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Hindawi Publishing Corporation Journal of Nanomaterials Volume 2011, Article ID 834139, 13 pages doi:10.1155/2011/834139

Review Article Application of Quantum Dots in Biological Imaging

Shan Jin, Yanxi Hu, Zhanjun Gu, Lei Liu, and Hai-Chen Wu

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China

Correspondence should be addressed to Lei Liu, [email protected]

Received 15 May 2011; Accepted 2 June 2011

Academic Editor: Xing J. Liang

Copyright © 2011 Shan Jin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Quantum dots (QDs) are a group of semiconducting nanomaterials with unique optical and electronic properties. They have distinct advantages over traditional fluorescent organic dyes in chemical and biological studies in terms of tunable emission spectra, signal brightness, photostability, and so forth. Currently, the major type of QDs is the heavy metal-containing II-IV, IV-VI, or III-V QDs. Silicon QDs and conjugated dots have also been developed in order to lower the potential toxicity of the fluorescent probes for biological applications. Aqueous is the common problem for all types of QDs when they are employed in the biological researches, such as in vitro and in vivo imaging. To circumvent this problem, ligand exchange and polymer coating are proven to be effective, besides synthesizing QDs in aqueous solutions directly. However, toxicity is another big concern especially for in vivo studies. Ligand protection and core/shell structure can partly solve this problem. With the rapid development of QDs research, new elements and new morphologies have been introduced to this area to fabricate more safe and efficient QDs for biological applications.

1. Introduction their practical use in biology and medicine. Indeed, several studies have reported that size, charge, coating ligands, and , or so-called quantum dots oxidative, photolytic, and mechanical stability, each can (QDs), show unique optical and electronic properties, contribute to the cytotoxicity of cadmium-containing QDs. including size-tunable emission, simultaneous excita- Another critical factor that determines the cytotoxicity of tion of multiple fluorescence colors, high signal brightness, QDs is the leakage of heavy metal ions from the core caused long-term photostability, and multiplex capabilities [1–4]. by photolysis and oxidation [23–25]. The long-term effects Such QDs have significant advantages in chemical and of these ions enrichment and durations within the body biological researches in contrast to traditional fluorescent raise concerns about the biocompatibility and safety of QDs organic dyes and green fluorescent proteins on account of [24, 26]. On the other hand, the rapid clearance of QDs from their photobleaching, low signal intensity, and spectral over- the circulation by the reticuloendothelial system (RES) or lapping [5–7]. These properties of QDs have attracted great entrapment of QDs in the spleen and liver [27, 28]canresult interest in biology and medicine in recent years. At present in the degradation of the imaging quality of the objective and QDs are considered to be potential candidates as luminescent the enhancement of background noise. probes and labels in biological applications, ranging from One promising solution to these problems, including molecular histopathology, disease diagnosis, to biological QDs sequestered in the liver/spleen and long-term retention imaging [8–10].Numerousstudieshavereportedtheuseof in the body, is to evade the recognition and uptake by QDs for in vitro or in vivo imaging of sentinel lymph nodes the reticuloendothelial system thus extending the circulation [11–17], tumor-specific receptors [18–20], malignant tumor time of the particles in the body. In addition, for biological detectors [21], and tumor immune responses [22]. studies, QDs should be aqueous soluble in order to adapt the However, the major concerns about potential toxicity of biological environment. Therefore, when QDs are employed II-IV QDs (such as CdTe and CdSe) have cast doubts on in biological imaging, many factors should be considered and 2 Journal of Nanomaterials

10 1

9 0.8 8 0.6 0.4 7 0.2 Absorption 6 0 600 800 1000 1200 1400

5 Wavelength (nm)

4 480 520 560 600 640 680 720 Qdot diameter (nm)

3

2

1 400 600 800 1000 1200 1400 Emission wavelength (nm) Luminescence

CdS CdTe/CdSe CdSe InP 480 520 560 600 640 680 720 CdTe InAs Wavelength (nm) CdHgTe/ZnS PbSe (a) (b)

FITC (x 5) GFP qdot qrod SAV MBP IgG 20 nm bead

5nm (c)

Figure 1: Emission maxima and sizes of quantum dots of different composition (with permission from [3]).

the system should be properly designed in order to meet a set toxic heavy metal ions can be easily leaked out into biological of requirements [29–31]. systems if the surfaces are not properly covered by the shells In this report, we first briefly review different types of or protected by ligands. QDs that have been fabricated so far and their synthesis The limitation of heavy metal-containing QDs stimulates methods, including surface functionalization. Next, we focus extensive research interests in exploring alternative strategies on their applications in biological imaging. Finally, the new for the design of fluorescent nanocrystals with high biocom- trends of QDs imaging are discussed. patibility. In this case, the practical strategy is to develop highly fluorescent based on nontoxic elements 2. Types and Characteristics of QDs or explore luminescent π-conjugated polymer dots. Silicon is another type of QDs which possess Traditionally, the typical QDs consist of a II-IV, IV-VI, or III- unique properties when their sizes are reduced to below V semiconductor core (e.g., CdTe, CdSe, PbSe, GaAs, GaN, 10 nm. Similar to their predecessors, silicon QDs also have InP, and InAs; Figure 1)[3, 32, 33], which is surrounded many advantages over traditional fluorescent organic dyes by a covering of wide bandgap semiconductor shell such as [35], including resistance to photobleaching and wide emis- ZnS in order to minimize the surface deficiency and enhance sion range from visible to region with relatively high the quantum yield [5, 34].The unique optical and elec- quantum yields. Moreover, owing to silicon’s nontoxic and tronic properties of QDs, including narrow light emission, environment-friendly nature, Si QDs are used as fluorescent wideband excitation, and photostability, provide them with probes for bioimaging [35]. It has been reported that for significant advantages in the applications of multiplexed in vivo applications, Si QDs mainly degrade to silicic acid molecular targets detection [21] and as optical bioimaging which can be excreted through urine [36], and for in vitro probes [8–10]. One major drawback which severely hinders use, Si QDs are considered 10-times safer than Cd-containing the application of II-IV, IV-VI, or III-V QDs for in vivo bio- quantum dots (Figures 2 and 3)[37]. However, the chal- logical research is the concern about the toxicity associated lenges present in employing Si QDs in bioimaging arise from with the cadmium, lead, or arsenic containing QDs. The their water solubility and biocompatibility. Usually, Si QDs Journal of Nanomaterials 3 are produced in nonpolar with hydrophobic ligands (such as styrene and octene) on their surface in order to protect the Si cores. Therefore, it is a common problem that Si QDs show poor solubility and unstable photolumines- cence (PL) in aqueous solutions [38–40]. PL degradation can occur gradually after modified Si QDs are transferred from organic solvents to aqueous solutions. Thus, fabricating good water-disperse Si QDs with stable optical characters is vital to their applications in bioimaging studies. Recently, Tilley and Yamamoto reported that water-soluble Si QDs covered by allylamine [41, 42] and poly(acrylic acid), respectively, had been successfully obtained and showed good performance in 50 μm fixed- labeling [40]. The semiconducting properties of conjugate (a) derive from their π-electron delocalization along the poly- mer chain. Mobile charges are allowed to move between π and π∗ orbitals. Highly bright fluorescence can be stable under one-photon or two-photon excitation. Polymers with (b) such structures are promising in the fields of LED and bi- osensors [43, 44]. Burroughes and coworkers first reported Figure 2: images of mesoporous silica beads (5-μm the luminescent conjugated polymers in 1990 [45]. Later on, diameter, 32 nm pore size) doped with QDs emitting light at 488 Friend and So successfully applied π-conjugated polymers (blue), 520 (green), 550 (yellow), 580 (orange), or 610 nm (red; with to light-emitting devices [46, 47]. Recently, related works permission from [37]). focused on fluorescent polymer encapsulation and func- tionalization to yield conjugated polymer dots (CP dots) for bioimaging and probes (Figure 4)[48, 49]. Compared In this process, ionic perchlorates such as Cd(ClO) ·6H O with other fluorescent probes, CP dots are convenient for 4 2 and Al Te are used as the precursors. In the presence of fluorescence microscopy and laser excitation because of their 2 3 ligands such as 3-mercaptopropionic acid (3-MPA), glutathi- absorption ranging from 350 to 550 nm. Also, fluorescence one (GSH), or other hydrosulfyl-containing materials, CdTe quantum yields can be slightly variable around 40%. More- QDs can be successfully obtained in aqueous medium. The over, CP dots can exhibit higher brightness than any other advantage of this method lies in its environment-friendly nanoparticles of the same size under certain conditions [50]. synthesis procedure and relatively low cost. QDs produced by this method can be directly applied in biological researches 3. Synthesis Methods of QDs without ligand exchange procedure. However, compared with the QDs produced by organometallic method, thiol- 3.1. Organometallic Method. Organometallic chemistry pro- capped QDs show low quantum yields, and broad full width vides an efficient pathway to produce monodisperse QDs in of half-maximum (FWHM), which can be attributed to its nonpolar organic solvents, such as CdSe nanocrystals cov- wide size distribution and poor stability in aqueous solution. ered by tri-n-octylphosphine oxide (TOPO) ligand. In a typical preparation, Me2Cd and Bis(trimethylsilyl)selenium 3.3. Hydrothermal Method/Microwave-Assisted Method. In ((TMS)2Se) can be used as organometallic precursors. Mon- order to reduce QDs surface defects generated during the odisperse CdSe with uniform surface derivatization and reg- growth process, these two methods are put forward to syn- ularity in core structure can be obtained based on the pyroly- thesize QDs with narrow size distribution and high quantum sis of organometallic reagents by injection into a hot coordi- ◦ ◦ yields. According to hydrothermal method, all reaction re- nating between 250 C to 300 C[51]. The adsorption gents are loaded in hermetic container and then heated to of ligands results in slow growth and annealing of the supercritical temperature; high pressure produced by the ff cores in the coordinating solvents. QDs with di erent sizes temperature can efficiently reduce reaction time and surface ff can be successfully obtained at di erent temperatures. The defects of QDs [53]. Moreover, because high reaction tem- advantage of this method is obvious because this method can perature can separate the processes of nucleus formation and produce a variety of QDs with high quantum yields and the crystal growth, this method has clear advantages over tradi- size distribution can be easily controlled by altering temper- tional aqueous synthesis, affording narrow size distribution ature or reaction time. Organometallic method is currently QDs. considered as the most important means to synthesize QDs. Microwave-assisted irradiation is another attractive method for QDs synthesis. This method was first introduced 3.2. QDs Synthesized in Aqueous Solution. Since Rajh and by Kotov group [54] and followed by Qian and coworkers to coworkers first reported the synthesis of thiol-capped CdTe demonstrate a fast and simple synthesis of CdTe and CdSe- QDs in aqueous solution, a great deal of efforts have CdS QDs by microwave-assisted irradiation in 2006 [55]. focused on synthesizing water-dispersed QDs directly [52]. Microwave irradiation can be considered as heating source 4 Journal of Nanomaterials

100 100 140 120 80 80 100 60 60 80 60 40 40 40 Cell viability (%) Cell viability (%) Cell viability (%) 20 20 20 0 0 0 1 10 100 1 10 10 100 Concentration of Si (μg/mL) Concentration of CdHgTe (μg/mL) Concentration of CdTe (μg/mL)

24 hr 24 hr 24 hr 48 hr (a) (b) (c)

Figure 3: Cytotoxicity of (a) Si, (b) CdTe, and (c) HgCdTe quantum dots toward Panc-1 cells. Error bars represent standard deviation (N = 3). Note that the concentration range is much higher in (a) than in (b) and (c) (with permission from [38]).

OC8H17

n n H13C6 C6H13 MeO PDHF MEH-PPV

OCH8 H17

PFPV n H C C H 17 8 8 17 MeO

(a) (b)

σ2pϕ

25 105

104 20

103 15 (GM) (nm)

10

5 2 200 nm 10 770 790 810803 850 870 0 Wavelength (nm)

PFPV PDHF MEH-PPV Rhodamine B (c) (d) Figure 4: (a) Chemical structures of the conjugated polymers. (b) Photograph of the fluorescence from aqueous CP dot dispersions under two-photon excitation of an 800 nm mode-locked Ti : sapphire laser. (c) A typical AFM images of the PFPV dots on a silicon substrate. (d) Semilog plot of two-photon action cross-sections (σ2pϕ) versus the excitation wavelength for CP dots and rhodamine B (reference compound; with permission from [49]). Journal of Nanomaterials 5

CdTe

ZnS Peptides COOH

= P O QD705-RGD C(RGDyK) = N + A O P H O = Ligand P B Tissue N + section exchange H Functionalization Aqueous solubility PMAL C Antibodies Organic : chloroFrom Aqueous phase D Coatin MSA InP/ZnS Overnight InP/ZnS OH − QDs stirring OH COO Cell HO membrane HO O O Proteins MSA coated Sialicase Hydrophobic ligands Nucleus QD

Scheme 1: Strategies to deal with QDs aqueous solubility problems and fabricate functionalized QDs for biological applications.

to optimize synthesis conditions, and in this system, water to cytotoxicity. Since nanoparticles within certain diameters is an excellent solvent for its tan δ is equal to 0.127. The (thisvaluemaybedifferent according to biological environ- whole solution can be quickly heated to above 100◦Ctoyield ment) are of similar size to certain cellular components and homogeneous QDs and the quantum yields can reach as high proteins, they may bypass natural mechanical barriers, thus as 17%. leading to adverse tissue reactions [26]. The application of QDs in biological imaging also re- 4. Toxicity, Solubilization, and quires QDs with good water solubility. Several strategies have been developed to circumvent this problem, which are sum- Functionalization of Quantum Dots marized as follows. In order to apply QDs to biological studies, safety, and bio- The first one is to synthesize QDs in aqueous solution compatibility are the issues that must be concerned. Toxico- directly, which has been depicted in this review (vide supra). logical studies have suggested that certain potential adverse The second is to obtain QDs in organic solvents, and then human health effects may be resulted from their exposure transfer them to aqueous solutions (Scheme 1). Nie and to some novel nanomaterials, such as gold nanoparticles Weiss groups pioneered the preparation of water-dispersed and QDs, but the fundamental cause-effect relationships are QDs by ligand exchange method (Figure 4)[5, 56]. In ad- ffi relatively obscure [23]. Thus, it is necessary to figure out dition, polymer and lipid coating is another e cient way to the interaction of QDs with biological systems and their render QDs with good water solubility [57, 58]. potentially harmful side effects in cells. Several studies reveal To improve in vitro and in vivo imaging quality and that the toxicity of QDs depends on many factors which detection sensitivity in biological fields, multiplex capabili- can summarized as inherent physicochemical properties and ties QDs have been developed. Recent efforts mainly focus on environmental conditions [23, 24, 26]. Not all QDs are alike, the functionalization of QDs to accommodate the demands each type of QDs should be characterized individually as to of imaging sensitivity and specificity [59]. Nie reported its potential toxicity. QD sizes, charges, concentrations, outer multicolor QD-antibody conjugates for the rapid detection coatings materials and functional groups, oxidation, and of rare Hodgkin’s and Reed-Sternberg (HRS) cells from mechanical stability all have been implicated as contributing infiltrating immune cells such as T and B lymphocytes [60]; factors to QD toxicity [24]. Derfus et al. studied the cytotox- Shi combined Fe3O4 with amine-terminated QDs to yield icity of CdSe QDs and pointed that without coating, the cy- fluorescent superparamagnetic QDs for in vivo and in vitro totoxicity of CdSe cores correlated with the liberation of free imaging (Figure 5)[61]. Cd2+ ions due to deterioration of QDs lattice. And in vivo, the liver is the primary site of acute injury caused by cadmium 5. QDs in Biological Imaging ions even though the concentration of Cd2+ is reduced to 100–400 μM[23]. In order to eliminate QD toxicity re- In order to apply QDs in biological imaging, recent studies sulted from heavy metal ions releasing, surface coating and have focused on developing near-IR luminescent QDs which core/shell structure are considered to be efficient solutions exhibit an emission wavelength ranging from 700 to 900 nm. to this problem. Many new types of QDs such as core/shell Light within this range has its maximum depth of penetra- (CdSe/ZnS) and core/shell/shell (CdSe/ZnS/TEOS) QDs tion in tissue and the interference of tissue autofluorescence with high luminescence are developed [34, 35]. Some re- (emission between 400 nm and 600 nm) is minimal [62, 63]. searchers also found that the quantum size effect which In addition, for the purpose of gathering more information resulted from the minute size of nanomaterials could lead during specific cellular processes in real time, QDs must be 6 Journal of Nanomaterials

O Polyethylene QDs oxide C Polystyrene − OH + − O + − + − PEG NH2 + NH

Amine-functionalized Fe3O4 (QD)

5nm 5nm Carboxylate-functionalized MNS QDs-conjugated MNSs (a)

O PLGA + PTX O

O m n O CH3 PLGA

O

O O OH CH3 H3C O NH H3C CH3 O H CH3 H QDs O PLGA O QDs OH O O CH OH 3 + PTX O 5nm O Paclitaxel (PTX) QDs-conjugated MNSs 5nm PTX-PLGA-QD -MNSs (b)

Figure 5: Schematic diagrams illustrating surface functionalization of magnetic nanospheres (MNSs): (a) conjugation of amine- functionalized quantum dots (QDs) to the surface of carboxylate-functionalized MNSs using conventional NHS/EDC coupling method; (b) PTX loading using a thin PLGA coat on the surface of QD-MNSs (with permission from [61]).

conjugated with which have the capabilities of rec- through endocytosis after washing away the excess QDs. Fur- ognizing the target. These surface modifications can also help ther, they also demonstrated the potential application of QDs prevent aggregation, reduce the nonspecific binding, and in cell tracking by using avidin-conjugated QDs to label cells. are critical to achieving specific target imaging in biological With the development of biomarkers in cell biology, the studies. Further functionalization of QDs can be achieved tracking of some specific cells (such as cells) becomes by the modification of protecting ligands [64–66], introduc- possible. Gac et al. have successfully detected apoptotic cells ing targeting groups such as apolipoproteins and peptides by conjugating QDs with biotinylated Annexin V, which (Scheme 1)[64]. In this part, we mainly introduce the recent enables the functionalized QDs to bind to phosphatidylserine development of conjugated QDs for in vitro and in vivo imag- (PS) moieties present on the membrane of apoptotic cells but ing; some new QDs related to bioimaging are also included. not on healthy or necrotic cells (Figure 6)[70]. The detection and imaging of apoptotic cells makes it possible to moni- 5.1. In Vitro Imaging. Early studies of in vitro imaging used tor specific photostable apoptosis. Wolcott reported silica- QDs to label cells. For instance, PbS and PbSe capped with coated CdTe QDs decorated with functionalized groups carboxylic groups had been obtained in aqueous solution by not only for labelling proteins, but also for preventing ligands exchange to label cells [67]; CdTe capped with 3- toxic Cd2+ leaking from the core [71]. Nie developed cell- mercaptopropionic acid (3-MPA) was used as imaging tool penetrating QDs coated with polyethylene glycol (PEG) to label Salmonella typhimurium cells [68]. Jaiswal et al. grafted polyethylenimine (PEI), which were capable of pene- reported labelling HeLa cells with acid-capped CdSe/ZnS trating cell membranes and disrupting endosomal organelles QDs [69]. It is found that the cells can store QDs in vesicles in cells [72]. Recently, in the demand of using biocompatible Journal of Nanomaterials 7

Light t = 0s t = 500 s t = 1000 s t = 1500 s Dox FITC FITC FITC FITC QD QD

QD Alex fluor 647 Alex fluor 647 Alex fluor 647 Alex fluor 647 QD-APt: “ON” QD-APt(DOX): “OFF” (a)

Qdots Qdots Qdots Qdots QD Target cancer cell “OFF”

Drug release (a) QD QD 120 Lysosome Dox: “ON” Nucleus QD: “ON” 100 (b) 80 Figure 7: (a) Schematic illustration of QD-Apt(Dox) Bi-FRET

10 (%) system. (b) Schematic illustration of specific uptake of QD- I/I 40 Apt(Dox) conjugates into target cancer cell through PSMA mediate endocytosis. The release of Dox from the QD-Apt(Dox) conjugates 20 induces the recovery of fluorescence from both QD and Dox (“ON” state), thereby sensing the intracellular delivery of Dox and enabling 0 the synchronous fluorescent localization and killing of cancer cells 0 200 400 600 800 1000 1200 1400 1600 1800 (with permission from [85]). Time (s) I/I10 (%) Alexa I/I10 (%) Qdots I/I10 (%) FITC (PS-PEG-COOH) with conjugation to biomolecules, such (b) as streptavidin and immunoglobulin G (IgG) to label cell surface receptors and subcellular structures in fixed cells [21, Figure 6: Comparison of the staining efficiency of Annexin V- 80], and other non-Cd-containing QDs such as Ge-QDs and functionalized Qdots (staining achieved in one step using 2 nM of near-IR InAs for imaging of specific cellular proteins [81, 82]. Qdots and 4 nM of biotinylated Annexin V, red), FITC-Annexin In order to broaden QDs applications, MRI detectable QDs V (0.5% v/v, green) and Alexa Fluor 647-Annexin V (0.5% v/v, were demanded. Many studies have set out to explore para- pink). (a) Light microscope pictures and pictures taken at t = magnetic QDs. Sun reported a new kind of magnetic fluores- 0, 500, 1000, 1500 s. (b) Intensity variation as a function of time for the three staining agents conjugated to Annexin V, FITC, Alexa cent multifunctional which contained silica- Fluor 647 and Qdots. Intensity values expressed as relative values coated Fe3O4 and TGA-capped CdTe QDs. This nanocom- I/I0,whereI0 is the intensity measured at the first irradiation of the posite was successfully employed for HeLa cell labelling and sample (with permission from [70]). imaging, as well as in magnetic separation [83]. Mulder et al. have successfully modified CdSe/ZnS with paramagnetic coating and arginine-glycine-aspartic acid (RGD) conjuga- and nontoxic QDs as nanoprobes, rare earth (RE) elements tion in order to combine the functions of specific protein are used to fabricate a new type of QDs, such as Gd-doped binding and contrast agent for molecular imaging [84]. ZnO QDs. RE-doped QDs have distinct advantages over The challenges encountered in cancer therapy stimulate heavy metal-containing QDs, not only because of avoiding comprehensive studies. Many efforts focus on the tracking the increase of by polymer or silica coating and diagnosis of cancer cells. Cao et al. used near-IR QDs in synthesis procedure, but also providing a simple, green with an emission wavelength of 800 nm (QD800) to label synthesis method. Liu et al. reported the development of squamous cell carcinoma cell line U14 (U14/QD800). The Gd-doped ZnO QDs with enhanced yellow fluorescence, and fluorescent images of U14 cells can be clearly obtained after these QDs can be used as nanoprobes for quick cell detec- 6hbycellendocytosis[62]. In 2007, Bagalkot et al. reported tion with very low toxicity [73]. Other progresses include a more complex QDs-aptamer- (Apt-) doxorubicin (Dox) CdSe/ZnS with PEG coating and CdSe/ZnS-peptide conju- conjugate system [QD-Apt(Dox)] to endow QDs with the gation for cytosol localization and nucleus targeting [74–77], capability of targeting, imaging, therapy, and sensing the CdS passivated by DNA to yield nontoxic QDs as new bio- prostate cancer cells that express the prostate-specific mem- logical imaging agent [78], CdSe/L-cysteine QDs designed brane antigen (PSMA) protein (Figure 7)[85]. Other types to label serum albumin (BSA) and cells [79], polymer QDs of QDs that have been applied in pancreatic cancer cells 8 Journal of Nanomaterials

Composite image of mouse, colored NIR on top of field

Tumor Tumor Cumulative histogram of tumor (left) and the mouse body (right)

0 255 0 255 0 2550 255 Count: 19116 Min: 10 Count: 501534 Min: 4 Count: 25448 Min: 37 Count: 535446 Min: 7 Mean: 62.164 Max: 106 Mean: 42.799 Max: 107 Mean:176.818 Max: 22 6 Mean: 127.063 Max: 255 StdDev: 16.051 Mode:61(763) StdDev : 23.450 Mode: 10 (29749) StdDev: 37.301 Mode: 197 (1034) StdDev: 63.694 Mode: 44 (7691) (a)

196 228.14 260.29 292.43 324.57 181 184.21 187.43 190.64 193.86 176.26 182.35 188.44 194.63 200.62

196 228.14 260.29 292.43 324.57 181 184.21 187.43 190.64 193.86 224.98 227.26 229.55 231.83 234.11 (b)

Figure 8: Targeting of lung tumor by injecting 2C5 QD-Mic in two mice. (a) Composite images (white field image superimposed with the fluorescence intensity), and cumulative histograms for the tumor region and the whole body of two mice; (b) composite ex vivo images (white field image superimposed with the fluorescence intensity) of lungs from mice bearing metastatic B16F-10 lung melanoma tumor (with permission from [94]).

imaging include CdSe/CdS/ZnS using transferrin and anti- targeting of living cells by transfection and RNA delivery. Biju Claudin-4 as targeting ligands, InP QDs conjugated with identified an insect neuropeptide, also-called allatostatin, cancer antibodies, and water-soluble Si-QDs micelles encap- which can transfects living NIH 3T3 and A431 human sulated with polymer chains [86–88]. Another important epidermoid carcinoma cells and transports quantum dots role played by QDs in biological application is transfection. (QDs) inside the cytoplasm and even the nucleus of the Many studies reported fluorescence imaging and nucleus cells [89]. This method showed the conjugation of QDs with Journal of Nanomaterials 9 allatostatin could achieve high transection efficiency, which other morphologies of QDs will interact with the biological was promising for DNA gene delivery and cell labelling. systems. Yong et al. reported CdSe/CdS/ZnS quantum rods Also, with specific conjugation with allatostatin, the QDs had (QRs) coated with PEGylated phospholipids and RGD another potential application in cancer, called photodynamic peptide for tumor targeting [101]. This conjugation was therapy [90]. demonstrated to be a bright, photostable, and biocompatible luminescent probe for the early diagnosis of cancer and was 5.2. In Vivo Imaging. In addition to their usage as nanop- believed to offer new opportunities for imaging early tumor robes and labels for in vitro imaging, QDs have also been growth. widely used as in vivo imaging agents (Figure 8)[91–94]. Cancer-specific antibody, coupled to near-IR QDs with pol- 6. Prospects ymer coatings is the most popular QDs agent for tumor- targeted imaging. Progress in this field has been reviewed in Owing to the initial success of QDs-biomolecule conjugates one literature [94, 95]. employed for in vitro and in vivo imaging, it is conceivable However, the depth of targets makes the QDs in vivo that future researches will continue to focus on the devel- imaging very challenging. This special application of QDs opment of QDs conjugations for cell labelling and cancer requires them with low toxicity, high contrast, high sensitiv- diagnoses. However, for bioapplications, biocompatibility of ity, and photostability. Dubertret et al. fabricated one type QDs remains the major challenge which must be carefully of CdSe/ZnS QDs which were encapsulated individually in dealt with. Many studies have showed the short-term stability phospholipid block-copolymer micelles. After further modi- and long-term breakdown of Cd-containing QDs in in vivo fication by DNA, this multifunctionalized DNA-QD-micelles imaging, which raise concerns about their chronic toxicity. can be developed to obtain Xenopus embryo fluorescent Some studies showed that even robust coatings on the images in PBS. Moreover, after injection into embryos, the surfaces of QDs cannot prevent their releasing of Cd2+ ions. QD micelles showed high stability and nontoxicity (<5 × 109 The emerging picture of Cd-containing QDs toxicity not nanocrystals per cell) [96]. Another study reported by only suggests that QDs are required to improve safety before Michalet showed that CdSe/ZnS QDs can exhibit high con- their wide biological applications as fluorescent nanoprobes, trast and imaging depth in two-photon excitation confocal but also a variety of noncadmium alternatives are in urgent microscopy by visualizing vessels in live mice [3, 97]. demand. At present, many efforts have focused on exploring TheyconfirmedthatcoatingssuchasPEGonthesurfaces new types of fluorescent nanomaterials such as upconver- of QDs could further reduce their toxicity and accumulation sion materials NaYF4 and NaGaF4. In addition, with the in liver. Some studies also pointed out that the increase development of new techniques and detection methods, QDs of the length of PEG coating polymer on the surface of are shown to have applications in wider fields. Zhang et al. QDs was able to slow down their extraction toward the successfully introduced Kelvin microscopy (KFM) as a liver, so coating polymer on the surfaces was another factor method to examine the binding of QDs with DNA in vitro worthy of considering when QDs are employed for in vivo and in vivo [102]; Huang et al. demonstrated a QDs sensor imaging [98]. Other reports that used low-toxicity QDs for quantitative visualization of surface charges on single include the development of non-Cd-containing QDs such living cell [103]. We believe that with the development of new as CuInS2/ZnS core/shell nanocrystals for in vivo imaging QDs with low toxicity and multiplex functionalities, QDs will [95]. In addition, sensitivity is another important factor that find more versatile applications in biological fields other than must be considered in QDs-related in vivo imaging. One bioimaging. study used nude mice for in vivo imaging after near-IR QD800-labeled BcaCD885 cells (BcaCD885/QD800) being Acknowledgment implanted [99]. Fluorescence signals of QDs accumulated in the tumor could be detected after 16 days of incubation at The authors are grateful to the “100 Talents” program certain concentrations. 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