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The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications Chapter 4 Cd-Containing Quantum Dots for Biomedical Imaging Jing Wang College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, 310014 P. R. China email@example.com Quantum dots (QDs), also termed semiconductor nanocrystals, are 1–10 nm inorganic particles with unique size-dependent optical and electrical properties due to quantum confinement. Since the two seminal reports on biological application of QDs in 1998, QDs have initiated a new realm of bioscience by combining nanomaterials with biology, which will profoundly influence future biological and biomedical research. This chapter summarizes the optical and structural properties, synthesis, bioconjugation, nanotoxicity and biomedical applications of Cd-containing QDs, with a look at the future challenges and prospects in the development of QDbased biomedical imaging. 1. Introduction In the field of semiconductor nanocrystals, also named colloidal quantum dots (QDs), research continues to grow apace with rapid advances in chemistry, bioscience, materials and device physics and fabrication techniques worldwide. Before 1998, QDs were only considered as materials for photonics or electronics applications, based upon their unique optical and electronic properties.1 In recent years, however, attention has increasingly turned to advanced fluorescence imaging applications of QD probes, such as multiplexed quantitative analysis of cellular phenotypes, real-time monitoring of intracellular processes 111 b2227_V3_Ch-04.indd 111 25-Jan-16 3:04:56 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF H; ONG KONG on 10/25/16. For personal use only. 112 J. Wang and in vivo molecular imaging.2–9 Exhibiting many important distinguishing characteristics compared to conventional fluorophores, including size-tunable and spectrally narrow light emission along with efficient light absorption throughout a wide spectrum, improved brightness with outstanding resistance to photobleaching and degradation and extremely large Stokes shift, QDs greatly expand the capabilities of fluorescence imaging. In particular, bio conjugated QDs have become indispensable tools for extended imaging of living cells and small animals. Therefore, synthesis, bioconjugation, nanotoxicity as well as imaging applications of QDs have emerged into great research areas. Because of QDs are of prime importance in biomedical applications, a variety of synthesis methods have been proposed to produce QDs with personalized characteristics. To date, the synthesis of QDs is achieved mainly through two synthetic routes. The first synthetic route, known as hightemperature organometallic procedure paved by Bawendi and co-workers in 1993,10 relies on the pyrolysis of various kinds of precursors. In the earlier studies, dimethylcadmium (CdMe2) and trioctylphosphine selenide (TOPSe) was used as cadmium and Se source, respectively. The synthesis was carried out in an inert atmosphere by injecting the cadmium and selenium precursors dissolved in TOP into hot (300°C) TOPO followed by growing the nanocrystals at ~230–260°C. Yet, utilization of a highly toxic and unstable Cd precursor (CdMe2) imposes restrictions on the equipment and reaction conditions and limits flexibility in the QD core design. A leap towards large scale preparation of high-quality QDs has been made by Peng et al. using alternative cheap precursor materials (such as cadmium oxide (CdO), cadmium acetate (CdAc2), cadmium carbonate (CdCO3)).11–13 Relatively mild and simple reaction conditions along with slower nucleation and growth rates offer extensive flexibility in engineering of QD chemical composition, geometry and photophysical properties. As the most mature products of this synthetic route, CdSe QDs, including those with core/shell (e.g. CdSe/CdS, CdSe/ZnSe), core/shell/shell (e.g. CdSe/CdS/ZnS) and alloyed structures (e.g. CdSeTe) constructed in combination with other types of II–VI semiconductors, have represented the most successful examples of fluorescent nanocrystals and become almost the symbol of fluorescent QDs as well. Colloidal aqueous synthesis of QDs, is an advantageous alternative to the widely used organometallic route.14 Compared with that route, the aqueous approach (1) does not require high temperatures and glove boxes, (2) employs the most widespread and biocompatible solvent — water, (3) is easily up-scalable up to industrial requirements and (4) provides various functionalization of QDs via applying an appropriate capping ligand, which in turn may be further functionalized by noncovalent or covalent linking.15 This method permits successful synthesis of a series of various QDs directly in aqueous medium employing mild b2227_V3_Ch-04.indd 112 25-Jan-16 3:04:56 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 113 preparative conditions. In general, the aqueous synthetic strategy consists of the reaction between metal and chalcogen containing precursors in the presence of an appropriate stabilizer with subsequent nucleation and growth of QDs, typically by heat treatment. In the case of aqueous synthesis, thiols are definitely the most used ligands for the stabilization of multifarious QDs. The formation of nanoparticles is a dynamic process, which is usually explained by an Ostwald ripening (OR) mechanism mainly consisting of the growth of larger particles at the expense of smaller ones present in an ensemble.16 The most successful example of the cadmium chalcogenides is CdTe owing to its unique optical properties, e.g. strong fluorescence in the visible region. Based on the synthesis of CdTe QDs, approaches for obtaining some alloyed materials such as CdSeTe, CdTeS, CdHgTe, as well as core/shell structures such as CdTe/CdS, CdTe/ CdSe, CdTe/ZnTe and even core/shell/shell CdTe/CdS/ZnS have been developed. The obtaining of different core/shell and core/shell/shell structures directly in water reported during the last decade is a great step forward toward a superior quality of QDs accessible from an organometallic route. In order to utilize high-quality QDs for biomedical applications, biofunctionality has to be added to otherwise inert nanoparticles. This is usually achieved by decorating QDs with proteins, peptides, nucleic acids, or other biomolecules that mediate specific interactions with living systems (like cells, virus and animals). Surface engineering is thus crucial not only for tuning the fundamental properties of nanoparticles and rendering them soluble and stable in different microenvironments, but also for creating nanoparticle–biomolecule hybrids capable of participating in biological behaviors. Systematic nanotoxicity assessment of QDs is also of critical importance for their practical biomedical applications. More recently, there have been intense concerns on nanotoxicity investigates of QDs. Most QDs are made of heavy metal ions (e.g. Cd2+ ions), which may result in potential in vitro toxicity that hampers their practical applications. Many questions remain to be answered before the QDs community moves into the clinical research. For safe application of QDs in a clinical context, understanding the response of humans to QDs is essential. In this chapter, we will describe the optical and structural properties, synthesis, bioconjugation, nanotoxicity and biomedical applications of Cd-containing QDs. A critical evaluation of barriers impacting current QDs technologies will be discussed and insights into the future outlook of the field will be explored. 2. Optical and Structural Properties of QDs QDs are nanocrystals composed of nanometer-sized crystalline clusters of a few hundred to a few thousand atoms, which have different properties compared b2227_V3_Ch-04.indd 113 25-Jan-16 3:04:56 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 114 J. Wang with the bulk crystals.1,17 They can emit different wavelengths over a broad range of the light spectrum from visible to near infrared (NIR), depending on their sizes and chemical compositions.18–21 The advantage of size-tunable fluorescence emission is that common protocols for surface modification or bioconjugation can be applied for obtaining multicolor probes. The fluorescence of QDs is intrinsic, bright and prolonged, surpassing the sensitivity and resolution capabilities of the traditional organic fluorophores (e.g. organic dyes and fluorescent proteins), which endows them with new possibilities in biomedical areas. Broad absorption bands of QDs provide two advantages: (i) freedom to select any excitation wavelength below the band gap energy and (ii) minimize background by increasing Stokes shift. Other bio medically attractive properties of QDs include 10–100 ns fluorescence lifetime22 (this permits QDs to be used in timeresolved fluorescence bioimaging), stability against photobleaching23 (this allows QDs to be used in monitoring biological events, such as protein tracking) and their large surface area1 (this permits multiple bioconjugation and the preparation of multifunctional and multimodal probes). Figure 1 presents the mainly optical properties of QDs with organic fluorophores.24,25 The unique optical and structural properties formulate QDs to be ideal alternatives for organic fluorophores, in particular, for multimodal and multiplexed imaging of single-molecules, cells, tissues and animals. Additional information on these optical properties of QDs that are relevant to bioimaging can be obtained from recent review articles.23–30 3. Synthesis of Cd-Containing\Based QDs Among CdX QDs, CdSe and CdTe have attracted much attention in biomedical imaging due to their tunable and stable fluorescence in the visible to NIR region. Therefore, we will introduce the synthesis of CdSe- and CdTebased QDs with popular methods. 3.1. Organometallic synthesis of CdSe-based QDs With entrance of nucleation and growth techniques to synthesize QDs in hightemperature organic solvents, synthesis of QDs made easier and more controllable. An organometallic system for synthesis QDs consists of three components: precursors, organic surfactants and solvents. Sometimes, surfactants also serve as solvents.31 In this process to create QDs, ionic sources of the component materials such as Cd2+ ions are needed. This method consists of pyrolysis of b2227_V3_Ch-04.indd 114 25-Jan-16 3:04:56 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 115 Figure 1. Unique optical properties of QD. (A) Narrow size-tunable light emission profile enables precise control over the probe color via varying the nanoparticle size. (B) Outstanding photostability of QDs enables real-time monitoring of probe dynamics and accurate quantitative analysis, whereas quick photobleaching of organic dyes limits such applications. (C) Capability of absorbing high-energy (UV-blue) light without damaging the probe and emitting fluorescence with a large Stokes shift enables efficient separation of the QD signal over the fluorescent background. Reprinted from Ref. 24. Copyright 2005, with permission from Elsevier. organometallic precursors into a hot coordinating solvent to manufacture monodisperse (<5% size dispersion) QDs composed of cadmium chalcogenides.32 The most successful example of the cadmium chalcogenides synthesized in organometallic procedure is CdSe QDs.10–13,33–38 For the first time, b2227_V3_Ch-04.indd 115 25-Jan-16 3:04:56 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 116 J. Wang controllable nucleation and growth of narrow size distribution QDs was demonstrated by Murray et al. in 1993.10 In this approach, CdMe2 was reacted at ca. 230–300°C with TOPSe in the presence of TOPO. Here, TOPO was heated to ca. 300°C under vacuum for 20 min followed by injection of a mixture of CdMe2 and the Se precursor in an atmosphere of Ar. The growth of CdSe QDs was carried out at ca. 230–260°C. The resultant sample contained a size distribution (ca. 1.2–11.5 nm) of QDs and the different sizes were separated by size-selective purification from a mixture of 1-butanol and methanol. Since the above-mentioned breakthrough reports on the colloidal synthesis of CdSe QDs from CdMe2, researchers were interested in replacing CdMe2 due to its toxicity, volatility and pyrophoric nature. A leap towards large scale preparation of high-quality QDs has been made by Peng et al. using alternative cheaper and greener precursor materials (such as CdO, CdAc2, CdCO3).11–13 Relatively mild and simple reaction conditions along with slower nucleation and growth rates offer extensive flexibility in engineering of QD chemical composition, geometry and photo-physical properties. TEM images of CdSe QDs prepared from CdO, showing size- and shape control as functions of time under reactions and precursor ratio is shown in Figures 2A–2F. At present, these are the mainly two synthetic procedures to produce CdSe QDs with the desired optical properties for biomedical research and applications. Figure 2. Temporal shape evolution of rice-shaped CdSe QDs from CdO. Reprinted with permission from Ref. 12 (A–F). Copyright 2002, American Chemical Society. b2227_V3_Ch-04.indd 116 25-Jan-16 3:04:57 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 117 To use QDs in biology, it is extremely important to passivate or cap the CdSe QDs with a layer of ZnS or CdS, usually called type-I core/shell QDs.39 The ZnS or CdS shell improves the fluorescence quantum yield (QY) of the QDs and protects them against photo-oxidation (which is important for minimizing cytotoxicity and for enhancing photostability).40–45 Until now, several groups have developed high-bandgap-energy inorganic shells (e.g. CdS and ZnS) several atomic layers thick that effectively passivate the photoactive core of CdSe QDs. For example, Peng et al. have observed confinement of the hole created during excitation within the CdSe core by a CdS shell.41 As a result of such confinement, hole-dependent photo-oxidative processes that cause QD degradation and result in the loss of fluorescence are impeded. Also, a thicker shell might significantly reduce QD blinking (intermittence in light emission) associated with charge trapping and un-trapping at surface defects of a nanomaterial or due to charge ejection from the QD (Auger ionization) followed by recombination process.46,47 The most studied type-I core/shell structure to date is CdSe/ ZnS, as evidenced by the number of publications dealing with this system. To produce a ZnS capping layer, a solution of dimethylzinc and hexamethyldisilathiane (in tri-N-octylphosphine) can be slowly dripped into the reaction vessel after isolating or obtaining CdSe QDs of a desired size. The low temperature and slow drip rate prevents the nucleation of ZnS QDs. The thickness of the ZnS shell is mediated by the amount of precursors injected into the reaction vessel. The ZnS shell has a larger bandgap energy than CdSe, eliminating the core’s surface defect states. Besides, the ZnS shell has a similar bond length to the CdSe, minimizing crystal-lattice strain and allowing for epitaxial growth. Another kind of core/shell materials, namely Type-II QDs, with the valence band edge or the conduction band edge of the shell material located in the band gap of the core, whose emission wavelength could be tuned across the visible to NIR region by controlling the thickness of the shell or the core size.39 Research on colloidal type-II systems was triggered by the seminal work of Bawendi et al.48 who described the synthesis and optical properties of CdTe/CdSe and CdSe/ZnTe core/shell QDs. The emission wavelength of CdTe/CdSe QDs could be tuned by changing the shell thickness and the core QD size from 700 to 1000 nm (Figure 3). This approach is an alternative possibility to shift the emission peak to higher wavelengths, which would not be attainable by simply increasing the size of the core QDs in a type-I core/shell system. At present, CdTe/CdSe QDs could be synthesized without the use of organometallic precursors by applying CdO, TOPTe and TOPSe. This approach leads to fluorescence QYs approaching 40% for small shell thicknesses below 0.5 nm. b2227_V3_Ch-04.indd 117 25-Jan-16 3:04:57 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 118 J. Wang Figure 3. (a) Normalized fluorescence spectra of CdTe/CdSe QDs having a core/shell radii of 1.6/1.9 nm, 1.6/3.2 nm, 3.2/1.1 nm, 3.2/2.4 nm and 5.6/1.9 nm (from left to right, respectively). (b) Normalized fluorescence decays of 3.2/1.1-nm CdTe/CdSe QDs and of the corresponding 3.2-nm CdTe core QDs (dotted line). Reprinted with permission from Ref. 48. Copyright 2003, American Chemical Society. 3.2. Aqueous synthesis of CdTe-based QDs Although the first colloidal material synthesized via the aqueous approach and thoroughly investigated was CdS, the most successful example of the cadmium chalcogenides is CdTe owing to its unique optical properties, in particular, its strong and tuneable fluorescence in the visible to NIR region. In general, the aqueous synthesis of thiol-capped CdTe QDs consists of the reaction between Cd2+ ions or their complexes and Te containing precursors in the presence of an appropriate stabilizer with subsequent nucleation and growth of the QDs, typically controlled by heating. As metal precursors water soluble salts are used, commonly perchlorates and chlorides.16 Te2− is introduced in the reaction in the form of H2Te gases49,50 generated by the decomposition of the corresponding Al2Te3, or in the form of NaHTe prepared by the reduction of Te powder51–55 or NaTe2O356–58 by NaBH4.59 An electrochemical generation of Te2− has also been applied in the synthesis of CdTe QDs.60 A variety of thiols as capping ligands employed in the aqueous synthesis of CdTe or other thiol-stabilized semiconductor nanocrystals is summarized in Ref. 16. Therefore, the typical synthetic protocol for obtaining aqueous thiol-capped CdTe QDs is as followed: The Cd precursor, such as Cd(ClO4)2, CdCl2 or CdAc2, is dissolved in water and stirred, an appropriate amount of thiol stabilizers is added, the pH of the solution is adjusted by addition of NaOH and then deaerated. Under stirring, H2Te or NaHTe is added to the solution, leading to the formation of CdTe precursors, which is b2227_V3_Ch-04.indd 118 25-Jan-16 3:04:57 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 119 accompanied by a change of color of the solution. The CdTe precursor solution is stable in the atmosphere and the formation and growth of QDs proceed upon refluxing. The fluorescence QY of CdTe QDs synthesized by the aqueous route is affected by many parameters during the synthesis, including precursor concentration, the ratio of Cd/Te and Cd/thiol stabilizer, pH value, etc.50 Careful optimization of these parameters allows CdTe QDs synthesized by the aqueous route to have fluorescence QY comparable to QDs synthesized by the organometallic route. CdTe QDs have been obtained directly in water firstly by Resch et al.61 in the presence of hexametaphosphate, then Rajh et al.62 employed a mixed stabilizer system consisting of hexametaphosphate and 3-mercapto-l, 2propanediol. Rogach et al.63 first reported the synthesis and characterization of fluorescent CdTe QDs in 1996 by using thioglycerol and mercaptoethanol as surface stabilizing agent. Although under optimized conditions, the fluorescence QY of obtained QDs remains lower than 3%. The first successful example of thioglycollic acid (TGA) as stabilizer for CdTe QDs was reported by Gao and co-workers.64 They demonstrated that the pH has a great influence on the fluorescence QY of QDs. It was observed that in acidic range, the TGA and Cd2+ excess in the mother-solution will deposit on the surface of CdTe QDs forming a shell layer structure comprised of Cd-TGA complexes due to the secondary coordination between the carbonyl group of TGA and the primary thiol-coordinating cadmium. Since the Cd-TGA complexes can effectively eliminate the nonradiative pathway for excitons, the fluorescence QY is greatly increased. Zhang et al.53 later improved the aqueous synthesis of CdTe QDs and studied the effect of carboxyl groups on fluorescence and stability of the mercaptocarboxylic acid capped CdTe QDs. In 2007, Rogach et al.50 reported on the state-of-the art synthesis and improved fluorescence properties of thiol-capped CdTe QDs prepared in aqueous phase (Figure 4). Their results demonstrated that the use of an optimized molar ratio of Cd2+ to thiol stabilizer indeed allows us to increase the room-temperature fluorescence QY of as-synthesized TGA-capped CdTe QDs to values from 40 to 60%. Furthermore, they stress several advantages of mercaptopropionic acid (MPA) as the capping agent in the aqueous synthesis of CdTe QDs. It is worth mentioning that NIR fluorescence has attracted much more interests in biomedical imaging and diagnostics applications because NIR emission offers several advantages, including minimal interferential absorption, low biological autofluorescence and high tissue penetration.65–67 To date, much effort has been donated to develop new methods for preparing high-quality NIR-emitting CdTe QDs due to the relatively narrow bandgap energy.68–70 Also, several improvements to the conventional aqueous synthetic method for b2227_V3_Ch-04.indd 119 25-Jan-16 3:04:57 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 120 J. Wang Figure 4. Photoluminescence (PL) (a) and absorption (b) spectra of TGA- and MPA-capped CdTe QDs demonstrate their tuneability over a broad spectral range in the visible and NIR (λex = 450 nm). TEM image of MPA-capped CdTe QDs, 5.5 nm average size, with a PL maximum at 780 nm (c) PL decays of MPA-capped CdTe QDs of increasing sizes (d) Adapted with permission from Ref. 50. Copyright 2007, American Chemical Society. thiol-capped CdTe QDs have been reported by various groups, e.g. hydrothermal synthesis,71–73 illumination,74 ultrasonic methods75 and microwave irradiation.76,77 Epitaxially growing wide bandgap inorganic semiconducting materials on the surface of fluorescent nanocrystal core with narrower bandgap has been demonstrated to be an effective approach for effectively eliminating the surface traps of the core nanocrystals and so lead to high fluorescence QY.39 Gao’s group firstly reported the preparation of CdTe/CdS core/shell QDs with fluorescence QY around 85% via an illumination-assisting process.74 It is revealed that the slowly released S2− from TGA will deposit on the surface of CdTe QDs in combination with Cd2+, forming a CdTe/CdS core/shell structure which can greatly increase the fluorescence QY of the resultant QDs. With these insights, thermally unstable sulfide compounds such as thioacetamide,78 thiourea79 or glutathione80 are also successfully used instead of TGA for slowly releasing sulfide ions in coating CdTe QDs with a CdS shell. Recently, microwave irradiation has been proposed as an advantageous b2227_V3_Ch-04.indd 120 25-Jan-16 3:04:58 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 121 technique for core/shell particle synthesis. For example, CdTe/CdS/ZnS core/shell/shell QDs with fluorescence QY up to 80% were achieved by microwave irradiation in coating MPA-stabilized CdTe QDs by a CdS shell followed by a ZnS shell.81 The resulting QDs were reported to be very stable in biological media and noncytotoxic, which makes them very promising candidates for biolabeling. Alternatively, ultrasonic irradiation was also applied for obtaining CdTe/CdS core/shell QDs by using thioglycerol capped CdTe QDs and cadmium acetate, thiourea as core and shell precursor, respectively.75 Thus far, a series of high-quality type-II core/shell NIR QDs have been successfully prepared in aqueous phases. Yan et al.82 reported a layer-by-layer colloidal epitaxial growth of L-cysteine-capped CdTe/CdSe type-II core/shell QDs in aqueous solution with the emissions between 600 and 850 nm. Inspired by this, Han’s group83 developed a hydrothermal technique for the NIR CdTe/CdSe QDs, which the fluorescence QY was further improved to 40% in aqueous phase. Recently, lattice-mismatch strain tuning theory has emerged as a fascinating strategy to convert standard type-I QDs into type-II heterostructures, resulting in the red-shift of emission spectrum and consequently the NIR QDs. Nie84 described a class of wurtzite CdTe core based on core/shell nanocrystals that were converted into type-II QDs by lattice strain in organic phase, the strain induced by the lattice mismatch could be used to tune the light emission — which displayed narrow line width and high fluorescence QY — across the visible to NIR part of the spectrum (500–1,050 nm). Deng et al.85 reported a two-step low-temperature aqueous method based on the lattice-mismatch strain between the CdTe core and CdS shell materials for synthesizing water-soluble CdTe/CdS coremagic/shellthick QDs, whose NIR emission (475–810 nm) could be tuned by varying the shell thickness (Figure 5). Following this line of thought, Chen and co-workers58 used air-stable and commercial Na2TeO3 as Te source to replace traditional NaHTe or H2Te and proposed a one-pot aqueous approach for producing highly luminescent NIR CdTe/CdS coresmall/shellthick QDs with the fluorescence QY up to 65%. In fact, achieving core/shell structured QDs in aqueous systems remains highly challenging. We have to admit, unfortunately, that the formation of core/shell structures is often not unambiguously proven owing to the small difference of electron density between CdTe and II–VI semiconductor shells. Therefore, more experimental proofs may be required to support the statements made for some of the aforementioned core/shell structures.86 In order to extend the emission of CdTe to longer wavelengths one uses alloying of the QDs with materials having narrower band gaps which have quite similar lattice parameters as CdTe.87 As an ideal candidate for b2227_V3_Ch-04.indd 121 25-Jan-16 3:04:58 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 122 J. Wang Figure 5. (A) Schematic diagram illustrating the formation of the CdTe/CdS magic-core/ thick-shell tetrahedral QDs. (B) Room-temperature PL emission profiles of a series of QD samples obtained by varying the CdS shell growth to overcoat the magic-sized CdTe core with thicker shells. (C) Room-temperature QD decay kinetics monitored at the maximum emission wavelengths of the samples in (B). The calculated excited state lifetimes are listed. Adapted with permission from Ref. 85. Copyright 2010, American Chemical Society. application in NIR emission, some NIR alloyed QDs were first synthesized in the organic phase by an organometallic method that restricted their direct applications in biosystems. Recently, several highly luminescent CdTe-based anionic alloyed QDs have been directly prepared in aqueous solutions. For example, Mao and co-workers88 presented a facile one-pot hydrothermal method to fabricate water-dispersed NIR CdTeS alloyed QDs with high fluorescence QY. By a different strategy, Zhu’s group89 prepared high-quality NIR CdSeTe alloyed QDs in an aqueous medium following a facile one-pot refluxing route by simultaneous reactions of NaHSe and NaHTe with Cd2+ in the presence of L-cysteine. By varying the ratio of NaHSe to NaHTe and the refluxing time, the absorption onset of the resultant CdSexTe1–x QDs were b2227_V3_Ch-04.indd 122 25-Jan-16 3:04:58 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications Cd-Containing QDs for Biomedical Imaging 123 The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. tuned over a wavelength region of 580–814 nm. In a similar way, CdTe-based cationic alloyed QDs can also be prepared using the classic aqueous synthetic route. The aqueous synthesis of NIR emitting alloyed CdHgTe and core/ shell CdHgTe/CdS along with pure HgTe QDs has been first reported by Harrison, Rogach and others.90,91 A similar route was also adopted in synthesizing CdxZn1−xTe nanocrystals with fluorescence QY up to 75%.92 3.3. Water solubilization and functionalization At present, the majority of studies involving an application of QDs in biomedical imaging were realized with CdSe/ZnS and CdTe QDs. CdSe/ZnS QDs are normally synthesized in organic phase. However, in order to be useful for biological applications QDs must be made water-soluble. In general, water-solubilization procedure should yield QDs soluble and stable in biological buffers, preserve the original photo-physical properties, retain relatively small particle size and provide reactive groups for subsequent conjugation to biomolecules. Over the years many scientists have discovered a wide array of surface coatings for solubilization of QDs satisfying these criteria. The techniques used to achieve solubilization include ligand exchange, surface silanization and phase transfer methods. The ligand exchange method is based on the exchange of the hydrophobic surfactant molecules with bi-functional molecules, which are hydrophyllic on one side and hydrophobic on the other, to bind to the ZnS shell on the QDs. Most often thiols as functional groups are served to bind to the ZnS and carboxyl groups are served as hydrophyllic ends.93 Examples include utilization of negatively-charged carboxy-terminated thiols, such as TGA94 and MPA and thiol-containing zwitterionic molecules, such as cysteine,95,96 for decoration of QD surface with hydrophilic moieties. This is by far the simplest method to achieve solubilization. Despite the simplicity of the process, ligand exchange with monodentate surface ligands often compromises the fluorescence QY, photochemical and colloidal stability of the QDs, as ligands tend to detach from the QD surface leaving behind surface trap sites and causing nanoparticle aggregation.97,98 In general, crosslinking of small ligands or substitution from mono-thio to di-thiol ligands substantially improves long-term stability. For example, Liu et al.99 have utilized di-thiol ligand dihydrolipoic acid (DHLA) conjugated to poly(ethylene glycol) (PEG) to prepare small and stable QDs with some loss of fluorescence efficiency (drop in QY from 65% to 43%). In an alternative approach, Sukhanova et al.100 have water-solubilized QDs with DL-Cysteine and further stabilized the particles with poly(allylamine), achieving improvement in QD colloidal stability and increase in fluorescence QY b2227_V3_Ch-04.indd 123 25-Jan-16 3:04:59 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 124 J. Wang (from 40% to 65%). Surface silanization involves the growth of a silica shell around the nanocrystal.101,102 This method not only produces water-soluble and biocompatible QDs, but also offers the facile introduction of functionality to a silica layer which enables further conjugation with biomolecules. For instance, Gerion et al.102 introduced a new method for the preparation of high-quality silica shells on CdSe/ZnS QDs. At first, they suspended QDs in methanol by adding (3-Mercaptopropyl)trimethoxysilane (MPS) and tetramethylammonium hydroxide and then silica shells were grown on the MPS layer by using (trihydroxysilyl)propyl methylphosphonate. Selvan et al.103 introduced a sol–gel process for the synthesis of silica-coated CdSe/ZnS QDs which retained bright fluorescence. They have shown that the number of QDs per silica shell and the thickness of the shell can be well-controlled by encapsulating QDs in reverse microemulsions followed by growing silica shells from tetraethyl orthosilicate. The phase transfer method is to retain the native TOPO coating and encapsulate the hydrophobic QDs with amphiphilic molecules such as polymers104,105 or phospholipids.106 The hydrophobic portion of this molecule intercalates within alkyl-chainterminated surface ligands while the hydrophilic portion (e.g. charged groups, PEG, etc.) faces outwards, interacting with the aqueous solvent and rendering the particle water-soluble. This method produces exceptionally stable water-soluble QDs with preserved optical properties, as the coating does not directly interact with the nanocrystal surface and does not disturb the surface passivation layer.107 However, coating with a polymer usually results in dramatic increase of the nanoparticle hydrodynamic size and this may be detrimental for quantitative biomarker detection in a crowded biological environment and hamper intracellular penetration of the QD probes. CdTe QDs are commonly synthesized in an aqueous medium,16 which are water soluble intrinsically and can be coated with various thiol capping ligands and consequently different functional groups. It is worth mentioning that organometallically synthesized CdSe/ZnS QDs are inherently larger than aqueously synthesized CdTe QDs.5 Indeed, apart from the presence of a shell made of semiconductor material of wider bandgap, which has been found to increase their fluorescence QY by reducing surface defects, supplementary shells have to be introduced on the surface of CdSe/ZnS QDs to make them water soluble. With aqueously synthesized CdTe QDs, this step is optional and aqueously synthesized CdTe QDs are smaller than CdSe/ZnS even with a coating ligand such as TGA. This smaller size has been shown to be a direct factor of interest in biomedical imaging. Once solubilization is achieved, QDs can be functionalized by conjugation to a number of biological molecules including avidin, biotin, b2227_V3_Ch-04.indd 124 25-Jan-16 3:04:59 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 125 oligonucleotides, peptides, antibodies, DNA and albumin through surface reactive groups for specific targeted action.93 Surface engineering is thus crucial not only for tuning the fundamental properties of nanomaterials and rendering them stable and soluble in different environments, but also for creating nanoparticle–biomolecule hybrids capable of participating in biological processes.25 Such hybrids should combine useful properties of both materials involved, i.e. optical properties of the nanocrystals and biological functions of ligands attached. Methods of bioconjugation broadly fall into two categories: noncovalent and covalent conjugation.108 Noncovalent coupling of QDs and biomolecules presumes the use of linkers between the nanoparticle and the biomolecule, mainly including absorption, electrostatic interaction and mercapto-exchange. Biomolecules like oligonucleotide and various serum albumins109 can be absorbed on the surface of the water-soluble QDs. This process is nonspecific and depends on pH, temperature, ionic strength and surface charge of the molecule.93 The surface charge plays an important role in the cellular interaction of QDs and is determined by the free surface reactive groups. It has been demonstrated that proteins engineered with positively charged domains, can interact with the negative charges on the QDs surface coated with DHLA through electrostatic interaction.110 These conjugates have greater fluorescence but are also more stable than unconjugated QDs. CdTe QDs have also been coupled with DNA through electrostatic interaction notably by the mean of a cationic polymer. Peng et al.111 used the poly(diallyldimethylammonium chloride) (PDDA) cationic polymer to coat thiol-capped CdTe QDs in order to obtain positively charged QDspolymers structures, able to interact electrostatically with a labeled DNA probe in a DNA hybridization assay based on fluorescence resonance energy transfer (FRET). In a similar experiment, Jiang et al.112 used another type of cationic polymer, poly[9,9-bis (3′-((N,N-dimethyl)-N-ethylammonium propyl)-2,7-fluorenealt-1,4-phenylene]dibromide (PDFD) to allow the dyelabeled DNA to interact with QDs. Nevertheless, electrostatic interactions are nonspecific and relatively weaker compared to covalent bonding and this may pose a problem in the biological environment. Many biological molecules have a thiol group, which can be tagged on to the surface of a QD by a mercapto-exchange process.93 However, the resulting bond between thiol and Zn is not only weak but also dynamic and this may lead to precipitation of the biomolecules in solution as they easily detach from the QD surface. Covalent linkage is the most stable of all the bioconjugation methods and utilizes functional groups on the QDs surface like primary amine, carboxylic acids, and thiols to form a covalent bond with similar groups present on biomolecules or through the use of cross-linker molecules. For example, linking b2227_V3_Ch-04.indd 125 25-Jan-16 3:04:59 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 126 J. Wang of proteins via primary amine groups to carboxylic acid-containing QDs can be achieved via carbodiimide-mediated amide formation (i.e. EDC, 1-ethyl3-(3-dimethylaminopropyl) carbodiimide, condensation reaction). As this reaction utilizes naturally occurring amine groups it does not require additional chemical modification of proteins, preserving their natural structure; but it lacks control over the molecular orientation of the attached proteins, thus allowing attachment at a point close to the ligand’s active site that might result in partial or complete loss of biological functionality of that ligand. Furthermore, the EDC reaction might result in QD aggregation due to crosslinking between multiple reactive sites on QDs and proteins. As a result, several groups have developed another covalent bonding procedure involving active ester maleimide-mediated amine and sulfhydryl coupling. Since free sulfhydryl groups are rare in native biomolecules, additional treatment of the ligands is often required (e.g. reduction of antibodies with dithiothreitol). This reaction yields stable QD-ligand complexes with often controlled ligand orientation. However, chemical treatment might compromise the biological activity of ligands and cause reduced sensitivity and/or specificity of the probe. Nonetheless, both approaches are widely used for variety of applications, including custom production of QD-antibody probes and preparation of QD-streptavidin conjugates. Also, additional information about QD-bio conjugates can be obtained from recent review articles.18,20,26,30,93,113 Preparation of bio conjugated QDs is a prerequisite for bioimaging. 3.4. Nanotoxicity Since 1998, QDs, especially Cd-containing QDs, have been extensively used in biomedical research, based upon the unique optical and structural properties. At the same time, a great deal of concern has been raised about the potential hazards of QDs because of their heavy-metal content.114 Even many researchers are convinced that QDs will never be used for treating patients because of their potential nanotoxicity. The perception that QDs are toxic originates from in vitro studies where Cd-containing nanoparticles killed cells in culture. Nanotoxicity of QDs has also been studied using small animal models. Despite the fact that no abnormal behavior or tissue damage was noticed in mice and rats over periods of months after the systemic administration of QDs, the correlation of these results with the potential for negative effects of QDs on humans remains unclear. For the past several years, concern about QD nanotoxicity has been a major roadblock to the translation of QDs toward clinical biomedical applications. Reports of QD nanotoxicity in the literature have been quite confusing b2227_V3_Ch-04.indd 126 25-Jan-16 3:04:59 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 127 due to the wide variation in the types of QDs being tested and in the biological models in which they have been tested. Each type of QD has its own unique physicochemical properties that determine its potential nanotoxicity. Along with composition, the physical and surface characteristics of the QDs play a major role in determining nanotoxicity. These characteristics include size, shape, surface charge and surface coatings. A vast number of QD formulations are possible, each with a unique combination of physicochemical properties that dictate the QD’s interaction with a biological entity. One must fully characterize the QDs prior to evaluating their nanotoxicity. In the following section, we summarize key findings from in vitro and in vivo studies, explore causes of the discrepancy in QD toxicological data, and finally provide our view on the future direction of this field. 3.5. In vitro nanotoxicity Mechanisms of QD-induced cytotoxicity have been elucidated by studying model cell culture systems exposed to group II-VI Cd-containing QDs. Derfus et al.44 demonstrated that, when oxidized in air or by ultraviolet light irradiation, TGA stabilized CdSe QDs released free Cd2+ into solution and caused primary liver cell (hepatocyte) death. Intracellular QD degradation with cadmium release has also been suggested. Microscopy studies have shown that QDs localize within cellular endosomes and lysosomes115 and are thereby exposed to an acidic or oxidative microenvironment. In a cell free assay, Mancini et al. found that hypochlorous acid, present in phagocytic cells, oxidized polymer-encapsulated CdS/ZnS-capped CdSe QDs with solubilization of cadmium, zinc, sulfur, and selenium species.116 Sometimes, a nanomaterial may not directly affect the viability of treated subjects but triggers a sequence of inflammatory mediator formation. Recent works found that the generation of reactive oxygen species (ROS) such as superoxide (O2), hydroxyl radicals (HO•), peroxide radicals (ROO•), hydrogen peroxide (H2O2) and singlet oxygen can also adversely affect cellular functions. Ipe et al.117 irradiated TGA-stabilized CdS, CdSe and CdSe/ZnS QDs and identified photogenerated radical species with electron paramagnetic resonance spectroscopy and a radical-specific fluorescence assay. It was found that redox potential was dependent on QD chemical composition. While CdS QDs had sufficient redox power to produce hydroxyl and superoxide radicals, CdSe QDs exclusively formed hydroxyl radicals. Additionally, the ZnS shell was an efficient inhibitor of QD reactivity as CdSe/ZnS QDs did not generate any ROS. Clift et al.118 studied the formation of oxidative stress caused by QDs with different surface coatings. By measuring the b2227_V3_Ch-04.indd 127 25-Jan-16 3:04:59 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 128 J. Wang reduced glutathione and oxidized glutathione levels in J774.A1 macrophage cell line, their study demonstrated that both carboxyl- and amine-terminated QDs can cause oxidative stress that leads to the modulation of intracellular Ca2+ signaling. A unique aspect of nanoparticle nanotoxicity is their size-dependent intracellular routing. Nanoscale particles are able to reach organelles that are inaccessible to metal ions. This may result in unique patterns of cytotoxicity compared to their constituent metals. Following uptake by a cell, QDs are packaged into small intracellular vesicles and transported from the cell periphery to the perinuclear region.119 Contrastingly, Cd2+ was predominantly located in the cytoplasm, where it was sequestered by metallothionein.120 The observation that QDs can localize to the cell’s nuclear compartment has led researchers to investigate their potential genotoxicity. If QDs cause DNA mutations without cell death, their effect is propagated through future generation of cells and can ultimately lead to disease. Green and Howman showed that biotin-coated CdSe/ZnS QDs could nick supercoiled DNA.121 Expression of p53 is a good indicator of DNA damage. p53 is a tumor suppressor that regulates the cell cycle and is described as a ‘‘guardian of the genome’’. It prevents the conversion of damaged DNA to genome mutation.122 Choi et al.123 revealed that uncoated CdTe QD activated the p53 genotoxic stress pathways and resulted in the upregulated transcription of Puma(p53-upregulated modifier of apoptosis) and Noxa(NADPH oxidase activator 1), which are involved in apoptosis. 3.6. In vivo nanotoxicity In vivo studies using animal models take into account an entire spectrum of biological interactions in living organisms and are the natural next step, after in vitro testing, in evaluating and understanding the nanotoxicity of QDs. While cytotoxicity can easily be measured in vitro via metabolic activity or membrane permeability assays, nanotoxicity is not as straightforward to quantify in animals. In addition to measuring organism viability, sublethal toxicity such as organ damage must be considered. Researchers observed that the organs of the reticuloendothelial system (RES) nonspecifically took up and retained the majority of injected QDs.124,125 The RES is part of the body’s defense system to eradicate foreign materials and consists of phagocytic cells located primarily in the liver, spleen and lymph nodes. Data then suggested that QDs are degraded within these organs. Fitzpatrick et al.126 b2227_V3_Ch-04.indd 128 25-Jan-16 3:04:59 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 129 followed BALB/c and nude mice for 2 years postinjection of CdSe/ZnS QDs and observed blue-shifted emission peaks in the liver, spleen, and lymph nodes, reflective of particle retention and breakdown within these organs. As QDs become smaller during the degradation process, the fluorescence emission shifts from red to blue and the excitonic fluorescence peak becomes broader. Yang et al.127 made similar observations. They have used ICP-MS analysis to show that after intravenous injection of cadmium-based QDs in mice, the cadmium level in the kidneys and liver eventually (over 28 days) reached nearly 10% and 40% of the injected dose, respectively. The redistribution of cadmium over time may signify the degradation of QDs in vivo, because the natural accumulation sites of Cd2+ ions are the liver and kidneys. Nanotoxicity of QDs is a very important issue to address in order to fully utilize the potential of QDs for healthcare and medical research applications.128,129 A great deal of concern has been raised about the possible healthcare risk of QDs, both in the press and by practicing physicians. Thus a critical assessment of risk versus benefit of use of engineered QDs for diagnostics and therapy is extremely vital for the advancement of QD use in medicine. 4. Biomedical Imaging In the last decade, surface engineering and bio-functionalization techniques have transformed QDs into complex cellular probes capable of interaction with biomolecules and direct participation in biological processes. Since Alivisatos101 and Nie94 simultaneously reported using QDs as labels in biological research in 1998, QDs-based biomedical fluorescent imaging has made the most progress and attracted the greatest interest. The ability of one or more ‘color/size’ of biofunctionalized QDs to label cells allows extended visualization by fluorescent imaging under continuous illumination as well as multicolor imaging. Biomedical research can be divided into two types,130 in vitro and in vivo. In vitro research generally refers to the manipulation of organs, tissues, cells, and biomolecules in a controlled, artificial environment. The characterization and analysis of biomolecules and biological systems in the context of intact organisms is known as in vivo research. The in vivo research involves experiments performed in the context of the large system of the body of an experimental animal. In this section, we discuss in detail the recent fluorescent imaging research performed in vitro and in vivo, which helps to indicate developing trends in the biomedical applications of QDs. b2227_V3_Ch-04.indd 129 25-Jan-16 3:04:59 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications 130 J. Wang 4.1. In vitro imaging The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 4.1.1. Molecular pathology Microscopy especially fluorescence and confocal is an established technique for evaluation of phenotypes of healthy cells as well as for detection of molecular signatures of diseases. Histological techniques, such as fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), enable detection of nucleic acids and protein biomarkers within cells and tissue specimens with a high degree of sensitivity and spatial resolution. Traditional fluorophores such as organic dyes and fluorescent proteins have been widely used in these applications, either as stains for highlighting cell structures or as specific probes for labeling biomarkers. However, applicability of traditional fluorophores in multiplexed and quantitative analysis for molecular profiling, is limited by rapid photobleaching, spectral cross-talking, and the need to excite fluorophores at unique wavelengths. In contrast, the novel optical properties of QDs131,132 overcome many of the problems and QDs have already proven to be a powerful tool for sensitive quantitative molecular profiling of cells and tissues, providing unique identification of individual cell lineages and uncovering molecular signatures of pathological processes.132,133 Wu et al.104 were among the first demonstrating that QDs can be used to specifically and effectively label molecular targets at the subcellular level (Figure 6). In this study, QDs encapsulated within a polymer-shell were biofunctionized with molecules (streptavidin and immunoglobulin) and could be used to label different types of targets (breast cancer (BC) marker Her2, actin and microtubule fibers and nuclear antigens) at different subcellular locations (surface, intracellular and inside the nucleus) and with different types of specimens (cultured live cells, fixed cells and tissue sections). Additionally, QDs of two different emissions (630 and 535 nm) were used simultaneously and compared to an Alexa dye. Their results showed that QD-based probes can be very effective in cellular imaging and offer substantial advantages over organic dyes in multiplex target detection. With the development of cell imaging, it became clear that the hydrodynamic size of the QD-ligand bioconjugate should be minimized in order to achieve good penetration of the probes within the cross-linked intracellular compartments of fixed cells. Tholouli et al. have employed the biotin-streptavidin linkage for preparation of QD-oligonucleotide probes for FISH-based studies of mRNA.134 Biotinylated DNA probes pre-incubated with QD-Streptavidin conjugates enable detection of 3 mRNA targets in a 1-step FISH procedure. Yet, pre-conjugation of multiple oligonucleotides to QDs significantly increases the overall size of the probe, thus requiring specimen b2227_V3_Ch-04.indd 130 25-Jan-16 3:04:59 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 131 Figure 6. Labeling of surface and intracellular targets with QD probes. In single-color examples membrane-associated Her2 receptors are detected with primary antibodies and QD-labeled secondary IgG (A, green), while intracellular nuclear antigens (B, red) and microtubules (C, red) are visualized with primary IgG/secondary IgG-biotin/QD-Streptavidin cascade. Both labeling routes can be applied simultaneously for a two-color staining (D). The nuclei are counterstained with Hoechst 33 342 (blue) in A and C. Reprinted by permission from Macmillan Publishers Ltd: Ref. 104. Copyright 2003. permeabilization with proteinase K, which necessarily degrades cell and tissue architecture and destroys most of the protein-based biomarkers useful for IHC studies. In another example, Ying135 employed different sized glutathione-capped CdTe QDs (4, 4.5 and 5 nm) to bind with histone, which is abundant in cell nuclei. Cells of a HepG2 cell line were incubated with the b2227_V3_Ch-04.indd 131 25-Jan-16 3:04:59 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 132 J. Wang QDs consecutively. It could be seen that the 4.0-nm-sized QDs could swiftly penetrate the cellular matrix and bind to dense areas, such as nucleoli, staining these regions green after less than 1 h of incubation. In contrast, 5.0-nmsized QDs could only gain access into the cytoplasms after 24 h of incubation, staining these regions red. The 4.5-nm-sized QDs could stain the nucleoli orange after 24 h instead of 2 h of incubation. These findings suggested that the accessibility of QDs to targets within the fixed cellular matrix was affected by size-dependent diffusion processes (the results indicated in Figure 7). Figure 7. Confocal fluorescence images of cells stained with QDs. (a) Fixed HepG2 cells with nucleoli and cytoplasm stained by GSH-CdTe517 QDs (green) and GSH-CdTe618 QDs (red). (b) Fixed NIH 3T3 cells with actin immunostained using biotin-labeled GSH-CdTe618 QDs. (c) Live MDA-MB-435 cells incubated with F3-labeled GSH-CdTe618 QDs (red). (d) Live macrophage RAW264.7 cells incubated with GSHCdTe618 QDs (red) and cell viability calcein dye (green). Reprinted from Ref. 135. b2227_V3_Ch-04.indd 132 25-Jan-16 3:05:00 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 133 Tissue staining with QD conjugates is another interesting area of research concerning in vitro molecular detection in clinical application.104,134,136–140 The detection of epidermal growth factor receptor 2 (Her2) is important for BC treatment and prognosis. Li and co-workers141 detected the expression of Her2 in BC in an automated, quantitative, sensitive and convenient way by using a QDs-IHC analysis system, which proved the potential of QDs-based probes in clinical practise. In 54 specimens, low expression of Her2 could be detected clearly (Figures 8a and 8b) by QDsIHC. All five IHC±cases could be definitely identified by QDs-IHC. For accurate qualification, tissue autofluorescence was removed by software. The researchers also observed photostability of QDs-IHC, because durable QDs fluorescence did not change much in 9 days, even after 75 days in some cases (Figures 8c and 8d). Compared with conventional IHC, the QDs based approach is more sensitive, accurate and economic for the detection of Her2 in clinical BC diagnosis, especially for cases of IHC (2+). Moreover, Figure 8. Accurate Her2 testing by QDs-IHC. (a) Specimens with different Her2 IHC scores detected by QDs-IHC. (b) Control for (a), performed by conventional IHC. Photobleaching and preservation of QDs fluorescence: QDs fluorescence photobleaching on day 1 (c) and day 75 (d). Scale bar 100 µm. Reprinted from Ref. 141. b2227_V3_Ch-04.indd 133 25-Jan-16 3:05:01 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 134 J. Wang the results detected by this system might be more suitable for selecting molecular targeted therapy of BC. Therefore, this new method may have potential for clinical application, especially in developing countries. The existence and quantity of tumor biomarkers represents the generation of tumor tissues and differentiation of tumor cells; thus biomarker detection and quantification is favorable for the early diagnosis, classification and therapy of cancer. Recent studies showed that a panel of biomarkers rather than a single one is needed to accurately determine the stage of the disease. Thus, multiplexing is becoming increasing important for cancer diagnosis due to the advantage of reducing variability between tissue slices. The high brightness and photostability of QD probes enables sensitive and robust measurement of the biomarker expression levels. However, quantitative comparison of different biomarkers in multiplexed staining might be compromised by the strong signal enhancement of larger (red) QD and reduction of smaller (green-blue) QD signals. For example, Ghazani and coworkers142 have demonstrated three-color staining of lung carcinoma xenografts for epidermal growth factor receptor (EGFR), E-cadherin and cytokeratin with 655, 605 and 565 nm QD-based assays and noticed significant enhancement of 655 nm signal over 565 nm one, attributing this phenomenon to FRET from smaller to larger QDs. Further, the discordance in fluorescence intensity of individual probes directly relates to light absorption properties of QDs, as larger QDs possess larger absorption cross-sections and thus collect light more effciently. The effect of FRET depends on the density and distribution of biomarkers, which is hard to predict and account for during quantitative analysis. However, differences in photo-physical properties of individual probes can be readily characterized in advance and incorporated into signal analysis algorithms. In a recent study, Yezhelyev et al.143 have demonstrated the use of QDs for multiplexed detection of five tumor biomarkers in cultured human breast cells and on single paraffin embedded clinical tissue sections. Simultaneous quantification of ER, PR and Her2 receptors correlated closely with the results from traditional methods including immunohistochemisty, western blotting and fluorescence in situ hybridization, suggesting that the QD-based technology is well suited for molecular profiling of tumor biomarkers in vitro. Future advancements in the area of QD-based molecular pathology will be centered around highly multiplexed quantitative molecular profiling. Engineering of more compact and sensitive QD probes with outstanding stability and nonfouling properties will, therefore, remain the major focus of research in this area. b2227_V3_Ch-04.indd 134 25-Jan-16 3:05:02 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications Cd-Containing QDs for Biomedical Imaging 135 The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 4.1.2. Real-time monitoring of dynamic molecular processes Staining of fixed cells and tissue specimens provides information on biomarker expression and distribution, nevertheless, the study of intracellular molecular pathways underlying the physiological and pathological processes is limited by the static nature of this technique. Real-time imaging of live cells, on the other hand, enables the study of highly complex and dynamic biological processes that occur at molecular level. While the relatively large size of QD probes often hampers cellular entry and intracellular targeting, access to the biomarkers expressed on the cell membrane is usually readily achievable. Consequently, the majority of applications reported in the literature describe dynamics of membrane proteins (e.g. receptor diffusion) and membrane-associated processes (e.g. endocytosis and intracellular traffcking) rather than monitoring of intracellular targets. Up to now, much work has been achieved concerning the study of cell receptor diffusion and interaction. In a single-molecule imaging study, Dahan et al.144 have used QDs for labeling of individual glycine receptors on the surface of cultured spinal neurons and tracking the receptor diffusion in and out of synaptic cleft (Figure 9). Differential 2D diffusion coeffcients of receptors have been measured over time spans 240 times longer than previously achieved using organic dyes as tags, with 4- to 8-fold better spatial resolution and with a signal to noise ratio almost an order of magnitude higher. While the steric effect of QD probes could not be assessed through this study, relative characterization of receptor diffusion patterns within the synaptic, perisynaptic and extrasynaptic regions was achieved. Pinaud et al.145 demonstrated that QD labeling enabled the high resolution and long-term tracking of individual glycosyl-phosphatidyl-inositol anchored avidin test probes (Av-GPI) and their various diffusion behaviors. It was revealed that cholesterol-/ sphingolipid-rich microdomains can segment the diffusion of GPI-anchored proteins in living cells and the dynamic partitioning raft model can precisely describe the diffusive behavior of some raft-associated proteins across the plasma membrane. In another study, QDs have been used to reveal a previously unknown receptor diffusion mechanism for recovery from synaptic depression in neurons.146 Tracking of the rapid lateral diffusion of QD-labeled AMPA glutamate receptors have shown diffusion behavior comparable to that of organic dye-labeled receptors, while providing a robust fluorescence signal for the duration of experiment. Murcia et al.147 have demonstrated that labeling of individual cell membrane lipids with QDs does not affect lipid diffusion (as compared to dye-labeled lipids), while enhanced brightness of the probe enables high-speed single molecule tracking at 1,000 frames per second. b2227_V3_Ch-04.indd 135 25-Jan-16 3:05:02 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 136 J. Wang Figure 9. Labeling of individual glycine receptors in cultured spinal neurons. QD probes label glycine receptors throughout somatodendritic compartment (A) and can be located adjacent to (B, arrowhead) or in front of (B, arrow) inhibitory synaptic boutons. TEM examination reveals QD clustering at the extrasynaptic (C), perisynaptic (D) and synaptic (E) regions. Reprinted from Ref. 144 with permission from AAAS. Copyright 2008. Besides providing insight on the molecular dynamics of cell membrane components, QD probes facilitate the detailed study of such important processes as endocytosis and intracellular traffcking. For example, Vu et al.148 successfully detected downstream signaling and neuronal differentiation in b2227_V3_Ch-04.indd 136 25-Jan-16 3:05:02 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 137 PC 12 cells by activating TrkA with QDs conjugated to the β subunit of nerve growth factor (βNGF). Here, functional bioassays of neurite growth indicated that the level of cell signaling by QD-βNGF is lower than that by βNGF. Similarly, by activating TrkA or p75 NGF receptor in PC 12 cells with QD-NGF or QD-ricin toxin A (RTA) conjugate, Rajan et al.149,150 evaluated endocytosis, cytoplasmic redistribution and shuttling of QD-NGFreceptor complexes at ensemble and single-molecule levels. In particular, they found that extended imaging of QD-NGF-TrkA single-molecule complexes can stage distinct endocytic phases and cytoplasmic transport of TrkA with high spatial and temporal resolutions. In another study Zhang et al.151 have utilized the unique size and pH-dependent fluorescence of QDs for the study of the dynamics of synaptic vesicles during multiple rounds of neuronal transmission without perturbing the vesicle cycling. Monitoring of individual QD-loaded synaptic vesicles has enabled characterization of complete vesicle fusion (full-collapse fusion) and transient fusion (so-called kissand-run behavior) with respect to time and frequency of impulse firing and uncovered new aspects of neurotransmitter release and replenishment mechanisms. Effcient specific interaction with cell components requires otherwise inert QD probes to possess biological functionality, which is usually conveyed by decoration of QDs with targeting biomolecules. Often such moieties are represented by the receptor ligands attached to QD surface either covalently or through a streptavidin-biotin linker. For instance, Lidke et al.152 utilized QD epidermal growth factor (QD-EGF) conjugates for targeting erb1-eGFP or erb3-mCitrine in Chinese Hamster Ovary (CHO) cells or A431cells. The QD-EGF conjugate, which was prepared by mixing QD-streptavidin conjugate with biotinylated-EGF, is an ideal candidate for the detection of heterodimerization between erb1 and erb2 (Figure 10). Also, owing to the exceptional photo stability of QDs, the QD-EGF conjugate could be used for detecting the courses of EGF-QD to erb1 binding and retrograde transport of QD-EGF-erb complexes from filopodia to the cell body, both at singlemolecule level. In a later report by the same group, antigen uptake and processing by dendritic cells have been studied using QDs functionalized with pathogen-specific ligands.153 Highly stable ligand-coated QDs mimicking viruses and pathogenic microorganisms provide a powerful model system for the detailed characterization of the immune response mechanisms. Future advances in continuous monitoring of dynamic molecular processes within living systems will rely on the expanded capabilities brought by highly bright and photostable QD probes. Having size comparable to proteins or small viruses, QDs are capable of carrying multiple biomolecules that mediate antigen recognition, receptor binding, endocytosis and intracellular b2227_V3_Ch-04.indd 137 25-Jan-16 3:05:02 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 138 J. Wang Figure 10. Fluorescence images of CHO cells expressing erbB1-eGFP. The cells were exposed to 7 nM solution of biotin-EGF for 10 min at 4°C and subsequently labeled with QDs. Lower panel, images after 5 min at 37°C. Scale bars, 20 µm. Reprinted by permission from McMillan Publishers Ltd: (Nature Biotechnology), Ref. 152. Copyright 2004. trafficking, thus facilitating the design of a variety of minimally invasive model systems for the study of cell physiology. 4.1.3. Labeling of intracellular targets in live cells Labeling of intracellular targets in live cells, which examines molecular processes occurring within living systems, is essential for extended imaging of the structures of subcellular organelles and the functions of intracellular molecules. As QDs cannot readily cross intact cell membrane and diffuse within the crowded intracellular environment, specific labeling of intracellular components is highly problematic. Therefore, a functional QD probe for live-cell intracellular labeling should employ effcient intracellular delivery mechanism including cell uptake and cytoplasmic release. Mechanical techniques represent the most straight-forward approach to QD intracellular delivery, as virtually no modification of QD probes already available for extracellular labeling is required. For example, peptide-functionalized QD probes delivered to the cytoplasm via microinjection successfully exploit active peptide-specific transport mechanisms to reach target compartments, nucleus and mitochondria, within several hours after delivery (Figure 11A).154 In b2227_V3_Ch-04.indd 138 25-Jan-16 3:05:02 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 139 Figure 11. Mechanical (A–C) and nonmechanical (D–F) routes for intracellular delivery of bio-functional QDs within live cells. (A) Microinjection enables intracellular loading of unmodified QD probes along with carrier solution on a cell-by-cell basis. (B) Delivery with nanotubes offers precise control over QD delivery location, but requires QD anchoring to nanotubes via reducible linkers. (C) High throughput microinjection via nanosyringe arrays delivers unmodified QDs within large cell population, but changes the surface topology for cell growth. (D) QDs functionalized with cell-penetrating peptides might employ endosomemediated and nonendosomal pathways (depending on the peptide structure), offering flexibility in tuning the QD-cell interaction. (E) Pinocytosis enables uptake of unmodified QD probes with consequent cytoplasmic distribution. (F) Utilization of active receptor-mediated QD uptake via endocytosis followed by endosomal escape via proton-sponge effect represents a highly efficient noninvasive delivery method with specific targeting capabilities. Adapted with permission from Ref. 25. another example, Yum et al. have utilized gold-coated boron nitride nanotubes (with a diameter of 50 nm) to deliver QDs within the cytoplasm or nucleus of live HeLa cells with consequent 30-minute monitoring of QD diffusion within those compartments (Figure 11B).155 Aiming at high-throughput intracellular delivery, Park et al. have engineered arrays of vertically aligned carbon nanosyringes that, upon cell growth on top of them, provide cytosolic access for injection of unmodified QDs (Figure 11C).156 b2227_V3_Ch-04.indd 139 25-Jan-16 3:05:03 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 140 J. Wang In contrast to mechanical techniques, nonmechanical approaches are gaining increasing popularity due to the potential for high-throughput robust QD intracellular delivery with minimal intrusion to cell physiology. Functionalization of QDs with engineered peptides, small versatile biomolecules, might provide great flexibility in tuning the QD interaction with cell components (Figure 11D).157 In general, highly cationic peptides facilitate enhanced interaction with the cell membrane and QD internalization, whereas additional targeting moieties govern intracellular distribution. For example, Delehanty et al.158 have modified QDs with HIS-tagged cell penetrating peptide based on the HIV-1 Tat protein motif, achieving effcient internalization of QDs via endocytosis, while Rozenzhak et al.159 have added the nuclear localization sequence for nuclear targeting and apoptotic GH3 domain for triggering cell death. Despite the versatility of QD-peptide conjugates for labeling of intracellular targets, this approach still suffers from the uncontrolled probe aggregation and lysosomal sequestration inside cells. Recent work on QD cell uptake and intracellular targeting has focused on employing endocytosis/pinocytosis as a universal delivery mechanism and endosome destabilization/lysis as a cytosolic access route.160 One strategy involves QD cell-loading using osmotic lysis of pinocytic vesicles (Figure 11E). For example, Courty et al.161 have utilized this approach to load QD-tagged kinesin motors to living HeLa cells and monitor single-motor movement within the cytoplasm. Aiming at containing all functionalities within single QD probes, Duan and Nie have coated QDs with hybrid poly(ethylene glycol)/polyethylenimine (PEG/PEI) polymers producing nanoparticles with a reasonably small HD (15–22 nm) and endosome-disrupting capacity and yet good stability and biocompatibility (Figure 11F). To improve QD photo-physical properties, Yezhelyev et al.162 have decorated negativelycharged polymer-coated QDs with tertiary amines, thus producing protonabsorbing QD probes that effciently achieve intracellular endosomal release while featuring bright fluorescence and good colloidal stability. A promising technology for real-time sensing of target recognition is based on the nonradiative energy transfer (FRET) from the QD to acceptor/ quencher molecules. McGrath et al.163 have taken advantage of FRET between QD-transferrin probes and dye-transferrin conjugates. During receptor mediated endocytosis, dimerization of transferrin receptors results in increased proximity between QDs and acceptor dyes, thus enabling FRET. Yet, accidental nonFRET excitation of acceptor dye was unavoidable with standard single-photon imaging modality in this study. To minimize this artifact, Clapp et al.164 have utilized a two-photon QD excitation route that significantly enhances the signal-to-noise ratio of intracellular FRET. b2227_V3_Ch-04.indd 140 25-Jan-16 3:05:04 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications Cd-Containing QDs for Biomedical Imaging 141 The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Since QDs have two-photon absorption cross sections several orders of magnitude larger than typical organic dyes, undesirable two-photon excitation (840 nm) of acceptor Cy3 dye is dramatically reduced in comparison to single-photon excitation (488 nm). 4.2. In vivo imaging The characterization and analysis of biomolecules and biological systems in the context of intact organisms is known as in vivo research. Compared with the imaging in vitro, QD imaging in vivo faces different challenges caused by the increase in complexity in going to a multicellular organism and with the accompanying increase in size. Fluorescence in vivo imaging with QD probes promises to greatly expand the capabilities of existing imaging modalities,7 providing access to high-resolution multiplexed biodistribution of QDs in vivo, in vivo vascular imaging, in vivo real-time cell tracking and in vivo targeted molecular imaging. Fluorescence imaging of live animals is limited by the poor transmission of visible light through the living tissues as well as by the intense autofluorescence of tissue chromophores. Fortunately, there is a NIR optical window (650–900 nm) in which major fluorophores in mammals, deoxy- and oxyhemoglobin (HbO2), and water have minimal absorption.65 One of the greatest advantages of QDs for imaging in vivo is that the fluorescence emission wavelengths of QDs can be tuned throughout the NIR window by adjusting their composition and size.165 Also QDs have a two photon absorption cross section several times greater than organic dyes and this property makes them more efficient at probing thick tissue specimens by multiphoton microscopy.166 4.2.1. Biodistribution of QDs in vivo For most investigations of in vivo imaging, QDs are usually directly injected into the live animal intravenously or subcutaneously and thereby are delivered into the bloodstream. The behaviors of QDs in vivo are very interesting because they have to interact with the components of plasma, blood cells and the vascular endothelium. The biodistribution and clearance of QDs in various organs are often the focus of research. Chan and co-workers167 scrutinized the behavior and suitability of QDs for in vivo biomedical applications by performing a thorough quantitative analysis of the kinetics, clearance and metabolism of semiconductor QDs in vivo, following intravenous dosing in Sprague–Dawley rats. Initially, Chan et al. studied the plasma clearance of b2227_V3_Ch-04.indd 141 25-Jan-16 3:05:04 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 142 J. Wang QDs to determine how fast the QDs leave the bloodstream and enter organs. The results demonstrated that two kinds of QDs, QD-LM and QDBSA (i.e. QDs capped with mercaptoundecanoic acid and crosslinked with lysine or BSA, respectively) decayed monoexponentially in plasma according to firstorder kinetics; however, the plasma clearance of the QD-LM, estimated as dose/area under the plasma concentration–time curve, was significantly lower than that for QD-BSA. The differences in the pharmacokinetics between QD-LM and QD-BSA likely originated from the described surface modification and the size of the QDs. The researchers also examined the uptake of QDs into various organs, after leaving the bloodstream. Their results revealed that the majority of the QDs dose was in the liver and not the spleen. The much larger uptake by the liver versus the spleen is partially due to the much larger size of the liver, resulting in a higher fraction of dose being sequestered. Also, there was a big quantitative difference in tissue distribution between the QD-LM and QD-BSA in the liver, spleen, lung and kidney. QD-LM was present in higher quantities in lung and kidney in comparison to QD-BSA. All these findings suggested that the elucidation of QD metabolism and clearance necessitate focused studies on the liver, kidney and the RES. This was the first quantitative report on the biodistribution and clearance of QDs in vivo, the quantitative findings of which are important for the advancement of QDs as contrast agents for cancer imaging and for improving the design of nanostructures for in vivo biomedical applications. Probably the safest and most desirable approach to addressing the toxicity issue is engineering of QD probes that are quickly and completely eliminated from the body via renal or bile excretion pathways without triggering uptake by the RES and avoiding degradation pathways. This approach seems especially favorable in light of sparse information on in vivo QD degradation mechanisms and long-term effect of QD accumulation in organs. Systematic investigation of QD biodistribution performed by Choi et al.95 in mice has identified a nanoparticle hydrodynamic size renal clearance threshold of 5.5 nm. When delivered systemically, small cysteine-capped QDs are readily excreted into the bladder with minimal accumulation in the liver (4.5%) and kidneys (2.6%), whereas larger particles exhibit significant liver uptake (26.5%). Further, the importance of nonfouling zwitterionic surface coatings in inhibiting the protein absorption and retaining the original nanoparticle hydrodynamic size has been emphasized. Prasad et al.96 have synthesized ultrasmall (3–5 nm in diameter) cysteine-coated CdTe/ZnTe QDs and tested biodistribution of these probes in mice. Three mice were injected with 5 mg/kg of CdTe/ZnTe QDs through the tail veins. After 2 weeks, the researchers examined the major organs (liver, spleen, kidney, lung and heart) b2227_V3_Ch-04.indd 142 25-Jan-16 3:05:04 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 143 to investigate for any sign of acute toxicity. They also investigated the biocompatible alloyed CdTeSe/CdS QDs as NIR optical probes for long-term biodistribution studies.168 A significant reduction in the amount of QDs in the liver and spleen was observed after 7 weeks post injection. From these studies, no signs of toxicity were observed in the tissues from the animals that received QDs, when compared with the control animals who received only buffer solution. These studies provide preliminary validation of the use of optical NIR QDs for short-term and long-term in vivo imaging. 4.2.2. In vivo vascular imaging One of the most common in vivo applications of QDs is fluorescence contrast imaging of the blood vasculature and lymphatic drainage system. For the former system, by 2003, Larson et al.166 had demonstrated that green-light emitting QDs could be used to image capillaries of adipose tissue and skin in living mice, following intravenous injection. In the same year, Lim and co-workers169 imaged the coronary vasculature of rat heart by using nearinfrared QDs. Recently, Stroh et al.170 have combined two photon intravital microscopy, blue-emitting QDs encapsulated in PEG-phospholipid micelles and a transgenic mice model with GFP-expressing perivascular cells to study the morphology of the tumor vasculature. Following systemic administration, QDs highlight the vessel boundary providing a clear picture of tumor vessel morphology while resisting extravasation for at least 30 min, whereas GFP fluorescence indicates the distribution of perivascular cells. Poor QD extravasation has been employed by Kim et al.171 for studying the patho-physiology of viral infection of the central nervous system in mice. Using intravital twophoton microscopy, QD extravasation from brain microvasculature has been monitored as a measure of disease-associated vascular injury and blood-brain barrier breakdown. Sentinel lymph node biopsy (SLNB) imaging is a means of detecting cancer metastasis and is now the standard of care in BC surgery. It is based on targeting the first draining lymph node, also called the sentinel lymph node (SLN) of a lymphatic basin at the cancer site to determine the extent of disease spread. A negative detection of metastasis in the SLN means that the disease is contained and extensive surgery can be avoided. Kim et al.165 intradermally injected NIR QDs with a hydrodynamic diameter of approximately 15–20 nm into large animals. They found that when only 400 pmol of NIR QDs were injected intradermally into the thigh of a 35-kg pig, a surgeon was able to follow lymphatic flow towards the SLN. The real-time images obtained included lymph channels that diverge from the injection site and b2227_V3_Ch-04.indd 143 25-Jan-16 3:05:04 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 144 J. Wang Figure 12. Multiplexed in vivo and ex vivo imaging of separate lymphatic networks with QD accumulation in SLNs. Reprinted with permission from Ref. 172. Copyright 2007, American Chemical Society. then coalesce into the SLN. Localization of the SLN required only approximately 3–4 min and the NIR QDs permitted image guidance throughout the procedure. Recently, the multiplexing capability of QDs has been exploited for in vivo imaging of 5 different lymphatic basins in mice (Figure 12). Following intracutaneous injection of 5 types of polymer-coated carboxyQDs ranging in emission wavelength from 565 to 800 nm into the paws, ears and chin of mice, Kobayashi et al.172 have monitored the transport of QDs through lymphatic networks and accumulation in SLNs. Further passage of QD probes to secondary draining lymph nodes was significantly inhibited, possibly due to nonspecific binding between negatively charged QD coating and proteins resulting in an increase in probe size. Cardiovascular and lymphatic angiography have been two of the most successful QD-based in vivo imaging applications. In combination with fluorescence reflectance imaging, QDs highlight macroscopic structures on a whole-animal or whole organ scale and serve as visual tags for image-guided b2227_V3_Ch-04.indd 144 25-Jan-16 3:05:04 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications Cd-Containing QDs for Biomedical Imaging 145 The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. surgery; two-photon intravital microscopy provides high-resolution examination of superficial vessels and their surrounding tissues; and emerging advanced imaging techniques, such as multiphoton microscopy with a needlelike gradient index lens for deep-tissue imaging,173 promise to enable detailed studies of intact vasculature deep within organs 4.2.3. In vivo real-time cell tracking Cells labeled with QDs in vitro exhibit fluorescence signals which allow the original cells and their progeny to be recognized while they are grafted in the living animals. Dubertret et al.106 reported the first research on tracking QD loaded cells. First, they microinjected QDs into the cytoplasms of single frog embryos. As the embryos grew, the progeny cells descended from the original labeled cell retained a portion of the fluorescent cytoplasm, which could be fluorescently imaged in real time under continuous illumination. Voura et al.174 have studied metastatic tumor cell extravasation into the lung tissue using DHLA coated QDs for cell tagging and two-photon emission scanning microscopy for post-mortem examination of excised tissue specimens. With the aid of lipofectamine, five groups of cells have been loaded ex vivo with QDs ranging in emission from 510 to 610 nm and then intravenously injected into live mice. High-resolution imaging of whole-mounted mouse lungs have enabled clear spectral separation of individual QD signals from each other and from tissue auto fluorescence, thus facilitating study of interaction between different tumor cell populations within the same animal. Simultaneously, Gao et al.107 delivered QDs linked to a translocation peptide (such as HIV Tat or polyarginine) into living cancer cells. Surprisingly, the level of QDs loading used did not affect cell viability and growth, as the implantation of QD-tagged cancer cells led to normal tumor growth in animal models. For example, tumor tissue could be observed fluorescently through the skin of a mouse after growth of transplanted cancer cells. The results showed that QD-tagged cells injected into a host mouse and the QDs signal observed in vivo provided a method for tracking cells (Figure 13). In another example, Stroh et al.170 have utilized two-photon intravital microscopy for tracking the transport of bone marrow-derived progenitor cells through tumor vessels in real time. Cells loaded with TAT-functionalized orange-emitting QDs have been injected into the carotid artery of live mice along with blue-emitting QDs for vessel contrast. As two spectrally distinct QD types can both be excited by 800 nm two-photon light, simultaneous highlighting of tumor vasculature and continuous cell tracking at B1 frame/s are possible. b2227_V3_Ch-04.indd 145 25-Jan-16 3:05:05 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 146 J. Wang Figure 13. In vivo imaging of implanted QD-tagged tumor cells. (A) Bright QD tags (B) enable visualization of tumor cells through skin with a noninvasive whole-animal fluorescence imaging, whereas organic dye (C) signal is indistinguishable from autofluorescence. (D) Imaging of subcutaneously implanted QD-loaded microbeads shows the potential for multiplexed in vivo cell detection and tracking. Reprinted by permission from Macmillan Publishers Ltd: Ref. 107. Copyright 2004. Steady advancements in vitro QD cell loading, promising initial results of in vivo QD cell tagging and development of sensitive in vivo fluorescence imaging techniques suggest that 3-dimensional multiplexed in vivo cell tracking for the study of dynamic cell migration phenomena might become available in the near future. 4.2.4. In vivo targeted molecular imaging QD-based fluorescence molecular imaging represents an attractive technique for the detection of specific biomarker expressing cells in vivo. While being simple and inexpensive in comparison to other targeted molecular imaging modalities, it provides a powerful tool for studying complex physiological phenomena (e.g. activation of immune response), detecting diseased cells and tissues (e.g. tumors) and evaluating the pharmacokinetics and biodistribution of targeted nanoparticle-based drug delivery vehicles in a whole-animal context. In a pioneering study performed by Akerman et al. green and red TGAcoated QDs coupled to peptides with affinity for lung endothelium and tumor vasculature have been intravenously injected into live mice.124 b2227_V3_Ch-04.indd 146 25-Jan-16 3:05:05 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. Cd-Containing QDs for Biomedical Imaging 147 Post mortem evaluation of tissue sections revealed remarkably specific QD targeting. Recently, Nie’s group175 developed single-chain anti-epidermal growth factor receptor (ScFvEGFR)-targeted QDs that specifically bind to and are internalized by EGFR-expressing tumor cells. EGFR is a potential marker for in vivo receptor targeted molecular imaging with excellent tumorto background contrast, which demonstrated upregulation in many cancer types and provided an opportunity for designing receptor-targeted approaches for cancer detection and treatment. The results elucidated that large numbers of QD-bound tumor cells in the tumor areas and QDs resided in the cytoplasm of the tumor cells, suggesting uptake of ScFvEGFR-QDs by the cells in the tumor mass possibly via receptor-mediated internalization. Owing to the limited absorption of light by tissues in the NIR region as compared with that in the ultraviolet and visible light spectra, NIR light can penetrate several centimeters below the body surface and internal fluorophores can be observed easily. Simultaneously, in the NIR region auto fluorescence from tissues is also Figure 14. In vivo active tumor targeting of the QD–folic acid (QD-FA) probes. Spectrally unmixed in vivo fluorescence images of liver-tumor-bearing nude mouse at (a) 0 h, (b) 30 min, (c) 2 h and (d) 4 h after injection of the QD–FA probes. The autofluorescence of the mouse was removed by spectral unmixing of the above images. Adapted with permission from Ref. 58 b2227_V3_Ch-04.indd 147 25-Jan-16 3:05:05 PM b2227 Nano Imaging: From Fundamental Principles to Translational Medical Applications The World Scientific Encyclopedia of Nanomedicine and Bioengineering I Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/25/16. For personal use only. 148 J. Wang limited, so that the fluorescence of an introduced fluorophore can be more clearly visualized. NIR tumor imaging in vivo is therefore more favorable than modalities that use the other aforementioned electromagnetic radiation. In 2006, Cai et al.176 have used polymer-encapsulated NIR CdTe/ZnS core/ shell QDs functionalized with cyclic arginine–glycine–aspartic acid (RGD) peptides for targeting integrinavb3 (a biomarker up-regulated in cancerous tissue during proliferation, metastasis and angiogenesis) following systemic administration in tumor-bearing mice. Specific in vivo tumor labeling was clearly detectable with a whole-animal hyper-spectral imaging. Also, Torchilin et al.177 used NIR QDs to image the whole body of mice with murine BC cells injected subcutaneously. One hour after the injection of QDs via the tail vein, QDs accumulated in the organs (liver, kidney and gastrointestine) and tumor was easily visible. After 2 h, spleen was also clearly visible; at 4 h, only liver and kidneys were viewed. For whole-animal tumor imaging, the tumor site was clearly identifiable at 1 h (with higher overall fluorescence) and 2 h and much less at 4 h. This work proved that QDs were an effective imaging agent both for tumor imaging and quantification, which will improve tumor imaging and diagnosis. The future development of imaging tumors with QDs can focus on producing actively targeted QDs by using tumor-specific antibodies, which should greatly accelerate early clinical tumor diagnosis and help to gain time for suitable cancer therapy. Most recently, Han’s group58 made an attempt to synthesize water-soluble carboxylic acid group terminated CdTe/ CdS coresmall/shellthick NIR QDs in aqueous solution by a one-step method and demonstrated their applicability in vivo active tumor targeting of nude mice (Figure 14). References 1. A. P. 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