gold nanoparticle combination for sequence-specific DNA detection.386 More recently, it has been shown that graphene oxide could bind dye-tagged ssDNA (5′-AGTCAGTGTGGAAAATCTCTAGC-FAM-3′), and quench its fluorescence with high efficiency.387 Hybridization with complementary target ssDNA (single stranded DNA) sequence (5′-GCTAGAGATTTTCCACACTGACT-3′, from the HIV-1 U5 long terminal repeat sequence) detached the formed dsDNA (double stranded DNA) from graphene oxide, thus recovering the dye fluorescence signal. This molecule beacon biosensor makes use of the distinct affinity of ssDNA and dsDNA to the graphene oxide. This is because the π-stacking interaction between the ring of nucleobases and the hexagonal cells of the graphene enables ssDNA to strongly adsorb onto the graphene oxide, while the nucleobases in dsDNA are shielded by the phosphate backbone, thus resulting in the deattachment.388,389 The molecular beacon technology has many other applications. For example, photodynamic molecular beacon has recently been presented to control the ability of photosensitizer to generate singlet 1O2, and thus the PDT activity.390 The activity of a photosensitizer is deactivated by 2853

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of localized surface plasmon resonance (LSPR) as the transduction mechanism to detect analytes. The home pregnancy test is an excellent example on how gold nanoparticles can be used to solve a real-world problem in colorimetric detection of hCG (human gonadotropic hormone) using LF immunoassay. The structure of the test LF strip is similar to Figure 20, but having only one test line and one control line. Release of the hCG hormone in urine starts after conception and increases during the first semester of pregnancy. When hCG is present in the urine, it will become colored by anti-hCG antibody-labeled gold nanoparticles, and then captured by immobilized biomolecules on the test line to produce a colored band for reading.404 The LSPR peak shift can also induce a variation of color for colorometric sensing. In the simplest scheme, the surface of a metallic nanoparticle is functionalized with a biorecognition element to capture the analyte to be detected. This binding changes the dielectric environment around the nanoparticle and thus shifts the LSPR peak used for detection. This method of sensing, also called “refractive index sensing”, can utilize metallic nanoparticles of different shapes, core−shell structures, and individually dispersed or immobilized on a substrate.405 A demonstrative scheme is shown in Figure 21. Here the gold or silver nanoparticles can be chemically tethered to a glass surface or electrostatically bound by creating opposite charges on the metal nanoparticles and the substrate. A method which provides control of the shape and size of the nanoparticle arrays on a solid substrate is nanosphere lithography (NSL).406 In this method, a metal is evaporated onto a self-assembled, close-packed monolayer of monodispersed polystyrene nanospheres. The metal nanostructures are deposited in the interspatial regions between the polystyrene nanospheres, which are subsequently removed to leave truncated tetrahedral-shaped metallic nanoparticle arrays with a hexagonal symmetry.3 Other variations of NSL using different types of masks have been proposed to produce metallic nanostructures of different shapes, such as nanodisks, nanorings, nanoshells, ellipsoids, nanorods, or any combinations of them. In one of the earlier works, the widely used biotin−streptavidin model was employed to demonstrate LSPR biosensing. Gold nanospheres immobilized on a glass substrate were coupled through a linker,407 and the binding with streptavidin was detected either by monitoring a change in the transmission or simply by monitoring the color change. Aggregation sensor is another type of LSPR sensor, which utilizes the aggregation of metallic nanoparticles caused by analyte binding. As the distance is shortened between nanoparticles, plasmonic coupling becomes dominant to produce a significant shift of the LSPR frequency, which is

assays to produce an improved detection limit over conventional assays. For example, an upconversion lateral flow (LF) immunochromatographic assay, schematically shown in Figure 20, provides a detection limit of 10 pg of human chorionic

Figure 20. Upconversion lateral flow immunochromatographic assay. The lateral flow strip is designed to accommodate up to 12 distinct test lines and two control lines. One control line acts as a test performance control, while the other one acts as a calibration line for the reader instrument. Reproduced with permission from ref 400. Copyright 2011 Elsevier.

gonadotropin, which is 10-fold lower as compared to conventional assays based on gold nanoparticles or colored latex beads.399 Moreover, it has been shown by Niedbala et al. that an LF-based UC strip assay is able to provide simultaneous detection of amphetamine, methamphetamine, phencyclidine, and opiates in saliva by using multicolor upconversion particles (Figure 20).400 In their design, the green-emitting (550 nm) particles were coupled to antibodies for phencyclidine and amphetamine, while the blue-emitting (475 nm) particles were coupled to antibodies for methamphetamine and morphine. Their differentiation is determined by the phosphor color and position appearing on the test strip. These sensors are typically based on particles with low upconversion efficiency. Recent progresses on the significant improvement of upconversion efficiency hold promise to produce pronounced improvement in the detection limit. 6.2. Plasmonic Sensors

Plasmonic biosensors are another major class of widely investigated and utilized optical biosensors. Both localized surface plasmon resonance (LSPR) in metallic nanoparticles and delocalized surface plasmon resonance (SPR) in metallic nanofilms have been utilized for biosensing. 6.2.1. Localized Surface Plasmon Resonance (LSPR) Colorimetric Sensors. Colorimetric detection is one of the easiest and most powerful sensing methods using metallic nanostructures. These sensors utilize a shift in the peak position

Figure 21. Biosensing scheme using refractive index sensing with LSPR of plasmonic nanostructures. (a) Substrate. (b) Metal nanoparticles are attached to substrate by chemical linkers or nanolithography. (c) Metal particles are modified with the sensor moiety. (d) Analytes attach specifically onto the recognition layer and (e) cause a change in the refractive index around the particles resulting in an LSPR shift. Reproduced with permission from ref 405. Copyright 2009 Elsevier. 2854

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Figure 22. Colorimetric sensing with polypeptide modified gold nanoparticles designed to allow folding, induced particle aggregation triggered by Zn2+, and a polypeptide-based synthetic receptor for binding of protein analytes. In the absence of the analyte, addition of Zn2+ triggers dimerization and folding, resulting in particle aggregation and a rapid color shift of the suspension from red to purple (left). Analyte binding precludes aggregation and the dispersion remains red (right). Reprinted with permission from ref 409. Copyright 2009 John Wiley & Sons.

discernible by the naked eye. Take gold nanospheres, for example: aggregation produces the color change from red to purple-blue to the naked eye. Mirkin et al. tethered thiolated single-stranded DNA (ssDNA) to a gold nanoparticle of ∼15 nm in diameter through the intense gold−sulfur interaction.408 The complex of ssDNA and gold particles dispersed well in solution and exhibited a well-defined LSPR, appearing a burgundy-red color to the eye. When the ssDNA was hybridized with its complementary DNA in the test solution, the formation of duplex resulted in an aggregation of gold nanoparticles, causing a color change into blue black. Colorimetric detection of proteins has been described by Liedberg et al. (Figure 22).409 In their design, polypeptidefunctionalized Au nanoparticles can specifically bind to the target analyte (human carbonic anhydrase II) through a polypeptide sensor scaffold. The extent of particle aggregation, induced by Zn2+-triggered dimerization and folding of a second polypeptide also present on the gold nanoparticles, is able to give a readily detectable colorimetic shift that is dependent on the concentration of human carbonic anhydrase II, with a detection limit of ≈10 nM. Halas, West, and co-workers used gold nanoshells with nanoshell-conjugated antibodies to demonstrate a rapid immunoassay, capable of detecting analytes within a complex blood biological media.410 Analyte-induced aggregation of these multiple metallic nanoparticles produces a red shift in the LSPR peak, along with a decrease in the extinction coefficient at ∼720 nm. Hirsch et al. demonstrated the detection of immunoglobulins. An interesting concept used for monitoring DNA conformational changes is that of a “plamonic molecular ruler”, introduced by Alivisatos’ group.411 In a plasmonic ruler, the change in separation between two metallic nanoparticles, due to DNA hybridization, produced a color change as well as a change in the plasmonic scattering intensity. 6.2.2. Localized Surface Plasmon Resonance (LSPR) Fluorescence Sensors. LSPR fluorescence sensors utilize analyte-induced enhancement in the luminescent intensity of a fluorophore, when its distance from a metallic nanoparticle is tuned by analyte interaction. Direct contact of fluorphore with the metal surface will result in dipole-induced quenching of fluorescence, as discussed earlier in the molecular beacon approach of section 6.1.3. When they are separated, the fluorescence will be fully recovered or enhanced due to elimination of quenching. On the other hand, if the fluorophore

is brought from a remote distance to the metallic nanostructure, the enhanced local plasmonic field at the metal surface enhances the fluorescence and the enhancement is maximum at an optimum nanoscale distance.3 Lakowicz et al. reported on metal-enhanced fluorescence to probe the hybridization of DNA.412 Thiolated oligonucleotides were bound to silver particles on a glass substrate. Addition of a complementary fluorescein-labeled oligonucleotide resulted in a dramatic timedependent 12-fold increase in the fluorescence intensity during the process of hybridization. This hybridization brought the fluorescein molecule close to the silver nanoparticles at an appropriate distance for surface plasmon enhancement of fluorescein’s intensity. Mirkin’s group has developed an aptamer nanoflare, a gold nanoparticle core functionalized with a dense monolayer of nucleic acid aptamers, that can directly quantify an intracellular analyte in a living cell.413 In the initial state, the fluorescence of the flare strand is quenched by the gold nanoparticle. When the ATP molecules bind to the aptamer, it causes a new folded secondary structure producing the releasing of fluorescence reporters. They demonstrated that these nanoconjugates are readily taken up by cells; whereby their signal intensity can be used to quantify intracellular ATP concentration. In analogy, Rotello et al. has created a sensor array containing six noncovalent gold nanoparticle−fluorescent polymer conjugates to detect, identify, and quantify protein targets.414 The polymer fluorescence is quenched by gold nanoparticles; the presence of proteins disrupts the nanoparticle−polymer interaction, producing distinct fluorescence response patterns. These patterns are able to discriminate individual proteins at nanomolar concentrations, and can be quantitatively differentiated by linear discriminant analysis (Figure 23). These novel nanomaterial-based protein detector arrays hold potential applications in medical diagnostics. More recently, Rotello and co-workers reported on using a similar strategy, a high-throughput multichannel sensor platform that can profile the mechanisms of various chemotherapeutic drugs in minutes.415 The sensor is made of a gold nanoparticle complexed with three different fluorescent proteins that can sense drug-induced physicochemical changes on cell surfaces. The presence of cells will rapidly displace fluorescent proteins from the gold nanoparticle surface and restore the fluorescence. Fluorescence “turn on” of the fluorescent proteins depends on the drug-induced cell surface changes, generating patterns that identify specific mechanisms of cell death induced by drugs. 2855

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dependent SERS nanoprobe in living biological systems, utilizing 2-aminothiophenol (2-aminobenzenethiol, 2-ABT) labeled silver nanoparticles.420 Through the variations in the SERS spectra of the attached 2-ABT molecules, the probe is capable of measuring pH in its vicinity over the range 3.0−8.0. The vibrational band of the amino group of 2-ABT was chosen as the Raman probe, as it is sensitive to pH and the amino group is a common functional moiety of many biomolecules. We showed that these nanoprobes are robust and their pH sensitivity can be used when the nanoparticles are incorporated inside living cells. Although earlier reports used silver nanoparticles and roughsurface structures for SERS, the emphasis has recently shifted to the use of metallic gold nanostructures due to their environmental stability.421,422 Another area of increased activity utilizes anisotropic metallic nanostructures, such as nanoprisms, which exhibit a significantly increased electromagnetic enhancement factor, when compared to spherical nanoparticles; aggregates such as dimers show a significantly large plasmonic field, called hot spots, in the regions in-between nanoparticles.31 The SERS signals obtained from these hot spot regions are thus many orders of magnitude higher, providing greatly improved sensitivity. Nie and co-workers used a Raman-based molecular beacon for DNA detection.416 The principle of the molecular beacon has been described above, except that a dramatic change in SERS is used as a probe for DNA hybridization. Moreover, they have realized spectroscopic detection of in vivo tumor with surface-enhanced Raman nanoparticle tags.423 The ability for nanoscale control of the distance between the Raman dye position and the SERS active site opens up new avenues for nanostructure-based single-molecule detection and bioassays. Suh et al. reported a single-target-DNA hybridization method to obtain SERS-active gold−silver core−shell nanodumbbell, where the separation between the Raman dye position and the nanodumbbell can be engineered with nanometer precision.424 Atomic-force-microscope-correlated nano-Raman measurements show stable Raman signals from a single-DNA-tethered nanodumbbell. Nam et al. showed highly uniform and reproducible surface-enhanced Raman scattering from DNA-tailorable nanoparticles with a 1 nm interior gap.425 The nanogap between gold nanobridged particles can be well-defined by the tethered DNA molecules on the surface; a quantifiable amount of Raman dyes can be loaded in the uniform and hollow gap (∼1 nm). Single-particle nano-Raman mapping analysis revealed that >90% of Au-NPs had enhancement factors greater than 108, which is sufficient for single-molecule detection. Tian et al. report on shell-isolated nanoparticle-enhanced Raman spectroscopy, in which the Raman signal amplification is provided by gold nanoparticles with an ultrathin silica or alumina shell.426 The ultrathin coating keeps the nanoparticles from agglomerating, separates them from direct contact with the probed material, and allows the nanoparticles to conform to different contours of substrates (Pt and Au single-crystal surfaces and Si, demonstrated in their work). Sensitive measurements of SERS of different spots of yeast cells and detection of pesticide residues on citrus fruits illustrate the flexibility of the shell-isolated SERS approach for its broad range of applications. 6.2.4. Nanomaterial-Enhanced Surface Plasmon Resonance (SPR) Sensors. Surface plasmon resonance (SPR) sensors are the most extensively utilized optical biosensors for real-time monitoring and label-free sensing of molecule binding events at a metal−dielectric interface. They have found wide

Figure 23. Fluorophore displacement protein sensor array. (a) Displacement of quenched fluorescent polymer (dark green strips, fluorescence off; light green strips, fluorescence on) by a protein analyte (in blue), with concomitant restoration of fluorescence. The particle monolayers feature a hydrophobic core for stability, an oligo(ethylene glycol) layer for biocompatibility, and surface charged residues for interaction with proteins. (b) Fluorescence pattern generation through differential release of fluorescent polymers from gold nanoparticles. The wells on the microplate contain different nanoparticle−polymer conjugates, and the addition of the protein analyte produces a fingerprint for a given protein. Reprinted with permission from ref 414. Copyright 2007 Nature Publishing Group.

The nanosensor can be generalized to different cell types and does not require processing steps before analysis, offering an effective way to expedite research in drug discovery, toxicology, and cell-based sensing. 6.2.3. Surface Enhanced Raman Scattering (SERS) Sensors. SERS is emerging as a powerful biodetection method that provides detailed information about the chemical structure and the conformation of an analyte, with the sensitivity approaching a single molecule detection level.31,416 The exceptional intensity enhancement provided by SERS enables detailed analysis of the vibrational fingerprints of proteins, thus leading to significant advances in SERS-based immunoassays.417 SERS-based sensing has the advantage over fluorescence-based detection, as the problem of photobleaching in a typically used organic fluorophore is not encountered. The SERS-based method can be label-free, with direct detection of a protein possible by adsorption onto a SERS platform.418 A SERS probe can be alternatively used where the SERS-enhanced Raman spectrum of a molecular species is linked to a metallic nanoparticle for the detection of a target analyte. For multiprotein detection, Han et al. developed a new label-free protein detection “Western SERS”, which combines SERS and Western blot by using colloidal silver staining, allowing multiprotein detection on a single nitrocellulose membrane.419 The silver nanoparticles are adsorbed along the net of the membrane, to which proteins have already adhered, to produce the SERS effect. We demonstrated the application of a pH2856

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magnitude by nanomaterials, thus entailing biosensing of hardto-detect molecules even within the concentration range between picomoles and femtomoles. Two main general approaches have been engaged for nanomaterials-enhanced SPRs. (i) Nanomaterials-enhanced SPR sensing using nanostructured metallic substrate. For example, Kabashin et al. created a novel SPR sensing film consisting of gold nanorod metamaterials, which is capable of supporting propagation of SPR mode in an arrayed gold nanorod layer. A detection limit of 10 mM was achieved for biotin, which has a small molecular weight of 244 Da.431 This detection limit is considerably lower than that from conventional label-free plasmonic devices used for detection of small analytes. This improvement benefits from two aspects: (i) a substantial space overlap between the probing electromagnetic field and the active biological substance incorporated between the nanorods; (ii) a strong plasmonmediated energy confinement inside the sensing layer. Graphene-coated metallic SPR sensing substrates have also emerged recently, which can produce a larger evanescent electric field compared to those with bare metallic thin films, thus entailing a higher SPR sensitivity.432−434 For example, Zhang et al. reported on an SPR biosensor with graphenecoated Au film as the substrate for detection of human immunoglobulin G (IgG).434 The graphene sheet was functionalized with goat antihuman IgG as a capture antibody. This sensing platform allows a minimum concentration of 0.3 mg mL−1 analyte to be detected, which is about 4 times lower than that of conventional SPR biosensor using pure metallic Au as the substrate. The improved sensitivity can be ascribed to charge transfer from graphene to the surface of the metal thin film, which results in a strong electric field enhancement at the sensing surface.432 In addition, the graphene-modified SPR sensing substrate has a unique advantage over the conventional bare metallic Au substrate for detection of aromatic ring structure molecules. This is because the strong π-stacking forces could attach these molecules to the graphene surface without the need for the recognition element immobilized on the substrate to capture them, thus providing exciting opportunities to detect trinitrotoluene (TNT), peptides, DNA, RNA, and siRNA molecules. (ii) Enhanced SPR sensing using nanomaterials as amplification tags. These nanomaterials encompass metallic Au nanostructures,435,436 metallic Ag nanostructures,437,438 magnetic nanoparticles of iron oxide,439 carbon nanotubes,440 and lipsome nanoparticles.441 A sandwich structure is commonly utilized to enhance the sensitivity, whereby the target analyte is first captured by the recognition element immobilized on Au film and then binds to secondary-antibodyconjugated nanomaterials. For this scheme, metallic gold nanostructures have been extensively investigated and show pronounced sensitivity enhancement. For example, Huang et al. investigated specific interactions between Au nanoparticle labeled carbamate inhibitors (ALC1 and ALC2) and the acetylcholinesterase (AChE) immobilized on sensor chip surface. With the signal amplification of Au nanoparticles, the detection limits are 7.0 pM for ALC1 (513 Da) and 12 pM for ALC2 (585 Da), while ALC1 without Au nanoparticle labeling cannot be detected even at a high concentration of 0.6 nM. In our recent studies, we used gold nanorods as an amplification tag for immunoassay detection of tumor necrosis factor-alpha (TNF-α) antigen using a sandwich structure.442 TNF-α has been recognized as a tumor promoter which may be involved in

applications in theranostics, pharmaceutics, food safety, environmental monitoring, and homeland security.427−429 SPR sensors are commercially available in the market. A typical experimental setup with the Kretschmann configuration for SPR sensors is shown in Figure 24.1,430 SPR sensors utilize

Figure 24. Typical setup for an SPR biosensor. Surface plasmon resonance (SPR) detects changes in the refractive index in the immediate vicinity of the surface layer of a sensor chip. SPR is observed as a sharp shadow in the reflected light from the surface at an angle that is dependent on the dielectric constant (related to mass of coverage of the analyte at the surface). The SPR angle shifts (from I to II in the lower left-hand diagram) when biomolecules bind to the surface and change the mass of the surface layer. This change in resonance angle can be monitored noninvasively in real time as a plot of resonance signal (proportional to mass change) versus time. Reprinted with permission from ref 430. Copyright 2007 Nature Publishing Group.

surface plasmon polaritons (SPPs) comprised of collective coherent oscillations of free electrons in the conduction band of a metal, which propagate laterally at the metal/dielectric interface. At a certain angle, a minimum value of reflectivity of the incident light will be manifested, when it couples to the interface as a surface plasmon. This SPR wave is confined to the interface and decays exponentially in the axial direction to the surrounding medium with a penetration depth in the hundreds of nanometers range. When the sample solution containing the analyte flows over the sensor chip, the analyte binds to the ligand immobilized on the sensor chip (the association phase) and causes a refractive-index change at the metal−dielectric interface, thus generating a response (amount of shift of the coupling angle). The magnitude of the angle shift levels off over time, as an equilibrium condition between the free and the bound analyte is reached. When the flow switches to that of a running buffer which washes out the analyte (leading to dissociation of it from the ligand), the magnitude of the angle shift will decrease during the dissociation phase. The response curve for the association and dissociation cycle is often called a sensorgram (Figure 24). Despite the success of SPRs in a wide range of research fields, their sensitivities are still inadequate to detect trace amounts of low molecular weight molecules (less than 500 Da) or to detect common entities in an extremely dilute concentration (less than 1 pM). However, several recent studies have demonstrated that the sensitivity of SPR could be improved by orders of 2857

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the tumor initiation process.443 We demonstrated that the TNF-α antigen at a concentration as low as the femtomolar range can be detected, which showed more than 40-fold sensitivity increase compared to the conventional SPR biosensing technique without the use of gold nanorods. The enhanced sensitivity produced by gold nanosturctures mainly arises from three factors:436 (i) an increase of the absolute mass in each binding event, (ii) an increase in the bulk refractive index of the analyte due to the complex refractive index of the nanoparticles, and (iii) coupling between the localized surface plasmon resonance (LSPR) of the metallic nanoparticles and the surface plasmon resonance (SPR) of the sensing film. There are indications from our recent work that the presence of plasmonic coupling between the nanoparticles and the sensing film plays an important role in the enhancement factor of the nanoparticle-based SPR biosensor.442 This is because maximum enhancement effect is achieved when the LSPR peak wavelength of gold nanorods matches the oscillation frequency of effective plasmon on the SPR film.

Figure 25. Benefits of nanopharmacotherapy.

biodispersion, thus enhancing the efficacy of a drug. Once inside the body, all types of drugs face enzymatic degradation, and also capture by the reticuloendothelial system (RES). By appropriately tuning the size, shape, and surface properties of nanoparticles, enzymatic degradation and RES metabolism of drug nanoformulations can be largely prevented. Thus, the PK profile of a drug can be significantly improved via enhancement in its biocompatibility, circulation time, and bioavailability. For example, the chemotherapeutic drug Paclitaxel used in the nanoencapsulated form exhibits enhanced absorption into the systemic circulation, thus leading to improved bioavailability.451 7.1.2. Targeted Delivery. A nanoparticle formulation can favorably manipulate the PD profile of a drug by increasing the local concentration using target-specific delivery to diseased sites. Depending on the extent of drug localization at target areas, targeting is divided into (a) first order, i.e. targeting to particular tissue, (b) second order, i.e. targeting to a particular cell type, and (c) third order, i.e. targeting to a particular organelle within a cell. Targeting can be achieved by a passive mechanism, where the enhanced permeability and retention (EPR) effect associated with tumor tissues leads to enhanced delivery of circulating nanoparticles in the tumor tissues.452 The EPR effect refers to the property by which certain sizes of molecules tend to accumulate in tumor tissue much more than they do in normal tissues.453 This mode of targeting is restricted to cancer and inflammatory sites, and it can reach a precision of second order targeting at most. The other mode of targeting, i.e. active targeting, relies on the specificity of appropriately surface modified nanoparticles for receptors expressed on target cells/tissues.446 This mode of targeting is not only applicable for all disease categories, but also can achieve the precision of third order targeting (e.g., delivery inside the nucleus of a cell using nanoparticles tagged with nuclear localization peptides). 7.1.3. Controlled Release. A unique advantage offered by nanoparticles is their ability to release drugs in a controlled manner.454 “Controlled release” is an umbrella term that denotes any kind of influence/restraint that nanoparticles may exert on the release pattern of incorporated drugs. These include sustained release, stimuli-sensitive release, and externally activated release, as illustrated in Figure 26. These release patterns are governed by the interaction of the nanoparticles with their surroundings. Sustained release signifies time-dependent release of entrapped drugs from their nanocarriers into the surroundings. The release usually takes place over an extended time period, though in some cases initial “burst release” does occur. As shown in Figure 26, sustained release can take place from both biodegradable and nonbiodegradable nanocarriers. When the carriers are biodegradable, drug molecules are released as the

7. NANOPARTICLE-BASED THERAPY AND CONTROLLED RELEASE Probably the most versatile application of nanoparticles in medicine is for therapy of diseases.444−448 This is because nanoparticles have several benefits that help them overcome the major drawbacks faced by conventional therapies, namely poor bioavailability, insufficient target specificity, and significant side effects.1 The initial reports of nanoparticles used in therapy involved inert nanomaterials that served as carriers of drug molecules for improved delivery and therapeutic outcome. These drugs included conventional chemotherapeutics, immunosuppressants, antibiotics, anesthetics, steroids, painkillers, vaccines, etc. With the advancement of nanoparticle research in the biomedical domain, newer nanoparticles were developed, which could themselves act as “drugs” owing to their unique surface energy and physical properties. This led to the development of novel therapies yet unprecedented in medicine. With concurrent advancements in imaging modalities involving nanoparticles, concepts such as multimodality, combination therapies (allowing more than one therapy to act synergistically), theranostics (allowing the combination of therapy and diagnostics in a single conduit), and image guided therapies emerged. Nanotherapies also showed promise in complementing traditional therapeutic avenues, such as surgery, chemotherapy, and radiation therapy. 7.1. Nanopharmacotherapy

Nanopharmacotherapy deals with the safe, appropriate, and economical use of drugs in patients and has the potential to optimize drug efficacy, while minimizing side effects, thereby dramatically improving health outcomes for patients at a lower overall cost. Important factors influencing nanopharmacotherpy are pharmacokinetics (PK), which describes the effect of a biological environment on a drug, and pharmacodynamics (PD), which involves details of the effect of a drug in the targeted tissues and cells.449,450 Nanopharmacotherapy has ushered significant improvements in the PK/PD profile of drugs. The key benefits of nanopharmacotherapy are listed in Figure 25. 7.1.1. Dispersibility and Protection in Vivo. Nanoparticles provide a formulation for stable aqueous dispersions of several hydrophobic drugs prior to delivery in the body. Nanoformulation of a hydrophobic drug improves its 2858

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Figure 26. Principal modes of controlled release.

Figure 27. Schematic illustration of the scope of general nanotherapies.

drug release.460 On the other hand, certain pathological conditions such as inflammation have elevated local temperature, where thermosensitive drug delivery is useful. Nanoparticles based on poly-N-isopropylacrylamide (pNIPAM) have a low critical solution temperature (LCST) of 32 °C, above which it undergoes charge reversal and water expulsion, leading to drug release. External stimuli-sensitive release involves a two-step process in which the drug/nanocarrier system is first allowed to biodistribute, resulting in their preferential accumulation at the target site. Then, drug-doped nanoparticles accumulated at the diseased site are directly activated by an external stimuli, to produce local drug release.461,462 The application time of an external stimulus should coincide with the maximum accumulation of drug/nanoparticles at the target for therapy to be most effective. For example, drugs doped within polymeric nanoparticles containing photolabile bonds, such as azo linkages, can be made to release under light-activated nanoparticle degradation. For further details on stimulisensitive drug delivery, readers can consult some excellent reviews on this topic.46,456,457,462

carrier matrix erodes in a time-dependent manner. Nanoparticles of biodegradable polymers (having a hydrolyzable heteroatom backbone, e.g., −C−O−, −C−N−) such as polyesters and some inorganic materials such as calcium phosphate are examples of such carriers.57,46 On the other hand, when the carriers are nonbiodegradable, drug molecules can be released via slow diffusion through voids or channels in the carrier matrix. Nanoparticles of mesoporous inorganic materials such as silica, or certain nonbiodegradable polymers (having nonhydrolyzable −C−C− backbone) with a porous network such as poly(methyl methacrylate) (PMMA), constitute such nanocarriers from which drugs have been reported to diffuse out.455 One major shortcoming of a sustained release system is that the release is not specific to target sites. To ensure optimal drug release at a target, a variety of “stimulus-responsive” nanoparticulate drug delivery systems have been engineered.456 The “stimulus” can be internal, such as a change in pH and temperature, or the presence of a specific biological agent.457,458 On the other hand, an external “stimulus” represents activation with an external energy source, such as light, magnetic or electric field, ultrasound, etc. The carrier matrix is destabilized under such a stimulus, leading to localized drug release, which can be manipulated to be target specific. Internal stimuli-sensitive release exploits altered physiological/biochemical processes occurring at or around target sites. For example, a tumor tissue has an extracellular pH between 6.5 and 7.2, which is slightly lower than the normal pH of 7.4.459 Polymeric nanoparticles based on pH sensitive residues such as poly(amino) esters, vinyl esters, diortho esters, hydrazones, etc., undergo swelling at a slightly low pH, leading to tumor-specific

7.2. Nanotherapies

The various types of therapies offered by nanoparticles are depicted in Figure 27. We discuss these therapies, with a special emphasis on recent developments and clinical developments. Gene therapy will be discussed in section 8. 7.2.1. Drug Delivery. The concept of drug delivery involves the incorporation of a drug within nanoparticulate carriers via encapsulation, absorption, adsorption, or conjugation, for safe and stable administration in the body. In the literature, they are also conveniently referred to as nano2859

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absorption, adsorption, or conjugation. They include semiconductor nanoparticles (quantum dots),472 gold nanoparticles,40 iron oxide nanoparticles,473 etc. The main advantage of such structures is that they can co-incorporate other therapeutic or diagnostic agents for combination therapy or theranostic applications. However, despite numerous recent publications in this above area, no inorganic nanoparticle based drug-delivery formulation has yet made progress into the clinical trial arena. 7.2.2. Photodynamic Therapy (PDT). PDT is a light activated therapy, with promising applications in the treatment of a variety of diseases, such as cancer, dermatological disorders, microbial infections, etc.474,475 Here, the drugs (photosensitizers) are first targeted to diseased sites. Several photosensitizers naturally accumulate in tumor because of the increased retention time derived from the greater affinity of a photosensitizer for malignant cells. This is followed by their irradiation with light, thereby energizing the photosensitizers. Photosensitizers are mainly porphyrin-based compounds, designed to have excellent light absorption and a propensity to undergo intersystem crossing (ISC) to an excited triplet state (Figure 28a). The photosensitizer at the triplet state can transfer its energy to molecular oxygen (O2) which is unique in being a triplet in its ground state, leading to the formation of the highly reactive singlet oxygen 1O2 (a type II process). A type I process can also occur, whereby the photosensitizer reacts directly with the constituents of the cellular microenvironment, acquiring a hydrogen atom or electron and eventually producing highly reactive peroxides or superoxides which destruct the malignant cell by oxidation.476 Both type I and type II reactions may occur simultaneously, and the ratio between these processes often depends on the type of sensitizer used, on the concentrations of oxygen, and on the cellular microenvironment. Because of the high reactivity and short half-life of the reactive oxygen species (ROS), only cells that are proximal to the area of the ROS production (areas of photosensitizer localization) are directly affected by PDT. The half-life of singlet oxygen in biological systems is