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Upconversion nanophosphores for bioimaging
Accepted Manuscript Title: Upconversion nanophosphores for bioimaging Author: Suying Xu, Sheng Huang, Qian He, Leyu Wang PII: DOI: Reference:
S0165-9936(15)00008-4 http://dx.doi.org/doi: 10.1016/j.trac.2014.11.014 TRAC 14371
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Trends in Analytical Chemistry
Please cite this article as: Suying Xu, Sheng Huang, Qian He, Leyu Wang, Upconversion nanophosphores for bioimaging, Trends in Analytical Chemistry (2015), http://dx.doi.org/doi: 10.1016/j.trac.2014.11.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Upconversion nanophosphores for bioimaging Suying Xu, Sheng Huang, Qian He, Leyu Wang* State Key Laboratory of Chemical Resource Engineering, Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029, P.R. China * Corresponding author. E-mail address: [email protected] (L. Wang)
HIGHLIGHTS Upconversion nanophosphores show great advantages in bioimaging applications Synthesis methods, surface-modification strategies for upconversion nanophosphores Upconversion nanophosphores with ultrasmall size and high luminescence We summarize recent applications of upconversion nanophosphores in bioimaging Challenges for future developments of upconversion nanophosphores in bioimaging
ABSTRACT Upconversion nanophosphore (UCNP)-based optical imaging is a promising technique in the field of bioimaging due to the unique optical properties of UCNPs. UCNPs can be excited by long wavelength light with low power density and have excellent biocompatibility. By carefully controlling the synthesis of UCNPs, nearinfrared emission can also be obtained. Such NIR-to-NIR bioimaging probes show great advantages in terms of bioimaging, such as deep penetration and low autofluorescence. Moreover, UCNPs can also offer a platform for fabricating multifunctional nanocomposites for multi-modal imaging by incorporating magnetic resonance imaging, X-ray computed tomography and single-photon emission computerized tomography techniques. In this review, we briefly introduce the fundamental points of UCNPs, highlight the methods of synthesis and surfacemodification strategies, and summarize recent progress. We also discuss the current issues faced by researchers. Keywords: Bioimaging Fluorescence Nanocomposite Nanoparticle Nanophosphore Near-infrared Optical imaging Surface modification Synthesis Upconversion luminescence Abbreviations: AEP, 2-aminoethyl dihydrogen phosphate; CB, Conduction band; CTAB, Cetyltrimethylammonuim bromide; EDTA, Ethylenediamine tetraacetic acid; LSS, Liquid-solidsolution; MRI, Magnetic resonance imaging; NIR, Near-infrared; NP, Nanoparticle; OA, Oleylamine; OCMC, O-carboxylmethyl chitosan; PAA, Poly(acrylic acid); PEI, Poly(ethylene imine); PET, Positron-emission tomography; PVP, Polyvinylpyrrolidone; UCNP, Upconversion nanophosphores; VB, Valence band; X-ray CT, X-ray computed tomography
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1. Introduction Bioimaging includes a wide range of imaging techniques, such as magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound and optical imaging. Optical imaging technology is of great importance for bioscience studies, such as imaging-guided surgery and post-surgery evaluations for complete removal of diseased sections [1–3]. Such techniques experienced flourishing development in recent years due to their high temporal and spatial resolutions and the wide availability of luminescent materials, including small organic fluorophores, fluorescent doping nanoparticles (NPs), fluorescent proteins, quantum dots (QDs) and luminescent metal complexes [4–6]. Despite great progress achieved with small organic fluorophores and fluorescent proteins in labeling and protein interaction studies, some drawbacks of these fluorophores [e.g., poor photostability, high background noise (arising from autofluorescence)} have limited their applications in bioimaging. To some degree, semiconducting QDs overcome these issues, but they also brought new problems, such as cytotoxicity and photo blinking (intermittency of fluorescence), since QDs are made from harmful heavy metals and cytotoxicity test results also indicated such issues [7]. There is therefore still high motivation to search for more suitable phosphores withdesirable characteristics, such as high chemical stability and photostability, long wavelength excitation and emission, and good biocompatibility. In recent years, rare-earth (RE)-metal doped luminescent nanophosphores, here specifically referred to as upconversion nanophosphores (UCNPs), were considered promising candidates for biomedical-imaging applications due to their unique optical properties, including photon upconversion, high photostability, low cytotoxicity and inertness to environment conditions, such as pH, temperature and coexisting chemicals [8–14]. The upconversion properties afford lots of benefits in terms of imaging as with lower power excitation light, normally at 980 nm, UCNPs generate UV/visible, in some cases, even near-infrared (NIR) luminescence. The long wavelength light can penetrate samples more deeply without inducing autofluorescence. Moreover, the luminescence of UCNPs is very stable without photobleaching and photoblinking issues. When UCNPs are applied in bioimaging, they therefore allow not only deeper penetration, no toxicity and low autofluorescence, but also continuous monitoring. These advantages make UCNPs outstanding for bioimaging [4,15–17]. However, UCNPs normally have larger particle size than other conventional fluorophores, which holds back their applications in in-vivo biological fields as nonspecific aggregation would occur. Besides, particles with size larger than 10 nm are hard for the body to clear. Efforts have been devoted to decrease the size of UCNPs, yet problems arise as more surface defects are produced with decreased size and, in turn, reduce the luminescence of the UCNPs. Ideally, UCNPs for bioimaging should have sub-10 nm particle size while maintaining high luminescence. UCNPs comprise a host matrix lattice and a mixture of RE metals as dopants. The luminescence of the UCNPs can be tuned by the host matrix used, the ratio of the doped metals, size and shape, and experimental conditions [15]. Careful control of synthesis conditions is therefore required in order to acquire the UCNPs with small particle size, uniform shape, and high luminescence. However, as most of the methods of synthesis applied use hydrophobic compounds as capping ligands, these UCNPs are always dispersible in non-polar organic solvents other than aqueous solutions or 2 Page 2 of 14
biological buffers. Surface modification is therefore required to endow hydrophilic and biocompatibility properties to these UCNPs being considered for use in biological systems. It is worth noting that the surface modification should not increase the size and or reduce the luminescence, giving more challenges. In terms of biomedical imaging, apart from optical imaging, certain techniques [e.g., X-ray computed tomography (CT), positron-emission tomography (PET), and MRI] are frequently employed, each having its own advantages and disadvantages. Considering their different spatial resolutions, penetration depths and sensitivities of, a combination of these techniques would endow significant benefits in generating high-resolution three-dimensional images. RE-doped UCNPs are promising platforms for incorporating typical elements for different imaging techniques to acquire multimodal imaging probes. Here, we summarize recent progress of UCNPs in bioimaging applications and highlight recently developed strategies for synthesis and surface functionalization. This review first describes the different mechanisms of luminescence, followed by a brief introduction to different methods of synthesis and surface-modification strategies. Then, we discuss the principles of modulating the upconversion luminescence (UCL) of UCNPs. Finally, we review recent newly developed platforms for bioimaging applications, especially those with multimodal imaging properties, with the outlook on the future trends in optical bioimaging.
2. Synthesis of upconversion nanophosphores 2.1. Mechanisms for down-/up-conversion, two-photon and bandgap emission Considering the changes of emission wavelength after excitation and the origination of the excited process, luminescence mechanisms are generally divided into downconversion fluorescence, UCL, two-photon emission and bandgap emission (Scheme 1). With regard to downconversion emission, the excitation wavelength is longer than that of emission due to the energy transfer in the internal conversion process, while, for UCL, with the assistance of energy transfer and/or sequential absorption of two or more photons, the emission wavelength is shorter than that of excitation. Two-photon emission involves the absorption of two photons simultaneously, which normally requires a far more complicated light source, such as a femtosecond laser, than that for the upconversion process. Unlike the these mechanisms, bandgap emission originates from the energy released when an electron jumps from the conduction band (CB) to the valence band (VB), which is modulated by particle size, the socalled quantum size-confinement effect. 2.2. The characteristic composition of upconversion nanophosphores Based on the function of the doped ions in UCNPs, those ions are named sensitizers and activators, with sensitizers absorbing excitation light and activators giving out emission. The host materials are more like a skeleton and afford a spatial layout for the other two components. However, host materials should be optically transparent in the range of interest so as not to interfere with the emission of dopants. In addition, the crystal lattice is required to have low phonon energies, otherwise nonradiative energy losses will increase, leading to the low upconversion efficacy [18,19]. At this point, fluorides are brought to the forefront, as they typically have low-phonon lattice energy in the matrices common for RE ions [20]. As for cations, the ionic radii of the chosen cation should be close to that of dopant ions, so that doping will not 3 Page 3 of 14
induce lattice stress and formation of crystal defects. NaF, CaF2 and YF3 are suitable host materials to meet these requirements. Another factor that needs to be considered is the crystal field of a host material, as the optical properties of a lanthanide ion are greatly affected by the symmetry of the crystal surrounding it. Taking NaYF4 as an example, the hexagonal type (-type) exhibits higher luminescent intensity than the cubic (-type) [21]. Currently, hexagonal NaYF4 (-NaYF4) is the most common host material for UCNPs, especially for green and blue emissions, which typically are an order of magnitude more efficient at photon upconversion. Nowadays, more and more new host materials are being discovered and evaluated, which offers more possibility to develop new types of UCNP [22]. Due to the low upconversion efficiency of the single-doping system, currently codoping systems are more popular in the design of high-luminescence UCNPs. The layout of energy-level structure is one of the most important considerations when choosing doped ions. For the activator, it normally requires sequential excitation to a higher excited state, so ions that have similar energy gaps between three or more subsequent levels are preferred. By screening the RE metal table, ions, such as Er3+, Ho3+ and Tm3+, could meet the requirements to get efficient UC emission when excited at around 980 nm. As for sensitizers, they need to have large absorption cross section in NIR spectral range. The transition of Yb3+ ion from 2F7/2→2F5/2 has good match of energy of 975 nm light, so the most studied co-doping systems at the moment are -NaYF4:Yb3+-Er3+ and -NaYF4:Yb3+-Tm3+ [1]. With the increased availability of the irradiation energy source, more and more other lanthanide ions are being explored for using as sensitizers. 2.3. Synthesis of upconversion nanophosphores Since the optical properties of UCNPs greatly depend on the synthesis procedures, to obtain UCNPs with small size, high luminescence, uniform shape and pure crystal phase will mainly depend on exploration of the methods of synthesis. Detailed discussion of methods of synthesis have been reported in several reviews [1,18,23,24]. Here, we briefly introduce the methods of synthesis, then focus on the synthesis of UCNPs with identified sizes, shapes and properties. 2.3.1. General methods Many methods of synthesis have been developed, such as thermal decomposition, hydrothermal, sol-gel, Ostwald-ripening processing, coprecipitation, and microemulsion. The thermal decomposition method was frequently used in UCNP synthesis, but the high reaction temperature and toxic by-products held back its applications. The liquid-solid-solution (LSS) strategy reported by Li’s group offered a general strategy for synthesizing monodisperse NPs with narrow size distribution at moderate temperatures [25]. This strategy is based on a general phase transfer and separation mechanism at the interface of the liquid, solid and solution phases by using polar solvents, such as ethanol, hydrophobic ligands and RE salts. Different shapes of UCNP could also be obtained based on the LSS strategy by varying the conditions of synthesis, such as dopant concentrations, reaction temperature and time [26]. However, one issue with this synthesis strategy is that the NPs obtained are hydrophobic and further surface-modification steps are required before bioimaging applications.
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Later, the hydrothermal methods were altered by incorporating hydrophilic capping ligands during the synthesis process. Consequently, the hydrophilicity of the nanophosphores obtained was greatly improved. The hydrothermal methods were solution-based, generally performed in a sealed environment under high pressure and moderate temperature. RE nitrates, chlorides, and oxides were typical RE precursors with HF, NH4F, NaF and NH4HF2 as fluoride source and citrate, ethylenediamine tetraacetic acid (EDTA), cetyltrimethylammonuim bromide (CTAB), and oleylamine (OA) as capping ligands. The coordination of hydrophilic ligands toward RE3+ could in one way tailor the crystal phases, shapes and sizes and, in another way, promote solubility in aqueous solutions. But, the limitations of this method are that the size and the shape of the UCNPs are hard to control, especially those with small particle sizes. Ostwald-ripening, coprecipitation and microemulsion methods process have also been explored frequently to fabricate UCNPs [27–30]. The Ostwald-ripening process is driven by the thermodynamic stability of the smaller crystals or particles being less than that of larger ones, resulting in dissolution of the smaller and growth of the larger. Such processes can also happen to the molecules on the surface of a particle, which are energetically less stable than those in the interior, leading to the formation of particles with hollow structure. The coprecipitation strategy is based on coprecipitation of mixture solutions of RE-metal salt by a suitable precipitator followed by annealing treatment. The microemulsion method makes use of the RE metal-containing water drop forming a micelle with the assistance of surfactant molecules in water-insoluble organic solvents and then the micelle being processed at high temperature to obtain the UCNPs. Currently, there is no general strategy for preparing UCNPs to meet different requirements with respect to various applications, since each of the methods of synthesis mentioned above has its own advantages and disadvantages. In another aspect, methods of synthesis with new techniques have also been explored, such as microwave-assisted synthesis. 2.3.2. Ultrasmall size and high luminescence As discussed above, the ideal size of nanophosphores for bioimaging is around 10 nm, as it would be easier for the body clear injected nanophosphores. One problem is that defects and ligands on the surface would increase as particle sizes reduced, leading to quenching of the luminescence. The synthesis of UCNPs with small particle size and strong luminescence is crucial but also challenging for the researchers. So far, great progress has been achieved using various strategies, which can generally be classified as: (1) variation of host materials and compositions; (2) adjustment of experimental conditions; and, (3) fabrication with core/shell structures. Table 1 briefly summarizes representative examples. Since the sizes and the optical properties of UCNPs greatly depend on the compositions and the experimental conditions, current efforts are devoted to optimizing these parameters in order to acquire ultrasmall, high-luminescence UCNPs [31–33,37,38]. The doping strategy is a widely applied method to modulate the size and the structural properties of the UCNPs. It is known that the -NaYF4 UCNP shows higher luminescence, and Liu’s group achieved simultaneous control of phase and size of NaYF4 through Gd3+ doping [39], resulting in a reduction in particle size together with cubic-to-hexagonal phase conversion in much less time. It was reasoned that, in the hexagonal phase of NaYF4, an ordered array of F- ions with two types of relatively low-symmetry cation sites results in significant electron-cloud distortion of cations to accommodate the structural changes. Thus, doping light lanthanides with 5 Page 5 of 14
large ionic radii exhibits a high tendency towards electron cloud distortion, and then the hexagonal structure is favored. The Gd3+ ion was chosen for doping into NaYF4 as the ionic radii of Gd3+ is larger than that of Y3+. The results indicated that ultrasmall NPs with strong UCL were successfully prepared using this strategy. Inspired by this, Li’s group obtained -NaLuF4 by doping Gd3+ [32]. Recently, our group developed a novel in-situ Gd3+exchange strategy to achieve small size (about 12-nm) particles with UCL greatly enhanced (up to 29 times) [40]. We need to note that some of the ultrasmall, high-luminescence UCNPs were obtained by combining the different strategies mentioned above. For example, Chen et al. developed a method that doping the lanthanide ion led to modifying the uneven size and shape of alkaline-earth fluoride nanophases to monodisperse ultrasmall nanospheres and both application of new host materials and the ion doping accounted for size modulation and luminescence enhancement [31]. UCNPs with core/shell structures were in the spotlight in recent years. The low UCL efficiency, resulting from the increased surface defects and deleterious crossrelaxation when the particle size reduced, could be improved by introducing a shell with a sufficient thickness, which may substantially inhibit quenching, thus maintaining the luminescence intensity of the UCNPs even at reduced particle size. According to the compositions of the shell, core/shell structures could be categorized into three types: (1) passive-shell coating (inert shell, normally a silica shell or a shell with the same composition as that of host materials); (2) active-shell coating (shell with the ability to absorb irradiation energy); and, (3) active core-active shell coating (both the core and shell). The passive-shell is merely used to protect the dopants from the surface quenchers, while active-shell containing lanthanide ions could reduce the cross-relaxation in the meantime [36,41,42]. A sub-10 nm UCNP with active-core/active-shell structure was recently reported by Wang’s group [36]. The growth of a Gd3+, Nd3+:SrF2 shell was introduced on the surface of Tm3+, Yb3+:BaF2 nanocrystal cores through a thermal decomposition process to form highly uniform Tm3+, Yb3+:BaF2@Gd3+, Nd3+:SrF2 active-core@active-shell nanocubes. The doping of Tm3+/Yb3+ and Nd3+ into the core and shell, respectively, could effectively suppress the adverse energy transfers between the lanthanide ions. Enhancement of NIR-to-NIR luminescence was acquired based on this fabrication method with potential further applications to in-vivo biomedical imaging. A recent study carried out by researchers in Berkeley Laboratory demonstrated that there are new rules for modulating the particle size and the luminescence [43]. Traditionally accepted rules for designing UCNPs are that to use a high concentration of sensitizer ions and a relatively low concentration of emitter ions in order to obtain enough energy while avoiding self-quenching. However, their findings showed that the rules for design of UCNP ensembles are not suitable for UCNPs with sub-10 nm size. According to their rules, activator concentration should be as high as possible for synthesizing UCNPs for single-particle imaging under higher excitation powers, indicating that there are new possibilities for designing sub-10 nm UCNPs with strong luminescence. 2.4. Principles of modulating the luminescence of upconversion nanophosphores Since the luminescence of UCNP is originated from the emission of the doped ion, manipulation of UCL mainly rely on the variations of the ion concentration and sorts of the doped ion. However, one problem with this strategy is that that may cause cross-relaxation quenching between different lanthanide ions. In another aspect, the 6 Page 6 of 14
multicolor emission can also be afforded by varying other precursors during the synthesis process. A recent study indicated that multicolor UCL of NaYF4:Yb3+/Er3+ NPs (NPs) were successfully performed simply by controlling the concentrations of NaF during the synthesis.[44] Moreover, the luminescence of UCNPs was reported to be tuned by the incorporation of organic fluorophores or QDs through the luminescence resonant energy transfer (LRET).[45] Considering the bioimaging applications of UCNPs, the NIR-to-NIR luminescent UCNPs are more desirable since both the excitation and emission wavelengths are in the optical transparent range of biological tissues. In order to achieve NIR luminescence, the Tm3+ ion, that with energy level transition (3H4→3H6) falling in the NIR range (750-900 nm) under excitation at 980 nm, was frequently chosen as the activator for designing NIR-to-NIR UCNPs.[46] Synthesis of Tm3+-doped UCNPs with various host materials including NaYF4, NaGdF4, and NaLuF4 has been reported.[5] Whereas, our group has ever developed for the first time a Er3+-based NIR-NIR UCL system.[47] Unlike the upconverting nanocrystals previously reported in the literature that emit visible (blue–green–red) UCL, these poly-(acrylic acid) functionalized YF3:Yb3+/Er3+ NPs emit strong NIR (NIR, 831 nm) UCL under 980 nm excitation. Apart from varying dopant ions and experimental conditions, the manipulation of energy transfer process could be another approach for extending the choices of constructing the new NIR-to-NIR systems.[48] 2.5. Synthesis of upconversion nanophosphores for multimodal imaging Thanks to the nanocomposite nature of most UCNPs, multifunctional UCNPs are ready to be achieved by incorporating elements with different properties. More importantly, some of the lanthanide ions themselves could serve for different purposes, which greatly simplified the fabrication procedures. For example, the lanthanide ion, Gd3+, has unpaired electrons, showing paramagnetic responses, and the Gd-containing UCNPs could be used as contrast agents for MRI. Considering the current available techniques for bioimaging, radioactive elements are often doped during the synthesis of UCNPs, then PET or SPECT imaging could be obtained by measuring the radioactivity signals. The Fe3O4 is another common components used for the additional paramagnetic properties, and the incorporation of it often involves the construction of a core/shell structure, which bring the extra benefits for constructing UCNPs, avoiding luminescence quenching. Except for ion doping and core/shell method, a physically combination of as-synthesized elements in one micelle or nanocontainer is another strategy for achieving multifunctional materials. For instance, a bifunctional NP has been fabricated by physical absorption of two as-synthesized components via electrostatic interaction of the two particles.[49]
3. Surface modification UCNPs obtained via thermal decomposition and hydrophobic ligands assisted hydrothermal methods always endow a hydrophobic nature for the UCNPs since ligands on the surface normally have a long alkyl chain. In terms of bioimaging, these UCNPs need be further modified to convert into hydrophilic ones, making them well dispersed into aqueous solution or biological buffer. In addition, the fabrication of nanocomposites with different functional groups are essential for biomedical applications as these functional groups like amines, thiols and carboxylic acids allow for further modification by many biological related moieties. With regard to optical bioimaging, those nanophosphores with specific binding affinity toward certain sites are especially desirable. 7 Page 7 of 14
To date, common surface modification methods were employed including ligand exchange, layer-by-layer fabrication, silanization (coating with silica layer), hydrophobic/hydrophobic interaction, and one-step synthesis of surface functionalized UCNPs. In recent years, the last one keeps drawing attention since this one-step method greatly simplifies the procedures for preparing hydrophilic UCNPs. The ligand exchange method is rather straight forward by replacing the original ligand such as oleylamine (OA) with other hydrophilic or amphiphilic ligand. Commonly used ligands normally contain either -COOH group or -NH2 moieties, which could further carry on bioconjugation by coupling reaction. One potential effect of this method is that the exchange process might induce surface defects to the UCNPs. Li's group have successfully prepared PAH/PSS/PADH-coated UCNPs via layer-by-layer methods, which were developed by employing the electrostatic interaction between different components.[50] This method allowed the preparation of NPs with different shapes and sizes by uniform layers and controllable thickness. In addition, due to the hydrophilicity and biocompatibility of silica, silanization is extensively employed in surface modification; however, one drawback of this method is that the size of UCNPs will increase during this process, normally over several nanometers. Alternatively, by employing the hydrophobic/hydrophobic interactions, Wang et al has developed a general strategy for transforming hydrophobic NPs to hydrophilic ones with one particle per micelle.[51-52] Specifically, the hydrophobic interactions between the original ligands and the hydrophobic parts of the employed amphiphilic carboxylated phospholipid promoted the formation of a micelle with the carboxylic acid head toward outside, resulting in a hydrophilic surface of UCNPs (Fig. 1). Furthermore, multiple particles could be fabricated into one micelle through manipulation of the conditions. Inspired by such strategy, multifunctional nanocomposites could be obtained if particles with different functions were included. Therefore, hydrophilic multifunctional UCNPs could be obtained directly from original hydrophobic NPs. One-step surface modification method has received considerable attention owing to the simplified synthetic process. Moreover, by utilizing some hydrophilic biocompatible polymers like polyvinylpyrrolidone (PVP), poly(ethylene imine) (PEI), poly(acrylic acid) (PAA), poly(amino acid),[53] O-carboxymethyl chitosan (OCMC),[54] and 2-aminoethyl dihydrogen phosphate (AEP) during the synthesis procedures, the UCNPs with hydrophilic and biocompatible properties have also been achieved.[28, 47, 55-58] Those polymers are supposed to have multiple purposes during the synthesis. In one of the poly(acrylic acid) coated YF3:Yb3+/Er3+ UCNPs reported recently, the obtained UCNPs are well dispersed in water due to the presence of carboxylate groups in PAA and with an emission around 831 nm. Compared with the synthesis of YF3:Yb3+/Er3+ without PAA, these PAA coated nanophosphores are smaller in size and longer in emission wavelength. Such properties implied that the PAA ligand might have effects on the crystal growth and thus modulation of emission. Though the one-step surface modification, lots of efforts were saved on post-treatment procedure, yet the monodispersed UCNPs obtained can hardly be obtained through such method. Therefore, there are still lots of challenges for researchers in this field.
4. Bioimaging of upconversion nanophosphores Optical imaging of biological samples based on UCNPs has received extensive attention, especially those with multi-functions which have the potentials to be employed in multimodal imaging. UCL could be used for visualizing living biosamples, but only in several millimeters depth. Such limitation can be compensated by MRI and X-ray CT, which offer the physiological information as 8 Page 8 of 14
well as the structures of biosamples without penetration depth limitation. PET and SPECT techniques show high sensitivity but had relatively low spatial resolutions. Since different bioimaging techniques provide different spatial resolutions, penetration depth and areas of applications, an enhanced quality of bioimaging could be achieved by combination of the advantages of each bioimaging technique. Generally based on the number of the techniques used, the imaging could be divided into dual modal and multimodal imaging. Dual modal imaging mainly includes UCL/FL, UCL/X-ray CT, UCL/MRI, UCL/PET, and UCL/SPECT. For the multimodal imaging, higher requirements of synthesis are necessary. Here several examples will be introduced to illustrate the usage of multimodal imaging. 4.1. Dual-modal imaging Many efforts were put on the design of UCNPs for dual modal imaging, of which one example was chosen to discuss in detail here.[16, 52, 59] The band-gap fluorescence ZnS:Mn2+ QDs and up-conversion luminescence NaYF4:Er3+/Yb3+ NPs were embedded into hydrophilic polymer matrixes through in situ cross-linking polymerization. The as-prepared multifunctional nanocomposites had orange fluorescence and green UC luminescence under UV and NIR (980 nm) irradiation respectively, which showed the capability for fluorescence and up-conversion luminescence dual modal imaging. These nanocomposites were further linked to folic acid moiety by bioconjugation, which enabled the specific binding toward cancer cell. In addition, this fabrication strategy could extend to synthesize multifunctional nanocomposites for MRI, CT and others by incorporating different elements. The X-ray CT technique is frequently included for UCNPs based multimodal imaging, because this technique mainly relies on the atomic number and electron density of the agents. There is a wide choices of lanthanide ions since many lanthanides are heavy atoms, providing higher attenuation coefficient for X-ray CT. Actually, some of the host materials themselves could be utilized for this technique without further modification, especially those with NaLuF4 as host matrix due to the Lu atom with the highest atomic number of 71. Moreover, by incorporating of paramagnetic Gd3+ and Mn2+ ions, the afforded UCNPs could be used for UCL/ MRI dual-modal imaging, 4.2. Multimodal imaging Recently reported examples for trimodal bioimaging mainly focus on the UCL/MRI/CT and UCL/MRI/SPECT strategies due to the bifunctional nature of some lanthanide such as Gd3+, and Lu3+ as mentioned above.[60-62] For those UCNPs with different doping ions for multimodal imaging, the rational design of the fabrication strategy is required otherwise a decrease of the original function would occur. Li's group reported a UCNPs-based four-modal probe for tumor angiogenesis imaging, including UCL, X-ray CT, MRI and SPECT techniques.[63] The RE metal radioisotope 153Sm has a similar atomic radius to other Ln3+ and a physical half-life of 46.3 h, making it suitable for doping into UCNPs and for the long-term SPECT imaging. A core/shell structure was constructed with NaLuF4:Yb,Tm as a core and 4 nm of 153Sm3+-doped NaGdF4 as a shell. The as-prepared UCNPs have been confirmed to be effective for four-modal based bioimaging (Fig. 2). One highlight of this four-modal imaging nanocomposite is that the increased imaging technique did not compromise its performance and sensitivity of each imaging mode, which are attributed for the smart arrangement of each element. Proper layout of Gd3+ at the out layer of the nanostructure significantly enhances its MRI signals since the Gd3+ at the 9 Page 9 of 14
inner layer only showed limited enhancement. And the 153Sm3+ was directly doped into the NaGdF4 shell via surface-exchange process due to the high affinity of lanthanide ions toward fluorides without further disruption of the structures. More importantly, the multimodal imaging probe was also successfully applied to the tumor angiogenesis imaging, which is of great importance for early diagnosis of cancer.
5. Conclusion and outlook With the development of technology, optical imaging is used more and more widely in various aspects, especially in clinical studies for localizing tumor site, preand post-surgery investigation as well as tracing of drug circulation. The development of UCNPs based optical imaging has been greatly highlighted owing to the unique properties like low excitation power for continuous monitoring, deep penetration and large Stoke's shift. With regard to bioimaging, there will be a great era waiting ahead for UCNPs. Meanwhile, many problems are also waiting to be solved, as follows. (1) The exploration of novel synthetic strategies as well as surface modification methods is still highly desirable, especially for achieving those ultrasmall UCNPs with high luminescence, hydrophilic surface and biocompatible properties. The quantum yield of UCNPs still needs to be improved by reducing the crystal defects and improving upconversion efficiency. (2) Lots of challenges are faced ahead for fabricating multifunctional UCNPs with small size and new functional elements without impairing the original performance. (3) Although the UCNPs are claimed to be low cytotoxicity, further investigations on biocompatibility are still required considering the clinical applications. (4) The exploration of combining UCNPs-based optical imaging with other functions including photo dynamic therapy and drug delivery is very intriguing, which offers the possibility for imaging as well as on-site treatment. Acknowledgements This research was supported in part by the National Natural Science Foundation of China (21475007 and 21275015), the State Key Project of Fundamental Research of China (2011CB932403 and 2011CBA00503), the Fundamental Research Funds for the Central Universities (ZZ1321 and YS1406), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1205). We also thank the “Public Hatching Platform for Recruited Talents of Beijing University of Chemical Technology” for support. References [1] G. Chen, H. Qiu, P. N. Prasad, X. Chen, Upconversion nanoparticles: design, nanochemistry, and applications in theranostics. Chem. Rev. (2014). [2] N. Phuong-Diem, S. J. Son, J. Min, Upconversion Nanoparticles in Bioassays, Optical Imaging and Therapy. J.Nanosci. Nanotech. 14 (2014) 157-174. [3] J. Shen, L. Zhao, G. Han, Lanthanide-doped upconverting luminescent nanoparticle platforms for optical imaging-guided drug delivery and therapy. Adv. Drug Deliv. Rev. 65 (2013) 744-755. [4] J. Niu, X. Wang, J. Lv, Y. Li, B. Tang, Luminescent nanoprobes for in-vivo bioimaging. Trac-Trends Anal. Chem. 58 (2014) 112-119. [5] M. Chen, X. He, K. Wang, D. He, X. Yang, H. Shi, Inorganic fluorescent nanoprobes for cellular and subcellular imaging. Trac-Trends Anal. Chem. 58 (2014) 120-129. 10 Page 10 of 14
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Captions
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Scheme 1. Schematic representations of different mechanisms for luminescence (a) Jablonski Energy diagram, illustrating the down-conversion fluorescence; (b) Upconversion process of lanthanide-doped crystals, ETU: energy transfer upconversion, ESA: excited state absorption; (c) Two-photon absorption based emission; (d) Emission process of semiconductors based on the bandgap emission
Fig. 1 Illustration of the surface modification strategies through hydrophobic/ hydrophobic interactions and the images for the mono- and multi-modal imaging
Fig. 2 (a-d) Four-modal imaging of the focused tumor from the tumor-bearing nude mouse 1 h after intravenous injection of NaLuF4: Yb,Tm@NaGdF4 (153Sm): (a) In vivo UCL-image, (b) X-ray CT image, (c) SPECT image, (d) NM imaging of tumor. (e) UCL confocal image of the paraffin section of tumor tissue. (f) Schematic illustration of tumor angiogenesis imaging using NaLuF4: Yb,Tm@NaGdF4 (153Sm) as the probe. [Reproduced with permission, Copyright 2013, American Chemical Society;]
Table 1. Representative ultrasmall upconversion nanophosphores strategies Average Host Strategies size material (nm) Variations in 5.0 SrF2 composition 7.8 -NaLuF4 5.3-8.0 Variations in experimental conditions
Doping ion
Remarks
Ref.
Gd3+, Yb3+ Yb3+, Er3+
New host materials Gd3+doping
[31] [32]
Yb3+, Er3+
Ln3+ doping
[17]
[33]
4.5-15.0
NaYF4
Yb3+, Er3+
Surfactant concentration
7.0-10.0
NaYF4
Yb3+, Tm3+
Concentration of Yb3+
[34]
Core NaYF4: Yb, Er 5 nm
Shell NaYF4 2 nm
11.1
KGdF4:Tm, Yb 3.7 nm
KGdF4 7.4 nm
Enhanced luminescence
[35]
10
Ln:BaF2 3 nm
Ln:SrF2 7 nm
Active core/active shell
[36]
7.0 Construction of core/shell structure
Ba2LaF7
with high luminescence obtained by different
Inert shell
[33]
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S0165-9936(15)00008-4 http://dx.doi.org/doi: 10.1016/j.trac.2014.11.014 TRAC 14371
To appear in:
Trends in Analytical Chemistry
Please cite this article as: Suying Xu, Sheng Huang, Qian He, Leyu Wang, Upconversion nanophosphores for bioimaging, Trends in Analytical Chemistry (2015), http://dx.doi.org/doi: 10.1016/j.trac.2014.11.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Upconversion nanophosphores for bioimaging Suying Xu, Sheng Huang, Qian He, Leyu Wang* State Key Laboratory of Chemical Resource Engineering, Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029, P.R. China * Corresponding author. E-mail address: [email protected] (L. Wang)
HIGHLIGHTS Upconversion nanophosphores show great advantages in bioimaging applications Synthesis methods, surface-modification strategies for upconversion nanophosphores Upconversion nanophosphores with ultrasmall size and high luminescence We summarize recent applications of upconversion nanophosphores in bioimaging Challenges for future developments of upconversion nanophosphores in bioimaging
ABSTRACT Upconversion nanophosphore (UCNP)-based optical imaging is a promising technique in the field of bioimaging due to the unique optical properties of UCNPs. UCNPs can be excited by long wavelength light with low power density and have excellent biocompatibility. By carefully controlling the synthesis of UCNPs, nearinfrared emission can also be obtained. Such NIR-to-NIR bioimaging probes show great advantages in terms of bioimaging, such as deep penetration and low autofluorescence. Moreover, UCNPs can also offer a platform for fabricating multifunctional nanocomposites for multi-modal imaging by incorporating magnetic resonance imaging, X-ray computed tomography and single-photon emission computerized tomography techniques. In this review, we briefly introduce the fundamental points of UCNPs, highlight the methods of synthesis and surfacemodification strategies, and summarize recent progress. We also discuss the current issues faced by researchers. Keywords: Bioimaging Fluorescence Nanocomposite Nanoparticle Nanophosphore Near-infrared Optical imaging Surface modification Synthesis Upconversion luminescence Abbreviations: AEP, 2-aminoethyl dihydrogen phosphate; CB, Conduction band; CTAB, Cetyltrimethylammonuim bromide; EDTA, Ethylenediamine tetraacetic acid; LSS, Liquid-solidsolution; MRI, Magnetic resonance imaging; NIR, Near-infrared; NP, Nanoparticle; OA, Oleylamine; OCMC, O-carboxylmethyl chitosan; PAA, Poly(acrylic acid); PEI, Poly(ethylene imine); PET, Positron-emission tomography; PVP, Polyvinylpyrrolidone; UCNP, Upconversion nanophosphores; VB, Valence band; X-ray CT, X-ray computed tomography
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1. Introduction Bioimaging includes a wide range of imaging techniques, such as magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound and optical imaging. Optical imaging technology is of great importance for bioscience studies, such as imaging-guided surgery and post-surgery evaluations for complete removal of diseased sections [1–3]. Such techniques experienced flourishing development in recent years due to their high temporal and spatial resolutions and the wide availability of luminescent materials, including small organic fluorophores, fluorescent doping nanoparticles (NPs), fluorescent proteins, quantum dots (QDs) and luminescent metal complexes [4–6]. Despite great progress achieved with small organic fluorophores and fluorescent proteins in labeling and protein interaction studies, some drawbacks of these fluorophores [e.g., poor photostability, high background noise (arising from autofluorescence)} have limited their applications in bioimaging. To some degree, semiconducting QDs overcome these issues, but they also brought new problems, such as cytotoxicity and photo blinking (intermittency of fluorescence), since QDs are made from harmful heavy metals and cytotoxicity test results also indicated such issues [7]. There is therefore still high motivation to search for more suitable phosphores withdesirable characteristics, such as high chemical stability and photostability, long wavelength excitation and emission, and good biocompatibility. In recent years, rare-earth (RE)-metal doped luminescent nanophosphores, here specifically referred to as upconversion nanophosphores (UCNPs), were considered promising candidates for biomedical-imaging applications due to their unique optical properties, including photon upconversion, high photostability, low cytotoxicity and inertness to environment conditions, such as pH, temperature and coexisting chemicals [8–14]. The upconversion properties afford lots of benefits in terms of imaging as with lower power excitation light, normally at 980 nm, UCNPs generate UV/visible, in some cases, even near-infrared (NIR) luminescence. The long wavelength light can penetrate samples more deeply without inducing autofluorescence. Moreover, the luminescence of UCNPs is very stable without photobleaching and photoblinking issues. When UCNPs are applied in bioimaging, they therefore allow not only deeper penetration, no toxicity and low autofluorescence, but also continuous monitoring. These advantages make UCNPs outstanding for bioimaging [4,15–17]. However, UCNPs normally have larger particle size than other conventional fluorophores, which holds back their applications in in-vivo biological fields as nonspecific aggregation would occur. Besides, particles with size larger than 10 nm are hard for the body to clear. Efforts have been devoted to decrease the size of UCNPs, yet problems arise as more surface defects are produced with decreased size and, in turn, reduce the luminescence of the UCNPs. Ideally, UCNPs for bioimaging should have sub-10 nm particle size while maintaining high luminescence. UCNPs comprise a host matrix lattice and a mixture of RE metals as dopants. The luminescence of the UCNPs can be tuned by the host matrix used, the ratio of the doped metals, size and shape, and experimental conditions [15]. Careful control of synthesis conditions is therefore required in order to acquire the UCNPs with small particle size, uniform shape, and high luminescence. However, as most of the methods of synthesis applied use hydrophobic compounds as capping ligands, these UCNPs are always dispersible in non-polar organic solvents other than aqueous solutions or 2 Page 2 of 14
biological buffers. Surface modification is therefore required to endow hydrophilic and biocompatibility properties to these UCNPs being considered for use in biological systems. It is worth noting that the surface modification should not increase the size and or reduce the luminescence, giving more challenges. In terms of biomedical imaging, apart from optical imaging, certain techniques [e.g., X-ray computed tomography (CT), positron-emission tomography (PET), and MRI] are frequently employed, each having its own advantages and disadvantages. Considering their different spatial resolutions, penetration depths and sensitivities of, a combination of these techniques would endow significant benefits in generating high-resolution three-dimensional images. RE-doped UCNPs are promising platforms for incorporating typical elements for different imaging techniques to acquire multimodal imaging probes. Here, we summarize recent progress of UCNPs in bioimaging applications and highlight recently developed strategies for synthesis and surface functionalization. This review first describes the different mechanisms of luminescence, followed by a brief introduction to different methods of synthesis and surface-modification strategies. Then, we discuss the principles of modulating the upconversion luminescence (UCL) of UCNPs. Finally, we review recent newly developed platforms for bioimaging applications, especially those with multimodal imaging properties, with the outlook on the future trends in optical bioimaging.
2. Synthesis of upconversion nanophosphores 2.1. Mechanisms for down-/up-conversion, two-photon and bandgap emission Considering the changes of emission wavelength after excitation and the origination of the excited process, luminescence mechanisms are generally divided into downconversion fluorescence, UCL, two-photon emission and bandgap emission (Scheme 1). With regard to downconversion emission, the excitation wavelength is longer than that of emission due to the energy transfer in the internal conversion process, while, for UCL, with the assistance of energy transfer and/or sequential absorption of two or more photons, the emission wavelength is shorter than that of excitation. Two-photon emission involves the absorption of two photons simultaneously, which normally requires a far more complicated light source, such as a femtosecond laser, than that for the upconversion process. Unlike the these mechanisms, bandgap emission originates from the energy released when an electron jumps from the conduction band (CB) to the valence band (VB), which is modulated by particle size, the socalled quantum size-confinement effect. 2.2. The characteristic composition of upconversion nanophosphores Based on the function of the doped ions in UCNPs, those ions are named sensitizers and activators, with sensitizers absorbing excitation light and activators giving out emission. The host materials are more like a skeleton and afford a spatial layout for the other two components. However, host materials should be optically transparent in the range of interest so as not to interfere with the emission of dopants. In addition, the crystal lattice is required to have low phonon energies, otherwise nonradiative energy losses will increase, leading to the low upconversion efficacy [18,19]. At this point, fluorides are brought to the forefront, as they typically have low-phonon lattice energy in the matrices common for RE ions [20]. As for cations, the ionic radii of the chosen cation should be close to that of dopant ions, so that doping will not 3 Page 3 of 14
induce lattice stress and formation of crystal defects. NaF, CaF2 and YF3 are suitable host materials to meet these requirements. Another factor that needs to be considered is the crystal field of a host material, as the optical properties of a lanthanide ion are greatly affected by the symmetry of the crystal surrounding it. Taking NaYF4 as an example, the hexagonal type (-type) exhibits higher luminescent intensity than the cubic (-type) [21]. Currently, hexagonal NaYF4 (-NaYF4) is the most common host material for UCNPs, especially for green and blue emissions, which typically are an order of magnitude more efficient at photon upconversion. Nowadays, more and more new host materials are being discovered and evaluated, which offers more possibility to develop new types of UCNP [22]. Due to the low upconversion efficiency of the single-doping system, currently codoping systems are more popular in the design of high-luminescence UCNPs. The layout of energy-level structure is one of the most important considerations when choosing doped ions. For the activator, it normally requires sequential excitation to a higher excited state, so ions that have similar energy gaps between three or more subsequent levels are preferred. By screening the RE metal table, ions, such as Er3+, Ho3+ and Tm3+, could meet the requirements to get efficient UC emission when excited at around 980 nm. As for sensitizers, they need to have large absorption cross section in NIR spectral range. The transition of Yb3+ ion from 2F7/2→2F5/2 has good match of energy of 975 nm light, so the most studied co-doping systems at the moment are -NaYF4:Yb3+-Er3+ and -NaYF4:Yb3+-Tm3+ [1]. With the increased availability of the irradiation energy source, more and more other lanthanide ions are being explored for using as sensitizers. 2.3. Synthesis of upconversion nanophosphores Since the optical properties of UCNPs greatly depend on the synthesis procedures, to obtain UCNPs with small size, high luminescence, uniform shape and pure crystal phase will mainly depend on exploration of the methods of synthesis. Detailed discussion of methods of synthesis have been reported in several reviews [1,18,23,24]. Here, we briefly introduce the methods of synthesis, then focus on the synthesis of UCNPs with identified sizes, shapes and properties. 2.3.1. General methods Many methods of synthesis have been developed, such as thermal decomposition, hydrothermal, sol-gel, Ostwald-ripening processing, coprecipitation, and microemulsion. The thermal decomposition method was frequently used in UCNP synthesis, but the high reaction temperature and toxic by-products held back its applications. The liquid-solid-solution (LSS) strategy reported by Li’s group offered a general strategy for synthesizing monodisperse NPs with narrow size distribution at moderate temperatures [25]. This strategy is based on a general phase transfer and separation mechanism at the interface of the liquid, solid and solution phases by using polar solvents, such as ethanol, hydrophobic ligands and RE salts. Different shapes of UCNP could also be obtained based on the LSS strategy by varying the conditions of synthesis, such as dopant concentrations, reaction temperature and time [26]. However, one issue with this synthesis strategy is that the NPs obtained are hydrophobic and further surface-modification steps are required before bioimaging applications.
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Later, the hydrothermal methods were altered by incorporating hydrophilic capping ligands during the synthesis process. Consequently, the hydrophilicity of the nanophosphores obtained was greatly improved. The hydrothermal methods were solution-based, generally performed in a sealed environment under high pressure and moderate temperature. RE nitrates, chlorides, and oxides were typical RE precursors with HF, NH4F, NaF and NH4HF2 as fluoride source and citrate, ethylenediamine tetraacetic acid (EDTA), cetyltrimethylammonuim bromide (CTAB), and oleylamine (OA) as capping ligands. The coordination of hydrophilic ligands toward RE3+ could in one way tailor the crystal phases, shapes and sizes and, in another way, promote solubility in aqueous solutions. But, the limitations of this method are that the size and the shape of the UCNPs are hard to control, especially those with small particle sizes. Ostwald-ripening, coprecipitation and microemulsion methods process have also been explored frequently to fabricate UCNPs [27–30]. The Ostwald-ripening process is driven by the thermodynamic stability of the smaller crystals or particles being less than that of larger ones, resulting in dissolution of the smaller and growth of the larger. Such processes can also happen to the molecules on the surface of a particle, which are energetically less stable than those in the interior, leading to the formation of particles with hollow structure. The coprecipitation strategy is based on coprecipitation of mixture solutions of RE-metal salt by a suitable precipitator followed by annealing treatment. The microemulsion method makes use of the RE metal-containing water drop forming a micelle with the assistance of surfactant molecules in water-insoluble organic solvents and then the micelle being processed at high temperature to obtain the UCNPs. Currently, there is no general strategy for preparing UCNPs to meet different requirements with respect to various applications, since each of the methods of synthesis mentioned above has its own advantages and disadvantages. In another aspect, methods of synthesis with new techniques have also been explored, such as microwave-assisted synthesis. 2.3.2. Ultrasmall size and high luminescence As discussed above, the ideal size of nanophosphores for bioimaging is around 10 nm, as it would be easier for the body clear injected nanophosphores. One problem is that defects and ligands on the surface would increase as particle sizes reduced, leading to quenching of the luminescence. The synthesis of UCNPs with small particle size and strong luminescence is crucial but also challenging for the researchers. So far, great progress has been achieved using various strategies, which can generally be classified as: (1) variation of host materials and compositions; (2) adjustment of experimental conditions; and, (3) fabrication with core/shell structures. Table 1 briefly summarizes representative examples. Since the sizes and the optical properties of UCNPs greatly depend on the compositions and the experimental conditions, current efforts are devoted to optimizing these parameters in order to acquire ultrasmall, high-luminescence UCNPs [31–33,37,38]. The doping strategy is a widely applied method to modulate the size and the structural properties of the UCNPs. It is known that the -NaYF4 UCNP shows higher luminescence, and Liu’s group achieved simultaneous control of phase and size of NaYF4 through Gd3+ doping [39], resulting in a reduction in particle size together with cubic-to-hexagonal phase conversion in much less time. It was reasoned that, in the hexagonal phase of NaYF4, an ordered array of F- ions with two types of relatively low-symmetry cation sites results in significant electron-cloud distortion of cations to accommodate the structural changes. Thus, doping light lanthanides with 5 Page 5 of 14
large ionic radii exhibits a high tendency towards electron cloud distortion, and then the hexagonal structure is favored. The Gd3+ ion was chosen for doping into NaYF4 as the ionic radii of Gd3+ is larger than that of Y3+. The results indicated that ultrasmall NPs with strong UCL were successfully prepared using this strategy. Inspired by this, Li’s group obtained -NaLuF4 by doping Gd3+ [32]. Recently, our group developed a novel in-situ Gd3+exchange strategy to achieve small size (about 12-nm) particles with UCL greatly enhanced (up to 29 times) [40]. We need to note that some of the ultrasmall, high-luminescence UCNPs were obtained by combining the different strategies mentioned above. For example, Chen et al. developed a method that doping the lanthanide ion led to modifying the uneven size and shape of alkaline-earth fluoride nanophases to monodisperse ultrasmall nanospheres and both application of new host materials and the ion doping accounted for size modulation and luminescence enhancement [31]. UCNPs with core/shell structures were in the spotlight in recent years. The low UCL efficiency, resulting from the increased surface defects and deleterious crossrelaxation when the particle size reduced, could be improved by introducing a shell with a sufficient thickness, which may substantially inhibit quenching, thus maintaining the luminescence intensity of the UCNPs even at reduced particle size. According to the compositions of the shell, core/shell structures could be categorized into three types: (1) passive-shell coating (inert shell, normally a silica shell or a shell with the same composition as that of host materials); (2) active-shell coating (shell with the ability to absorb irradiation energy); and, (3) active core-active shell coating (both the core and shell). The passive-shell is merely used to protect the dopants from the surface quenchers, while active-shell containing lanthanide ions could reduce the cross-relaxation in the meantime [36,41,42]. A sub-10 nm UCNP with active-core/active-shell structure was recently reported by Wang’s group [36]. The growth of a Gd3+, Nd3+:SrF2 shell was introduced on the surface of Tm3+, Yb3+:BaF2 nanocrystal cores through a thermal decomposition process to form highly uniform Tm3+, Yb3+:BaF2@Gd3+, Nd3+:SrF2 active-core@active-shell nanocubes. The doping of Tm3+/Yb3+ and Nd3+ into the core and shell, respectively, could effectively suppress the adverse energy transfers between the lanthanide ions. Enhancement of NIR-to-NIR luminescence was acquired based on this fabrication method with potential further applications to in-vivo biomedical imaging. A recent study carried out by researchers in Berkeley Laboratory demonstrated that there are new rules for modulating the particle size and the luminescence [43]. Traditionally accepted rules for designing UCNPs are that to use a high concentration of sensitizer ions and a relatively low concentration of emitter ions in order to obtain enough energy while avoiding self-quenching. However, their findings showed that the rules for design of UCNP ensembles are not suitable for UCNPs with sub-10 nm size. According to their rules, activator concentration should be as high as possible for synthesizing UCNPs for single-particle imaging under higher excitation powers, indicating that there are new possibilities for designing sub-10 nm UCNPs with strong luminescence. 2.4. Principles of modulating the luminescence of upconversion nanophosphores Since the luminescence of UCNP is originated from the emission of the doped ion, manipulation of UCL mainly rely on the variations of the ion concentration and sorts of the doped ion. However, one problem with this strategy is that that may cause cross-relaxation quenching between different lanthanide ions. In another aspect, the 6 Page 6 of 14
multicolor emission can also be afforded by varying other precursors during the synthesis process. A recent study indicated that multicolor UCL of NaYF4:Yb3+/Er3+ NPs (NPs) were successfully performed simply by controlling the concentrations of NaF during the synthesis.[44] Moreover, the luminescence of UCNPs was reported to be tuned by the incorporation of organic fluorophores or QDs through the luminescence resonant energy transfer (LRET).[45] Considering the bioimaging applications of UCNPs, the NIR-to-NIR luminescent UCNPs are more desirable since both the excitation and emission wavelengths are in the optical transparent range of biological tissues. In order to achieve NIR luminescence, the Tm3+ ion, that with energy level transition (3H4→3H6) falling in the NIR range (750-900 nm) under excitation at 980 nm, was frequently chosen as the activator for designing NIR-to-NIR UCNPs.[46] Synthesis of Tm3+-doped UCNPs with various host materials including NaYF4, NaGdF4, and NaLuF4 has been reported.[5] Whereas, our group has ever developed for the first time a Er3+-based NIR-NIR UCL system.[47] Unlike the upconverting nanocrystals previously reported in the literature that emit visible (blue–green–red) UCL, these poly-(acrylic acid) functionalized YF3:Yb3+/Er3+ NPs emit strong NIR (NIR, 831 nm) UCL under 980 nm excitation. Apart from varying dopant ions and experimental conditions, the manipulation of energy transfer process could be another approach for extending the choices of constructing the new NIR-to-NIR systems.[48] 2.5. Synthesis of upconversion nanophosphores for multimodal imaging Thanks to the nanocomposite nature of most UCNPs, multifunctional UCNPs are ready to be achieved by incorporating elements with different properties. More importantly, some of the lanthanide ions themselves could serve for different purposes, which greatly simplified the fabrication procedures. For example, the lanthanide ion, Gd3+, has unpaired electrons, showing paramagnetic responses, and the Gd-containing UCNPs could be used as contrast agents for MRI. Considering the current available techniques for bioimaging, radioactive elements are often doped during the synthesis of UCNPs, then PET or SPECT imaging could be obtained by measuring the radioactivity signals. The Fe3O4 is another common components used for the additional paramagnetic properties, and the incorporation of it often involves the construction of a core/shell structure, which bring the extra benefits for constructing UCNPs, avoiding luminescence quenching. Except for ion doping and core/shell method, a physically combination of as-synthesized elements in one micelle or nanocontainer is another strategy for achieving multifunctional materials. For instance, a bifunctional NP has been fabricated by physical absorption of two as-synthesized components via electrostatic interaction of the two particles.[49]
3. Surface modification UCNPs obtained via thermal decomposition and hydrophobic ligands assisted hydrothermal methods always endow a hydrophobic nature for the UCNPs since ligands on the surface normally have a long alkyl chain. In terms of bioimaging, these UCNPs need be further modified to convert into hydrophilic ones, making them well dispersed into aqueous solution or biological buffer. In addition, the fabrication of nanocomposites with different functional groups are essential for biomedical applications as these functional groups like amines, thiols and carboxylic acids allow for further modification by many biological related moieties. With regard to optical bioimaging, those nanophosphores with specific binding affinity toward certain sites are especially desirable. 7 Page 7 of 14
To date, common surface modification methods were employed including ligand exchange, layer-by-layer fabrication, silanization (coating with silica layer), hydrophobic/hydrophobic interaction, and one-step synthesis of surface functionalized UCNPs. In recent years, the last one keeps drawing attention since this one-step method greatly simplifies the procedures for preparing hydrophilic UCNPs. The ligand exchange method is rather straight forward by replacing the original ligand such as oleylamine (OA) with other hydrophilic or amphiphilic ligand. Commonly used ligands normally contain either -COOH group or -NH2 moieties, which could further carry on bioconjugation by coupling reaction. One potential effect of this method is that the exchange process might induce surface defects to the UCNPs. Li's group have successfully prepared PAH/PSS/PADH-coated UCNPs via layer-by-layer methods, which were developed by employing the electrostatic interaction between different components.[50] This method allowed the preparation of NPs with different shapes and sizes by uniform layers and controllable thickness. In addition, due to the hydrophilicity and biocompatibility of silica, silanization is extensively employed in surface modification; however, one drawback of this method is that the size of UCNPs will increase during this process, normally over several nanometers. Alternatively, by employing the hydrophobic/hydrophobic interactions, Wang et al has developed a general strategy for transforming hydrophobic NPs to hydrophilic ones with one particle per micelle.[51-52] Specifically, the hydrophobic interactions between the original ligands and the hydrophobic parts of the employed amphiphilic carboxylated phospholipid promoted the formation of a micelle with the carboxylic acid head toward outside, resulting in a hydrophilic surface of UCNPs (Fig. 1). Furthermore, multiple particles could be fabricated into one micelle through manipulation of the conditions. Inspired by such strategy, multifunctional nanocomposites could be obtained if particles with different functions were included. Therefore, hydrophilic multifunctional UCNPs could be obtained directly from original hydrophobic NPs. One-step surface modification method has received considerable attention owing to the simplified synthetic process. Moreover, by utilizing some hydrophilic biocompatible polymers like polyvinylpyrrolidone (PVP), poly(ethylene imine) (PEI), poly(acrylic acid) (PAA), poly(amino acid),[53] O-carboxymethyl chitosan (OCMC),[54] and 2-aminoethyl dihydrogen phosphate (AEP) during the synthesis procedures, the UCNPs with hydrophilic and biocompatible properties have also been achieved.[28, 47, 55-58] Those polymers are supposed to have multiple purposes during the synthesis. In one of the poly(acrylic acid) coated YF3:Yb3+/Er3+ UCNPs reported recently, the obtained UCNPs are well dispersed in water due to the presence of carboxylate groups in PAA and with an emission around 831 nm. Compared with the synthesis of YF3:Yb3+/Er3+ without PAA, these PAA coated nanophosphores are smaller in size and longer in emission wavelength. Such properties implied that the PAA ligand might have effects on the crystal growth and thus modulation of emission. Though the one-step surface modification, lots of efforts were saved on post-treatment procedure, yet the monodispersed UCNPs obtained can hardly be obtained through such method. Therefore, there are still lots of challenges for researchers in this field.
4. Bioimaging of upconversion nanophosphores Optical imaging of biological samples based on UCNPs has received extensive attention, especially those with multi-functions which have the potentials to be employed in multimodal imaging. UCL could be used for visualizing living biosamples, but only in several millimeters depth. Such limitation can be compensated by MRI and X-ray CT, which offer the physiological information as 8 Page 8 of 14
well as the structures of biosamples without penetration depth limitation. PET and SPECT techniques show high sensitivity but had relatively low spatial resolutions. Since different bioimaging techniques provide different spatial resolutions, penetration depth and areas of applications, an enhanced quality of bioimaging could be achieved by combination of the advantages of each bioimaging technique. Generally based on the number of the techniques used, the imaging could be divided into dual modal and multimodal imaging. Dual modal imaging mainly includes UCL/FL, UCL/X-ray CT, UCL/MRI, UCL/PET, and UCL/SPECT. For the multimodal imaging, higher requirements of synthesis are necessary. Here several examples will be introduced to illustrate the usage of multimodal imaging. 4.1. Dual-modal imaging Many efforts were put on the design of UCNPs for dual modal imaging, of which one example was chosen to discuss in detail here.[16, 52, 59] The band-gap fluorescence ZnS:Mn2+ QDs and up-conversion luminescence NaYF4:Er3+/Yb3+ NPs were embedded into hydrophilic polymer matrixes through in situ cross-linking polymerization. The as-prepared multifunctional nanocomposites had orange fluorescence and green UC luminescence under UV and NIR (980 nm) irradiation respectively, which showed the capability for fluorescence and up-conversion luminescence dual modal imaging. These nanocomposites were further linked to folic acid moiety by bioconjugation, which enabled the specific binding toward cancer cell. In addition, this fabrication strategy could extend to synthesize multifunctional nanocomposites for MRI, CT and others by incorporating different elements. The X-ray CT technique is frequently included for UCNPs based multimodal imaging, because this technique mainly relies on the atomic number and electron density of the agents. There is a wide choices of lanthanide ions since many lanthanides are heavy atoms, providing higher attenuation coefficient for X-ray CT. Actually, some of the host materials themselves could be utilized for this technique without further modification, especially those with NaLuF4 as host matrix due to the Lu atom with the highest atomic number of 71. Moreover, by incorporating of paramagnetic Gd3+ and Mn2+ ions, the afforded UCNPs could be used for UCL/ MRI dual-modal imaging, 4.2. Multimodal imaging Recently reported examples for trimodal bioimaging mainly focus on the UCL/MRI/CT and UCL/MRI/SPECT strategies due to the bifunctional nature of some lanthanide such as Gd3+, and Lu3+ as mentioned above.[60-62] For those UCNPs with different doping ions for multimodal imaging, the rational design of the fabrication strategy is required otherwise a decrease of the original function would occur. Li's group reported a UCNPs-based four-modal probe for tumor angiogenesis imaging, including UCL, X-ray CT, MRI and SPECT techniques.[63] The RE metal radioisotope 153Sm has a similar atomic radius to other Ln3+ and a physical half-life of 46.3 h, making it suitable for doping into UCNPs and for the long-term SPECT imaging. A core/shell structure was constructed with NaLuF4:Yb,Tm as a core and 4 nm of 153Sm3+-doped NaGdF4 as a shell. The as-prepared UCNPs have been confirmed to be effective for four-modal based bioimaging (Fig. 2). One highlight of this four-modal imaging nanocomposite is that the increased imaging technique did not compromise its performance and sensitivity of each imaging mode, which are attributed for the smart arrangement of each element. Proper layout of Gd3+ at the out layer of the nanostructure significantly enhances its MRI signals since the Gd3+ at the 9 Page 9 of 14
inner layer only showed limited enhancement. And the 153Sm3+ was directly doped into the NaGdF4 shell via surface-exchange process due to the high affinity of lanthanide ions toward fluorides without further disruption of the structures. More importantly, the multimodal imaging probe was also successfully applied to the tumor angiogenesis imaging, which is of great importance for early diagnosis of cancer.
5. Conclusion and outlook With the development of technology, optical imaging is used more and more widely in various aspects, especially in clinical studies for localizing tumor site, preand post-surgery investigation as well as tracing of drug circulation. The development of UCNPs based optical imaging has been greatly highlighted owing to the unique properties like low excitation power for continuous monitoring, deep penetration and large Stoke's shift. With regard to bioimaging, there will be a great era waiting ahead for UCNPs. Meanwhile, many problems are also waiting to be solved, as follows. (1) The exploration of novel synthetic strategies as well as surface modification methods is still highly desirable, especially for achieving those ultrasmall UCNPs with high luminescence, hydrophilic surface and biocompatible properties. The quantum yield of UCNPs still needs to be improved by reducing the crystal defects and improving upconversion efficiency. (2) Lots of challenges are faced ahead for fabricating multifunctional UCNPs with small size and new functional elements without impairing the original performance. (3) Although the UCNPs are claimed to be low cytotoxicity, further investigations on biocompatibility are still required considering the clinical applications. (4) The exploration of combining UCNPs-based optical imaging with other functions including photo dynamic therapy and drug delivery is very intriguing, which offers the possibility for imaging as well as on-site treatment. Acknowledgements This research was supported in part by the National Natural Science Foundation of China (21475007 and 21275015), the State Key Project of Fundamental Research of China (2011CB932403 and 2011CBA00503), the Fundamental Research Funds for the Central Universities (ZZ1321 and YS1406), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1205). We also thank the “Public Hatching Platform for Recruited Talents of Beijing University of Chemical Technology” for support. References [1] G. Chen, H. Qiu, P. N. Prasad, X. Chen, Upconversion nanoparticles: design, nanochemistry, and applications in theranostics. Chem. Rev. (2014). [2] N. Phuong-Diem, S. J. Son, J. Min, Upconversion Nanoparticles in Bioassays, Optical Imaging and Therapy. J.Nanosci. Nanotech. 14 (2014) 157-174. [3] J. Shen, L. Zhao, G. Han, Lanthanide-doped upconverting luminescent nanoparticle platforms for optical imaging-guided drug delivery and therapy. Adv. Drug Deliv. Rev. 65 (2013) 744-755. [4] J. Niu, X. Wang, J. Lv, Y. Li, B. Tang, Luminescent nanoprobes for in-vivo bioimaging. Trac-Trends Anal. Chem. 58 (2014) 112-119. [5] M. Chen, X. He, K. Wang, D. He, X. Yang, H. Shi, Inorganic fluorescent nanoprobes for cellular and subcellular imaging. Trac-Trends Anal. Chem. 58 (2014) 120-129. 10 Page 10 of 14
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Captions
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Scheme 1. Schematic representations of different mechanisms for luminescence (a) Jablonski Energy diagram, illustrating the down-conversion fluorescence; (b) Upconversion process of lanthanide-doped crystals, ETU: energy transfer upconversion, ESA: excited state absorption; (c) Two-photon absorption based emission; (d) Emission process of semiconductors based on the bandgap emission
Fig. 1 Illustration of the surface modification strategies through hydrophobic/ hydrophobic interactions and the images for the mono- and multi-modal imaging
Fig. 2 (a-d) Four-modal imaging of the focused tumor from the tumor-bearing nude mouse 1 h after intravenous injection of NaLuF4: Yb,Tm@NaGdF4 (153Sm): (a) In vivo UCL-image, (b) X-ray CT image, (c) SPECT image, (d) NM imaging of tumor. (e) UCL confocal image of the paraffin section of tumor tissue. (f) Schematic illustration of tumor angiogenesis imaging using NaLuF4: Yb,Tm@NaGdF4 (153Sm) as the probe. [Reproduced with permission, Copyright 2013, American Chemical Society;]
Table 1. Representative ultrasmall upconversion nanophosphores strategies Average Host Strategies size material (nm) Variations in 5.0 SrF2 composition 7.8 -NaLuF4 5.3-8.0 Variations in experimental conditions
Doping ion
Remarks
Ref.
Gd3+, Yb3+ Yb3+, Er3+
New host materials Gd3+doping
[31] [32]
Yb3+, Er3+
Ln3+ doping
[17]
[33]
4.5-15.0
NaYF4
Yb3+, Er3+
Surfactant concentration
7.0-10.0
NaYF4
Yb3+, Tm3+
Concentration of Yb3+
[34]
Core NaYF4: Yb, Er 5 nm
Shell NaYF4 2 nm
11.1
KGdF4:Tm, Yb 3.7 nm
KGdF4 7.4 nm
Enhanced luminescence
[35]
10
Ln:BaF2 3 nm
Ln:SrF2 7 nm
Active core/active shell
[36]
7.0 Construction of core/shell structure
Ba2LaF7
with high luminescence obtained by different
Inert shell
[33]
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