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Lanthanide-doped upconversion nanoparticles electrostatically coupled with photosensitizers for near-infrared-triggered photodynamic therapy† Meng Wang,ac Zhuo Chen,b Wei Zheng,a Haomiao Zhu,a Shan Lu,a En Ma,a Datao Tu,a Shanyong Zhou,b Mingdong Huangb and Xueyuan Chen*ab Lanthanide-doped upconversion nanoparticles (UCNPs) have recently shown great promise in photodynamic therapy (PDT). Herein, we report a facile strategy to fabricate an efficient NIR-triggered PDT system based on LiYF4:Yb/Er UCNPs coupled with a photosensitizer of a b-carboxyphthalocyanine zinc (ZnPc-COOH) molecule via direct electrostatic interaction. Due to the close proximity between UCNPs and ZnPc-COOH, we achieved a high energy transfer efficiency of 96.3% from UCNPs to ZnPc-

Received 4th April 2014 Accepted 6th May 2014

COOH, which facilitates a large production of cytotoxic singlet oxygen and thus an enhanced PDT

DOI: 10.1039/c4nr01826e

inhibition of tumor growth both in vitro and in vivo, thereby revealing the great potential of the UCNP-

www.rsc.org/nanoscale

based PDT systems as noninvasive NIR-triggered PDT agents for deep cancer therapy.

efficacy. Furthermore, we demonstrate the high efficacy of such a NIR-triggered PDT agent for the

1

Introduction

Photodynamic therapy (PDT) based on the photochemical reactions of the photosensitizers has gained growing acceptance as a noninvasive medical technique for cancer treatment in recent years, due to its high therapeutic efficacy and less side effects in comparison with radiotherapy and chemotherapy.1,2 It is generally established that, the cytotoxic reactive oxygen species in PDT are produced by the excited photosensitizer under light with an appropriate wavelength, which cause the oxidative damage of tumor tissues.3–6 However, in conventional PDT, most of the photosensitizers are activated by visible or even ultraviolet (UV) light with poor tissue-penetration capacity, which thus limits the application of PDT in the treatment of large or internal tumors.7,8 Recently, lanthanide (Ln3+)-doped upconversion nanoparticles (UCNPs) have received much attention in versatile bioapplications due to their ability of converting near-infrared (NIR) light to visible light.9–17 Along with the remarkable light penetration depth and the absence of auto-uorescence in biological specimens under NIR excitation, these UCNPs have a

Key Laboratory of Optoelectronic Materials Chemistry and Physics, Key Laboratory of Design and Assembly of Functional Nanostructures, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China. E-mail: [email protected]irsm.ac.cn; Fax: + 86 591 8764-2575; Tel: + 86 591 8764-2575

b

State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China

c

University of Chinese Academy of Sciences, Beijing 100049, China

† Electronic supplementary information (ESI) available: Tables S1 and S2 and Fig. S1–S13. See DOI: 10.1039/c4nr01826e

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shown great promise as photosensitizing nanoplatforms in NIR-triggered PDT to overcome the above-mentioned drawbacks under UV/visible light.18–23 In addition, nanoprobes based on UCNPs may enable targeted bioimaging of tumor cells or tissues through either the enhanced permeability and retention effect or surface modication with special biomolecules that can specically recognize the tumor markers.24 These features make UCNPs promising multifunctional nano-bioprobes for cancer theranostics. Nonetheless, there is still a long way to go before the commercialization of these UCNPs for clinical PDT in view of their low therapeutic efficacy under NIR light.19 In the UCNP-based PDT system, cytotoxic singlet oxygen (1O2) is generated through the excited photosensitizers that are activated via a F¨ orster resonance energy transfer (FRET) process from UCNPs upon NIR excitation. Thus, the production of 1O2 and the efficacy of PDT are critically dependent on the efficiency of energy transfer from UCNPs to the photosensitizer, which relies on the distance between the donor and the acceptor.25 Several strategies have been developed to load the photosensitizer onto the UCNPs to facilitate the FRET process, including silica coating26–28 and polymer encapsulation.29,30 However, all of these loading protocols introduced an interlayer between UCNPs and the loaded photosensitizers, which would inevitably increase the distance between them and thus reduce the distance-dependent FRET efficiency. We anticipate that a higher 1O2 production with a higher energy transfer efficiency can be achieved if the photosensitizers are directly attached onto the surface of UCNPs without any interlayer. So far, a vast majority of UCNP-based PDT agents are restricted to the systems of Ln3+-doped NaYF4.31–35 LiYF4, as another efficient host material for Ln3+ doping to achieve

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desirable upconversion luminescence (UCL), to the best of our knowledge, had not been explored for PDT applications before. Herein, we synthesize monodisperse LiYF4:Yb/Er UCNPs of different sizes via a facile thermal decomposition route. Through electrostatic attraction, we fabricate an efficient NIRtriggered PDT system by direct conjugation of the UCNPs with a spectrally matchable photosensitizer of the b-carboxyphthalocyanine zinc (ZnPc-COOH) molecule we recently developed.36 We measure the photosensitizers' loading capability and stability of such direct conjugation and the 1O2 production in the as-designed PDT system. Furthermore, we examine the safety of the PDT system to human embryo lung broblast cells (HELF) and the in vitro PDT efficacy on human mammary cancer cells (MDA-MB-231). Finally, we assess the therapeutic efficacy of a UCNP-based PDT agent on tumor growth in vivo by intratumorally injecting them into Kunming mice bearing H22 hepatocarcinoma tumors.

2 Experimental section 2.1

Chemicals and materials

NaOH, NH4F, polyvinylpyrrolidone (PVP), cyclohexane, N,Ndimethylformamide (DMF), ethanol and hydrogen chloride (HCl) were purchased from Sinopharm Chemical Reagent Co., China. Ln(CF3COO)3 was prepared as reported in the literature.37 CF3COOLi$H2O, CF3COONa, YCl3$6H2O (99.99%), YbCl3$6H2O (99.99%), ErCl3$6H2O (99.99%), oleic acid (OA), oleylamine (OM), 1-octadecence (ODE), and 40 ,6-diamidino-2phenylindole (DAPI) were purchased from Sigma-Aldrich (China). Monosubstituted b-carboxyphthalocyanine zinc (ZnPcCOOH) was synthesized as previously reported.36 All the chemical reagents were of analytical grade and used as received without further purication. 2.2

Synthesis of LiYF4:Yb/Er UCNPs

Monodisperse LiYF4:Yb/Er UCNPs were synthesized via a facile thermal decomposition route. In a typical procedure for the synthesis of LiYF4:20% Yb3+,2% Er3+ UCNPs with a mean size of 47 nm, 1 mmol of CF3COOLi$H2O, 0.78 mmol of Y(CF3COO)3$4H2O, 0.2 mmol of Yb(CF3COO)3$4H2O and 0.02 mmol of Er(CF3COO)3$4H2O were mixed with 8 mL of OA and 2 mL of OM in a 50 mL three-neck round-bottom ask. The resulting mixture was heated to 110  C under N2 ow with constant stirring for 30 min to form a transparent solution. Subsequently, the resulting solution was heated to 310  C under N2 ow with vigorous stirring for 1 h and then cooled down to room temperature (RT) naturally. The resulting UCNPs were precipitated by addition of ethanol, collected by centrifugation, washed with ethanol several times, and nally re-dispersed in cyclohexane. 2.3

Synthesis of b-NaYF4:Yb/Er UCNPs

b-NaYF4:Yb/Er UCNPs was synthesized via a modied hightemperature coprecipitation method.38 In a typical synthesis, 0.312 mmol of YCl3$6H2O, 0.08 mmol of YbCl3$6H2O and 0.008 mmol of ErCl3$6H2O were mixed with 3 mL of OA and 7 mL of This journal is © The Royal Society of Chemistry 2014

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ODE in a 50 mL three-neck round-bottom ask. The resulting mixture was heated to 150  C under N2 ow with constant stirring for 30 min to form a clear solution and then cooled down to RT. Thereaer, 10 mL of methanol solution containing 1.6 mmol of NH4F and 1 mmol of NaOH was added and the solution was stirred at 50  C for 30 min. Aer methanol was evaporated, the solution was heated to 290  C under N2 ow with vigorous stirring for 90 min and then cooled down to RT. The obtained UCNPs were precipitated by addition of 15 mL of ethanol, collected by centrifugation, washed with ethanol several times, and nally re-dispersed in cyclohexane. 2.4

Synthesis of b-NaLuF4:Gd/Yb/Er UCNPs

b-NaLuF4:Gd/Yb/Er UCNPs was synthesized via a modied thermal decomposition method.39 In a typical process, 1 mmol of Ln(CF3COO)3 (4 mol% of Lu, 30 mol% of Gd, 20 mol% of Yb, 2 mol% of Er) and 2 mmol of Na(CF3COO)$H2O were added to a 50 mL ask containing 20 mL of OM at RT. The obtained mixture was heated at 110  C for 30 min under magnetic stirring in a N2 atmosphere, in order to dissolve triuoroacetate and simultaneously to remove the residual water and oxygen. Subsequently, the resulting transparent solution was heated to 330  C under N2 ow with vigorous stirring for 130 min and then cooled to RT naturally. The resulting UCNPs were precipitated by the addition of ethanol, collected via centrifugation, washed with ethanol several times, and re-dispersed in cyclohexane. 2.5

Synthesis of ligand-free UCNPs

The ligand-free LiYF4:Yb/Er UCNPs were obtained by removing the surface ligands of the oleate-capped UCNPs through acid treatment.40 In a typical process, 30 mg of the as-synthesized oleate-capped UCNPs were dispersed in 15 mL of acidic ethanol solution (pH 1; prepared by adding 112 mL of concentrated hydrochloric acid to 15 mL of absolute ethanol) and ultrasonicated for 15 min to remove the surface ligands. Aer the reaction, the UCNPs were collected via centrifugation at 16 500 rpm for 20 min, and further puried by adding an acidic ethanol solution (pH 4). The resulting products were washed with ethanol and deionized water several times, and redispersed in deionized water. The ligand-free b-NaYF4:Yb/Er and b-NaLuF4:Gd/Yb/Er UCNPs were prepared by using the same method with LiYF4:Yb/Er UCNPs. 2.6

Synthesis of UCNP–ZnPc-COOH

In a typical procedure, 5 mL of DMF solution containing 30 mg of ligand-free UCNPs was added into 5 mL of DMF solution containing 3 mg of ZnPc-COOH. The mixture solution was stirred for 30 min at RT. Then the UCNP–ZnPc-COOH nanocomposite was collected by centrifugation at 16 500 rpm for 5 min, washed with DMF, and re-dispersed in 15 mL of DMF, into which 0.2 g of PVP was added and stirred for another 3 h. Thereaer, PVP-capped UCNP–ZnPc-COOH was collected by centrifugation at 16 500 rpm for 5 min, washed with ethanol several times, and re-dispersed in deionized water.

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2.7 Measurement of the loading capacity of photosensitizers in UCNP–ZnPc-COOH The content of ZnPc-COOH capped on the surface of LiYF4:Yb/ Er UCNPs was determined by measuring the characteristic absorbance of ZnPc-COOH peaking at 680 nm for the DMF solution of UCNP–ZnPc-COOH (1 mg mL1). The weight amount of ZnPc-COOH loaded on LiYF4:Yb/Er UCNPs can be calculated based on the standard curve derived from the absorption spectra of DMF solutions of pure ZnPc-COOH. The loading capacity of ZnPc-COOH was calculated as follow: loading capacity (%) ¼ (weight amount of ZnPc-COOH in UCNP–ZnPc-COOH)/(weight amount of UCNP–ZnPc-COOH)  100%. 2.8 Measurement of the release of photosensitizer from UCNP–ZnPc-COOH Firstly, 2 mg of UCNP–ZnPc-COOH was soaked in 1 mL of aqueous solution of different pHs (pH ¼ 3, 5, 7, 9, and 11) or in 1 mL of phosphate-buffered saline (PBS, pH 7.2) under continuous stirring at 37  C. Aer soaking for 7 days, the solution of UCNP–ZnPc-COOH was centrifuged at 16 500 rpm for 10 min. The corresponding supernatant was then assessed to determine whether the photosensitizers were leached out into solution by measuring their absorption spectra. 2.9 Cell culture and confocal laser scanning microscopy imaging MDA-MB-231 and HELF cell lines were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and were routinely maintained in RPMI-1640 (GIBCO BRL), supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), penicillin (100 U mL1), and streptomycin (100 mg mL1) at 37  C under humidied air containing 5% CO2. MDAMB-231 cells were seeded into culture plates and allowed to adhere for 24 h. Aer washing with PBS, the cells were incubated in culture medium containing 0.5 mg mL1 of ligand-free UCNPs or UCNP–ZnPc-COOH at 37  C for 2 h under 5% CO2 and then washed with PBS sufficiently to remove excess UCNPs. The cells were subsequently incubated with DAPI at RT for 5 min and washed with PBS. The cell imaging was performed with a modied Olympus FV1000 laser scanning upconversion luminescence microscope equipped with a continuous-wave (CW) laser at 980 nm (Connet Fiber Optics, China). A 60 oilimmersion objective lens was used and the CW laser diode at 980 nm provided the excitation source. The luminescence signals were detected in the green channel (500–560 nm), red channel (620–680 nm) and blue channel (450–490 nm). 2.10

Cell viability assay

Cells subjected to the aforementioned photosensitization experiments were returned to a 5% CO2 atmosphere at 37  C for another 24 h round of incubation before they were subjected to cell viability assay, upon which, Cell Counting Kit-8 (CCK-8) was subsequently applied to the cells followed by incubation at 37  C under 5% CO2 for 4 h. The OD450 value of

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each well was measured on a multimodal microplate reader (Synergy 4, BioTek). The following formula was applied to calculate the percent inhibition rate of cell growth: cell viability (%) ¼ (mean of absorbance value of treatment group/ mean of absorbance value of control)  100%. Four replicates were run per UCNP dose in each cell line, and each experiment was repeated three times.

2.11 Real-time phototoxicity assay of UCNP–ZnPc-COOH based on electric cell-substrate impedance sensing The real-time phototoxicity of UCNP–ZnPc-COOH was monitored by means of electric cell-substrate impedance sensing (ECIS) measurement. An 8-well plate 8W10E (Applied Biophysics, Inc., NY, USA) was used in this assay. Each well has a surface area of 0.8 cm2 and holds 600 mL of culture medium. Each well contains ten individual interdigitated gold microelectrodes (250 mm diameter) to measure electric current ow through the medium. These individual electrodes are connected to an ECIS Z system (Applied Biophysics, Inc., NY, USA). The frequency of electric impedance response for each cell type was optimized by running a simultaneous measurement at multiple frequencies of the applied voltage. In a typical ECIS experiment, the 8W10E plates were equilibrated with cell medium in an incubator (37  C, 100% humidity and 5% CO2) for at least 2 h before the cells (2  105) were seeded into each well of the array plates with fresh medium (200 mL). The plates were then connected to an ECIS instrument and put back into the incubator. The cells were allowed to attach to the plates for 5 h until the electric impedance response of cells on the plates was stabilized and became a straight line. The ECIS measurement was paused and the culture medium of wells in the 8W10E plates was replaced with fresh medium only (as a control) or the medium containing UCNP–ZnPc-COOH (at a nal concentration of 0.125, 0.25, 0.5, and 1 mg mL1). Aer 2 h incubation, the cells were washed with fresh culture medium to remove the unbound photosensitizers. The 980 nm NIR laser was applied to irradiate the selected wells at a power density of 0.5 W cm2 for 5 min. The electric impedance data collection was continued throughout the incubation and extended for 16 h aer illumination.

2.12

In vivo PDT

Mice bearing H22 hepatocarcinoma tumors with a diameter of 0.6 cm were randomly assigned into two groups (n ¼ 5). All mice were treated with 30 mL of UCNP–ZnPc-COOH (20 mg mL1) via intratumoral injection. At 12 h post-injection, the mice in the experiment group were irradiated with a 980 nm laser (power density of 0.5 W cm2) at the tumor site for 20 min. To avoid any tissue damage by heating, the laser treatment was done with 5 min interval for every 1 min of light exposure. The mice in the control group were treated without any laser irradiation. The therapeutic efficacy of UCNP–ZnPcCOOH on the mice was assessed for every two days by measuring the body weight and the tumor volume which can be calculated by length  (width)2/2.

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2.13

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Histology examination

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To conrm the PDT efficacy of UCNP–ZnPc-COOH, histology analysis of tumor tissues was performed aer the treatment. Tumor tissues in the control group and the experiment group were separately xed with 10% neutral buffered formalin and embedded in paraffin (n ¼ 5). The sliced organs were stained with hematoxylin–eosin staining (H&E) and examined under a microscope. 2.14

Structural and optical characterization

Powder X-ray diffraction (XRD) patterns of the samples were collected on an X-ray diffractometer (MiniFlex2, Rigaku) with Cu Ka1 radiation (l ¼ 0.154187 nm). Both the low- and highresolution transmission electron microscopy (TEM) measurements were performed on a JEOL-2010 TEM equipped with the energy-dispersive X-ray spectrum. Fourier transform infrared (FTIR) spectra were recorded in KBr discs on a Magna 750 FTIR spectrometer. The hydrodynamic diameter distribution and zpotentials of UCNPs were determined by means of dynamic light scattering (DLS) measurement (Nano ZS ZEN3600, Malvern). UV-Vis absorption spectra were measured with a PerkinElmer Lambda 900 UV/Vis/NIR spectrometer. Upconversion emission spectra were collected under 980 nm laser excitation at 10 W cm2 provided by a continuous-wave laser diode. The UCL photographs of corresponding UCNPs dispersed in cyclohexane solution (0.5 mg mL1) were taken by using a Nikon D90 under 980 nm laser irradiation at a power density of 20 W cm2. UCL lifetimes were measured with a customized ultraviolet (UV) to mid-infrared steady-state and a phosphorescence lifetime spectrometer (FSP920-C, Edinburgh) equipped with a digital oscilloscope (TDS3052B, Tektronix) and a tunable mid-band Optical Parametric Oscillator (OPO) pulse laser as the excitation source (410–2400 nm, 10 Hz, pulse width of 5 ns, Vibrant 355II, OPOTEK). The absolute upconversion quantum yield of LiYF4:Yb/Er UCNPs was measured with a self-made UCL spectroscopy system at RT upon a 980 nm diode laser excitation at a power density of 60 W cm2. Confocal imaging of cells was performed with a modied Olympus FV1000 laser scanning confocal microscope (60 oil-immersion objective lens).

3 Results and discussion 3.1 Controlled synthesis and optical properties of LiYF4 UCNPs The LiYF4 crystal has a tetragonal structure (space group I41/a) with single site symmetry of S4 for all Y3+ ions.41 Monodisperse LiYF4:Yb/Er UCNPs with different sizes were synthesized via a modied thermal decomposition method by varying the molar ratio of solvent oleic acid/oleylamine (see Table S1 in the ESI†). The as-synthesized UCNPs were hydrophobic and can be readily dispersed in a variety of nonpolar organic solvents, such as cyclohexane. TEM images show that the morphology of the resulting UCNPs changed from sphere to rhombus with an increase in the mean size from 8.1  1.0 nm, 22  1.5 nm to 47  2.5 nm, respectively, as the ratio of oleic acid to oleylamine decreased from 8/6, 8/4 to 8/2 (Fig. 1a–c). High-resolution TEM

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Fig. 1 TEM images of LiYF4:Yb/Er UCNPs with a mean size of (a) 8.1 nm, (b) 22 nm, and (c) 47 nm. (d)–(f) show the corresponding HRTEM images. (g)–(i) show the UCL photographs of the corresponding UCNPs dispersed in cyclohexane under 980 nm NIR laser irradiation. (j) UCL spectra of LiYF4:Yb/Er UCNPs of different sizes. (k) UCL decays from 4S3/2 of Er3+ in LiYF4:Yb/Er UCNPs of different sizes.

(HRTEM) images show clear lattice fringes with an observed dspacing of 0.41 nm, which is in good agreement with the lattice spacing of the (101) plane of tetragonal-phase LiYF4 (Fig. 1d–f), thus verifying the high crystallinity of the UCNPs. The high crystallinity and increasing size of UCNPs were further corroborated by their more intense and narrower XRD peaks (see Fig. S1 in the ESI†). Upon 980 nm NIR laser excitation, the colloidal cyclohexane solution of the resulting UCNPs displayed intense green UCL, which became brighter with the increasing particle size (Fig. 1g– i). UCL spectra (Fig. 1j) show that all UCNPs exhibited characteristic and sharp emission peaks, which can be exclusively attributed to the intra-4f electronic transitions of Er3+. Particularly, the overall UCL intensity was remarkably enhanced with approximately enhancement factors of 116 and 1956 for UCNPs with mean sizes of 27 and 47 nm, respectively, as compared to that of 8.1 nm UCNPs. The corresponding absolute upconversion quantum yield, dened as the ratio of the number of emitted photons to the number of absorbed photons, was determined to be 0.03% (8.1 nm), 0.08% (22 nm) and 0.6% (47 nm), respectively, upon 980 nm NIR laser excitation at a power density of 60 W cm2, which are higher than that of NaYF4 counterparts (Table S2†).42 The improved upconversion quantum yield in larger UCNPs can be attributed to the reduced surface quenching effect,43 which is consistent with their increased UCL lifetimes (Fig. 1k and S2†); for instance, the UCL lifetime of 4S3/2 of Er3+ increased from 26 ms (8.1 nm) and 77 ms (22 nm) to 377 ms (47 nm) (Fig. 1k).

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3.2

Fabrication of the UCNP–ZnPc-COOH PDT system

To make the UCNPs hydrophilic and biocompatible, we removed oleate ligands from their surface by acid treatment.40 The successful removal of surface ligands was corroborated by thermogravimetric analysis (TGA) and Fourier transform infrared (FTIR) spectra for UCNPs before and aer acid treatment (Fig. S3 and S4†). These ligand-free UCNPs showed much better hydrophilicity and biocompatibility (Fig. S5 and S6†). Due to the removal of surface ligands, positively charged Ln3+ ions were exposed on the surface of ligand-free UCNPs, leaving their colloidal solution a positive z-potential of +49.5 mV at pH 6.5 (Fig. S7†). As a result, these ligand-free UCNPs are enabled for direct conjugation in water with electronegative groups of hydrophilic and biocompatible molecules for various bioapplications. Aer conjugation with specic photosensitizers such as ZnPc-COOH, these ligand-free LiYF4:Yb/Er UCNPs can be further designed for NIR-triggered PDT (Fig. 2a). Considering their higher upconversion quantum yield, we purposely selected 47 nm UCNPs for the following PDT experiments. Thanks to the bare Ln3+ ions on the surface of ligand-free UCNPs, the electronegative groups of ZnPc-COOH can be easily conjugated to the surface of UCNPs via electrostatic attraction.40,44–46 As compared to other strategies for loading photosensitizers onto UCNPs ever reported, for example, silica coating and polymer encapsulation, such direct conjugation of photosensitizers onto UCNPs is operationally simpler. More importantly, it facilitates the distance-dependent energy transfer from UCNPs to

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photosensitizers and thus enhances the efficacy of PDT as will be demonstrated below. It should be noted that the p–p stacking interaction of the phthalocyanine aromatic ring of ZnPc-COOH may result in aggregation of the UCNPs in aqueous solution. To avoid this, ZnPc-COOH-capped UCNPs (UCNP– ZnPc-COOH) were further decorated with a long-chain molecule polyvinylpyrrolidone (PVP).44 The appearance of the phthalocyanine aromatic ring of ZnPc-COOH bands and C]O bonds of PVP in FTIR spectra, and the different decomposition temperatures, weight losses and z-potentials for UCNPs before and aer surface modication, revealed the successful conjugation of ZnPc-COOH and PVP to the surface of UCNPs (Fig. S3, S4, and S7†). Aer surface modication with PVP, UCNP–ZnPc-COOH recovered excellent water solubility and can be readily dispersed in distilled water (0–1 mg mL1), forming a clear blue colloidal solution (inset of Fig. 2b). In comparison with ligand-free UCNPs, the hydrodynamic diameter of PVP-capped UCNP– ZnPc-COOH increased from 50.7 nm to 63.5 nm (Fig. S5†), owing to the formation of the PVP layer on the surface of the UCNPs. The cytotoxicity of UCNP–ZnPc-COOH was measured on HELF cells by using a CCK-8 assay. The cell viability was determined to be larger than 95% even at a high concentration of 1 mg mL1 for UCNP–ZnPc-COOH (Fig. S6†). The high cell viability infers that UCNP–ZnPc-COOH is biocompatible and nearly nontoxic to live cells. In order to evaluate the loading capability and stability of the as-designed PDT system, we performed a loading and release study of the photosensitizer in UCNP–ZnPc-COOH. From the

Fig. 2 (a) Schematic illustration of multifunctional LiYF4:Yb/Er UCNPs for cancer theranostics. (b) Absorption spectrum of the ZnPc-COOH solution and UCL spectra of ligand-free UCNPs and UCNP–ZnPc-COOH, respectively. Note that the UCL spectra were normalized at 540 nm. The insets (from left to right) show UCL photographs of the corresponding UCNPs dispersed in aqueous solution under 980 nm NIR laser irradiation. UCL decays from (c) 4F9/2 and (d) 4S3/2 of Er3+ in either ligand-free UCNPs or UCNP–ZnPc-COOH by monitoring their emissions at 669 and 540 nm, respectively. (e) Time-dependent bleaching of DPBF caused by 1O2 generation in the presence of 0.5 mg mL1 of UCNP–ZnPcCOOH under 980 nm NIR laser irradiation. All the absorbances were normalized at the zero point of irradiation time.

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absorption spectrum of UCNP–ZnPc-COOH soaked in phosphate-buffered saline (PBS, 0.25 mg mL1) (Fig. S8†), the content of ZnPc-COOH on the surface of ligand-free UCNPs was quantied to be 1.1% (w/w). Moreover, it was found that a negligibly low content of ZnPc-COOH was released from UCNP– ZnPc-COOH even for 7 days soaked in aqueous solution with varying pH (3–11) or in PBS (Fig. S9 and S10†), which veries the high stability of our PDT system. As shown in Fig. 2b, the red UCL emission band at 669 nm of LiYF4:Yb/Er UCNPs spectrally overlaps very well with the absorption band of ZnPc-COOH on the surface of UCNPs, which enables an efficient FRET process from UCNP to ZnPc-COOH. As a consequence, upon 980 nm NIR laser excitation, the red UCL intensity of UCNP–ZnPcCOOH was observed to decrease markedly relative to that of the ligand-free counterparts by normalizing the green UCL emission at 541 nm where no FRET occurred (Fig. 2b). Accordingly, the UCL lifetime of 4F9/2 monitored at 669 nm was found to decrease from 488 to 138 ms (Fig. 2c), whereas the UCL lifetime of 4S3/2 monitored at 541 nm only decreased slightly from 385 to 345 ms (Fig. 2d), which further veries the occurrence of an efficient FRET from UCNPs to ZnPc-COOH. The energy transfer efficiency, dened as (I0  I1)/I0, where I0 and I1 are the integrated intensities of red UCL bands for the ligand-free UCNPs and UCNP–ZnPc-COOH in the normalized UCL spectra, respectively, was determined to be 96.3%.47,48 To the best of our knowledge, this value is the highest among the energy transfer efficiency in UCNP-based PDT systems ever reported.25,26 The excellent performance can be attributed to the much shortened distance between the donor (UCNP) and the acceptor (ZnPcCOOH) beneting from the strategy of direct electrostatic coupling that facilitated the distance-dependent FRET process. More importantly, the facile strategy can be easily extended to the construction of other efficient UCNP-based PDT systems, such as NaYF4 and NaLuF4 UCNPs, where the efficiency of energy transfer from the UCNP to the photosensitizer was determined to be 88.7% and 92.4%, respectively (Fig. S11 and S12†). To determine the production of 1O2 in our PDT system, we measured the bleaching of 1,3-diphenylisobenzonfuran (DPBF), whose absorbance at 420 nm would be diminished in the presence of 1O2.49,50 As shown in Fig. 2e, the absorbance of DPBF incubated with UCNP–ZnPc-COOH decreased exponentially with the time under 980 nm NIR laser excitation at a power density of 1 W cm2, with approximately 91% decrease in 26 min, indicating a high 1O2 production. By contrast, in the control experiments where the measurement was conducted without NIR excitation or ligand-free UCNPs instead of UCNP– ZnPc-COOH were used under otherwise identical conditions, the absorbance of DPBF remained nearly unchanged, indicative of the absence of 1O2. These results unambiguously demonstrate that the generation of 1O2 was triggered by energy transfer from UCNP to ZnPc-COOH upon NIR laser excitation. In addition, owing to the high energy transfer efficiency, the production of 1O2 was greatly enhanced as compared to the other NIR-triggered PDT system previously reported.21,25,28 For example, in NaYF4:Yb/Er UCNP-based PDT systems fabricated by doping merocyanine 540 in a silica shell, the highest

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consumption of 1O2 probes was only 3% which was achieved under 16 min continuous NIR irradiation at a power density of 1 W cm2, indicating a much lower 1O2 production than that in our PDT system.21 These results reveal the great potential of LiYF4:Yb/Er UCNP–ZnPc-COOH as an efficient NIR-triggered PDT agent. 3.3

In vitro PDT

By means of CCK-8 assay and electric cell-substrate impedance sensing (ECIS) measurement,51 we examined the phototoxicity and the real-time phototoxicity of MDA-MB-231 cells aer incubation with UCNP–ZnPc-COOH for 24 h, in order to assess their in vitro PDT efficacy. As shown in Fig. 3a, upon 980 nm NIR laser irradiation at a relatively low power density of 0.5 W cm2 for 10 min, a signicant reduction in cell viability was observed for MDA-MB-231 cells incubated with UCNP–ZnPc-COOH, in stark contrast to those incubated with either UCNP–ZnPcCOOH without NIR irradiation or ZnPc-COOH-coupled LiYF4 NPs without the doping of Yb/Er (LiYF4–ZnPc-COOH). Accordingly, in the ECIS measurement (Fig. 3b), the electric impedance declined gradually with the time and the concentration of the co-incubated UCNP–ZnPc-COOH for MDA-MB-231 cells aer the 980 nm NIR laser irradiation for 10 min, indicating a gradual cell death induced by the NIR-triggered PDT agent. By contrast, in the control experiment where cells incubated without UCNP–ZnPc-COOH were used under otherwise identical conditions, the electric impedance showed only a slight decrease. The corresponding microscopic images of MDA-MB231 cells clearly showed an increased cell death with the increasing concentration of UCNP–ZnPc-COOH incubated (Fig. 3c), which is consistent with the results of cell viability acquired from CCK-8 and ECIS assays. By using a tissue phantom with a thickness of 10 mm, we demonstrated that the 980 nm NIR light has a deeper tissue penetration than the 660 nm red light that is commonly used for the excitation of

Fig. 3 (a) Phototoxicity of UCNP–ZnPc-COOH and LiYF4–ZnPcCOOH for MDA-MB-231 cells. Each data point represents the average of quadruplicate measurements. (b) The real-time phototoxicity of MDA-MB-231 cells incubated with different concentrations of UCNP– ZnPc-COOH. As a control, cells incubated without UCNP–ZnPcCOOH were used under otherwise identical conditions. (c) Microscopic images of MDA-MB-231 cells after PDT treatment.

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ZnPc in conventional PDT (Fig. S13†). We also conrmed that the PDT system constructed with the larger UCNPs (47 nm) has a higher PDT efficacy against cancer cells than those constructed with smaller counterparts (Fig. S14†). These results reveal the great potential of the LiYF4:Yb/Er UCNP–ZnPc-COOH system as an efficient PDT agent for deep cancer therapy.

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3.4

In vivo PDT

To investigate the in vivo therapeutic efficacy of UCNP–ZnPcCOOH, we intratumorally injected H22 tumor-bearing mice with 30 mL of UCNP–ZnPc-COOH (0.5 mg mL1). Aer 12 h of post-injection, the mice were irradiated with a 980 nm NIR laser at a power density of 0.5 W cm2 at the tumor site for 20 min. To avoid any tissue damage induced by the heating effect of the laser irradiation, the laser treatment was done with a 5 min interval for every 1 min of irradiation and the laser power density used here (0.5 W cm2) is well below the conservative limit set for human skin exposure to 980 nm NIR light (0.7 W cm2).52 Mice that received intratumoral injection of UCNP–ZnPc-COOH without subsequent laser exposure were applied as control. The in vivo PDT efficacy of UCNP– ZnPc-COOH was then assessed by measuring the tumor volumes over a period of 2 weeks. The tumor volume in the control group increased remarkably from 248 mm3 to 1282 mm3 during the 14-day treatment; in sharp contrast, the growth rate of tumor in the experiment group was signicantly attenuated as evidenced by the tumor volume change from 240 mm3 to 501 mm3 during the same treatment period (Fig. 4a and c). Furthermore, the body weight of the mice in the control group remarkably decreased by 15% aer 14-day treatment, while that in the experiment group kept essentially unchanged (Fig. 4b), indicating that

Fig. 4 Variation of (a) tumor volumes and (b) body weights of mice in

experiment and control groups, respectively. Each data point represents the average value of 5 mice. (c) Representative photos of a mouse showing tumors at 14 days after treatment in experiment and control groups, respectively. (d) Images (left) and the corresponding high-resolution images (right) of H&E stained tumor tissues harvested from the experiment and control groups after 14 days.

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the health condition of the mice in the experiment group was greatly improved by the treatment. Histological analysis of tumor tissues at 14 days post-treatment showed that no damage was found in the tumor tissues from the control group whereas obvious apoptotic and necrotic tumor cells were observed in tissues from the experiment group, as evidenced by the images of hematoxylin–eosin (H&E) staining of tumor slices (Fig. 4d). The corresponding high-resolution images clearly showed that most of the tumor cells were damaged in the experiment group. These results reconrm the high in vivo therapeutic efficacy of UCNP–ZnPc-COOH, and thus show great promise to develop LiYF4:Yb/Er UCNP– ZnPc-COOH as an efficient NIR-triggered PDT agent in biological or clinical applications. Besides PDT, these ZnPc-COOH loaded UCNPs can be also used for cancer cell imaging by utilizing their intense UCL. Aer incubation with MDA-MB-231 cancer cells, intense green UCL of Er3+ from the UCNPs was clearly visualized in the cells upon 980 nm NIR laser irradiation (Fig. 5). In comparison with those incubated with ligand-free UCNPs, the red UCL signal of Er3+ from the cells incubated with UCNP–ZnPc-COOH was nearly quenched, due to the strong absorption of ZnPc-COOH in the red spectral region. These results show that the ZnPc-COOH loaded UCNP may serve as an imaging-guided and NIR-triggered PDT agent for cancer theranostics, where the green UCL of the UCNPs can be monitored to image cancer cells and the red UCL can be utilized to activate the photosensitizer to generate 1O2 for cancer therapy.

Fig. 5 Confocal laser scanning microscopy images of MDA-MB-231 cells after incubation with LiYF4:Yb/Er UCNPs with a mean size of (a) 8.1 nm, (b) 22 nm, (c) 47 nm, and with (d) UCNP–ZnPc-COOH for 2 h at 37  C. Er red emissions are shown in panel 1 (lem ¼ 500–560 nm, lex ¼ 980 nm). Er green emissions are shown in panel 2 (lem ¼ 620–680 nm, lex ¼ 980 nm). DAPI blue images (lem ¼ 450–490 nm, lex ¼ 405 nm) that indicate the nuclear regions are shown in panel 3. Panel 4 is the overlay images of panels 1, 2, and 3.

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4 Conclusions In summary, we have fabricated an efficient NIR-triggered PDT system based on LiYF4:Yb/Er UCNPs by direct coupling with the photosensitizer of ZnPc-COOH via electrostatic attraction. Such direct electrostatic coupling may result in a close distance between the energy donor of the UCNP and the acceptor of ZnPc-COOH, and thus promote the energy transfer from UCNP to ZnPc-COOH, which gave rise to the highest energy transfer efficiency (96.3%) among UCNP-based PDT systems ever reported. As a result, an extremely high 1O2 production, namely, 91% consumption of 1O2 probes, has been achieved in our PDT system upon 26 min NIR irradiation at a low power density of 1 W cm2. By taking advantages of both the green and red UCL of the ZnPc-COOH loaded LiYF4:Yb/Er UCNPs, we have demonstrated their potential applications in imaging-guided and NIRtriggered PDT for cancer theranostics. Finally, we have shown the high efficacy of the UCNP-based PDT agent for the inhibition of tumor growth both in vitro and in vivo. Our results reveal the great potential of inorganic UCNPs electrostatically coupled with specic photosensitizers like ZnPc-COOH as an efficient and noninvasive NIR-triggered PDT agent in deep cancer therapy.

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Acknowledgements This work is supported by the 973 program of MOST (No. 2014CB845605), the Special Project of National Major Scientic Equipment Development of China (No. 2012YQ120060), the NSFC (Nos. 11104266, 11204302, 11304314, 51002151, U1305244, and 21325104), and the Strategic Priority Research Program and Scientic Equipment Development Project of the CAS (Nos. XDA09030307 and YZ201210).

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Lanthanide-doped upconversion nanoparticles electrostatically coupled with photosensitizers for near-infrared-triggered photodynamic therapy.

Lanthanide-doped upconversion nanoparticles (UCNPs) have recently shown great promise in photodynamic therapy (PDT). Herein, we report a facile strate...
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