Nanoscale REVIEW. 1. Introduction. Ming-Hsien Chan a and Ru-Shi Liu. View Article Online View Journal View Issue

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1 REVIEW View Article Online View Journal View Issue Cite this:, 2017, 9, Received 8th September 2017, Accepted 27th October 2017 DOI: /c7nr06693g rsc.li/nanoscale Advanced sensing, imaging, and therapy nanoplatforms based on Nd 3+ -doped nanoparticle composites exhibiting upconversion induced by 808 nm near-infrared light Ming-Hsien Chan a and Ru-Shi Liu a Department of Chemistry, National Taiwan University, Taipei 106, Taiwan. rsliu@ntu.edu.tw b Department of Mechanical Engineering and Graduate Institute of Manufacturing Technology, National Taipei University of Technology, Taipei 106, Taiwan * a,b Malignant tumors are currently the leading cause of death worldwide, followed by cardiovascular and cerebrovascular diseases. Although various methods, such as blood examination, tissue biopsy, and radiography, for tumor detection, exist, these techniques still require further refinement. Researchers have recently explored the use of novel adjuvant methods, specifically luminescence imaging detection, for the detection of tumors. The light-triggered approach is less invasive and induces fewer side effects than traditional detection methods. This paper highlights recent advances in the design, property tuning, and applications of nanoparticles that exhibit upconversion under 808 nm excitation. When doped with neodymium ions, upconverted nanoparticles gain the ability to absorb 808 nm light. The advantageous unique features of 808 nm light include deep tissue penetration and limited thermal side effects. The 808 nm-excited upconverted nanoparticles exhibit superior potential for use in biosensing, bioimaging, therapy, and three-dimensional display. Thus, innovative theranostic nanoplatforms can be developed by incorporating 808 nm-excited upconverted nanoparticles with phototherapy agents. Such a composite technique is expected to possess the individual advantages of each material. 1. Introduction In 1959, the well-known American physicist R. Feynman delivered his famous speech titled There s Plenty of Room at the Bottom during the Annual Meeting of the American Physical Society. This speech has since motivated other scientists to Ming-Hsien Chan Ming-Hsien Chan received his bachelor s and master s degrees in the Department of Bioscience and Biotechnology from the National Taiwan Ocean University in Currently, he is a Ph.D. candidate in Prof. Liu s group at the National Taiwan University. His current research interests include the synthesis of biological nanomaterials and their applications in vitro and in vivo. Ru-Shi Liu is currently a professor at the Department of Chemistry, National Taiwan University. He received his bachelor s degree in Chemistry from Shoochow University (Taiwan) in He received his master s degree in nuclear science from the National TsingHua University (Taiwan) in He obtained two Ph.D. degrees in chemistry one from Ru-Shi Liu the National TsingHua University in 1990 and one from the University of Cambridge in His research concerns the field of Materials Chemistry. This journal is The Royal Society of Chemistry 2017,2017,9,

2 Fig. 1 (a) Nanotechnology could improve the synthesis, properties, and functions of biomedicinal compounds. (b) Nanocomposite systems could provide alternative answers to the original clinical question. explore nanotechnology. Offering advantageous physical and chemical properties, nanotechnology is widely applied in the industrial, constructional, biological, physical, and chemical fields (Fig. 1a). 1 3 Nanotechnology is used in two major aspects of the biomedical field, namely, diagnosis and therapy. In this paper, we first discuss the development of multifunctional nanocomposites and then expound their diagnostic and therapeutic properties. In addition, we introduce the novel term theranostic, which refers to the combined application of therapeutic and diagnostic platforms in personal nanomedicine; however, the direct application of these multifunctional nanoplatforms in biological environments requires further modifications. 4 7 Therefore, the convenient utilization of multifunctional nanomaterials must be realized for cancer treatment (Fig. 1b). Upconversion (UC), or anti-stokes, is a special phenomenon wherein long wavelengths are absorbed by a material and are emitted as short wavelengths. Stokes law posits that materials excited by high-energy light emit low-energy light. When a material is excited by a light source with a short wavelength and a high frequency, the material emits long wavelengths and low-frequency light. In a typical Stokes emission, ultraviolet light stimulates the emission of visible light, and blue light stimulates the emission of yellow light. However, several materials can demonstrate the aforementioned behavior but with the opposite light effect. Thus, this phenomenon was called the anti-stokes effect. It states that the materials can absorb the low energy light to emit high energy light, which can achieve the opponent conversion. For anti-stokes phenomena, the ideal host matrix of the luminescent material with UC should have highly susceptible radiating centers and low phonon energy to decrease the excitation energy caused by non-radiation. Moreover, the material should exhibit a good near-infrared (NIR) light transmittance alongside a high chemical and thermal stability to maintain the structural integrity of the bulk crystal grid. In order to promote the UC process, the materials also need to have strong absorption in a suitable NIR region for the biological application ( nm). Rare earth elements in the trivalent state exhibit similar ionic and chemical properties. Therefore, inorganic compounds have been the preferred host matrices for nanomaterials that exhibit UC. 8 For example, Ca 2+,Sr 2+, and Ba 2+ with certain transition metal elements, such as Zr 4+ and Ti 4+, are favored in UC applications because of the similarity of their ionic radii to those of rare earth elements; yttrium ions (Y 3+ ) are the most commonly used lanthanide compounds. Inorganic compounds containing these elements are also suitable for the synthesis of the host matrix for materials that exhibit UC. 9,10 Previous studies have focused mainly on the identification of compounds that contain rare earth oxides and fluorides. Highly stable oxides can easily produce a stretching vibration with phonon energy that exceeds 500 cm 1 in the main lattice. By contrast, halogen compounds, such as chloride, bromide, and iodide, generally possess phonon energy less than 300 cm 1. These hygroscopic elements easily introduce defects associated with water vapor, making them unsuitable for application in the fabrication of materials with UC. Thus, fluoride, which has high chemical stability and a low phonon energy of 350 cm 1, is ideal for the fabrication of nanomaterials that exhibit UC. The excitation band of UC luminescent materials is located in the NIR light region. The NIR light window is the wavelength range in which the penetration depth of light in biological tissue reaches the maximum value. The penetration depth of UV and visible light in biological tissues is mainly hampered by tissue absorption. The lower (i.e., short wavelength region, UV and visible light) and upper limits (i.e., long wavelength region, far infrared light) of light penetration are determined by blood and water absorption, respectively. The appropriate light source for optical imaging and identification must be selected within the appropriate wavelength range of the optical window to improve the imaging efficiency and penetration depth and to avoid tissue damage. This review article aims at providing a brief introduction to the future development of luminescent materials that exhibit UC for their applications in biomedical testing and bioimaging. Innovative strategies that utilize alternative sensitizers have been recently designed to exploit the excitation wavelengths of UC nanoparticles (UCNPs). This review highlights the advances in the design and therapeutic applications of multifunctional nanoplatforms that exhibit UC under 808 nm excitation. The future challenges and prospects in this field are also discussed. 2. The luminous principle and composition of upconversion nanoparticles (UCNPs) UC, which is also referred to as a nonlinear optical route, is the anti-stokes process of transducing low energy into high energy. UCNPs could successively emit light through the UC process by absorbing two or more pump photons via an intermediate and then emitting multi-radiation in the visible or even in the UV or NIR regions, at a shorter wavelength than the pump wavelength. This fundamental concept was first 18154,2017,9, This journal is The Royal Society of Chemistry 2017

3 independently formulated and proposed by Auzel in the 1960s. Anti-Stokes emission was initially difficult to investigate given that it involves emission energies that exceed excitation energies by only a few kt. 11 These energies are linked to the thermal population of energy states above excitation states by an equivalent energy amount. In the past, UC was conducted with bulk or micro-materials, thus resulting in low UC emission efficiency. Consequently, the detailed nanoscale properties of Stokes emission were difficult to assess. Moreover, these materials were strictly developed as fluorescent materials for bioimaging because of scale limitations. However, the advances achieved in recent years have enabled the synthesis of UCNPs with high stability and effective emission. Optical probes based on UCNPs have a larger anti-stokes shift, sharper emission bandwidth, wider multicolor and tunable emission, longer lifetime, higher photostability, and lower cytotoxicity than traditional down-conversion fluorescent organic dyes and quantum dots (QDs). Moreover, UCNP-based optical probes lack background interference from autofluorescence. These characteristics make UCNPs attractive contrast agents in fluorescent biomedical imaging. However, the UC effect limits energy intensity, indicating that UCNPs should be embedded in an appropriate main lattice sensitizer. In addition to the wide absorption cross-section in the NIR range, the ideal sensitizer must match the co-doped activator to facilitate the energy transfer process. Among all lanthanide metal elements, Yb 3+ and Nd 3+ are the most suitable sensitizers with excitation at 980 and 808 nm near the NIR band and a considerable absorption cross-section. The order distribution of Yb 3+ at 2 F 7/2 to 2 F 5/2 is also resonant with that of the other commonly used UC lanthanide, which has only a 4F order of the 2 F 5/2 single excited state. The f f transitions of elemental ions (e.g., Er 3+, Tm 3+, and Ho 3+ ) promote efficient energy conversion. Therefore, for in vivo experiments, Nd 3+ is a suitable sensitizer that can act as an excitation light source at 808 nm, thereby avoiding the thermal damage caused by the 980 nm laser to biological tissues. Yb 3+ and Nd 3+ are the most commonly used ions for sensitizer conversion given their deep tissue penetration. 12,13 The activator plays a role in the release of light from the luminescent material embedded in the host matrix. The activator promotes follow-up and then transitions to a higher energy level, thereby decreasing the probability that non-radiative energy relaxation would occur. Excessive doping with an activator; however, may lead to inactivation. The energy dissipation associated with cross-relaxation can be minimized by adjusting the doping proportions of the different activators to control light emission Energy transfer UCNPs are composed of rare earth elements that consist of 15 lanthanides, as well as scandium (Sc) and yttrium (Y). The latter two elements are considered rare-earth elements given their similar radii to trivalent ions and chemical properties. Lanthanides with 4f energy levels that are close to one another are responsible for the special UC process via multi-photon transfer (Fig. 2). Lanthanides are found in the sixth period and Fig. 2 Partial energy level diagrams of trivalent lanthanide ions. 16 (Reprinted with permission from ref. 16. Copyright 2005, Royal Society of Chemistry.) IIIB groups in the periodic table and encompass elements between lanthanum (La) and lutetium (Lu). These elements have electronic configurations of 14 4f n 1 5d 0 1 6s 2 (n =1 15). Trivalent lanthanide ions (Ln 3+ ) are the most common and stable oxidized lanthanides, except for Ce 4+,Tb 4+, and Yb 2+, for which the f orbitals are empty, half-, or full-occupied, respectively. The unique properties of lanthanides result from their 4f electrons. The number of unpaired electrons in 4f orbitals reflects strong unquenched angular momentum and can reach up to seven, thus promoting effective spin orbit coupling and conferring significant paramagnetic properties (except for La 3+ and Lu 3+ ). Magnetic moment, magnetic susceptibility, and Ln 3+ electron relaxation time are determined by 4f electron configurations, which significantly differ along the series Host matrix UCNPs are fabricated from inorganic host nanocrystals that exhibit UC at room temperature. UC behavior is induced by doping low concentrations of the selected trivalent lanthanide ion into the host nanocrystals. Dopant ions are classified as sensitizers or activators on the basis of their function. The ion that emits radiation is called the activator, whereas the synthesizer absorbs energy. The first step in the synthesis of UCNPs is the selection of a suitable host material, which should possess high chemical stability and low lattice mismatch with the dopant ions and phonon energies. Phonons will decrease the energy efficiency of the transfer process between the sensitizer and the activator, and most of the energy would be lost as heat during the non-radiative process. In addition, the crystal phase of the host materials also significantly influences the efficiency of UC. For common UCNPs, such as NaYF 4 :Yb/Er, the hexagonal phase of NaYF 4 :Yb/Er enhances UC efficiency by several orders compared with the cubic phase of NaYF 4 :Yb/Er. This enhancement occurs because compared with the high symmetry of the cubic-phase host, the low symmetry of the This journal is The Royal Society of Chemistry 2017,2017,9,

4 hexagonal-phase hosts can apply a crystal field that contains a greater number of uneven components around the dopant ions. 17 According to the literature, uneven components can enhance electronic coupling between 4f energy levels, thereby leading to high electronic configurations and subsequently increasing the f f transition probabilities of the dopant ions. Another approach to improving the efficiency of UC is to decrease the size of the cation components in the host materials while increasing the strength of the crystal field. 18 As of now, the host matrix of most of the UCNPs is an oxide and the halogen compound. Except for oxides and halogen compounds, the latest emerging halogen oxygen compounds, which usually include lanthanide oxyfluorides and oxychlorides, are showing a different perspective. 19,20 The lanthanide oxyfluorides and oxychlorides are considered to be attractive candidates for UC host materials, because they combine the advantages of both the fluoride/chloride and the oxide counterparts, for example, a low phonon energy, excellent mechanical strength, and good chemical and thermal stability. 21 The oxyfluorides and oxychlorides offer as a new host system for UCNPs a means to understand the impact of crystal structure on UC photoluminescence properties Sensitizers Yb 3+ -Sensitized upconversion (980 nm-excited). The host materials of UCNPs are designed to prevent any possible adverse conditions that may hinder UC luminescence, and the sensitizers and activators are selected to emit specific UC emissions. The trivalent Yb ion (Yb 3+ ) is the most suitable element for UCNP sensitizers because Yb 3+ takes an extremely simple 2 F 7/2 2 F 5/2 transition at the 4f energy level, as denoted by the absorption band at 980 nm. In addition, Yb 3+ reveals the largest absorption cross-section among the lanthanide ions. Activators, which are selected from 15 lanthanides, are core elements that determine the region of emission. Fig. 3a shows the UC processes of Er 3+ and Tm 3+ doped with Yb 3+ as the sensitizer in UC nanocrystals; the major emission colors are blue, green, and light red ( 650 nm) for the Er 3+ system, and blue, orange, and dark red ( 800 nm) for the Tm 3+ system. In addition, UC emission can be simply adjusted by changing the sensitizer: the activator ratio, as indicated by the major emission colors of red for Yb 3+ and blue for Tm 3+ (Fig. 3b). 22, Nd 3+ -Sensitized upconversion (808 nm-excited). The 808 nm laser light source encompasses the optical window. In addition to high tissue penetration, imaging and treatment efficiency are enhanced in this region, and the absorption of water molecules is avoided because of the heat effect. 24,25 In recent years, many scientists have intensively studied the light mechanism and biological applications of different optical excitation wavelengths. 26,27 The lanthanide element Nd 3+ can be excited at 730, 808, 865, and 980 nm when the photons in the lattice of the main body are changed. Water molecules are weakly absorbed in these regions (<900 nm). In addition, Nd 3+ exhibits considerable absorption and cross-sectional areas in the 808 nm region. Nd 3+ -doped UCNPs commonly need to use Fig. 3 (a) Schematic of the typical UC processes of Er 3+ and Tm 3+. (b) Multi-color tuning of UC by doping NaYF 4 NPs with various ratios of lanthanides. 22 (Reprinted with permission from ref. 22. Copyright 2008, American Chemical Society.) other lanthanide elements such as Yb 3+ to convert nanomaterials under 808 nm excitation. The co-doped Yb 3+ in the system acts as the bridge responsible for the adsorption of NIR light energy, and it facilitates energy transfer between Nd 3+ and Yb 3+ in different types of matrixes, which can then activate energy transfer (Fig. 4a). 13 Highly efficient light conversion can be realized by utilizing 808 nm NIR light excitation. The thermal effect is effectively reduced, consequently avoiding tissue damage. In theory, the excitation wavelength can be engineered by varying the dopants, because lanthanide ions can absorb light of varied wavelengths that range from UV to NIR. Therefore, the traditional sensitizer, Yb 3+, can be replaced by other lanthanide ions that can absorb excitation energies at 808 nm. Nd 3+ is an ideal alternative sensitizer because of its large absorption cross-section at 808 nm, which is one order of magnitude larger than that of Yb 3+ at 980 nm (Fig. 4b). The UC of Nd 3+ -sensitized photons, including Nd 3+ -sensitized Tm 3+ and Nd 3+ -sensitized Er 3+, in bulk materials, has been known for more than a decade (Fig. 4c and d). 28 In addition, with the discovery of advanced approaches for NP synthesis, several groups have independently demonstrated the efficient UC of Nd 3+ -sensitized photons in NaYF 4 NPs using core shell nanostructures. 29 Nd 3+ ions are co-doped with Yb 3+ and activator ions in Nd 3+ -sensitized UCNPs. Under excitation by 808 nm radiation, Nd 3+ ions transfer excitation energy to the nearby Yb 3+ ions, which in turn serve as energy migrators that transfer energy to emitting ions, resulting in UC emission. Although the Nd 3+ ion can directly transfer energy to activators, its transfer efficiency is low. Other lanthanide ions apart from 18156,2017,9, This journal is The Royal Society of Chemistry 2017

5 Fig. 4 (a) Absorption of water in the NIR and the integration scheme of the Nd 3+ Yb 3+ energy transfer process through the introduction of Nd 3+ -doped UCNPs. The red line marks the absorption of large amounts of water. (b) Energy transfer pathway from the shell (Nd 3+ to Yb 3+ ) to the core (Yb 3+ to Er 3+ ) UC emission in core shell nanostructures under 808 nm excitation. (c) UC emission spectra of core shell UCNP under 980 and 808 nm excitation. (d) UC excitation spectrum of core shell UCNP under 540 nm emission. 13 (Reprinted with permission from ref. 13. Copyright 2013, American Chemical Society.) Nd 3+ and Yb 3+ are also potential sensitizers for photon UC. However, these lanthanide ions exhibit weak UC emission possibly because of their small absorption cross-section and crossrelaxation. When the size of the single-lanthanide-ion-doped material is decreased to the nanoscale, additional surface quenching caused by the enlarged surface area will further decrease UC emission. 30 Thus, UC mechanisms must be comprehensively understood and novel nanostructure designs must be formulated to develop new lanthanide sensitizers Activators The activator plays a role in the release of light from the luminescent material embedded in the host matrix. The life cycle of the activator is extended at the intermediate level as the activator absorbs the energy of the adjacent excited state and then temporarily stores the excited electrons. The activator thus promotes follow-up and then transitions to a higher energy level, thereby decreasing the probability that non-radiative energy relaxation would occur. Lanthanides, such as Tm 3+ and Er 3+, possess a considerable energy gap and are commonly used for converting the nano-activated agent. However, excessive doping with an activator may lead to the inactivation or fluorescent quenching of UC emission such as causing interion cross-relaxation energy loss or decreasing the efficiency of light conversion. UC nanocrystals are usually doped with less than 2% dopant, a concentration that is well below that of the sensitizer (20%). The energy dissipation of cross-relaxation can be minimized by adjusting the doping proportions of the different activators to control light emission Modifications Another important aspect of the fabrication of UCNPs is surface modifications, which can be categorized as surface passivation or functionalization. The goal of surface passivation is to coat UCNPs with a protective shell to enhance the efficiency of UC given that the nature of the 4f 4f electronic transitions in the UC process is Laporte-forbidden. 11 Numerous studies have attempted to enhance the photoluminescence intensity by coating the surface of UCNPs with an inner or active shell with similar crystalline structures that decrease surface defects and interference by the solvent quencher from non-radiative decay. 31 Moreover, the active shell can better enhance photoluminescence than the inner shell because co-doping the sensitizer in the core and the outer shell will increase the number of pumping photons. 32 Most UCNPs are protected by long organic ligands, UCNPs are synthesized at a high reaction temperature in an organic solvent with a high boiling point. However, such UCNPs cannot be directly applied in a biological system without further modifications, as shown in Fig. 5. Typical strategies for transferring UCNPs into an aqueous solution include the removal of the capping ligand, the layer-by-layer method, coating with amorphous silica or amphiphilic polymers, and ligand oxidation and exchange. 33 Furthermore, UCNPs exchanged or coated with another hydrophilic ligand, such as antibodies, aptamers, or peptides, with high bio-affinity can offer further functionality Upconverted nanomaterials in biological research Traditional nanomaterials with medical applications are limited by several disadvantages, including leakage, which causes interstitial damage, poor solubility, rapid degradation in vivo, unfavorable pharmacokinetics, non-specificity, dose limitation, and other side effects. The main objective of the development of nanomaterials for cancer therapy is to overcome these problems. Drugs must be capable of transmission Fig. 5 Typical strategies for the surface modification of UCNPs. 34 (Reprinted with permission from ref. 34. Copyright 2014, American Chemical Society.) This journal is The Royal Society of Chemistry 2017,2017,9,

6 through blood circulation and of accumulating in and exerting therapeutic effects on tumor tissue. Thus, prolonging the halflife of drugs in blood circulation is a key objective of drug development. Nano-sized drug carriers for cancer treatment are mainly distributed in the nm range primarily because of the body s structure. The wall size of the human glomerular capillary is approximately 10 nm; drugs with a size of less than 10 nm will initiate renal metabolism and thus be filtered out of the body. Drugs that are less than 200 nm in size do not accumulate in the spleen and will be degraded through phagocytosis by the reticuloendothelial system. The reticuloendothelial system is an important component of the immune system. Phagocytic cells are produced by bone marrow stem cells and differentiate into monocytes, which then differentiate into macrophages upon leaving blood circulation to infiltrate body tissue. Nanomaterials can facilitate the effective accumulation of drugs in the tumor environment because the vascular endothelial cells in the tumor region are irregularly arranged relative to that in normal tissues. Specifically, the gap between each endothelial cell is approximately from 200 nm to 2 μm in tumor and normal tissues, respectively. Therefore, nanomaterials can directly enter the tumor region via passive accumulation. Moreover, the tumor region lacks lymphatic drainage. 35 Consequently, nanomaterials will be retained in the tumor and are not easily removed. This effect is known as the enhanced permeability and retention (EPR) effect. 36 If nanomaterials are modified with a targeting ligand, they could further interact with a specific receptor on the surface of the cancer tumor and improve the extent of drug accumulation Sensors The main objective of biosensing is to provide a biomedical signal based on applying the theories and methods of information science to assess and extract disturbances and noise in the submerged data. These signals are analyzed, classified, displayed, stored, and transmitted. The main purpose of medical diagnosis is to effectively detect an illness before it occurs. Even the samples used in these tests can be used to forecast the patient s long-term conditions. UC can be efficiently applied in bioimaging because they can convert two or more low-energy photons into high-energy photons. Furthermore, the UC materials can absorb infrared light to produce fluorescence signals, which can effectively avoid the unnecessary background data since biological tissue has lower noise in the infrared region. However, the poor efficiency of the fluorescence signal must first be overcome to enable signal detection and realize effective light conversion. Scientists have proposed the use of various UC nanocomposites, such as composite materials that consist of UCNPs and gold nanoparticles (AuNPs), as multifunctional platforms for biomedical applications. 38 Surface plasmon resonance (SPR) refers to the absorbance of light by precious metals on the surface of a planar material or NPs, resulting in the coupling of electronic holes. This phenomenon is the basis of many standardized bioimaging tools. Many biosensors based on fluorescence imaging can be used in biochip detection platforms. 39 In particular, AuNPs and other metal materials possess unique optical properties; that is, total reflection occurs when the incident light hits the metal surface. The evanescent wave will be coupled with the resonance of the surface plasma wave. When combined with UCNPs, the resonant wave can greatly enhance the upconverted fluorescence efficiency. 40 In addition, when doped with different lanthanide metals (Yb 3+, Nd 3+, and Er 3+ ), UCNPs can change the color of the emitted light from green to red, thereby facilitating instrument detection by providing light and heat effects that can be detected by another instrument (Fig. 6a). Furthermore, Nd 3+ -doped UCNPs prevent the heat effect in the water solution. 41 The changes in the temperature and quality of the water solution can be confirmed by a series of heat efficiency enhancements through UC (Fig. 6b and c). 42 Thus, this low-toxicity nanocomposite material is the most suitable for new nano-sensors in future medical testing. 3.2 Multiple imaging Biomedical imaging refers to the non-invasive technique of obtaining images of internal tissue of the human body or parts of the human body for medical purposes. The development of biomedical imaging involves different scientific fields, such as diagnostics, radiology, and thermal, optical, acoustic, and magnetic sciences; these areas could be applied individually or in combination. Scientists must develop efficient signal extraction and storage technologies, as well as develop Fig. 6 (a) Schematic of the proposed ET quenching mechanisms in Nd 3+ -sensitized core shell UCNCs for water detection. 42 (b) The absorption spectrum of distilled water (DI water) over the visible and the NIR ranges. The red arrows indicate the low and high water absorption bands, where the alternative excitation bands should be extended to avoid overheating biological tissues. (c) Comparison of the temperature increases associated with YVO 4 :Er 3+ and Yb 3+ core/shell NPs. 41 (Reprinted with permission from ref. 41 and 42. Copyright 2017, Royal Society of Chemistry and OSA Publishing.) 18158,2017,9, This journal is The Royal Society of Chemistry 2017

7 methods for the interpretation, analysis, and diagnosis of the obtained images to effectively apply imaging technology in medicine, medical engineering, medical physics, and health information science. Commonly used clinical bioimaging methods include X-ray, nuclear magnetic resonance imaging (NMRI), and computer tomography (CT). The rapid development of nanotechnology in recent years has motivated the use of nanomaterials in biological calibration and imaging. Nanobiopsy is used for in vitro and in vivo drug delivery studies and clinical trials, and it is expected to be more sensitive on the microscopic scale. Fluorescence-based methods are the most common and most widely studied bioimaging methods. For example, organic fluorescence and semiconductor quantum dots have been developed as bioimaging tools. Although organic fluorescent materials can be applied to biomolecular calibration through excitation, their absorption wavelength is narrow, and their wavelength range is broad. Consequently, mutual interference is observed between the spectra of upconverted fluorescent materials and their light stability is poor, making them easily affected by the light energy quenching effect. 43 By contrast, semiconductor QDs are characterized by high quantum yield, high molar extinction coefficient, adjustable size, extensive absorption spectrum, symmetrical and narrow emission, and good chemical optical stability, suggesting that semiconductor QDs are suitable for applications in single-molecule tracking and efficient drug screening through fluorescence imaging Nonetheless, the use of semiconductor QDs in biomedical applications remains hindered by certain limitations and disadvantages, including potential biological toxicity, poor compatibility, and environmentally hazardous bulk parent materials. In addition, organic fluorescent and semiconductor QD materials are down-conversion materials, which require ultraviolet or visible light as the excitation light source. Biological tissues easily Fig. 7 (a) Construction and operating principles of nanoplatforms for biological imaging. (b) UC emission spectra at room temperature (inset: UC multicolor tuning through control over dopant concentrations in NaYF 4 : Yb, Er, and Tm NPs and applied to multi-imaging). (c) UCL 650 nm images and specificity of UCNPs under 808 nm excitation. 48 (Reprinted with permission from ref. 48. Copyright 2015, Royal Society of Chemistry.) autofluorescence, thus interfering with the accuracy of bioimaging techniques (Fig. 7a and b). 47 Exposure to lowwavelength ultraviolet light prevents the detection of deep biological tissues. In addition, prolonged exposure to ultraviolet light also makes tissues vulnerable to high-energy light burning, leading to cell damage and adverse effects. Fe 3+, Gd 3+, and other magnetic ions may also be added to NPs to provide magnetic properties in addition to fluorescent properties. Magnetic developers are mainly classified as paramagnetic, ferromagnetic, and superparamagnetic types. These developers affect the nature of contact with water molecules, enhance tissue image contrast, and improve the reliability of NMRI diagnosis. Common examples of magnetic developers include dextran-modified iron-oxide NPs. Glucan on the binding sites of antibodies enables viral particles to adhere to the antibody, thus facilitating the aggregation of NPs. The image is then scanned on the basis of the particles magnetic properties. However, magnetic resonance imaging (MRI) requires a relatively large amount of sample buildup to promote signal integrity. The degree of contrast provided by the developers also easily varies because of different tissue organizations. Composite NPs provide a new approach for NMR imaging and fluorescence detection for cancer cell calibration and at the same time show the distribution of carcinogenic molecules. These tools can help clinicians identify the most effective treatment method on the basis of the analysis of cancer lesions. The use of NPs in bioimaging not only shows a tumor s stereotaxic position but also enables the development of several imaging methods for the timely detection of tumors (Fig. 7c). 3.3 Phototherapy Phototherapy is a light treatment method in which a specific wavelength is used to stimulate PSs for heat ablation and/or produce reactive oxygen species (ROS) to induce cell apoptosis. Hyperthermia, a physical form of phototherapy, has been widely used in the treatment of drug-resistant cancers. Effective energy conversion is the most important factor for the development of heating materials for tumor treatment. Traditionally, gold nanomaterials (AuNMs), such as gold nanorods (AuNRs), gold nanoshells (AuNSs), and AuNCs, are used to induce hyperthermia. Au NMs can accumulate in tumor tissue through the EPR effect. Direct laser irradiation is used to produce heat for photothermal therapy (PTT). The photothermal effect is an efficient, strong energy conversion that occurs when AuNMs are irradiated by radiation derived from the surface of electrons with strong oscillations. As mentioned above, the NIR laser is the best option in this case; hence, AuNMs must convert their SPR to the NIR region. 49 Isothermal materials can also be directly used as a heat generator in a hybrid UCN and plasmonic material system for PTT. 50 Photodynamic therapy (PDT) refers to the therapeutic effect of the optical drive on the local environment. To drive PDT, a photosensitizer (PS) plays the key role of using the oxygen molecules and the specific excitation light to generate ROS. Non-toxic light perception substances commonly play a role in This journal is The Royal Society of Chemistry 2017,2017,9,

8 Fig. 8 PDT. Principles of PDT. (a) Mechanism and (b) reaction pathways of these elements to achieve efficient PDT. A specific wavelength of light is emitted by the light source system and the light-sensitive substances are activated through oxygen molecules to produce cytotoxic single-base oxygen 1 O 2 (singlet oxygen), free radicals, and other ROS substances. This activation is accompanied by the photochemical reaction induced by the excitation light source; the release of light-sensitive substances from the high concentrations of active oxygen species that have accumulated in tumor cells causes the cells to produce an immune response. This process may lead to DNA damage and cell necrosis or apoptosis. 51 To date, PDT has been successfully used to treat early-stage lung, Barrett s esophageal, 30 bladders, head and neck, 52 and skin cancers PDT. PDT is based on the production of ROS, which damage organelles, tissues, or organs. PDT is an emerging therapeutic modality that utilizes PS and light irradiation to eradicate cancer tissues. PDT is a type of optical-drive treatment that achieves the localized treatment effect and is driven by a PS, oxygen molecules and an excitation light source. Light-sensitive substances are not toxic in the absence of light and must be involved with the above elements to achieve the desired anti-tumor effect. The principles of PDT are shown in Fig. 8a and b. Specifically, photosensitive electrons in the ground state are excited by a specific wavelength of light. Electrons remain in the single-state excitation state for a very short duration (lifetime = 1 ns to 100 ns) before returning to the ground state. Alternatively, after excitation, two pairs in the electronic orbit of the excited triplet state are formed. The triple state exists for an extended duration (lifetime 500 ns) and hence easily reacts with the environment. 51 Triplet reactions can be divided into Types I and II reactions. In Type I reactions, electrons are generated or protons are transferred and radicals are formed when the PS is excited by light of a specific wavelength to form a triplet. Then, the radicals collide with the surrounding single acceptor molecules, such as a cell membrane molecules or a typical molecule. Free radicals include single base oxygen, hydroxyl, hydrogen peroxide, and superoxides. Meanwhile, in Type II reactions, the PS is excited to the triplet state, and triplet oxygen molecules are formed by the reaction of singlet oxygen. 54 Singlet oxygen significantly damages proteins, nucleic acids, or esters in tissue cells given its extreme instability and reactivity with other molecules. The short lifetime (<0.04 μs) and travel distance (<0.02 μm) of singlet oxygen ensure that only tumor tissues are damaged. Most PDTs involve the Type II reaction in an oxygen environment and use specific light wavelengths to stimulate the PS, the light source, and oxygen molecules to produce the photochemical effects of singlet oxygen, which causes the necrosis or apoptosis of cancer cells by causing DNA damage. The choice of PS is an important factor that determines the effect of PDT. First-generation PSs are poorly selective for tumor tissues, reside for prolonged durations in the body, and cause cytotoxicity in normal tissue. Moreover, given that the PS requires an excitation wavelength in the visible-light band, its tissue penetrability is limited and the localization of PDT in deep tumors is difficult to achieve. 30 Therefore, second-generation PSs that involve a short light period and a long excitation wavelength were developed. These PSs penetrate tissues more deeply and yield a single base oxygen molecule than first-generation PSs. However, the efficiency of PDT remains restricted by its absorption band that cannot penetrate deep tissue and is poorly selective for deep tumors (only targets shallow lesions). At present, many constraints are present in PDT, particularly, PS in vivo transmission, tumor selectivity, and the development of an appropriate excitation light source. PS transmission in the body, regardless of the use of intravenous injection or blood transmission, encounters biological metabolism, enzyme degradation, non-specific distribution, poor drug accumulation in tumors, cytotoxicity, and other issues. The development of a light source involves a single wavelength of laser light because the cost of light production is very high and has become the bottleneck of PDT. Thus, current PDT urgently needs to overcome these shortcomings and consequently improve the success rate of cancer treatment. After the conversion of NPs, AuNRs can be used in PDT in the biological window. This step can solve the problem of tissue penetration and cause the PS to remain for a long time and accumulate at high concentrations at the tumor site. Through these strategies, the native restrictions on PDT may be addressed, and the method s therapeutic effect may be strengthened PTT. Tumor tissue is characterized by (a) rapid hyperplasia leading to vascular distortion, blood flow resistance, and thrombotic tendency; (b) increased rupture risk at high blood pressures during high fever because of vessel wall fragility; (c) alterations in the distance of the gaps between vascular endothelial cells, tumor cell encroachment, and luminal hyperplasia caused by obstruction; and (d) vascular nerve receptor abnormalities and poor temperature sensitivity. Thus, increasing the ambient temperature to 41 C 43 C can damage cancer cells through the following mechanisms: Heat increases the fluidity of tumor cell membranes, thus causing structural and functional damage to tumor cell membranes. The lysosomal membrane and the endoplasmic reticulum are destroyed due to the substantial release of dissolved hydrolases. Ultimately, cell membranes rupture, cytoplasmic the content is spilled, and cancer cells die. High temperatures can cause cancer death by inhibiting the actions of DNA polymerases and ligases in tumor cells. Under irradiation, numerous free electrons on the surfaces of AuNRs absorb light energy and jump from the ground state to the excited state (Fig. 9a). When excited electrons revert to their stable ground 18160,2017,9, This journal is The Royal Society of Chemistry 2017

9 Fig. 9 Schematic of (a) surface plasmon polariton; (b) the possible population of UC Yb 3+ and Er 3+ ions, as well as the energy transfer process of AuNRs; (c) localized SPR for heat generation; and (d) properties of multifunctional hybrid nanocomposites. 56 (Reprinted with permission from ref. 56. Copyright 2015, American Chemical Society.) state, part of the energy is released in the form of heat. As a result, the environmental temperature increases. This phenomenon describes the role of light and heat in cancer therapy (Fig. 9c). AuNRs exhibit excellent light stability and can efficiently convert light energy into heat energy for long time periods. Thus, AuNRs are more suitable than organic PSs for use as PSs in biomedicine. The application of AuNRs is roughly divided into the following steps: Aspect ratio control refers to the control of the plasma resonance of the long axis of the metal surface in the NIR light range. 55 Such manipulation is conducted to avoid heme organization and the water absorption range and hence prevent heat damage to normal cells. The above-mentioned conversion of NPs is achieved through the fluorescence excitation of AuNRs. In this case, SPR produces the photothermal effect under NIR light excitation and the UC of fluorescent probes based on multifunctional composites (Fig. 9b and d) UCNP carbon nanocomposites. Carbon materials possess high biocompatibility and unique optical properties. Graphene and graphite-phase carbon nitride are among the most suitable carbon materials for use as PSs. Graphite oxide is composed of various ratios of carbon, hydrogen, and oxygen (Fig. 10a). 57 The chemical properties of oxidized graphite are related to the functional group modified on graphene. Graphene oxide exhibits enhanced photoconductivity, low resistivity, fast-moving electrons, and good electron transport properties. Graphitic carbonitride is mainly composed of carbon and nitrogen and is polymerized from N-bridged tri-s-triazine monomers that form a graphite-like arrangement. This material belongs to a new type of non-metallic semiconductor material with broad application prospects (Fig. 10b). 51 The constituent structures of graphitic carbonitride are combined by sp 2 and form a conjugated system under the interaction of adjacent π-electron orbitals. In addition to its rich surface electronic properties, the material is also composed of a few hydrogen atoms that provide hydrogen bonds and Brønsted base properties. The carbon material is highly resistant to hightemperature environments and acidic, alkaline, and organic solvents. The absorption spectrum of the material lies in the UV Vis spectrum. Under energy irradiation, electrons in the 4. Nd 3+ -Sensitized UCNP nanocomposites 4.1 UCNP inorganic material nanoplatforms Neodymium-doped UCNPs can absorb 808 nm NIR light. The use of NIR light ( nm) in anti-cancer treatment has become popular with the rapid development of physical and electronic technologies, as well as nanotechnology. NIR light falls in the optical biological window of human tissue. In this window, tissue scattering and absorption are minimized and water molecules are absorbed the least. The UC material per se does not affect the effectiveness of light treatment. Hence, most phototherapy effects rely on the means by which two or more kinds of materials are combined to form nanocompounds, as well as the manner by which energy transfer stimulates the PS to produce the phototherapeutic effect. Fig. 10 (a) Application of UCNPs and g-c 3 N 4 in PDT and the rich surface functional properties of g-c 3 N (b) Manufacturing process and structural state of g-c 3 N (c) Application of UCNP@g-C 3 N (Reprinted with permission from ref Copyright 2013 and 2016, Elsevier and American Chemical Society.) This journal is The Royal Society of Chemistry 2017,2017,9,

10 valence electron band are excited and jump to the conduction band. At this point, the valence band produces a charge hole to form a group of the electron-hole pair. Oxidation in the hole and the surface in contact with water and oxygen produces free radicals, such as the superoxide radical [O 2 ], hydrogen peroxide, and the hydroxyl radical [ OH], and other ROS, with a strong oxidizing force (Fig. 10c). Given their unique electronic structure, excellent photocatalytic activity, and surface functionality, carbon materials are widely used as photoelectric conversion materials, photocatalysts, and energy storage materials. In recent years, these materials have been employed in biosensing, cell imaging, drug release studies, and PDT. 60 Wang et al. reported a multifunctional nanoplatform developed from a UCNP-grafted core shell structure combined with nanographene oxide (NGO) via bifunctional polyethylene glycol (PEG). The surface of NGO was then loaded with phthalocyanine. The multifunctional nanocomposite can be applied in combined PDT/PTT and serves as a tracker for UC fluorescence imaging. Notably, the imaging and treatment nanoscale platform is photoexcited and stimulated by 808 nm NIR light. This characteristic decreases the dose-limiting toxicity of the nanoplatform and the tissue damage caused by overheating. Feng et al. explored the applications of graphitic carbon nitride (g-c 3 N 4 ) integrated with UCNPs in PDT. Through excitation with 808 nm NIR light, the emitted UV and visible light can activate g-c 3 N 4 to produce substantial amounts of ROS. 59 Nd 3+ -doped UCNP can effectively convert energy and produce a significant thermal effect in combined PDT/PTT to enhance anti-tumor efficiency UCNP TiO 2 nanocomposites. Titanium dioxide (TiO 2 ), which is commonly used in cosmetics, is excited by UV light and even visible light. TiO 2 catalyzes the production of hydrogen and oxygen and can be used as a photocatalyst. 61 TiO 2 nanomaterials are wide-bandgap semiconductor materials with important applications in electronic devices and solar cells. TiO 2 nanocrystals can be synthesized through different synthesis methods, including the wet-chemistry route (hydrothermal 62 and nonhydrolytic), crystallization from amorphous TiO 2, and epitaxial growth. Among these methods, the hydro(solvo)thermal approaches are the most widely used given their versatility in manipulating the nucleation and growth behavior of crystals. 63 The shape of the growing crystal can be changed in a given environment largely by minimizing the total surface energy of the intrinsic driving requirement. Under natural or equilibrium conditions, the adsorption capacity of the material is determined with high selectivity by the distance from the coordinated titanium atom and/or under two adjacent coordinated titanium atoms on the surface. Therefore, the choice of the capping agent determines the surface crystal growth. The various crystal phases and morphologies of TiO 2 NPs have three major applications, namely, as solar cells, as photocatalysts in hydrogen evolution, and as PS in lithium-ion batteries. 64 Under UV light exposure, TiO 2 NPs can generate ROS, such as O 2, H 2 O 2, or OH; these species can damage intracellular DNA and membranes (Fig. 11a and b) In addition, as an inorganic PS, TiO 2 NPs Fig. 11 (a) Encapsulation of Nd 3+ -sensitized UCNP in TiO 2 nanoparticles applied in PDT. 63 (b) Use of TiO 2 photocatalyst as light-sensitive drugs to load into the mesoporous silica layer. The TEM results show the UCNPs combined with TiO 2. (c) The in vivo results show the brilliant effect with this nanosystem. (d) 808 nm NIR irradiation could prevent the skin-burning and make the tumor shrink down. 72 (Reprinted with permission from ref. 63 and 72. Copyright 2016 and 2015, Elsevier and American Chemical Society.) have potential applications in tumor PDT given their better biocompatibility, more stable performance (not easily bleached), and longer cycle time than traditional organic PSs. Therefore, the design and preparation of TiO 2 -based nanoprobes for tumor visualization, diagnosis, and treatment are highly valuable. Hou et al. improved the efficiency of core shell UCNP TiO 2 NPs by avoiding low-energy defects. They then applied the NPs in photodynamic chemotherapy under 808 nm excitation. Yang et al. constructed a core shell structured UCNP@mSiO 2 nanocomposite by coating a PS/ photocatalyst with a layer of TiO 2 excited under 808 nm NIR to UV/visible light for simultaneous bioimaging and PDT (Fig. 11c and d) UCNP gold nanocomposites. In addition, Au NMs have many advantages for enhancing the organic fluorophores emitting light by the SPR effect. Here, we focus on discussing this effect for plasmon-enhanced UC emitting efficiency, because UC luminescence usually is low emitting in nature. Through numerical calculations, previous studies showed that geometry is the most influential factor that changes the peak site of SPR exhibited by Au NMs. The SPR peak only slightly shifts from approximately 520 nm to 540 nm as the particle sizes of AuNMs increase from 5 nm to 100 nm such as AuNRs, AuNSs, and gold nanocages (AuNCs) with different geometric structures. 73 UV and visible light poorly penetrate the skin because most of the light is absorbed by some species. 74 For example, water and oxy/deoxyhemoglobin exhibit widely distinct absorption ranges of nm and nm, respectively. 75 Therefore, the most notable SPR range lies 18162,2017,9, This journal is The Royal Society of Chemistry 2017

11 within nm. Thus, the SPR peak should be red-shifted to the NIR region because energy could be absorbed with high efficiency in this region. AuNRs with anisotropic structures exhibit two SPR bands at 520 nm or nm that are assigned to the transverse and longitudinal axes, respectively. The long axis of the SPR band is readily red-shifted by increasing the aspect ratio of AuNRs. AuNSs and AuNCs that exhibit SPR peaks in the NIR region can be fabricated by individually altering the shell thickness or gold content. He et al. assembled the Au nanoclusters, Au 25 (Capt) 18 (Au 25 ), into the mesoporous silica shell coating outside of Nd 3+ -sensitized UCNPs. 76 The Au 25 shell exhibits extensive PTT effects, bringing about the three light-induced imaging ( photothermal, photoacoustic and upconverted fluorescence imaging) effects which can be simultaneously achieved by exciting with a single NIR light (808 nm). This is also the triggering factor for the photothermal cancer therapy. Li et al. established an ultrasensitive dual microrna detection strategy based on nanopyramids assembled from UCNPs, silver sulfide particles, and Au Cu 9 S 5 particles (Fig. 12). 77 Given its excellent photostability, narrow size distribution, high signal-to-noise ratio, and biocompatibility, Chen et al. used Nd 2 O 3 as the host material to absorb 808 nm NIR light and then transfer the energy to its surrounding AuNRs. 50 The resonance phenomenon of AuNRs can produce thermal energy and concurrently enhance the conversion of fluorescent light. Nd 2 O 3 /Au composites mainly contribute to the thermal effect, which dramatically increases the density of holes in the valence band UCNP phosphor nanocomposites. Black phosphor, as a stable chemical component, is being utilized as a metalfree semiconductor with a suitable band gap. The bulk shape phosphors can disrupt the layer shape by using the liquidphase exfoliation. Recently, Lv s group reported that black phosphorus nanosheets with a high quantum yield could not only be a PS but also could load the Nd 3+ -sensitized UCNPs as a nano-vehicle. 78 Herein, to achieve 808 nm NIR lightmediated PDT, they designed and fabricated a novel UCNP black phosphorus nanocomposite. The efficiency of the generated ROS was measured with different (650, 808, and 980 nm) laser sources using the same pump power for comparison. The previous description has mentioned that 808 nm is the finest biological optical window. The authors demonstrate the superior applicability of UCNP black phosphorus nanocomposite as an antitumor agent under a single 808 nm irradiation. 4.2 UCNP organic molecule nanoplatforms Although inorganic PS exhibits good photothermal conversion efficiency under NIR excitation, inorganic materials are usually not biodegradable and may persist in the body for a long time, particularly in the reticuloendothelial system. In recent years, other organic PSs with a high light energy conversion, such as rose bengal (RB), 79 Chlorin e6 (Ce6), 80 merocyanine 540 (MC540), 81 methylene blue (MB) 54 and pyropheophorbide a (Ppa), 82 have attracted considerable attention. These organic Fig. 12 (a) UCNPs and AuNPs are combined into pyramidal composites. The combined UNCP AuNP image system can be used to track the residence time of materials in vivo. (b) UCNP luminescence and (c) NIR fluorescence images of a tumor-bearing mouse. (d) PA, (e) CT, and (f) whole-body CT images of a tumor-bearing mouse. 77 (Reprinted with permission from ref. 77. Copyright 2017, Wiley.) materials are generally highly biocompatible and easily decomposed. Most organic PSs, such as porphyrins, chlorophylls, and dyes, contain benzene ring structures. 83 Organic PS are differentiated on the basis of their target cells. Organic PS molecules enter the cells in a targeted manner, but each PS acts differently in each cell organelle. For example, mthpc is directed against DNA in the nucleus, ALA localizes in the mitochondria, and methylene blue localizes in the lysosome UCNP photosensitizer nanocomposites. Rose bengal (RB) molecules are the first introduced photosensitive organic molecule. Visible emissions in the green spectrum are used to trigger 1 O 2 generation from RB for in vitro and in vivo PDT. Li et al. employed Nd 3+ -sensitized UCNPs with dual-band visible and NIR emissions under a single 808 nm excitation. UC emissions in the green spectrum can trigger covalently linked RB molecules for efficient PDT, and the NIR emissions derived from Yb 3+ and MRI are used for imaging. 79 The majority of the PSs absorb light in the visible region, particularly in the green This journal is The Royal Society of Chemistry 2017,2017,9,

12 and red wavelength segments that encompass the absorption zone of the main PS. Chlorin e6 (Ce6, red-light-excited PS) and merocyanine 540 (MC540, green-light-excited PS) are the most commonly used PSs in recent studies. 81 Idris 84 and his coworkers demonstrated the special multicolor emission of UCNPs based on the simultaneous activation of two individual PSs, such as MC540 and zinc(ii) phthalocyanine, under NIR excitation. In this dual PS system, MC540 and zinc(ii) phthalocyanine are loaded in mesoporous silica holes to enhance the efficiency of PDT (Fig. 13). Moreover, pyropheophorbide a (Ppa) is also suitable as a PS, which can absorb the red region of visible light emitting from the UCNPs (red-light-excited PS). 82 The most significant point is that Ppa, as second-generation PS, has a higher quantum yield of 1 O 2 and lower dark toxicity than the general PS UCNP cyanine dye (Cy3) nanocomposites. UCNPscomplex nanocomposites can also act as a kind of biological sensor. Zou et al. conjugated a cyanine dye (hcy3) with Nd 3+ - sensitized UCNPs by long alkyl chains to detect the hypochlorite (ClO ) molecules. 85 Hypochlorous acid (HOCl) or hypochlorite (ClO ) has received increasing attention because of its important role in animal immune systems, especially during phagocytosis. The polymer on the surface of UCNPs could embed hcy3 and make it absorb the green fluorescence emitted from UCNPs. As a ClO -sensitive chromophore, hcy3 could detect the molecules via reacting irreversibly with ClO. Through the increase of the luminescence intensity of UCNPs, the ClO molecules could be detected by using a confocal microscope in the hypochlorite pre-treated HeLa cells UCNP Indocyanine Green (ICG) nanocomposites. Most traditional dyes can only be excited to emit visible light. Some novel organic molecules such as Indocyanine Green (ICG) exhibit a strong photoacoustic signal at low concentration. 86 Recently, Liu and co-workers synthesized Gd 3+ and Nd 3+ -doped UCNPs and conjugated them with ICG. The versatile applications of the Nd 3+ -doped UCNPs as photosensitizing nanoplatforms for NIR-triggered UC luminescence have received much attention. This method allows achieving three biological imaging goals in one nanocomposite, namely 808 nm remarkable light penetration depth, the absence of autofluorescence in biological specimens under NIR excitation, and the ability to overcome the drawbacks associated with excitation under UV/visible light. Furthermore, magnetic resonance imaging provided by Gd 3+ and lanthanide ions can generate a high-resolution signal to be detected. Finally, photoacoustic imaging combines the merits of a rich optical contrast and deeper penetration in biological tissue as compared with other optical imaging techniques (Fig. 14) UCNP glycan nanocomposites. The metabolic labeling strategy is a kind of targeting method which could be Fig. 13 (a, b) UCNP-loaded RB as a nanotherapeutic platform. 79 (c, d) Energy transfer system emitting green and red fluorescence. (e) Loading of Ce6 and MC540 to achieve PDT. 81 (f) Ce6 and MC540 cross-sections with the emitted fluorescence of Er 3+ -doped UCNPs under 808 nm. (Reprinted with permission from ref. 79 and 81. Copyright 2016 and 2017, American Chemical Society.) Fig. 14 (a) Application of ICG combined with UCNPs as diagnostic nanotools. (b) Multifunctional composites can achieve the functions of UCL, MRI, and PAI in vivo. (c, d) UCNP composites can be imaged thermally and by fluorescence in vivo. (e) NIR light can penetrate tissue to a depth of 2.5 cm. 86 (Reprinted with permission from ref. 86. Copyright 2016, Wiley.) 18164,2017,9, This journal is The Royal Society of Chemistry 2017

13 applied for controlling cellular regulation at a specific position. Ai and co-workers modified a monosaccharide precursor, peracetylated Nazidoacetyl-mannosamine (Ac4ManNAz), on the surface of Nd 3+ sensitized-ucnps. 87 As an azido tag, Ac4ManNAz can anchor the UCNPs on the cell membrane by glycoconjugates. Upon 808 nm light excitation, the upconverted emission at 480 nm from UCNPs could activate channelrhodopsin-2 (ChR2), the light-gated membrane protein, and therefore manipulate cation influx (e.g., Ca 2+ ) across the cell membrane to remotely regulate physiological processes under living conditions (Fig. 15a). The authors also demonstrate the metabolic glycan labeling in zebrafish to study the feasibility of metabolic glycan labeling for localized UCP attachment in vivo (Fig. 15b). This illustrates that UC light can even be used as a high-efficiency photocatalytic nanoplatform for biological applications. 5. Toxicity assessment of UCNPs Although many innovative techniques have been developed in nanobiotechnology and nanomedicine, nanomaterials with novel physical and chemical properties exhibit unpredictable interactions at biological interfaces. The consequences of these interactions are potentially dangerous and toxic. Hence, a thorough understanding of the toxicological properties of UCNP is necessary for designing safe composites. Researchers must conduct in-depth investigations to determine the properties of lanthanide elements. The main element produced by the UCNPs is lanthanide metal ions. All rare earth elements of the trivalent compounds are close to the toxicity of iron ions, which have non-significant toxicity. 88 At high concentrations, the toxicity of lanthanides is through biological metabolism. The lanthanide binds with the nucleic acid and nuclear protein interfering with cell metabolism, which inhibits phosphatase and affects hematopoiesis. The recent literature confirms that lanthanide ions do not have significant acute toxicity at concentrations less than 1 mm. Only several lighter rare earth elements such as La 3+,Ce 3+, and Pr 3+ may induce fatty liver at high concentrations and cause hepatotoxicity. 89 The lanthanide metals used for the UCNPs are mainly Y 3+,Yb 3+,Nd 3+,Gd 3+,Tm 3+,Er 3+, and Ho 3+. The toxicity of these elements is relatively low, indicating that the UCNPs can be used as a candidate for biological nanotechnology. The toxicity of nanomaterials does not only depend on the composition of the parent material. Moreover, the toxicity of nanomaterials is affected by the physical and chemical properties of the nanomaterials themselves. These properties include their sizes, surface chemistries, shapes, protein absorption gradients, and surface morphologies. Therefore, nanostructures that are biocompatible in vitro must be further modified prior to in vivo applications, such as the surface ligand modification of UCNPs. The results of in vitro experiments can be used to model the biomechanics and in vivo behavior of nanomaterials, as well as provide relevant data. According to recent research, lanthanide nanoparticles with controlled morphology (e.g. the sphere or square), size (<50 nm), and intense emission have easy access to the intracellular compartment, fast kidney filtration and induce less toxicity. Some of the ultrasmall Nd 3+ -sensitized nanoparticles can be smaller than 10 nm. 90 This size benefit includes longer blood half-times due to reduced uptake by the reticuloendothelial system, which is more beneficial for the particles delivered by intravenous injection to the targeted sites. For now, the scientific community must study further the acute and chronic physiological effects of UCNP in vivo toxicity. To understand the metabolism of UCNPs, the biological interactions between the UCNPs and cells need to be considered. The initial point of contact will be affected by surface modifications. Moreover, the toxic effects of UCNP exposure via skin contact and other non-ingestion routes must be explored. The toxic effects of nanomaterials are identified by characterizing the nanomaterials and through in vitro and in vivo evaluation. Finally, the toxicity of the material can be attenuated by adjusting the nanocomposite synthesis process. Fig. 15 (a) Schematic illustration of the metabolic labeling strategy for site-specific covalent localization of NIR-light-responsive UCNPs on the cell membrane. (b) In vivo fluorescence imaging with and without NIR light treatment (0.8 W cm 2 for 2 h) in zebrafish 87 (Reprinted with permission from ref. 87. Copyright 2017, Wiley.) 6. Conclusions and prospects NPs and biomolecule detection technology have matured and have been applied in medical testing equipment, disease screening, and environmental pollutant testing. However, detecting trace ions or very small viruses, such as SARS and This journal is The Royal Society of Chemistry 2017,2017,9,