Near-Infrared Excited Orthogonal Emissive Upconversion

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Near-Infrared Excited Orthogonal Emissive Upconversion Nanoparticles for ImagingGuided On-Demand Therapy Ming Tang,†,‡ Xiaohui Zhu,*,† Yuehong Zhang,† Zeping Zhang,‡ Zhiming Zhang,† Qingsong Mei,§ Jing Zhang,† Minghong Wu,† Jinliang Liu,*,† and Yong Zhang*,†,∥ Downloaded via UNIV OF GOTHENBURG on August 26, 2019 at 23:15:28 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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School of Environmental and Chemical Engineering, Shanghai University, Shanghai, China 200444 School of Life Sciences, Shanghai University, Shanghai, China 200444 § School of Biological and Medical Engineering, Hefei University of Technology, Hefei, China 230009 ∥ Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117583 ‡

S Supporting Information *

ABSTRACT: Photodynamic therapy (PDT) has been considered as a promising and noninvasive strategy for clinical cancer treatment. Nonetheless, building a smart “off−on” theranostic PDT platform to spatiotemporally control the generation of reactive oxygen species in the PDT treatment still remains challenging. Here, we have rationally developed photoswitching upconversion nanoparticles (UCNPs) with orthogonal emissive properties in response to two distinct near-infrared (NIR) emissions at 808 and 980 nm, i.e., red emission with 980 nm excitation and green emission with 808 nm excitation. Unlike traditional photoswitching UCNPs, these specially designed core−shell−shell structured UCNPs do not require complicated multilayer doping as their red and green upconversion luminescence both originate from the same activator Er3+ ions in the core structure. As a proof of concept, we have demonstrated the capability of these orthogonal emissive UCNPs for imaging-guided PDT in a real-time manner, where the red emission excited by 980 nm light is used to trigger PDT and the green emission with 808 nm excitation is to diagnose and monitor the therapeutic treatment. Our study suggests that such specially designed UCNPs with orthogonal emissions hold great promise for NIR light-targeted and imaging-guided therapy under precisely spatiotemporal control. KEYWORDS: upconversion, photoswitch, on-demand therapy, near-infrared, nanoparticle

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the intermediate energy states and luminescence is emitted with shorter wavelength (i.e., higher energy) than the incident light.8 Due to their high photostability, long luminescence lifetime, low autofluorescence background, and deep tissue penetration depth (up to 10 mm),9−13 UCNPs are promising candidates for various bioanalytical and biomedical applications.14−16 Particularly, benefiting from the capability of upconverting near-infrared (NIR) incident light to ultraviolet and visible light, UCNPs have also been extensively employed as light transducers to activate photosensitizers for PDT treatment in the deep lesions. Since Zhang et al. first demonstrated the feasibility of combining UCNP and

hotodynamic therapy (PDT) is a clinically approved and noninvasive treatment that utilizes photosensitizing chemicals, called photosensitizers, along with excitation light to treat cancer and other malignant diseases.1,2 In a PDT treatment, upon irradiation by the light with a certain wavelength, the photosensitizers are activated and generate reactive oxygen species (ROS) that can cause oxidative stress and cellular damages. However, due to the fact that most photosensitizers need to be activated by ultraviolet or visible light that has poor tissue penetration depth, current PDT treatments are still limited to superficial lesions. Recently, lanthanide-doped upconversion nanoparticles (UCNPs) have attracted considerable attentions for applications in chemical sensing, data storage, anticounterfeiting, and optogenetics.3−7 Generally, the upconversion luminescence of UCNPs involves an “anti-Stokes” optical process in which two or more low-energy photons are sequentially absorbed through © XXXX American Chemical Society

Received: May 29, 2019 Accepted: August 22, 2019

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DOI: 10.1021/acsnano.9b04200 ACS Nano XXXX, XXX, XXX−XXX

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Figure 1. TEM image of NaErF4:Yb/Tm core (a), NaErF4:Yb/Tm@NaYF4:Yb core−shell (b), and NaErF4:Yb/Tm@NaYF4:Yb@NaNdF4:Yb core−shell−shell (c) nanoparticles. (d). High magnification TEM image of NaErF4:Yb/Tm@NaYF4:Yb@NaNdF4:Yb core−shell−shell nanoparticles and corresponding STEM elemental mapping of Er3+, Y3+, Yb3+, Tm3+, and Nd3+ ions.

merocyanine (M-540) photosensitizer for NIR-triggered PDT to treat bladder cells,17 many other photosensitzers such as zinc phthalocyanine (ZnPc), chlorin e6 (Ce6), rose bengal, and TiO218−22 have been integrated into UCNPs and these theranostic platforms have been extensively used for PDT in vitro and in vivo.23,24

Despite significant progress in the development of UCNPbased photodynamic therapy, building a smart “off−on” theranostic platform to spatially and temporally control the singlet oxygen (1O2) release during the PDT treatment still remains challenging. Ideally, an optimum theranostic system should rapidly identify the location of lesion areas and efficiently initiate the treatment on the targeted localizations. B


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utilized for the real-time imaging and the red emissions upon 980 nm laser excitation was to generate ROS for the PDT treatment on demand. Our studies showed that the green emissions with 808 nm excitation had negligible effects on cell viability while the red emissions with 980 nm excitation could effectively kill the cancer cells and greatly inhibit the tumor growth. This suggests that the specially designed orthogonal emissive UCNPs can hold great potential for NIR lighttriggered and targeted theranostic platforms for imagingguided therapy under spatiotemporal control.

Taking the advantages of endogenous differences between normal and tumor cells, a great deal of research has been carried out on stimuli-responsive materials which are sensitive to specific endogenous stimuli (e.g., lowered interstitial pH, higher glutathione concentration, and increased level of certain chemical species) and selectively target the cancer cells so as to maximally lower the adverse damage to healthy cells.25−29 However, this approach is rather complex and largely relies on the recognition of certain biomarkers in cancer cells, which, in turn, requires that stimuli-responsive materials must be able to undergo a specific surface charge change, degradation of the encapsulating materials, or hydrolytic cleavage or molecular conformational change in response to the desired stimulations.25 Another method to realize the on-demand treatment is via a bioimaging-guided approach, which offers prior diagnostic information on lesion areas and then guides the subsequent treatments. Recently, UCNPs have been engineered by doping or surface coupling to afford multiple imaging modalities such as magnetic resonance imaging (MRI), photoacoustic (PA), positron emission tomography (PET), and single photon emission computed tomography (SPECT)30,31 to provide the “all-in-one” theranostic platform by combining diagnostic and therapeutic modalities into one system. Although multimodality imaging is a powerful tool to offer diagnostic imaging and therapeutic treatments, some of the modalities still require specific imaging agents or sophisticated instruments. For example, PET modality uses positron-emitting radionuclides such as 18F and 11C, while SPECT uses gamma-emitting radiotracers such as 153Sm for imaging,32 which requires that those radioisotopes need to be well conjugated into the UCNPs and thus makes the synthesis of radioisotope-labeled UCNPs more complicated and challenging. In addition, as the diagnoses and treatments are typically separated, multimodality imaging sometimes cannot guarantee the real-time imagingguided treatment. An alternative approach to achieve real-time bioimaging is to develop photoswitchable UCNPs, which have been applied in imaging-guided PDT and synergistic PDT/ chemotherapy.33,34 However, in previous studies, in order to achieve the orthogonal emissions under different excitation wavelengths, the fabrication of photoswitchable UCNPs always involves the multishell synthesis with four or even more layers, which makes the fabrication work rather complicated and timeconsuming. Besides, the orthogonal emissions always originate from different activators in different layers in traditional photoswitchable UCNPs, and it still remains challenging to obtain orthogonal emissions from the same activator in the same shell. In this study, we report an alternative approach to realize imaging-guided “switch on−off” photodynamic therapy based on photoswitchable UCNPs. A specially designed orthogonal emissive UCNP composed of NaErF4:Yb/Tm@NaYF4:Yb@ NaNdF4:Yb with a simple core−shell−shell architecture was synthesized via a facile layer-by-layer deposition approach. By precisely controlling energy migration processes, the synthesized NaErF4:Yb/Tm@NaYF4:Yb@NaNdF4:Yb UCNPs were demonstrated to release orthogonal upconversion emissions both from the same Er3+ activators in the core structure, i.e., red signals upon 980 nm laser excitation and green signals with 808 nm laser irradiation. A mesoporous silica shell was further deposited onto UCNPs and photosensitizer (zinc phthalocyanine, ZnPc) was incorporated into the mesoporous silica to fabricate an imaging-guided PDT theranostic nanoplatform, in which the green emissions upon 808 nm laser irradiation was

RESULTS AND DISCUSSION Synthesis of Core−Shell−Shell Structured UCNPs with Orthogonal Emissions. A general strategy to achieve the photoswitchable functions in UCNPs is to design a multilayer structure. In this study, the as-designed NaErF4:Yb(19.5%)/Tm(0.5%)@NaYF4 :Yb(10%)@NaNdF4:Yb(10%) UCNPs with core−shell−shell architecture were synthesized by one pot successive layer-by-layer (SLBL) coprecipitation approach. To gain the red and green orthogonal upconversion emissions, the Yb3+ and Tm3+ ions were codoped into the core NaErF4 host lattice since Er3+ activators are known to produce the green and red luminescence and Tm3+ ions were previously shown to further enhance the red upconversion luminescence of Er3+ through energy trapping.35 As for the sensitizers, the Yb3+ sensitizers were confined in the core, inner shell, and outer shell to harvest 980 nm light while Nd3+ sensitizers were only doped in the outer shell to sensitize 808 nm laser light. As shown in Figure 1a, the core NaErY4:Yb/Tm nanoparticles are roughly spherical, become slightly cylindrical after coating the NaYF4:Yb inner shell (Figure 1b), and finally change to a dumbbell shape after coating the NaNdF4:Yb outer shell (Figure 1c). It can be observed that these dumbbell shaped nanoparticles have a uniform size with a width of approximately 30 nm and length of about 70 nm. Note that X-ray diffraction patterns (XRD) confirm that the core, core− shell and core−shell−shell nanoparticles all exhibit the hexagonal phase (Supporting Information, Figure S1). The dumbbell shaped morphology is possibly due to the comprehensive effects of oleate anion (OA−) induced dissolution of the core−shell nanocrystals (NaErF4:Yb/Tm@ NaYF4:Yb) and subsequent promotion of the longitudinal growth of NaNdF4:Yb outer shell.36 It is assumed that the high concentration of OA− ions adsorbed on the nanoparticle surface help to selectively dissolve the NaErF4@NaYF4 nanocrystals and the dissolved F− ions, in turn, participate in the epitaxial growth of NaNdF4:Yb nanocrystals. In addition, the crystallographic mismatch between the core NaErF4 and outer shell NaNdF4 nanocrystals has also probably prevented the transversal migration growth of the NaNdF4 on the NaErF4 core crystals.36 The lattice fringes in the inset in Figure 1c are determined to be 0.18 nm which corresponds to the (0002) plane of NaNdF4:Yb phase, confirming the NaNdF4:Yb shell mainly distributes at the ends of the dumbbell structure.37 Figure 1e represents the scanning transmission electron microscopy (STEM) elemental mapping of Er3+, Y3+, Yb3+, Tm3+, and Nd3+ ions according to the TEM image of the UCNPs in Figure 1d. Clearly, the elemental mapping in Figure 1e shows that most of Er3+ ions are localized in the middle while Nd3+ and Y3+ ions are mainly at the ends of UCNPs. In order to gain insights into the correlation between Nd3+ doping and morphology change of UCNPs, a series of core− shell−shell UCNPs with variable Nd3+ doping in the outer C


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Figure 2. (a) Schematic of the orthogonal emissive properties of the specially designed UCNPs, i.e., red emission with 980 nm laser light and green emission with 808 nm laser light excitation. (b) Upconversion luminesce spectra of the UCNPs under irradiation with 980 or 808 nm laser light. The inset in each panel is the luminescence photo of the same UCNP solution under irradiation by different excitation wavelengths.

population of 4F9/2 state and enhances the red upconversion emission. Upon excitation with 808 nm light, the Nd3+ ions act as the sensitizer and transfer the excitation energy to the Yb ions and then to the Er3+ activators in the core. However, as the Nd3+ sensitizer ions are only localized in the outer shell, the absorbance of the 808 nm excitation light is significantly lower compared with 980 nm light. In addition, as the energy migration distance between sensitizers (i.e., Nd3+ ions in the outer shell) and activators (Er3+ ions in the core) is much longer under excitation by 808 nm light, fewer photons reach the luminescent core and the energy transfer to the Tm3+ ions is also limited. Meanwhile, as fewer Er3+ ions are under excitation states, the cross-relaxation effect in the case of 980 D


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Figure 3. Schematic illustration of energy migration pathways of UCNPs under 980 and 808 nm excitations.

nm light excitation is greatly suppressed as well. As a result, when excited by 808 nm light, the UCNPs demonstrate the green colored luminescence of Er3+ ions, in which the green upconversion emission via 2H11/2, 4S3/2 → 4I15/2 transition (Figure 4b) dominates the luminescence process. Furthermore, as shown in Figure S2d, the intensity ratio of green to red upconversion emission with 808 nm excitation dramatically increases with Nd3+ doping in the outer shell, which also suggests that a heavy Nd3+ doping in the outer shell is critical for acquiring dominant green upconversion emissions at 808 nm. Synthesis of Mesoporous-Silica-Coated UCNPs. As the obtained UCNPs were capped with oleic acids in the synthesis procedures, they were insoluble in water and could not be directly used for biological applications. Therefore, another hydrophilic mesoporous SiO2 shell was subsequently coated onto these OA− capped UCNPs to render them water-soluble. The use of mesoporous SiO2 has several advantages, such as transferring the hydrophobic UCNPs to the aqueous phase, protecting the UCNPs from the harsh biological environments, and facilitating the release of reactive oxygen species through the porous structures. Figure 5a demonstrates the size and morphology of mesoporous coated UCNPs (UCNPs@ mSiO2). It clearly shows that the UCNPs are well encapsulated by a uniform mesoporous SiO2 shell with thickness of about 35 ± 2 nm. The N2 adsorption/desorption isotherm (Figure 5b) indicates that the isotherms of UCNPs@mSiO2 can be classified as type-IV with a hysteretic loop according to the IUPAC classification scheme for mesoporous materials. Calculated from the isotherms, the surface area of the UCNPs@mSiO2 is determined to be 605 m2·g−1 using the Brunauer−Emmett−Teller (BET) method and the average pore diameter is determined to be 2.28 nm (inset in Figure 5b), suggesting the as-synthesized UCNPs@mSiO2 can be

Figure 4. Proposed upconversion mechanism for red emission generated under 980 nm laser excitation (a) and green emission under 808 nm laser excitation (b).

used to store large amount of photosensitizers because of their high surface area and porous structures. Construction of UCNPs@mSiO2-ZnPc Nanophotosensitizers. Figure 5c presents the upconversion luminescence spectra of mesoporous SiO2 decorated UCNPs (UCNPs@ mSiO2) under excitation with 980 and 808 nm laser light, respectively. Similar to bare UCNPs, the UCNPs@mSiO2 nanocomposites still emit a dominant red fluorescence (650 nm) and much weaker green fluorescence (540 nm) upon excitation by a 980 nm laser, and generate much stronger green emission than the red one under 808 nm laser irradiation. This indicates that the surface function of bare UCNPs with mesoporous SiO2 does not affect the switchable emission properties of UCNPs as shown in Figure 2. Taking advantage of the near orthogonal emissive properties of UCNPs@mSiO2 nanocomposites under two excitation wavelengths, a theranostic nanoplatform for imaging-guided photodynamic therapy (PDT) is constructed by incorporating photosensitizers into the UCNPs@mSiO2 nanocomposites. Specifically, we have chosen zinc phthalocyanine (ZnPc) as the photosensitizer for the PDT treatment as it has a strong absorption cross section in the spectral range of 600−700 nm which overlaps well with the dominant red emission (650 nm) of UCNPs@mSiO2 nanocomposites under 980 nm laser irradiation (Figure 5c). Meanwhile, the bright green emission (540 nm) upon 808 nm light irradiation can be used for imaging-guided diagnostic purpose. In order to load ZnPc photosensitizers into the E


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between the red emission at 660 nm of UCNPs@mSiO2 and the absorption at 600−700 nm of ZnPc photosensitizers (Figure 5c), the nonradiative energy transfer is expected to occur from UCNPs@mSiO2 to ZnPc photosensitizers. Moreover, the release of ZnPc from UCNPs@mSiO2−ZnPc was also studied. The UCNPs@mSiO2−ZnPc nanoparticles were soaked in deionized water, PBS buffer, ethanol, and cell culture medium (DMEM) for 24 h, and the supernatants after centrifugation were collected. The UV−vis absorption spectra of the supernatants are shown in Figure S4. It shows there are no significant absorbance changes in the solution of deionized water, PBS buffer, and DMEM except ethanol, suggesting that ZnPc can well remain in mesoporous silica when incubated with cells in culture medium. Single Oxygen Production from UCNPs@mSiO2− ZnPc. The ability of the UCNPs@mSiO2−ZnPc nanophotosensitizers to generate single oxygen (1O2) under 980 and 808 nm laser excitations was evaluated using the dye 1,3diphenylisobenzofuran (DPBF) as an acceptor of 1O2. DPBF has a highly specific reactivity toward singlet 1O2, and the quenching reaction between DPBF and singlet 1O2 leads to a decrease in absorption intensity of DPBF.38 Therefore, the generation of the singlet 1O2 can be well monitored by measuring the decrease in the absorption signal of DPBF under laser irradiations. Figure 6a shows the absorption spectra of DPBF solution irradiated by 980 nm laser light in the presence of UCNPs@mSiO2−ZnPc nanophotosensitizers. It can be seen that the absorbance of DPBF at about 417 nm decreases rapidly within 30 min, while the absorbance of ZnPc at 660 nm stays almost unchanged. Figure 6b compares the absorbance changes of DPBF at 417 nm over time due to the singlet 1O2 generation under different conditions. The results show that the absorbance of DPBF solution changes little with either 980 nm (blue triangle) or 808 nm (red square) laser irradiations when UCNPs@mSiO2−ZnPc nanophotosensitizers are not present. When UCNPs@mSiO2−ZnPc nanophotosensitizers are added into the DPBF solution, the absorbance intensity of DPBF at 417 nm decreases by about 30% upon 980 nm laser irradiation for 30 min (purple rhombus) while it only decreases by 5% with 808 laser irradiation (dark square), indicating that much more singlet 1O2 species are generated upon 980 nm laser excitation than at 808 nm. This is particularly important, as singlet 1O2 species can cause oxidative damage to DNA, mitochondria, and cell membranes and lead to cell death. However, the results in Figure 6b clearly indicate that the UCNPs@mSiO2−ZnPc nanophotosensitizers upon 808 nm laser irradiation produced very few singlet 1O2 species and probably cause negligible effects on cell activities, which thus makes the green emission by 808 nm laser excitation a suitable tool for real-time diagnose or monitoring the therapeutic treatment. Therefore, real-time imaging-guided PDT can be achieved using orthogonal emissive UCNPs@ mSiO2−ZnPc nanocomposites, since photosensitizers, as the green emission with 808 nm laser light, can be used for diagnostic purposes and the red emission upon 980 nm laser light can be used to kill cells in lesions through generation of adequate reactive oxygen species. Imaging-Guided Photodynamic Therapy for Cancer Cells. Before in vitro and in vivo PDT, it is necessary to evaluate the cytotoxicity of the UCNPs@mSiO2 and UCNPs@ mSiO2−ZnPc nanoparticles. The standard CCK-8 assay was performed on human non-small cell lung cancer cells (A549). Figure 7a presents the viability of A549 cells incubated with

Figure 5. (a) TEM image of SiO2 mesoporous coated UCNPs (UCNPs@mSiO2). (b) N2 adsorption/desorption isotherm of UCNPs@mSiO2. The BET surface area is 605.62 m2 g−1. The inset is the pore size distribution of UCNPs@mSiO2 nanoparticles, showing the nanoparticles have a narrow Barrett−Joyner−Halenda (BJH) pore size distribution with a mean size of 2.28 nm. (c) Upconversion luminescence spectrum of UCNPs as donor under excitation of 980 nm laser light (red) and under excitation of 808 nm laser light (green), and the absorption spectrum of ZnPc (black) as acceptor.

mesoporous SiO2 shell, the UCNPs@mSiO2 nanoparticles were soaked into the pyridine solution of ZnPc for 24 h. In the specially constructed nanophotosensitizers, denoted as UCNPs@mSiO2−ZnPc, the adsorbing quantity of the ZnPc was further determined to be 5.15 wt % based on the UV−vis absorption spectra of standard ZnPc solutions with different concentrations (Figure S3). Due to the efficient overlap F


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Figure 7. (a) Viability of A549 cells incubated with UCNPs@ mSiO2 (blue) and UCNPs@mSiO2−ZnPC (red) at different concentrations. (b) Viability of A549 cells incubated with UCNPs@mSiO2−ZnPc at different concentrations for 24 h. The incubated cells were divided into three groups: with 808 nm laser irradiation (235 mW/cm2) for 10 min (green), with 980 nm laser irradiation (608 mW/cm2) for 10 min (purple), and with no light irradiation (yellow).

Figure 6. (a) UV−vis absorption spectra of UCNPs@mSiO2−ZnPc containing DPBF after different irradiation times with 980 nm irradiation. (b) Comparison of DPBF consumption for five different groups: (1) DPBF + 808 nm irradiation (235 mW/ cm2), (2) DPBF + 980 nm irradiation (608 mW/cm2), (3) UCNPs@mSiO2−ZnPc + DPBF, (4) UCNPs@mSiO2−ZnPc + DPBF with 808 nm irradiation (235 mW/cm2), and (5) UCNPs@ mSiO2−ZnPc + DPBF with 980 nm irradiation (608 mW/cm2).

dye. Cellular uptake and distribution of nanoparticles in cells were visualized using confocal laser scanning microscopy (CLSM). As shown in Figure 8, the DAPI is used to enhance the visualization of the cell nucleus (blue). The green upconversion luminescence (UCL) image originates from the upconversion emission signal at 545 nm with the excitation of the 808 nm laser, while the red UCL image is obtained by collecting signals at 650 nm with the excitation of the 980 nm laser. The DAPI image and the upconversion emission images are further merged, which clearly shows that UCNPs@ mSiO2−ZnPc nanoparticles are mainly aggregated around the nucleus in the cytoplasm. Moreover, in order to visualize the ROS signals inside cancer cells, the 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) probe was later used to characterize the distribution of ROS among the cells. It is reported that once DCFH-DA encounters with singlet oxygen, it is oxidized to DCF and emits a green fluorescence signal.39 Figure 9a demonstrates the bright field, generated ROS, and merged images of the A549 cancer cells incubated UCNPs@ mSiO2−ZnPc nanoparticles after irradiation by the 980 and 808 nm laser light for 10 min, respectively. It can be obtained that the ROS signal uniformly distributes almost all over the A549 cells under 980 nm laser irradiation and the morphology of the cells is severely damaged. On the contrary, under 808 nm laser irradiation, only very weak ROS signals can be

different concentrations (12.5, 25, 50, and 100 μg/mL) of UCNPs@mSiO2 and UCNPs@mSiO2−ZnPc nanocomposites. It shows that the cell viability is still over 90% when up to 100 μg/mL of UCNPs@mSiO2 (blue) and UCNPs@mSiO2−ZnPc (red) nanocomposites are incubated with the cells, suggesting that the nanoparticles do not influence A549 cells when their concentration is less than 100 μg/mL. In order to evaluate the PDT effects, Figure 7b presents the viability of A549 cancer cells incubated with different concentrations of UCNPs@ mSiO2−ZnPc irradiated without laser light (yellow), with 980 nm laser light (purple), and with 808 nm laser light (green). It shows an evident drop in the cell viability under 980 nm laser excitation while there is no significant decrease in cell viability under 808 laser light. For example, when the A549 cancer cells are incubated with 100 μg/mL UCNPs@mSiO2−ZnPc nanophotosensitizers, the cell viability is only 20% under 980 laser light while it can still remain at about 80% under 808 laser light. This is also consistent with the ROS detection results in Figure 6 that only 980 nm laser treatment can generate a large amount of ROS and cause cell death. In order to study the imaging-guided PDT treatment on A549 cancer cells, live A549 cells were incubated with UCNPs@mSiO2−ZnPc for 24 h and then stained with DAPI G


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shown in Figure S6. After in vivo imaging, the PDT treatments were carried out. The UCNPs@mSiO2−ZnPc nanoparticles were redispersed in PBS, and then 100 μL of PBS or UCNPs@ mSiO2−ZnPc (10 mg/mL) was injected into each tumorbearing mouse for PDT. Subsequently, the mice groups were divided into four groups as follows: group 1, only injection of PBS with 980 nm laser irradiation (the control group, n = 5); group 2, only injection of UCNPs@mSiO2−ZnPc (n = 5); group 3, injection of UCNPs@mSiO2−ZnPc with 808 nm laser irradiation (n = 5); and group 4, injection of UCNPs@ mSiO2−ZnPc with 980 nm laser irradiation (n = 5). The mice in groups 1, 3, and 4 were irradiated on day 0 and day 3 after injection with nanophotosensitizers. The power density of the 980 nm laser was 608 mW/cm2, and that of the 808 nm laser was 235 mW/cm2. During the treatment, the tumor sizes and body weights were measured every 2 days after the initial treatments. As seen in Figure 10a, the tumor volume of the mice in group 1 (PBS + 980 laser light), group 2 (only UCNPs@mSiO2−ZnPc), and group 3 (UCNPs@mSiO2− ZnPc + 808 nm laser light) apparently increases over time while the tumor growth in group 4 (UCNPs@mSiO2−ZnPc + 980 nm laser light) is greatly inhibited. Compared with the control group 1 (PBS + 980 nm laser), the tumor inhibitory ratio in group 4 is estimated to be about 82%. Figure 10b presents the body weight of the mice in four groups with treatment time, which shows a relatively slow increase in the body weight in groups 1, 2, and 3 while the body weight in group 4 (i.e., PDT treated group) shows an evident increase during the 14 days of treatment. This suggests that the health condition of the mice without PDT treatment is somewhat affected by the tumor growth while the UCNPs@mSiO2− ZnPc nanoparticles have no significant systemic toxicity and no adverse effects on the body weight following PDT treatment. In addition, the typical photographs of tumor-bearing mice (Figure 11a) and excised tumors of respective mice (Figure 11b) also indicate that the treatment of UCNPs@mSiO2− ZnPc with 980 nm irradiation dramatically inhibits the tumor growth and the PDT treated mouse has the lowest tumor size. In order to understand the main mechanisms of PDT treatment, histological analysis on tumor and main organs was carried out for different groups after 14 days of post-treatment, as shown in Figure 12a. It can be observed that the group treated with UCNPs@mSiO2−ZnPc + 980 nm laser presents the most severe apoptosis or necrosis, which can be further evidenced by the distinct cell shrinking and chromatin condensation in hematoxylin and eosin (H&E) stained samples. Figure 12b presents the H&E staining of major organs (heart, liver, spleen, lung, and kidney) from mice in different treatment groups, which shows no obvious organ damage or significant abnormalities for all the treated mice. These results all suggest that the orthogonal UCNPs@mSiO2− ZnPc exhibit no short-term toxicity in mice and show great potential to become a biocompatible PDT agent.

Figure 8. Confocal imaging of A549 cells incubated with medium containing UCNPs@mSiO2 for 24 h. The cell nuclei were stained with DAPI (blue). The green UCL image originated from the upconversion fluorescence (500−600 nm) with the excitation of 808 nm light, and the red UCL image originated from the upconversion fluorescence (600−700 nm) under 980 nm excitation.

detected and the morphology of the cells stills remains intact. The fluorescence intensity of DCF is further analyzed by the flow-cytometry method to quantitatively identify intracellular ROS signals. The bar graph in Figure 9b shows a summary of data from the flow-cytometry analysis. It is apparent that the fluorescence intensity of A549 cells treated with 980 nm laser irradiation shows much stronger ROS signals than that of untreated cells and cells treated with 808 nm laser irradiation. The detailed fluorescence intensity of each sample is displayed in Figure S5. These results further verify the feasibility of using the orthogonal emissive UCNPs as the real-time imagingguided PDT reagent since the dominant green emission under 808 laser light does little harm to the cells and can be used for diagnostic purposes while the red emission under 980 laser light can effectively kill cancers by producing large amounts of ROS. In Vivo PDT. The in vivo photodynamic therapy efficacy of the UCNPs@mSiO2−ZnPc was then investigated on the mouse model. As a proof of principle study, the A549 tumorbearing mice were intratumorally injected with UCNPs@ mSiO2−ZnPc nanophotosensitizers. The mice were evaluated when the tumor grew to a volume about 100 mm3. Initially, in vivo UCL fluorescence images were obtained at 2 h postinjection to identify the location of nanophotosensitizers in tumor. The in vivo upconversion fluorescence signals can be clearly identified by 808 and 980 nm laser irradiations, as

DISCUSSION In general, the emissive properties of photoswitching upconversion nanoparticles (UCNPs) can be tailored by modulating excitation wavelengths, power density, repetition rate, and pulse width.40−43 Although the external stimuli are varied in previous studies, a common strategy to make photoswitching UCNPs capable of releasing orthogonal emissions in response to these stimulations is to dope various sensitizers and activators in different layers, which is rather H


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Figure 9. (a) Confocal imaging of in vitro PDT. A549 cells were incubated with UCNPs@mSiO2−ZnPc for 24 h and then with DCFH-DA probe for 30 min and finally exposed to 980 nm (608 mW/cm2) and 808 nm (235 mW/cm2) laser light for 10 min, and no laser irradiation, respectively. (b) Relative intensity of DCF fluorescence in the flow cytometry measurement for A549 cells with different treatments. Note the control group that means the A549 cells were only incubated with DCFH-DA indicator.

developed in this work can also be extended to regulate the emission properties of other rare earth activators (e.g., Tm3+, Eu3+, and Ho3+) by precisely manipulating the energy migration pathways at different excitation wavelengths. As a proof of concept, we have demonstrated the feasibility of using these orthogonal-emissive UCNPs for imaging-guided PDT treatment, in which the green emission by 808 nm excitation is for real-time monitoring and the red emission upon 980 nm excitation is for on-demand PDT treatment. As clearly shown in Figures 9 and 11, the UCNP-based nanophotosensitizer shows efficient capability to kill tumor cells with 980 nm excitation while it causes negligible effects on cell activities under 808 nm excitation. Taken together, these results demonstrate that our specially designed UCNP-based theranostic nanoplatform has great implications for interactive diagnoses and treatment. Meanwhile, it should be notable that these photoswitching UNCPs may offer intriguing oppor-

challenging and time-consuming as it involves multishell synthesis of a few active and inert layers. Besides, the orthogonal emissions in traditional photoswitchable UCNPs always rely on different activators, such as Tm3+ ions for UV and Er3+ ions for visible emission. It still remains difficult to acquire orthogonal emissions from the same emitter in the same shell. In this work, we present a facile and convenient strategy to synthesize the orthogonal-emissive UCNPs with a simple core−shell−shell architecture. Unlike traditional photoswitching UCNPs, the specially designed UCNPs do not require complicated multilayer doping as the red and green emissions both originate from the same emitter, i.e., Er3+, in the same NaErF4:Tm core. As shown in Figures 2 and 3, by carefully regulating the energy transfer process, the dominant red and green upconversion emission can be obtained with 980 and 808 nm excitation, respectively. It is believed the methodology I


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tunities in other applications such as energy transfer techniques, super-resolution microscopy, programming optogenetics, etc. For example, Förster resonant energy transfer (FRET) combined with these excitation-responsive UCNPs as energy donors and multiple fluorophores as energy acceptors may be a promising probe for biosensing applications. Moreover, if the NIR excitations (i.e., 980 and 808 nm) are alternated to illuminate the sample, the respective upconversion emissions (i.e., red and green) can further function as a switchable light source to excite donor and acceptor fluorophores in the single-molecular FRET scheme, which may provide simultaneous information about the structure and stoichiometry of single molecules with possibly reduced photobleaching and photodamage.44 In addition, the ability to control red and green emissions via excitation wavelength may potentially be used in dual-color super-resolution imaging by separating the emission signals from two color channels.

CONCLUSION In this study, the specially designed orthogonal emissive UCNPs composed of NaErF4:Yb/Tm@NaYF4:Yb@NaNdF4:Yb with a simple core−shell−shell architecture are reported. By precisely controlling energy migration processes, these specially designed UCNPs could release orthogonal upconversion emissions both from the same Er3+ activators in the core structure, i.e., red signals upon 980 nm laser excitation and green signals with 808 nm laser irradiation. As a proof of concept, the mesoporous-silica shell was coated onto UCNPs and photosentitizer (ZnPc) was further incorporated into the mesoporous silica to fabricate an imaging-guided PDT theranostic nanoplatform, in which the green emission with

Figure 10. Relative tumor growth (a) and body weight (b) in different groups after various treatments.

Figure 11. Representative photographs of mice (a) and photos of segregated tumors after 14 days of PDT treatment (b). The white-dashed circle in (a) indicate the location of tumor in each mouse. J


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Figure 12. H&E stained images of tumors (a) and organs (heart, liver, spleen, lung, and kidney) (b) collected from representative mice after various treatments for 14 days. Yb(CH3CO2)3, and 840 mg of NaF were added into 25 mL of OA/ ODE mixture (v/v = 2:3), degassed, and stirred at 110 °C in vacuum for 0.5 h. Then, the temperature was rapidly increased to 300 °C and kept for about 1 h in Ar atmosphere to grow core nanoparticles. Meanwhile, 0.9 mmol Y(CH3CO2)3 and 0.1 mmol Yb(CH3CO2)3 were dissolved in the 8 mL mixture of OA/ODE (v/v = 1:1) to obtain the inner shell precursor solution and then stirred at 150 °C in Ar atmosphere. Once the synthesis of core nanoparticles in the previous step was complete, the inner shell precursor solution was slowly injected into the core reaction solution and the reaction was maintained at 300 °C for 40 min to grow the core−shell nanoparticles. Then, at the end of the synthesis of core−shell nanoparticles, the outer shell precursor solution of 0.9 mmol of Nd(CH3CO2)3 and 0.1 mmol of Yb(CH3CO2)3 in 8 mL of OA/ODE (v/v = 1:1) mixture that was preheated at 150 °C was added dropwise into the core−shell reaction solution and maintained the reaction at 300 °C for another 50 min. Once the reaction was complete, the solution was mixed with equal volume of ethanol to precipitate the products. Then, the products were collected by centrifugation and washed with the hexane/ethanol mixture. Synthesis of Mesoporous Silica-Coated Upconversion Nanoparticles. Mesoporous SiO2 coated UCNPs, denoted as UCNPs@mSiO2, were prepared via a modified approach according to the previous work.45 Briefly, 3 mL of the as-prepared UCNP

808 nm laser excitation was for real-time imaging for diagnosis purposes and the red emission upon 980 nm laser excitation was for on-demand PDT treatment. Our results suggest that the specially designed orthogonal emissive UCNPs can be expected to be a valuable tool in future clinical diagnoses and treatment.

EXPERIMENTAL SECTION Materials. Sodium fluoride (NaF), Nd(CH3CO2)3 (99.99%), Yb(CH3CO2)3 (99.99%), Y(CH3CO2)3 (99.99%), Tm(CH3CO2)3 (99.99%), Er(CH3CO2)3 (99.99%), oleic acid (OA), 1-octadecene (ODE), hexadecyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), zinc(II)-pythalocyanine (ZnPc), pyridine, and 1,3-diphenylisobenzofuran (DPBF) were purchased from SigmaAldrich. 4′,6-Diamidino-2-phenylindole (DAPI) and the ROS assay kit were purchased from Beyotime Biotechnology. Cell Counting Kit8 was purchased from Dojindo. All reagents were used as purchased, unless specified. Cell culture medium, FBS, penicillin−streptomycin, and trypsin-EDTA were purchased from Gbico. A Milli-Q water system (18.2 MΩ·cm, Thermo Fisher) provided the ultrapure water in the experiments. Synthesis of Tm3+-Doped Orthogonal Emissive UCNPs. First, 0.8 mmol Er(CH3CO2)3, 0.005 mmol Tm(CH3CO2)3, 0.195 mmol K


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ACS Nano cyclohexane solution was added to an aqueous solution containing 0.1 g of CTAB and 20 mL of deionized water. The mixture was then stirred vigorously overnight to evaporate cyclohexane solvent, resulting in the formation of a UCNPs−CTAB water solution. Next, 6 mL of ethanol, 300 μL of NaOH solution (2 M), and 400 μL of TEOS were added in sequence into 60 mL of UCNPs−CTAB water solution and refluxed at 70 °C for 2 h under vigorous magnetic stirring. The products were collected by centrifugation and washed with ethanol. In order to remove the pore-generating template CTAB and acquire the mesoporous structures, the as-obtained nanoparticles were redispersed in acidic ethanol solution (pH ∼ 1.5) at 60 °C for 3 h. The whole removal process was repeated twice. Finally, the obtained UCNPs@mSiO2 nanoparticles were dispersed in 20 mL of ethanol. Loading Photosensitizers into UCNPs@mSiO2 Nanoparticles. The photosensitizer, zinc phthalocyanine (ZnPc), was loaded into the pores of the mesoporous silica by soaking UCNPs@mSiO2 nanoparticles (10 mg) in a 1 mL solution of ZnPc in pyridine (0.2 mg/mL) for 24 h at room temperature. The nanoparticles were then centrifuged and washed with deionized water to remove the nonabsorbed ZnPc photosensitizers. The obtained UCNPs@ mSiO2−ZnPc was redispersed in water, and its UV−vis absorption spectrum was measured. To calculate the loading capacity, the UV− vis absorption spectra of standard ZnPc solutions with different concentrations were first prepared and then the absorbance and concentration data were plotted in a calibration curve (Figure S3). The loading capacity was determined to be 5.15% according to the calibration curve in Figure S3. Release Study of Photosensitizer from UCNPs@mSiO2 Nanoparticles. An amount of 1 mg of the ZnPc-loaded UCNPs@ mSiO2 nanoparticles (UCNPs@mSiO2−ZnPc) was soaked in either 1 mL of deionized water, 1× PBS, cell culture medium (DMEM), or ethanol for 24 h. The UCNPs@mSiO2−ZnPc nanoparticles were centrifuged out of the solution at 10 000 rpm for 15 min, and the supernatant was collected to determine the existence of photosensitizers that released out into the solution by measuring the UV− vis absorbance. Detection of Singlet Oxygen (1O2). The generation of singlet oxygen was determined by using a DPBF probe. In a typical 1O2 detection experiment, 10 μL of ethanol solution of DPBF (10 mmol/ L) was added to 2 mL of a UCNPs@mSiO2−ZnPc solution and then transferred into a 10 mm cuvette. The solution was protected from room light and irradiated with a 980 nm (608 mW/cm2) or 808 nm (235 mW/cm2) laser for 30 min. The absorption intensity of DPBF at 417 nm was recorded every 5 min. For the control experiments, DPBF absorption was also recorded for comparison at the same conditions in the absence of UCNPs@mSiO2−ZnPc or 980 nm irradiation. Cell Cytotoxicity and Photodynamic Therapy of Cancer Cells. The A549 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown in DMEM medium supplemented with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL streptomycin. Cells were maintained at 37 °C in a humidified 95% atmosphere containing 5% CO2. The cytotoxicity was measured using CCK-8 kits. The A549 cells were seeded in 96-well plates (8000 cells per well). After cultivation for 24 h, 100 μL of UCNPs@mSiO2 or UCNPs@mSiO2−ZnPc nanoconjugates was added into the culture medium at different concentrations, with five parallel wells for each concentration (0, 12.5, 25, 50, and 100 μg/mL). The CCK-8 assay was carried out to determine the cell viability relative to the control cells. For the in vitro PDT experiment, the cells were incubated with different concentrations of UCNPs@mSiO2− ZnPc (0, 12.5, 25, 50, and 100 μg/mL) for another 24 h in an incubator and then washed at least twice with PBS before being exposed to NIR irradiation. A power adjustable 980 nm fiber laser was collimated and employed as an area light source to irradiate the 96well plate. After 10 min irradiation with the 980 nm laser, the cells were allowed to incubate for another 24 h. Typically, 10 μL of CCK-8 kit was added into each well and incubated for 2 h at 37 °C. The absorbance in each well was measured at 450 nm by Tecan Spark

multifunctional microplate reader. Cell viability was then calculated as follows: [A ]laser /[A ]no‐ laser × 100 where [A]laser is the absorbance of the laser irradiated sample and [A]no‑laser is the absorbance of the respective no-laser irradiated sample. Upconversion Luminescent Imaging of Cells. A549 cells were seeded in the confocal dishes at a concentration of 1.5 × 105 cells per dish. After 24 h of cell attachment, cells were incubated with 100 μg/ mL UCNPs@mSiO2 for 24 h at 37 °C. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature. Then, cells were washed with PBS for three times. Cell nuclei were later stained with DPAI for 15 min at room temperature followed by washing twice with PBS for 5 min each. The A549 cells were imaged by a confocal laser microscope (Olympus FV 3000, Japan). Dark field, DAPI, and UCL imaging of UCNPs@mSiO2 was performed by using confocal microscope equipped with external 808 and 980 nm lasers. Intracellular ROS Detection. ROS generated inside cells was detected using the DCFH-DA Reactive Oxygen Species Assay Kit. A549 cells were seeded in confocal dishes at a density of 1.5 × 105 cells per dish. After incubation with UCNPs@mSiO2−ZnPc for 24 h, DCFH-DA probe was loaded into the cells for 30 min. Cells were washed with PBS three times and then exposed to the 980 nm (608 mW/cm2) or 808 nm (235 mW/cm2) laser for 10 min. After irradiation, fluorescent images of treated cells were acquired using a laser confocal microscope by excitation at 488 nm. The control dishes were treated with UCNPs@mSiO2−ZnPc but without laser irradiation. To further determine the generation of ROS, the fluorescence intensity of DCF was quantified by flow cytometry (Beckman CytoFLEX, USA). The A549 cells were loaded with 5 μM DCFH-DA probe at 37 °C for 30 min with gentle shaking. Then, the samples were washed with mild PBS. After different treatments, the cells were collected via trypsin and ROS signals were detected immediately by flow cytometry. Tumor Model and in Vivo PDT. BALB/c mice (4 weeks old, ∼20 g) were provided by the Shanghai Laboratory Animals Center (SLAC, Shanghai). All animal experiments were performed with the guidelines of the Institutional Animal Care and Use Committee of Shanghai University. A549 cells were harvested by incubation with 0.05% trypsin-EDTA and then collected by centrifugation and suspended in serum-free medium. Cells (107 cells per mouse) were subcutaneously implanted into the right oxter of each mouse. When the tumor volume reached about 100 mm3, A549-tumor-bearing mice were ready for in vivo imaging and PDT was then performed. The in vivo UCL imaging was performed with IVIS Lumina, equipped with 808 and 980 nm lasers as the excitation source. The mice were randomly divided into four groups, which were treated by intratumoral injection of (1) PBS (100 μL) + 980 nm laser, (2) UCNPs@mSiO2−ZnPc (10 mg/mL, 100 μL), (3) UCNPs@mSiO2− ZnPc (10 mg/mL, 100 μL) + 808 nm laser, and (4) UCNPs@ mSiO2−ZnPc (10 mg/mL, 100 μL) + 980 nm laser. The power densities of the 808 and 980 nm lasers were 235 and 608 mW/cm2, respectively. The total time of laser irradiation was 20 min, but, in order to avoid any skin tissue damage by heating, the mouse was exposed to the laser irradiation for 5 min with a 3 min interval each time. After first treatment, the tumor size and body weight were recorded every 2 days. The tumor volumes (mm3) were estimated by the formula: V = (LW 2)/2 , and the relative tumor volumes were calculated as V /V0 (where V0 is the initial tumor volume before treatment). The inhibition efficacy of PDT was

Vc − Vt Vc

× 100%, where

Vt and Vc stand for the tumor volumes of the treated mice and control mice, respectively. Histological Analysis. After treatment for 14 days, the mice were dissected. The main organs (heart, liver, spleen, lung, and kidney) and tumors from the treatment group and control group were collected for H&E staining and then examined by fluorescence microscopy. L


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(8) Wilhelm, S. Perspectives for Upconverting Nanoparticles. ACS Nano 2017, 11, 10644−10653. (9) Carlos, L. D.; Ferreira, R. A. S.; De Zea Bermudez, V.; Ribeiro, S. J. L. Lanthanide-Containing Light-Emitting Organic-Inorganic Hybrids: A Bet on the Future. Adv. Mater. 2009, 21, 509−534. (10) Wang, F.; Liu, X. Recent Advances in the Chemistry of Lanthanide-Doped Upconversion Nanocrystals. Chem. Soc. Rev. 2009, 38, 976−989. (11) Gu, B.; Zhang, Q. Recent Advances on Functionalized Upconversion Nanoparticles for Detection of Small Molecules and Ions in Biosystems. Adv. Sci. 2018, 5, 1700609. (12) Yang, Y.; Shao, Q.; Deng, R.; Wang, C.; Teng, X.; Cheng, K.; Cheng, Z.; Huang, L.; Liu, Z.; Liu, X.; Xing, B. In Vitro and in Vivo Uncaging and Bioluminescence Imaging by Using Photocaged Upconversion Nanoparticles. Angew. Chem., Int. Ed. 2012, 51, 3125−3129. (13) Chatterjee, D. K.; Yong, Z. Upconverting Nanoparticles as Nanotransducers for Photodynamic Therapy in Cancer Cells. Nanomedicine 2008, 3, 73−82. (14) Wang, F.; Banerjee, D.; Liu, Y.; Chen, X.; Liu, X. Upconversion Nanoparticles in Biological Labeling, Imaging, and Therapy. Analyst 2010, 135, 1839−1854. (15) Chen, G.; Qiu, H.; Prasad, P. N.; Chen, X. Upconversion Nanoparticles: Design, Nanochemistry, and Applications in Theranostics. Chem. Rev. 2014, 114, 5161−5214. (16) Zhou, J.; Liu, Z.; Li, F. Upconversion Nanophosphors for Small-Animal Imaging. Chem. Soc. Rev. 2012, 41, 1323−1349. (17) Zhang, P.; Steelant, W.; Kumar, M.; Scholfield, M. Versatile Photosensitizers for Photodynamic Therapy at Infrared Excitation. J. Am. Chem. Soc. 2007, 129, 4526−4527. (18) Xia, L.; Kong, X.; Liu, X.; Tu, L.; Zhang, Y.; Chang, Y.; Liu, K.; Shen, D.; Zhao, H.; Zhang, H. An Upconversion Nanoparticle−Zinc Phthalocyanine Based Nanophotosensitizer for Photodynamic Therapy. Biomaterials 2014, 35, 4146−4156. (19) Dou, Q. Q.; Teng, C. P.; Ye, E.; Loh, X. J. Effective NearInfrared Photodynamic Therapy Assisted by Upconversion Nanoparticles Conjugated with Photosensitizers. Int. J. Nanomed. 2015, 10, 419−432. (20) Sabri, T.; Pawelek, P. D.; Capobianco, J. A. Dual Activity of Rose Bengal Functionalized to Albumin-Coated Lanthanide-Doped Upconverting Nanoparticles: Targeting and Photodynamic Therapy. ACS Appl. Mater. Interfaces 2018, 10, 26947−26953. (21) Lucky, S. S.; Muhammad Idris, N.; Li, Z.; Huang, K.; Soo, K. C.; Zhang, Y. Titania Coated Upconversion Nanoparticles for NearInfrared Light Triggered Photodynamic Therapy. ACS Nano 2015, 9, 191−205. (22) Gai, S.; Yang, G.; Yang, P.; He, F.; Lin, J.; Jin, D.; Xing, B. Recent Advances in Functional Nanomaterials for Light−Triggered Cancer Therapy. Nano Today 2018, 19, 146−187. (23) Idris, N. M.; Gnanasammandhan, M. K.; Zhang, J.; Ho, P. C.; Mahendran, R.; Zhang, Y. In Vivo Photodynamic Therapy Using Upconversion Nanoparticles as Remote-Controlled Nanotransducers. Nat. Med. 2012, 18, 1580−1585. (24) Idris, N. M.; Jayakumar, M. K. G.; Bansal, A.; Zhang, Y. Upconversion Nanoparticles as Versatile Light Nanotransducers for Photoactivation Applications. Chem. Soc. Rev. 2015, 44, 1449−1478. (25) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991−1003. (26) Wang, C.; Cheng, L.; Liu, Y.; Wang, X.; Ma, X.; Deng, Z.; Li, Y.; Liu, Z. Imaging-Guided pH-Sensitive Photodynamic Therapy Using Charge Reversible Upconversion Nanoparticles Under NearInfrared Light. Adv. Funct. Mater. 2013, 23, 3077−3086. (27) Pan, W.; Shi, M.; Li, Y.; Chen, Y.; Li, N.; Tang, B. A GSHResponsive Nanophotosensitizer for Efficient Photodynamic Therapy. RSC Adv. 2018, 8, 42374−42379. (28) Hu, P.; Wang, R.; Zhou, L.; Chen, L.; Wu, Q.; Han, M.-Y.; ElToni, A. M.; Zhao, D.; Zhang, F. Near-Infrared-Activated Upconversion Nanoprobes for Sensitive Endogenous Zn2+ Detection

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b04200. XRD patterns of NaErF4:Yb/Tm core, NaErF4:Yb/ Tm@NaYF4:Yb core−shell and NaErF4:Yb/Tm@NaYF4:Yb@NaNdF4:Yb core−shell−shell nanoparticles; TEM images of NaErF 4 :Yb/Tm@NaYF 4 :Yb@ NaYF4:10%Yb, x%Nd UCNPs; intensity ratio of green to red upconversion emission at 808 nm with different Nd3+ concentrations in the NaYF4:10%Yb, x%Nd outer shell; UV−vis absorption spectra of standard ZnPc solutions with different concentrations; calibration curve (concentration vs absorbance) fitted by unweighted least-squares linear regression; UV−vis absorption spectra of supernatants of UCNPs@mSiO2-ZnPc nanophotosensitizers; flow cytometric analysis of intracellular ROS for different groups; in vivo UCL imaging of A549 tumor-bearing mice with UCNPs@mSiO2−ZnPc under 808 and 980 nm laser lights (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xiaohui Zhu: 0000-0001-9146-8909 Qingsong Mei: 0000-0003-4327-6931 Minghong Wu: 0000-0002-9776-671X Yong Zhang: 0000-0002-1303-0458 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We acknowledge the financial support from Shanghai Sailing Program (19YF1415200), National Natural Science Foundation of China (No.31671011), and Innovative Research Team of High-Level Local Universities in Shanghai. REFERENCES (1) Hopper, C. Photodynamic Therapy: A Clinical Reality in the Treatment of Cancer. Lancet Oncol. 2000, 1, 212−219. (2) Huang, Z. A. Review of Progress in Clinical Photodynamic Therapy. Technol. Cancer Res. Treat. 2005, 4, 283−293. (3) Christ, S.; Schäferling, M. Chemical Sensing and Imaging Based on Photon Upconverting Nano-and Microcrystals: A Review. Methods Appl. Fluoresc. 2015, 3, 034004. (4) Zhang, Z.; Shikha, S.; Liu, J.; Zhang, J.; Mei, Q.; Zhang, Y. Upconversion Nanoprobes: Recent Advances in Sensing Applications. Anal. Chem. 2019, 91, 548−568. (5) Gu, M.; Zhang, Q.; Lamon, S. Nanomaterials for Optical Data Storage. Nat. Rev. Mater. 2016, 1, 16070. (6) Chen, S.; Weitemier, A. Z.; Zeng, X.; He, L.; Wang, X.; Tao, Y.; Huang, A. J. Y.; Hashimotodani, Y.; Kano, M.; Iwasaki, H.; Parajuli, L. K.; Okabe, S.; Loong Teh, D. B.; All, A. H.; Tsutsui-Kimura, I.; Tanaka, K. F.; Liu, X.; McHugh, T. J. Near-Infrared Deep Brain Stimulation via Upconversion Nanoparticle−Mediated Optogenetics. Science 2018, 359, 679−684. (7) Xu, J.; Gulzar, A.; Yang, P.; Bi, H.; Yang, D.; Gai, S.; He, F.; Lin, J.; Xing, B.; Jin, D. Recent Advances in Near-Infrared Emitting Lanthanide-Doped Nanoconstructs : Mechanism, Design and Application for Bioimaging. Coord. Chem. Rev. 2019, 381, 104−134. M


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ACS Nano and Selective On-Demand Photodynamic Therapy. Anal. Chem. 2017, 89, 3492−3500. (29) Feng, L.; Xie, R.; Wang, C.; Gai, S.; He, F.; Yang, D.; Yang, P.; Lin, J. Magnetic Targeting, Tumor Microenvironment-Responsive Intelligent Nanocatalysts for Enhanced Tumor Ablation. ACS Nano 2018, 12, 11000−11012. (30) Sun, T.; Ai, F.; Zhu, G.; Wang, F. Upconversion in Nanostructured Materials: From Optical Tuning to Biomedical Applications. Chem. - Asian J. 2018, 13, 373−385. (31) Liu, Q.; Feng, W.; Li, F. Water-Soluble Lanthanide Upconversion Nanophosphors: Synthesis and Bioimaging Applications in Vivo. Coord. Chem. Rev. 2014, 273−274, 100−110. (32) Zhang, L.; Villalobos, A. Recent Advances in the Development of PET and SPECT Tracers for Brain Imaging. Annu. Rep. Med. Chem. 2012, 47, 105−119. (33) Li, X.; Guo, Z.; Zhao, T.; Lu, Y.; Zhou, L.; Zhao, D.; Zhang, F. Filtration Shell Mediated Power Density Independent Orthogonal Excitations-Emissions Upconversion Luminescence. Angew. Chem., Int. Ed. 2016, 55, 2464−2469. (34) Zuo, J.; Tu, L.; Li, Q.; Feng, Y.; Que, I.; Zhang, Y.; Liu, X.; Xue, B.; Cruz, L. J.; Chang, Y.; Zhang, H.; Kong, X. Near Infrared Light Sensitive Ultraviolet−Blue Nanophotoswitch for Imaging-Guided “Off−On” Therapy. ACS Nano 2018, 12, 3217−3225. (35) Chen, Q.; Xie, X.; Huang, B.; Liang, L.; Han, S.; Yi, Z.; Wang, Y.; Li, Y.; Fan, D.; Huang, L.; Liu, X. Confining Excitation Energy in Er3+-Sensitized Upconversion Nanocrystals through Tm3+-Mediated Transient Energy Trapping. Angew. Chem., Int. Ed. 2017, 56, 7605− 7609. (36) Liu, D.; Xu, X.; Du, Y.; Qin, X.; Zhang, Y.; Ma, C.; Wen, S.; Ren, W.; Goldys, E. M.; Piper, J. A.; Dou, S.; Liu, X.; Jin, D. ThreeDimensional Controlled Growth of Monodisperse Sub-50 nm Heterogeneous Nanocrystals. Nat. Commun. 2016, 7, 10254. (37) Xu, B.; Zhang, X.; Huang, W.; Yang, Y.; Ma, Y.; Gu, Z.; Zhai, T.; Zhao, Y. Nd3+ Sensitized Dumbbell-like Upconversion Nanoparticles for Photodynamic Therapy Application. J. Mater. Chem. B 2016, 4, 2776−2784. (38) Carloni, P.; Damiani, E.; Greci, L.; Stipa, P.; Tanfani, F.; Tartaglini, E.; Wozniak, M. On the Use of 1,3-Diphenylisobenzofuran (DPBF). Reactions with Carbon and Oxygen Centered Radicals in Model and Natural Systems. Res. Chem. Intermed. 1993, 19, 395−405. (39) Rastogi, R. P.; Singh, S. P.; Häder, D.-P.; Sinha, R. P. Detection of Reactive Oxygen Species (ROS) by the Oxidant-Sensing Probe 2′,7′-Dichlorodihydrofluorescein Diacetate in the Cyanobacterium Anabaena Variabilis PCC 7937. Biochem. Biophys. Res. Commun. 2010, 397, 603−607. (40) Lai, J.; Zhang, Y.; Pasquale, N.; Lee, K.-B. An Upconversion Nanoparticle with Orthogonal Emissions Using Dual NIR Excitations for Controlled Two-Way Photoswitching. Angew. Chem., Int. Ed. 2014, 53, 14419−14423. (41) Boyer, J.-C.; Carling, C.-J.; Gates, B. D.; Branda, N. R. TwoWay Photoswitching Using One Type of Near-Infrared Light, Upconverting Nanoparticles, and Changing Only the Light Intensity. J. Am. Chem. Soc. 2010, 132, 15766−15772. (42) Gainer, C. F.; Joshua, G. S.; De Silva, C. R.; Romanowski, M. Control of Green and Red Upconversion in NaYF 4:Yb3+,Er3+ Nanoparticles by Excitation Modulation. J. Mater. Chem. 2011, 21, 18530−18533. (43) Dawson, P.; Romanowski, M. Excitation Modulation of Upconversion Nanoparticles for Switch-Like Control of Ultraviolet Luminescence. J. Am. Chem. Soc. 2018, 140, 5714−5718. (44) Santoso, Y.; Hwang, L. C.; Le Reste, L.; Kapanidis, A. N. Red Light, Green Light: Probing Single Molecules Using Alternating-Laser Excitation. Biochem. Soc. Trans. 2008, 36, 738−744. (45) He, Q.; Gao, Y.; Zhang, L.; Bu, W.; Chen, H.; Li, Y.; Shi, J. One-Pot Self-Assembly of Mesoporous Silica Nanoparticle-Based pHResponsive Anti-Cancer Nano Drug Delivery System. J. Mater. Chem. 2011, 21, 15190−15192.

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Near-Infrared Excited Orthogonal Emissive Upconversion

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