In situ decorating of ultrasmall Ag2Se on upconversion nanoparticles as novel nanotheranostic agent for multimodal imaging-guided cancer photothermal therapy

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In situ decorating of ultrasmall Ag2 Se on upconversion nanoparticles as novel nanotheranostic agent for multimodal imaging-guided cancer photothermal therapy Kaimin Du a,b , Pengpeng Lei a , Lile Dong a,b , Manli Zhang a,b , Xuan Gao a,b , Shuang Yao a , Jing Feng a,b,∗ , Hongjie Zhang a,b,∗ a b

State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China University of Science and Technology of China, Hefei 230026, China

a r t i c l e

i n f o

Article history: Received 14 July 2019 Received in revised form 30 October 2019 Accepted 1 November 2019 Keywords: Nanocomposites In situ growth Photoluminescence Tetra-modal imaging Photothermal therapy

a b s t r a c t A novel multifunctional theranostic nanoplatform was fabricated via in situ growth of ultrasmall Ag2 Se nanodots on the surface of chitosan (CS) coated NaYF4 :Yb/Er@NaLuF4 :Nd/Yb@NaLuF4 upconversion nanoparticles (UCNPs). NaYF4 :Yb/Er@NaLuF4 :Nd/Yb@NaLuF4 @CS@Ag2 Se (labeled as UCNPs@CS@Ag2 Se) nanocomposites can provide downshifting (DS) and upconversion (UC) luminescence in NIR biological window I and II under 808-nm continuous-wave laser excitation simultaneously. Meanwhile, the attached Ag2 Se nanodots could produce hyperthermia and photoacoustic energy upon 808-nm laser irradiation due to its strong near-infrared (NIR) absorbance. Together with the X-ray absorbance feature of lanthanide components, the nanocomposites with excellent luminescent properties, high X-ray attenuation coefficient and strong NIR absorbance could be utilized as a smart contrast agent for upconversion luminescence (UCL)/downshifting luminescence (DSL)/computer tomography (CT)/photoacoustic (PA) multimodal imaging in vitro and in vivo. Furthermore, the as-synthesized nanocomposites possess superior photothermal performance, good biocompatibility, and negligible toxicity, showing great potential as an ideal photothermal therapy (PTT) agent. These outstanding properties revealed that UCNPs@CS@Ag2 Se nanocomposites are promising theranostic agents for tetra-modal imaging-guided PTT of cancer. © 2019 Elsevier Ltd. All rights reserved.

1. Introduction Diagnosis and treatment of cancer have always been the urgent problems to be solved, which require scientists to develop effective theranostic agents. Nowadays, the design and fabrication of hybrid nanocomposites that combine multiple nanomaterials have drawn immense research interest in biomedical fields, since such kind of composites integrate several different functions into a single nanoplatform and overcome the limitations of each single component counterparts [1–4], so as to realize more accurate diagnosis and therapy of cancer [5–11]. As known, lanthanide ion (Ln3+ )-doped upconversion nanoparticles (UCNPs) have particularly attracted attention due to the distinguished merits, including large anti-Stokes shifts, negligible auto-fluorescence, superior stability, excellent detection sensitiv-

∗ Corresponding authors at: State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China. E-mail addresses: [email protected] (J. Feng), [email protected] (H. Zhang).

ity, high penetration depth, low toxicity and good biocompatibility [12–19], which make them promising candidates for potential applications in biological labeling [13,20–24] sensing [25–27] drug delivery [28–30], biological imaging [31–35], and clinical therapeutics [36,37]. Despite the unique advantages, most of the applications widely use Yb3+ ions as sensitizers which can only be excited by a conventional 980-nm laser. However, the 980-nm laser will produce overheating in biological environments due to strong absorption of water and hemoglobin around 980 nm, which will lead to cell death and tissue damage [38–40]. In addition, fluorescence imaging in the visible region is usually limited by tissue penetration depth owing to the scattering of photons as well as the absorption of water and blood in biological tissues [41,42]. Fortunately, Nd3+ -sensitized UCNPs with the absorption around 808 nm has many charming merits not only can conquer the overheating problem of normal cells but also improve the penetration depth for deep tissue contrast imaging [43–45]. Meanwhile, surface modification of UCNPs for further biological applications is still an attractive and important topic. Until now, considerable effort has been devoted to explore efficient surface functionalization approaches to improve the stability and biocompatibility of

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Please cite this article as: K. Du, P. Lei, L. Dong et al., In situ decorating of ultrasmall Ag2 Se on upconversion nanoparticles as novel nanotheranostic agent for multimodal imaging-guided cancer photothermal therapy, Appl. Mater. Today, https://doi.org/10.1016/j.apmt.2019.100497

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UCNPs in vitro and in vivo [46–49]. For example, Shi et al. applied mesoporous silica (m-SiO2 ) shell coating on the UCNPs, because of its high surface, good biocompatibility, inimitable porous structure [50]. Lin et al. modified the UCNPs with a thin layer of poly(acrylic acid) (PAA), facilitating their potential application in the biological field [51]. Yang et al. transferred hydrophobic UCNPs to hydrophilic ones through the intervention of dopamine (DP) due to its excellent biocompatible and biodegradable properties [52]. Impressively, chitosan (CS) with a large amount of glucosamine and hydroxyl groups is economical, non-toxic and biodegradable, which can efficiently improve the stability, water dispersibility, and biocompatibility of UCNPs, as well as chelate for many metal ions, such as Ag+ , Cu2+ , Cd2+ , and Ni2+ [53–58]. Particularly, inspired by the tremendous progress of controllable synthesis of uniform UCNPs and subsequent surface functionalization, considerable efforts have been devoted to combine UCNPs with other functional materials, such as photothermal agents Au [2,59], and CuS [1,52,60], photodynamic reagents black phosphorus sheets (BPS) [61], and super-paramagnetic Fe3 O4 [62,63] in one hybrid system, which would provide potentials for synergistic diagnosis and treatment of cancer. Those pioneering works give us design inspiration to synthesize multimodal imaging-guided anti-cancer theranostic agents, however, most of them use the hybrid method to obtain nanocomposites. As known, there are some advantages of the in-situ growth method such as the simple synthesis process and mild reaction conditions compared with the hybrid method. And the obtained nanoplatform can be more uniform and stable. It is worth noting that Ag2 Se nanodots with high stability, low toxic, good NIR absorbance and effective photothermal conversion efficiency, have been extensively explored for potent PTT. It could be expected that the combination of UCNPs and Ag2 Se in a system by in situ growth routine has great potential for multimodal imaging-guided PTT of cancer. Herein, we report a facile in situ growth strategy to combine ultrasmall Ag2 Se nanodots with CS coated NaYF4 :Yb/Er@NaLuF4 :Nd/Yb@NaLuF4 (Er@Nd@Lu) UCNPs for multimodal imaging-guided PTT in vitro and in vivo. The optimal core-shell-shell Er@Nd@Lu can not only exhibit pronounced UCL (Yb3+ -Nd3+ -Er3+ ) for in vitro UCL imaging, but also exhibit intense DSL in the “NIR biological window”, showing great penetration depth for in vivo fluorescence imaging. Moreover, due to high X-ray attenuation coefficient of lutecium ions (Lu3+ ) and ytterbium ions (Yb3+ ), they can also be applied for CT imaging, facilitating excellent spatial resolution and deep tissue penetration. The layer of CS coating on the surface of UCNPs is employed to improve the stability, water dispersibility, and biocompatibility of UCNPs as well as chelate for Ag+ ions to grow Ag2 Se further. The attached Ag2 Se nanodots could be used for PA imaging and anti-cancer photothermal therapy owing to its strong NIR absorbance and excellent photothermal conversion efficiency. By employing this design, several different functions could be integrated into a single nanoplatform. The intracellular UCL imaging and in vivo DSL/CT/PA imaging, biodistribution, photothermal effect as well as long-term toxicology of NaYF4 :Yb/Er@NaLuF4 :Nd/Yb@NaLuF4 @CS@Ag2 Se (UCNPs@CS@Ag2 Se) nanocomposites were investigated systematically. The multifunctional UCNPs@CS@Ag2 Se nanocomposites could provide insight for further biomedical applications of inorganic nanocomposites.

2. Experimental section 2.1. Materials Yttrium chloride (YCl3 ·6H2 O, 99.9%), ytterbium chloride (YbCl3 ·6H2 O, 99.9.%), erbium chloride (ErCl3 ·6H2 O, 99.9.%),

lutetium chloride (LuCl3 ·6H2 O, 99.9.%), neodymium chloride (NdCl3 ·6H2 O, 99.9.%), oleic acid (OA, 90 %), 1-octadecene (ODE, 90 %), chitosan (CS, deacetylation degree ≥95 %, viscosity: 100–200 mPa s), selenium powder (Se), poly(vinylpyrrolidone) (PVP, MW = 58,000 g/mol) were purchased from Aladdin Reagents. Cetyl trimethyl ammonium bromide (CTAB, 99 %), sodium hydroxide (NaOH, > 98 %), ammonium fluoride (NH4 F), methanol (CH3 OH), ethanol (C2 H5 OH), cyclohexane, sodium borohydride (NaBH4 ), silver nitrate (AgNO3 ), were obtained from Beijing Chemical Reagents Materials. All chemical reagents are of analytical grade and used without any further purification. 2.2. Characterization The phase and crystallography of the products were characterized by Bruker D8 Focus powder X-ray diffractometer at a scanning rate of 0.5◦ min-1 in the 2␪ range from 20 to 80◦ using Cu K␣ radiation (␭ =1.5418 Å) operating at an operation voltage and current maintained at 40 kV and 40 mA. Transmission electron microscope (TEM), high resolution (HR) TEM, bright-field TEM image and the corresponding EDX elemental mappings were captured on a FEI Tecnai G2S-Twin instrument with a field emission gun operating at 200 kV. The composition of the samples were studied using a field emission scanning electron microscope (FESEM, S-4800, Hitachi) equipped with an energy-dispersive X-ray (EDX) spectrometer. The hydrodynamic size and ␰-potential values were recorded by dynamic light scattering technology on a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). Fourier transform infrared (FTIR) spectra were recorded using a PerkinElmer 580B IR spectrophotometer with a KBr pellet technique. X-ray photoelectron spectroscopy (XPS) were conducted with a VG ESCALAB MKII spectrometer. The vis–NIR absorption spectra were measured using a Shimadzu UV-3600 spectrophotometer (Shimadzu Co., Japan). Inductively-coupled plasma optical emission spectrometer (ICP-OES) was taken on an iCAP 6000 of Thermo scientific. Inductively-coupled plasma mass spectrometry (ICP-MS) was tested by an ELAN 9000/DRC ICP-MS system (PerkinElmer, USA). The UCL and DSL spectra were recorded by using a 808 nm laser diode and a triple grating monochromator (Spectra Pro-2758, Acton Research Corporation) equipped with a photomultiplier (Hamamatsu R928). 2.3. Synthesis 2.3.1. Synthesis of OA-stabilized ˇ-NaYF4 :Yb/Er nanoparticles OA-capped ␤-NaYF4 :Yb/Er nanoparticles were synthesized according to the literature methods [44]. Typically, YCl3 ·6H2 O (0.8 mmol), YbCl3 ·6H2 O (0.18 mmol) and ErCl3 ·6H2 O (0.02 mmol) were mixed in a 100 mL three-necked flask charged with oleic acid (5 mL) and 1-octadecene (15 mL). The mixture solution was heated to 165 ◦ C under argon atmosphere and kept for 60 min to obtain clear solution and then cooled down to room temperature. Subsequently, a methanol solution (10 mL) of NH4 F (4 mmol) and NaOH (2.5 mmol) was added and stirred at 50 ◦ C for 30 min to remove methanol, followed by heating to 100 ◦ C to degas fully, and remove oxygen and water under vacuum. After that, the mixed solution was heated to 100 ◦ C and kept for another 20 min. Then, the solution was heated to 300 ◦ C and kept for 60 min. Finally, the resulting nanoparticles were purified by centrifugation after the addition of ethanol, washed several times with cyclohexane and ethanol, and finally re-dispersed in cyclohexane for further experiments. 2.3.2. Synthesis of OA-stabilized core–shell structured NaYF4 :Yb/Er@NaLuF4 :Yb/Nd nanoparticles Briefly, as-prepared NaYF4 :Yb/Er core NPs (1 mmol), CF3 COONa (1 mmol), Lu(CF3 COO)3 (0.5 mmol), Yb(CF3 COO)3 (0.1 mmol) and

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Nd(CF3 COO)3 (0.4 mmol) were added to the mixture of OA (6.5 mL) and ODE (6.5 mL) in a three-necked flask at room temperature. The solution was pre-degassed for 10 min under vigorous stirring and then heated to 100 ◦ C in a temperature-controlled electromantle for 30 min to form a transparent solution and remove residual water under N2 flow. After that, the temperature was increased to 300 ◦ C and kept for 60 min under N2 atmosphere. Finally, the resulting nanoparticles were purified by centrifugation, washed several times with cyclohexane and ethanol, and finally re-dispersed in cyclohexane for further experiments.

2.3.3. Synthesis of OA-stabilized core–shell-shell structured NaYF4 :Yb/Er@NaLuF4 :Yb/Nd@NaLuF4 nanoparticles (UCNPs) The synthesis procedure of NaYF4 :Yb/Er@NaLuF4 :Yb/Nd@NaLuF4 is similar to that of NaYF4 :Yb/Er@NaLuF4 :Yb/Nd, except for using NaYF4 :Yb/Er@NaLuF4 :Yb/Nd as the core and Lu(CF3 COO)3 (1 mmol) and CF3 COONa (1 mmol) as precursors for epitaxial shell growth and shortening the reaction time to 30 min at 300 ◦ C. The final core-shell-shell structured NaYF4 :Yb/Er@NaLuF4 :Yb/Nd@NaLuF4 were designated as UCNPs, which was re-dispersed in cyclohexane (5 mL) for further use.

2.3.4. Synthesis of CS-functionalized UCNPs (UCNPs@CS) To obtain water-soluble UCNPs, 0.2 g of CTAB and 40 mL of water were successively added into 100 mL beaker, and the pellucid solution was obtained after magnetic stirring. Then, 5 mL of the UCNPs cyclohexane solution was added into the beaker, the mixture was then kept stirring for 2 h to evaporate cyclohexane, resulting in the formation of UCNPs-CTAB water solution. UCNP@CS was successfully synthesized according to the literature method [55]. In detail, 20 mg of CS was added to the above UCNPs–CTAB solution and the UCNPs@CS was obtained under bath ultrasonic treatment for 30 min. The product was centrifuged at 8000 rpm for 5 min and washed with deionized water for 3 times, finally dispersed in 10 mL of deionized water for further use.

2.3.5. Synthesis of UCNPs@CS@Ag2 Se nanocomposites by the in situ growth routine First, 2 mL of the as-synthesized UCNPs@CS nanoparticles were diluted to 10 mL with deionized water, followed by addition of AgNO3 solution (10.0 mM, 1 mL) and then the mixture was stirred at room temperature for 2 h. After that, 10 mg of PVP dissolved in 2 mL of deionized water were added to the mixture (solution A). In a typical synthesis, 7.896 mg (0.1 mmol) of Se powder was reduced by 11.35 mg (0.5 mmol) of NaBH4 in 20 mL deionized water under magnetic stirring at room temperature with the protection of inert gas (solution B). Finally, 1 mL of solution B was injected directly into the solution A to immediately generate a black solution, which indicated the formation of Ag2 Se nanodots on the surface of UCNPs@CS. 2.4. In vitro cytotoxicity assay of UCNPs@CS@Ag2 Se Typical methyl thiazolyl tetrazolium (MTT) reduction assay were performed to assess the cytotoxicity of UCNPs@CS@Ag2 Se. In detail, A549 cells were first seeded in 96-well plates (about 5000 per well) and cultured at 37 ◦ C and 5 % CO2 for 24 h in DMEM supplemented with 10 % FBS. Then, the cells were washed with PBS and incubated with the sample at different concentrations at 37 ◦ C for 24 h. After that, 15 ␮L of MTT with a concentration of 5 mg/mL was added and allowed to react with the cells for 4 h before the addition of dimethyl sulfoxide (150 ␮L) to dissolve the precipitates. The absorption of each solution was measured at 570 nm on a microplate reader (Thermo, Varioskan Flash).

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2.5. In vitro UCL imaging of UCNPs@CS@Ag2 Se The in vitro upconversion luminescence imaging of A549 cells was carried out by a custombuilt instrument for upconversion luminescence microscopy, which was rebuilt from an inverted fluorescence microscope (Nikon Ti-S) with an external 808 nm laser for illuminating the samples. A549 cells were seeded in 24-well culture plates (4 × 104 cells per well) and incubated (37 ◦ C, 5 % CO2 ) overnight. Then the original cell culture medium was discarded and fresh culture medium containing UCNPs@CS@Ag2 Se solution (Ag+ concentration = 200 ␮g/mL, 0.5 mL) was added into each well and incubated at 37 ◦ C for 1 h and 3 h, respectively. Then, the cells were carefully washed with PBS three times, fixed with 4 % poly formaldehyde PBS solution for 15 min, and then washed with PBS for three times.

2.6. Hemolysis assay Blood samples obtained from volunteers were diluted with 10 mL of PBS, and red blood cells (RBCs) were separated from the serum by centrifugating at 1200 rpm for 10 min. After being washed at least four times, the RBCs suspension was then diluted with 10 mL of PBS. Subsequently, 200 ␮L of diluted RBC suspension was mixed with 1 mL of PBS (as a negative control), deionized water (as a positive control), and UCNPs@CS@Ag2 Se nanocomposites at different concentrations (12.5, 25, 50, 100, and 200 ␮g/mL). After incubation for 4 h at 37 ◦ C, the mixtures were centrifuged at 12,000 rpm for 10 min. All of the obtained upper supernatants were added to a 96-well plate, and the absorbance was measured using a multifunction microplate reader. Finally, the percentage hemolysis of the RBCs was calculated by the following equation: hemolysis ratio (%) = (Asample - Acontrol (-) )/(Acontrol (+) - Acontrol (-) ) × 100 %.

2.7. Animal experiments Kunming mice were purchased from Laboratory Animal Center of Jilin University (Changchun, China). Animal care and handing procedures were in adherence with the guidelines of the Regional Ethics Committee for Animal Experiments. The tumor models were established by subcutaneous injection of H22 cells in the left axilla of each mouse. The tumor-bearing mice were used for experiments when the tumors size had reached about 100−200 mm3 .

2.8. Penetration depth tests and in vivo optical imaging Penetration depth tests were measured in lean pork. By covering with different depth pork layers above the tube of UCNPs@CS@Ag2 Se (10 mg/mL), the DSL images were captured using a CRi Maestro in vivo optical imaging system equipped with an 850 nm long-pass filter and excited with an 808 nm continuouswave laser diode. The UCL images was imaged on a CRi Maestro in vivo optical imaging system equipped with an external 808 nm laser as the excitation source. The 680 nm short-pass filter was used to prevent the interference of the excitation. All the parameters were set the same for clarity. For in vivo NIR Imaging, the mouse was first anesthetized by intraperitoneal injection of chloral hydrate solution (10 wt%), and then 100 ␮L of UCNPs@CS@Ag2 Se nanocomposites (RE3+ concentration =0.22 mmol/kg) was subcutaneously injected into a mouse. The images were captured using a CRi Maestro in vivo optical imaging system equipped with an 850 nm long-pass filter and excited with an 808 nm continuouswave laser diode.

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2.9. In vitro and in vivo photoacoustic imaging A preclinical photoacoustic computed tomography scanner (Endra Nexus 128, Ann Arbor, MI, USA) was used to obtain PA images. For in vitro PA imaging, UCNPs@CS@Ag2 Se nanocomposites with different Ag+ concentrations (0, 6.25, 12.5, 25, 50, 100 and 200 ␮g/mL) were embedded in agar gel tubes to produce photoacoustic imaging phantoms on a multispectral optical tomography (MSOT) imaging system. For in vivo PA imaging, H22 tumor-bearing mice with hair removed were anesthetized by 1.5. % isoflurane and then were injected with UCNPs@CS@Ag2 Se nanocomposites (Ag+ concentration =10 mg/kg) via the tail vein. The administered mice were imaged using the preclinical photoacoustic computed tomography scanner at different time points (preinjection, 0.5, 1, 2 and 24 h). The excitation wavelength was set from 700 to 950 nm with a 10 nm interval, and regions of interest were fixed at 20 mm.

where h is the heat transfer coefficient, S is the surface area of the container, Tmax, nanocomposites and Tmax,solvent are maximum steady-state temperature for UCNPs@CS@Ag2 Se nanocomposites solution and water, which are 70.2 and 28.0 ◦ C, respectively. I is the incident laser power (1.3 W/cm2 ), and A808 is the absorbance of UCNPs@CS@Ag2 Se nanocomposites at 808 nm (A808 = 0.25). ␶s is the sample system time constant, and md and Cd are the mass (0.25 g) and heat capacity (4.2 J/g) of the deionized water used as the solvent, respectively. ␪ is the dimensionless driving force temperature, Tsurr is the ambient temperature of the surroundings, T is a temperature for UCNPs@CS@Ag2 Se aqueous solutions at a constant cooling time (t), and the ␶s was determined to be 207.07 s (Fig. 3i). The photothermal conversion efficiency of UCNPs@CS@Ag2 Se nanocomposites was calculated according to Eq. (5): ␩= md Cd (T max, nanocomposites –T max,H2 O )/I(1 − 10

−A808

2.10. In vitro and in vivo X-ray CT imaging

) s = 37.6% (5)

For in vitro CT imaging, the CT contrast efficacy of UCNPs@CS@Ag2 Se nanocomposites was compared with clinical iopromide using the same element concentrations of 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mg/mL. For in vivo CT imaging, the tumorbearing mice were anesthetized by intraperitoneal injection of 100 ␮L of 10 % chloral hydrate. Then, UCNPs@CS@Ag2 Se nanocomposites (RE3+ concentration =0.22 mmol/kg) were intravenously injected for CT imaging. In vitro and in vivo CT images were collected using a Philips 256-slice CT scanner. The parameters were as follows: 120 kVp, 300 mA; thickness, 0.9 mm; pitch, 0.99; field of view, 350 mm; gantry rotation time, 0.5 s; table speed, 158.9 mm/s. 2.11. Photothermal effect, photostability, and photothermal conversion efficiency of UCNPs@CS@Ag2 Se nanocomposites To determine the photothermal effect of the nanocomposites, 250 ␮L of UCNPs@CS@Ag2 Se nanocomposites aqueous solutions with different Ag+ concentrations (0, 12.5, 25, 50, 100, 200 ␮g/mL) in 96-well plates were exposed to the NIR laser (808 nm, 1.3 W/cm2 ) for 10 min. The solution temperature was measured every 10 s by a thermocouple microprobe. The temperature of aqueous dispersion with Ag+ concentration of 200 ␮g/mL irradiated by different power density of NIR laser was also recorded. Moreover, the photostability of UCNPs@CS@Ag2 Se nanocomposites was evaluated using on-off cycles of NIR laser irradiation. To evaluate the photostability, a solution containing the nanocomposites (200 ␮g/mL) was irradiated by an 808-nm laser (1.3 W/cm2 ) for 10 min (LASER ON) and then natural cooling to room temperature without irradiation (LASER OFF). Subsequently, the additional five LASER ON/OFF cycles were further repeated to test the photostability. To evaluate the photothermal conversion efficiency (), UCNPs@CS@Ag2 Se nanocomposites aqueous solution (200 ␮g/mL, 250 ␮L) was irradiated by the 808-nm laser with the power density of 1.3 W/cm2 until the temperature was steady. Then, the laser was turned off and the system temperature was cooled naturally to the room temperature with measuring the temperature every 10 s. The photothermal conversion efficiency () was calculated using Eqs. (1)–(4): [65] ␩= hS(Tmax,NPs –Tmax,solvent )/I1 − 10−A808 )

(1)

 s = md Cd /hs

(2)

t = - s ln

(3)

␪ = T–Tsurr /(Tmax,nanocomposites –Tmax,solvent )

(4)

2.12. In vitro and in vivo photothermal effect For in vitro photothermal imaging 1 mL of UCNPs@CS@Ag2 Se aqueous solutions with different Ag+ concentrations (0, 12.5, 25, 50, 100, 200 ␮g/mL) in cuvette was irradiated for 0, 1, 2, 4, 6, 8 and 10 min (808 nm, 1.3 W/cm2 ). An infrared thermal camera was used to record the temperature of the solution. For in vivo photothermal imaging, the tumor-bearing mouse was intravenously injected with UCNPs@CS@Ag2 Se nanocomposites (Ag+ concentration =10 mg/kg). After 24 h, the tumor sites were exposed to the 808-nm laser (1.3 W/cm2 , 6 min). During the NIR irradiation, the infrared thermal camera was used to monitor the temperature changes of the tumor sites. 2.13. In vitro photothermal therapy of cancer cells To study the photothermal cytotoxicity of UCNPs@CS@Ag2 Se nanocomposites, A549 cells were incubated with five different concentration of UCNPs@CS@Ag2 Se aqueous solutions (12.5, 25, 50, 100, and 200 ␮g/mL) for 24 h, and then the cells were washed with PBS, followed by laser irradiation (808 nm, 1.3 W/cm2 ) for 6 min. After another 24 h incubation, the cell viability was evaluated using the standard MTT assay. A live/dead staining kit was employed to differentiate the living cells and dead cells on a fluorescence microscope (Leica). 2.14. In vivo photothermal therapy assays When the tumor volume reached about 100−200 mm3 , the tumor-bearing mice were randomly divided into four groups (n = 6) as follows: (1) control group (5 % glucose solution, 100 ␮L), (2) NIR laser only group (808 nm, 1.3 W/cm2 , 6 min), (3) UCNPs@CS@Ag2 Se group (10 mg/kg Ag+ , 100 ␮L), and (4) UCNPs@CS@Ag2 Se (10 mg/kg Ag+ , 100 ␮L) plus NIR laser (808 nm, 1.3 W/cm2 , 6 min). During the NIR irradiation, an infrared thermal camera was used to monitor the temperature changes of the tumor sites. Tumor size and body weight of the mice before and after treatment were measured using a caliper and an electronic balance, respectively. The tumor volume can be calculated according to the normal equation (volume = width2 × length/2). The relative tumor volume was calculated as V/V0 , where the V0 was the corresponding tumor volume before the treatment. After 14 days, the tumors were dissected and weighed to evaluate the therapeutic efficacy.

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Scheme 1. Schematic illustration of the fabrication process of UCNPs@CS@Ag2 Se nanocomposites and UCNPs@CS@Ag2 Se nanocomposites for multimodal imaging guided PTT of cancer.

2.15. In vivo biodistribution analysis of UCNPs@CS@Ag2 Se The tumor-bearing Kunming mice (n = 3) were injected with 100 ␮L of UCNPs@CS@Ag2 Se at a dosage of 10 mg/kg Ag+ . The main organs of the mice, such as heart, liver, spleen, lung, kidney and tumor, were collected and weighted at different time points (1 h, 2 h, 24 h, 3 d, and 7 d). Then organ section was treated with HNO3 and H2 O2 (v/v = 1:1) at 60 ◦ C until to form the clear solution. The concentration of Ag+ was measured by ICP-MS. 2.16. Histology analysis For the in vivo toxicity studies, healthy Kunming mice were injected with 100 ␮L of UCNPs@CS@Ag2 Se at a dosage of 10 mg/kg Ag+ . The mice without any treatment were used as the blank control. Over 45 days, the mice were executed, and the main organs of the mice (heart, liver, spleen, lung, and kidney) were harvested and fixed using 4 % paraformaldehyde. Tissue samples were then embedded in paraffin, sliced, and stained using H&E. The histological sections were observed under an optical microscope. 3. Results and discussion 3.1. Fabrication and characterization of UCNPs@CS@Ag2 Se nanocomposites The schematic procedure for the fabrication of UCNPs@CS@Ag2 Se nanocomposites was illustrated in Scheme 1. First, Ln3+ -doped upconversion nanoparticles with core–shell-

shell structure (NaYF4 :Yb/Er@NaLuF4 :Yb/Nd@NaLuF4 UCNPs) were synthesized. The active shell (NaLuF4 :Yb/Nd) coated on the surface of NaYF4 :Yb/Er core enable the upconversion emission under 808 nm excitation and provide the UCNPs with potential CT imaging ability. The inert NaLuF4 layer was further coated on the core-shell nanoparticles to enhance the UCL intensity of UCNPs. Subsequently, with the assistance of CTAB, CS was deposited on the surface of the UCNPs and hydrophobic UCNPs were transferred to the hydrophilic phase. Finally, ultrasmall Ag2 Se nanodots were attached and well distributed on the surface of UCNPs@CS by in situ growth method. This multifunctional nanoplatform with excellent multimodal imaging and anti-tumor performance could be used as a promising candidate for imaging-guided photothermal therapy. The X-ray diffraction (XRD) patterns of all the obtained nanoparticles are shown in Fig. S1. All the characteristic diffraction peaks of NaYF4 :Yb/Er, NaYF4 :Yb/Er@NaLuF4 :Yb/Nd, and NaYF4 :Yb/Er@NaLuF4 :Yb/Nd@NaLuF4 in Fig. S1a-c can match well with the standard hexagonal phase of NaYF4 (JCPDS 16–0334). In Fig. S1d, the XRD pattern of UCNPs@CS@Ag2 Se not only shows the diffraction peaks of the hexagonal-phased fluorides, but also displays the diffraction peak of Ag2 Se (JCPDS 24–1041). The transmission electron microscopy (TEM) images of the products obtained at different synthetic steps are shown in Fig. 1a-f. The prepared NaYF4 :Yb/Er core, core-shell NaYF4 :Yb/Er@NaLuF4 :Yb/Nd and the core–shell–shell NaYF4 :Yb/Er@NaLuF4 :Yb/Nd@NaLuF4 nanoparticles with the high structural uniformity and excellent monodispersity have mean diameters of 36 nm, 43 nm, and 52 nm, respectively (Fig. 1a-c). As presented in Fig. 1d and e, typical TEM image (Fig. 1d) indicates that UCNPs coated with a pale layer

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Fig. 1. TEM images of (a) NaYF4 :Yb/Er, (b) NaYF4 :Yb/Er@NaLuF4 :Yb/Nd, (c) NaYF4 :Yb/Er@NaLuF4 :Yb/Nd@NaLuF4 (UCNPs), (d) UCNPs@CS, (e) HRTEM image of UCNPs@CS@Ag2 Se and (f) TEM image of UCNPs@CS@Ag2 Se, and (g) HAADF-STEM image of UCNPs@CS@Ag2 Se and the corresponding element mappings.

has good dispersity without obvious shape change. Besides, from high resolution TEM (HRTEM) image (Fig. 1e), clear lattice fringes with interplanar spacing of 0.297 nm can be attributed to the (110) plane of ␤-NaYF4 and the lattice fringe with the distance of 0.333 nm can be in good agreement with the (111) plane of Ag2 Se. For UCNPs@CS@Ag2 Se (Fig. 1f), it can be clearly observed that ultrasmall Ag2 Se nanodots with the average size of ∼3 nm is conjugated on the surface of UCNPs@CS uniformly, forming a typical planet–satellite structure through the in-situ growth routine. Energy-dispersive X-ray (EDX) spectrum (Fig. S2) and X-ray photoelectron spectroscopy (XPS) (Fig. S3) of UCNPs@CS@Ag2 Se nanocomposites were performed to confirm the presence of Na, F, Y, Yb, Er, Lu, Nd, Ag and Se elements. Moreover, the brightfield TEM image and the corresponding elemental mapping images in Fig. 1g suggest the actual distribution of Y, Lu, Nd, F, Ag and Se elements in UCNPs@CS@Ag2 Se nanocomposites. The above results fully confirm that Ag2 Se nanodots were successfully linked with UCNPs@CS nanoparticles by facile in-situ growth method. In addition, the dynamic light scattering (DLS) measurements of UCNPs@CS and UCNPs@CS@Ag2 Se in water are shown in Fig. S4

and the corresponding intensity-average diameters are 91.2 nm and 141.8 nm, respectively. The zeta potentials of UCNPs@CS and UCNPs@CS@Ag2 Se NCs were measured to be +7.2 and -14.0 mV, respectively, indicating that CS and Ag2 Se were successfully bonded to UCNPs and resulted in UCNPs@CS@Ag2 Se nanocomposites (Fig. S5). 3.2. Luminescence properties and mechanism To evaluate the luminescent properties of as-synthesized OAUCNPs, UCNPs@CS, and UCNPs@CS@Ag2 Se, we measured the UCL and DSL spectra of the obtained samples excited by 808-nm laser at room temperature. As shown in Fig. 2a, under 808 nm excitation, the obtained nanomaterials exhibit both strong DSL and UCL over a wide range of emission spanning from visible to NIR II region. All of the nanomaterials exhibits three distinct characteristic sharp UC emission peaks in the range of 500–700 nm, which can be assigned to 2 H11/2 →4 I15/2 (525 nm), 4 S3/2 →4 I15/2 (545 nm) and 4 F9/2 →4 I15/2 (660 nm) transitions of Er3+ , respectively (Figs. 2a left and S6a). When hydrophobic UCNPs were transferred to the hydrophilic

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Fig. 2. (a) UCL/DSL spectra of UCNPs@CS and UCNPs@CS@Ag2 Se in water excited by 808-nm laser, (b) Schematic illustration of the UCL/DSL processes in a multilayer NaYF4 :Yb/Er@NaLuF4 :Yb/Nd@NaLuF4 nanoparticle system excited at 808 nm and (c) the corresponding energy-level diagram.

phase by coating CS layer, the UCL emission intensity is decreased indicating that the upconversion efficiency was decreased in aqueous environments (Fig. S6a). Note that the UCL emission is further weakened when conjugating Ag2 Se nanodots. This is attributed to the absorption of UCNPs@CS@Ag2 Se nanocomposites covered the UCL spectrum of UCNPs@CS, resulting in the luminescence resonance energy transfer (LRET) process (Fig. S6b). However, the bright green emission of UCNPs@CS@Ag2 Se nanocomposites could still be detected under 808 nm irradiation (inset, Fig. S6a). What’s more, the emission intensity is still high enough to be applied as a bioimaging probe. In DSL spectrum (Figs. 2a right and S6c), under 808 nm excitation, an overwhelmingly strong emission of Yb3+ (2 F5/2 →2 F7/2 ) centered at 980 nm along with a weak emission originating from Er3+ (4 I13/2 →4 I15/2 ) centered at 1532 nm could be observed. Meanwhile, four weak emission bands of Nd3+ centered at 862, 892, 1060, and 1340 nm were found corresponding to the transitions of 4F 4 4 4 4 4 4 4 3/2 → I9/2 , F3/2 → I11/2 , F3/2 → I13/2 , and F3/2 → I15/2 , respectively. This result indicates the energy could effectively transfer from Nd3+ to Yb3+ upon 808 nm excitation, which is consistent with the absorption spectrum of UCNPs (Fig. S6d). A larger absorption cross section of Nd3+ in the NIR region than that of Yb3+ could be found, manifesting that 808-nm laser as the excitation source has more superiority compared with 980-nm laser [39,44]. Moreover, when the UCNPs were converted to water through the introduction of CS, the decrease of DSL emission intensity is much less than that of UC emission intensity (Fig. S6a and b), indicating that the DSL process has less energy loss than the UCL process in the aqueous

solution. It is worth noting that the DSL intensity was not significantly reduced when Ag2 Se nanodots were uniformly covered on the surface of UCNPs@CS, which is attributed to the NIR emission of Ag2 Se centered at 1100 nm promotes the DSL of UCNPs@CS under 808 nm excitation. The luminescent spectra of as-prepared Ag2 Se nanodots were shown in the inset of Fig. S6b. The proposed UC and DS emission mechanisms of the coreshell-shell Er@Nd@Lu nanoparticles under 808 nm excitation are shown in the schematic illustration (Fig. 2b) and the corresponding energy-level diagrams of Nd3+ , Yb3+ and Er3+ were presented (Fig. 2c). The Nd3+ in the Shell 1 transits to its 4 F5/2 state by harvesting 808 nm photons, followed with non-radiative relaxation to the 4 F3/2 state, and then depart from the 4 F3/2 excited state drops back to its different 4 IJ terminal states (4 I15/2 , 4 I13/2 , 4 I11/2 , and 4I 9/2 ), corresponding to different emission bands centered at 1340, 1060, 892 and 862 nm. Due to the emissions of Nd3+ (4 F3/2 →4 I9/2 , 4F 4 3/2 → I11/2 ) show excellent superposition on the absorption spectrum of Yb3+ at 1060 nm, the energy can be efficiently transferred from Nd3+ to Yb3+ through interionic cross-relaxation to populate 2F 3+ and Yb3+ in the excited state can directly return 5/2 state of Yb to the ground state and emit DSL at 980 nm [64]. Simultaneously, the energy harbored by the Yb3+ ions in the shell 1 could transfer to nearby Yb3+ ions crossing the shell further to Yb3+ ions in the core and finally excite the Er3+ ions embedded in the core. The 4 I11/2 state of Er3+ can be populated by getting energy from Yb3+ . Then, a second photon transferred from Yb3+ will populate the 4F 3+ 7/2 state of Er , which could relax non-radiatively to the greenemitting states (2 H11/2 and 4 S3/2 ) for green UC emission around

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Fig. 3. (a) Visible (vis)–NIR absorption spectra of UCNPs@CS@Ag2 Se in water with different concentrations. Inset: the digital photograph of UCNPs@CS@Ag2 Se aqueous solution with different concentrations (6.25, 12.5, 25, 50, 100, and 200 ␮g/mL). (b) A linear relationship for the optical absorbance at 808 nm as a function of the concentration of UCNPs@CS@Ag2 Se. (c) Temperature elevation of water and UCNPs@CS@Ag2 Se aqueous solutions with different concentrations as a function of irradiation time exposure to 808-nm NIR laser (1.3 W/cm2 ). The temperature was measured every 10 s using a thermocouple microprobe. (d) Plot of temperature change (T) over a period of 600 s versus the concentration of UCNPs@CS@Ag2 Se. (e) Temperature elevation of water and UCNPs@CS@Ag2 Se aqueous solution (200 ␮g/mL) under 808-nm NIR laser with different irradiation power densities as a function of irradiation time. (f) Temperature variation of UCNPs@CS@Ag2 Se aqueous solution (200 ␮g/mL) under “on-off” cycles of NIR laser irradiation. (g) The vis-NIR spectra of UCNPs@CS@Ag2 Se dispersion before and after NIR laser irradiation, the absorption value of dispersion at 808 nm during cycles of irradiation were expressed by blue coordinate axis (right and top); inset pictures represented the well-dispersed UCNPs@CS@Ag2 Se aqueous solution before and after irradiation. (h) The photothermal response of UCNPs@CS@Ag2 Se aqueous solution (200 ␮g/mL) for 600 s with an NIR laser (808 nm, 1.3 W/cm2 ), and then the laser was shut off. (i) Linear time data versus -ln␪ obtained from the cooling period of (h).

525 and 540 nm. The 4 I11/2 state of Er3+ could also be attenuated to the 4 I13/2 state through non-radiation, generating DS emission at 1532 nm. The 4 F9/2 state of Er3+ can be populated through the 4 I13/2 state by absorbing a photon from Yb3+ to generate red UC emission around 650 nm. It is obvious that all the UCL and DSL processes discussed above can be achieved with introduction of Nd3+ in the core-shell-shell structure as shown in Fig. 2b. 3.3. Photothermal conversion performance The aqueous solutions of UCNPs@CS@Ag2 Se nanocomposites (Fig. 3a, inset) show broad vis-NIR absorption ranging from 400 to 1000 nm. With increasing Ag+ ion concentration, the spectrum demonstrates a steady increase and the absorbance at 808 nm is linearly increased as shown in Fig. 3a and b, indicating the nanocomposites have good dispersibility in water. Owing to its high NIR absorbance, we expect UCNPs@CS@Ag2 Se nanocomposites to exhibit excellent photothermal property, thereby acting as a potential theranostic agent for in vivo photoacoustic imaging-guided PTT. To test the photothermal effect, different concentrations of UCNPs@CS@Ag2 Se nanocomposites in aqueous solutions (12.5, 25, 50, 100, and 200 ␮g/mL) and pure water as the control were

exposed to an 808-nm NIR laser (1.3 W/cm2 ) for 10 min. It is noteworthy that the temperature of UCNPs@CS@Ag2 Se aqueous dispersion was rapidly increased, exhibiting a concentrationdependent photothermal conversion behavior (Fig. 3c). As shown in Fig. 3d, the temperature change (T) after irradiation for 10 min calculated from Fig. 3c were 11.9, 15.9, 21.9, 33, and 50.2 ◦ C, respectively, while the temperature change for pure water increased by less than 8.1 ◦ C under the same experimental conditions. Moreover, to study the effect of laser power on temperature, UCNPs@CS@Ag2 Se nanocomposites with the concentration of 200 ␮g/mL Ag+ was exposed to an 808-nm NIR laser at various laser power densities, it can be clearly observed that the effect of temperature elevation was power-dependent (Fig. 3e). It is well known that another prerequisite for photothermal agents during PA imaging and/or PTT treatment is high photostability. As shown in Fig. 3f, UCNPs@CS@Ag2 Se aqueous solution (200 ␮g/mL) was irradiated by continuous 808-nm NIR laser (1.3 W/cm2 ), the cycle of irradiation/cooling processes was repeated for six times, and the temperature change was constant during switching cycles test, demonstrating the excellent photothermal stability of UCNPs@CS@Ag2 Se nanocomposites. In addition, the absorption value of the solution at 808 nm is sub-

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Fig. 4. (a) Viability of A549 cells incubated with UCNPs@CS@Ag2 Se at varying concentrations (0−200 ␮g/mL) before and after irradiation by an 808-nm laser for 6 min at power density of 1.3 W/cm2 . (b) Fluorescence images of A549 cells costained cell with calcein AM (live cells, green) and PI (dead cells, red) after in vitro photothermal ablation under NIR laser irradiation with and without the addition of UCNPs@CS@Ag2 Se aqueous solutions with different Ag concentrations. (c) Hemolysis of UCNPs@CS@Ag2 Se after incubation with red blood cells at various concentrations (0−200 ␮g/mL) for 4 h, using PBS and deionized water as a negative and positive control, respectively. Inset: Hemolysis photo after centrifugation. (d) Inverted fluorescence microscope images of A549 cells incubated with UCNPs@CS@Ag2 Se for 1 h and 3 h at 37 ◦ C. Each series can be classified into the bright field image, luminescence image and overlay of the above two. The scale bar in each image is 50 ␮m.

stantially consistent under cycles of irradiation, and the vis-NIR spectra, the color of solution and the morphology exhibit no distinct change after six laser on/off cycles (Figs. 3g and S7), further suggesting that UCNPs@CS@Ag2 Se nanocomposites possess satisfactory photostability. The photothermal conversion efficiency () was further evaluated using the reported method [5,65]. The  value was calculated to be 37.6 % according to the obtained data (Fig. 3h and i), which is comparable and even more excellent than the current reported photothermal agents, such as UCNPs@Bi@SiO2 (∼28 %) [66], Cu2-x Se (∼22 %) [67], and Bi2 Se3 (∼26 %) [68]. Furthermore, the IR thermal images of the various concentrations of UCNPs@CS@Ag2 Se aqueous solution in cuvette irradiated for 0, 1, 2, 4, 6, 8 and 10 min with an 808-nm laser (1.3 W/cm2 ) were vividly shown in Fig. S8. As the concentration of the UCNPs@CS@Ag2 Se aqueous solution increases, the temperature rises rapidly, showing by the intensity bar ranging from 20 ◦ C to 60 ◦ C. As known, the tumor cells could be effectively killed at temperature higher than 42 ◦ C, because cancer cells are more sensitive to hyperthermia than normal cells [59]. In addition, the UCNPs@CS@Ag2 Se aqueous solution (200 ␮g/mL) could be quickly heated over 50 ◦ C after exposure to an 808-nm laser, indicating it holds a great prospect as promising candidate for photothermal therapy.

3.4. Cytotoxicity and UCL imaging-guided photothermal ablation study towards tumor cells Prior to biological applications, it is necessary to evaluate the biocompatibility of the as-prepared nanocomposites. The standard methyl thiazolyl tetrazolium (MTT) assay was first used to detect the cytotoxicity of UCNPs@CS@Ag2 Se nanocomposites. As presented in Fig. 4a (blue bars), the viability was maintained up to 82 % after A549 cells were incubated with different concentrations of UCNPs@CS@Ag2 Se nanocomposites (Ag+ concentration ranging from 0 to 200 ␮g/mL) for 24 h, indicating the nanocomposites has no obvious cytotoxicity on normal cells. The photothermal ablation efficacy of UCNPs@CS@Ag2 Se nanocomposites against tumor cells at different concentrations was investigated. Benefiting from the commendable photothermal performance of UCNPs@CS@Ag2 Se nanocomposites, the relative viabilities of A549 cells decrease obviously with the increased concentration of UCNPs@CS@Ag2 Se nanocomposites after laser irradiation for 6 min (808 nm, 1.3 W/cm2 ), indicating that high concentration of UCNPs@CS@Ag2 Se nanocomposites could enhance photothermal killing efficacy (Fig. 4a, red bars). Ultimately, the viability of A549 cells was calculated to be 26 % (Ag+ concentration =200 ␮g/mL). In addition, the therapeutic effect of

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Fig. 5. In vitro and in vivo multimodal imaging performances of UCNPs@CS@Ag2 Se nanocomposites. (a) In vitro PA images of UCNPs@CS@Ag2 Se nanocomposites at different concentrations. (b) Concentration-dependent PA signals of UCNPs@CS@Ag2 Se nanocomposites. (c) Time-dependent PA imaging in tumor-bearing mice before and after intravenous injection of UCNPs@CS@Ag2 Se nanocomposites. (d) PA signal intensities of the tumor area at different time points. (e) In vitro CT images of iobitridol (1) and UCNPs@CS@Ag2 Se nanocomposites (2) at different concentrations. (f) Concentration-dependent CT signals of iobitridol (red line) and UCNPs@CS@Ag2 Se nanocomposites (black line) in vitro. (g) Time-dependent CT imaging in tumor-bearing mice before and after intravenous injection of UCNPs@CS@Ag2 Se nanocomposites. (h) CT signal intensities of the tumor area at different time points. (i) In vitro NIR (Yb3+ emission) images of the nanocomposites at different concentrations. (j) Concentration-dependent NIR (Yb3+ emission) signals of nanocomposites in vitro. (k) Time-dependent NIR (Yb3+ emission) imaging in tumor-bearing mice before and after intravenous injection of UCNPs@CS@Ag2 Se nanocomposites. (l) NIR (Yb3+ emission) signal intensities of the tumor area at different time points.

UCNPs@CS@Ag2 Se nanocomposites will also be affected by the power density of NIR laser. To further confirm the photothermal effect of UCNPs@CS@Ag2 Se nanocomposites, cell costaining with calcein acetoxymethyl ester (AM) and propidium iodide (PI) was conducted after parallel treatments, and imaged by fluorescence microscopy. We investigate the photothermal elimination capabilities of different concentrations of UCNPs@CS@Ag2 Se nanocomposites to cancer cells at NIR laser power density of 0, 0.8, 1.0, and 1.3 W/cm2 and the live-dead cell staining was performed after different treatments. As shown in Fig. 4b, no obvious cytotoxicity was found for A549 cells when they were treated only with UCNPs@CS@Ag2 Se nanocomposites or only with different laser power groups (green fluorescence). In contrast, for the UCNPs@CS@Ag2 Se + NIR laser groups, the cells show severe apoptosis with increasing concentration and/or power intensity of NIR laser (red fluorescence). Moreover, the influence of UCNPs@CS@Ag2 Se on hemolysis of red blood cells (RBCs) was also investigated. As shown in Fig. 4c, after incubating with different concentrations of UCNPs@CS@Ag2 Se nanocomposites for 3 h, the calculated hemolysis ratio is less than 5.6.% at the maximum concentration (Ag+ concentration = 200 ␮g/mL). The RBCs hemolysis is negligible, which implied UCNPs@CS@Ag2 Se nanocomposites possess excellent hemocompatibility and could be applied intravenously for in vivo imaging and cancer treatment. Based on the above positive results, it can be predicted that the low cytotoxicity and favorable biocompatibility of UCNPs@CS@Ag2 Se nanocomposites would be beneficial for future biological applications. The inverted fluorescence microscope images of A549 cells incubated with UCNPs@CS@Ag2 Se nanocomposites at 37 ◦ C for 1 h and 3 h are shown in Fig. 4d. An obvious enhanced green emission could be observed under NIR laser excitation, implying that most nanocomposites were internalized into the A549 cells over the course of incubation time. The result indicates that UCNPs@CS@Ag2 Se

nanocomposites could be served as an excellent luminescence probe for cell imaging and monitoring endocytosis process of the cells. The above results demonstrate that UCNPs@CS@Ag2 Se nanocomposites have great potentials for in vivo cancer photothermal therapy. 3.5. In vitro and in vivo PA imaging, X-ray CT imaging and NIR fluorescence imaging Compared with fluorescence imaging, PA imaging is a burgeoning imaging technique, which is based on the photoacoustic effect of light absorbers, with significantly increased imaging depth and spatial resolution [11,69]. Thus, UCNPs@CS@Ag2 Se nanocomposites could also be used for PA imaging owing to the strong NIR absorbance and excellent thermal conversion efficiency. To assess the in vitro PA imaging performance, UCNPs@CS@Ag2 Se nanocomposites with different Ag+ concentrations were embedded in agar gel tubes to produce photoacoustic imaging phantoms on a preclinical photoacoustic computed tomography scanner (Endra Nexus 128, Ann Arbor, MI, USA). As expected, the PA signal exhibits concentration-dependent properties under 808 nm excitation, and the signal intensity was linearly increased with increasing Ag+ concentrations (Fig. 5a and b), which suggest that UCNPs@CS@Ag2 Se nanocomposites hold great potentials for PA imaging. Successively, UCNPs@CS@Ag2 Se nanocomposites (Ag+ concentration =10 mg/kg) were intravenously injected into H22-tumor-bearing mice, and then the cross-sectional PA images were collected at different times (pre-injection, 0.5, 1, 2, and 24 h post-injection). The average PA signal intensities derived from the tumor area were shown in Fig. 5c. It is worth noting that only weak PA signal in the tumor tissue could be observed before injection and the signal gradually increased after injection of UCNPs@CS@Ag2 Se nanocomposites within 2 h, indicating that many nanocomposites homogenously accumulate in the

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tumor site and then the signal decreased with increasing time. Quantitative analysis in Fig. 5d clearly demonstrate that the PA signal intensity in tumor at 2 h post-injection was increased by 5-fold compared with that before injection, which may be attributed to the enhanced permeability and retention (EPR) effect during blood circulation [70,71], and then decreased until 24 h post-injection due to metabolism of a portion of UCNPs@CS@Ag2 Se nanocomposites. X-ray CT imaging technique could provide an excellent image resolution and deep tissue penetration in vivo, and has been extensively used in modern clinical diagnosis and prognosis [72,73]. The atomic number and electron density of the CT agent determines the X-ray attenuation coefficient [74]. Since the atomic numbers and electron densities of lutetium (Z = 71, 9.85 g/cm3 ) and ytterbium (Z = 70, 6.98 g/cm3 ) are superior to the extensively applied iobitridol contrast agent (Z = 53, 4.9 g/cm3 ), which endows UCNPs@CS@Ag2 Se nanocomposites with a remarkable imaging ability in X-ray CT imaging. To assess in vitro CT contrast efficacy of UCNPs@CS@Ag2 Se nanocomposites, its X-ray absorption coefficient was compared with that of iobitridol. As shown in Fig. 5e, the CT signals of both contrast agents were enhanced with increasing their concentrations. Meanwhile, a good linear relationship between the Hounsfield unit (HU) values and the concentration of both contrast agents were observed (Fig. 5f). The HU value of UCNPs@CS@Ag2 Se NPs (180.1 HU) is ∼1.6 times higher than that of iobitridol (112.8 HU) at equivalent concentrations, indicating that the obtained nanocomposites have superior X-ray CT imaging ability and could be served as a promising CT imaging agent in clinical applications. Encouraged by the surprising results in vitro, we then assess the feasibility of UCNPs@CS@Ag2 Se nanocomposites as CT contrast agent in vivo. We first intratumorally injected UCNPs@CS@Ag2 Se (RE3+ =10 mg/mL, 50 ␮L) into the tumor-bearing mice. As shown in Fig. S9, the CT signal around the tumor site is significantly enhanced after injection of the nanocomposites and the HU values of the tumor site are increased from 47 (Pre) to 432 (Post). Based on the excellent CT contrast effect in tumor site, the timedependent CT imaging of tumor-bearing mice by intravenous injection of UCNPs@CS@Ag2 Se nanocomposites (RE3+ concentration =0.22 mmol/kg) was studied subsequently. The CT images of tumor area were collected at different time points (pre-injection, 0.5, 1, 2, and 24 h post-injection). As shown in Fig. 5g, the CT signal at tumor site was enhanced sustainably within 2 h compared with the pre-injection image due to the passive accumulation of UCNPs@CS@Ag2 Se nanocomposites. From the quantitative measurement shown in Fig. 5h, the mean HU value was gradually increased from 22.7 HU (pre-injection) to 47.2 HU (0.5 h), 54.5 HU (1 h), 76.3 HU (2 h) and 32.8 HU (24 h) after intravenous injection. These desirable results indicate that UCNPs@CS@Ag2 Se nanocomposites could accumulate well at the tumor site through the EPR effect. Therefore, the nanocomposites could be served as promising CT contrast agent in vivo. UCNPs@CS@Ag2 Se nanocomposites containing Nd3+ not only can act as sensitizers for the UC process, but also can serve as 808 nm-to-NIR (DS) fluorescent emitters, indicating that such nanocomposites have great potential to achieve dual-modal UCL/DSL fluorescence imaging. As known, visible light is not conducive to deep-tissue imaging due to light scattering and light absorption in living biological tissues such as blood [38,41]. NIR light has minimum tissue absorption and maximum penetration depth compare with visible and ultraviolet light, which is particularly desirable for higher contrast imaging. We performed a quantitative tissue penetration depth experiment of UCNPs@CS@Ag2 Se nanocomposites excited at 808 nm using different thicknesses of lean pork ranging from 0 to 15 mm. Both UCL (visible light region of Er3+ ) and DSL (NIR region of Yb3+ ) signal for each thickness at 808 nm excitation are shown in Fig. S10a and b. It

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can be clearly found that even at the tissue thickness of 9 mm, the NIR light of Yb3+ is also discernible, however, the visible light can hardly be detected even when the thickness was increased to 3 mm. The corresponding fluorescent signal was captured using the camera + filter system with the lean pork on the top of the centrifuge tube containing UCNPs@CS@Ag2 Se nanocomposites under 808 nm excitation. It could be found that the NIR image exhibits a higher signal-to-noise ratio compared with visible image, strongly indicating that such nanocomposites are promising candidates for NIR bioimaging. As shown in Fig. 5i and j, the NIR signal of Yb3+ exhibits concentration-dependent properties under 808 nm irradiation, and the fluorescence signal intensity was linearly increased with increasing the concentrations. Then, UCNPs@CS@Ag2 Se nanocomposites (RE3+ concentration =0.22 mmol/kg) was intravenously injected into tumor-bearing mice and the NIR fluorescence imaging at designated time intervals (0, 0.5, 1, 2, and 24 h) were recorded. As shown in Fig. 5k, fluorescence signals were visible in the tumor site at 0.5 h post-injection and enhanced gradually within 2 h due to the gradual accumulation of UCNPs@CS@Ag2 Se nanocomposites in the tumor site through the EPR effect. The NIR signal reached its maximum at 2 h post-injection and then decreased until 24 h postinjection (Fig. 5l), which is in good agreement with the previous CT/PA imaging results. Therefore, the above results demonstrate that UCNPs@CS@Ag2 Se nanocomposites are promising to offer contrasts under multimodal imaging for accurate tumor diagnosis. 3.6. Biodistribution, photothermal effect, photothermal therapy, and long-term toxicity of UCNPs@CS@Ag2 Se nanocomposites in vivo In order to investigate the in vivo biodistribution of UCNPs@CS@Ag2 Se nanocomposites, the tumor-bearing mice were sacrificed after intravenous injection of UCNPs@CS@Ag2 Se nanocomposites (Ag+ concentration =10 mg/kg) at different time points (1 h, 2 h, 24 h, 3 d and 7 d) and the Ag content in the tumor and the major organs (heart, liver, spleen, lung, kidney) was measured by ICP-OES. As shown in the distribution diagram (Fig. 6a), UCNPs@CS@Ag2 Se nanocomposites were mainly distributed in organs of the reticuloendothelial system, such as liver and spleen, in the first 2 h after intravenous injection, followed by the heart, lung, kidney, and tumor. At 2 h post-injection, the accumulation efficiency of UCNPs@CS@Ag2 Se nanocomposites in the tumor reached the maximum value due to the EPR effect, which is consistent with the PA/CT/NIR imaging results. With increasing time, the decrease of Ag in all the tested organs was observed, indicating the gradual excretion of UCNPs@CS@Ag2 Se nanocomposites from mice. Inspired by the outstanding in vitro photothermal killing effect of UCNPs@CS@Ag2 Se nanocomposites and their superior passively targeted accumulation properties at tumor sites, we further studied the feasibility of in vivo multimodal imagingguided photothermal elimination of cancer cells. The in vivo photothermal effect of UCNPs@CS@Ag2 Se nanocomposites was monitored using an infrared thermal camera. UCNPs@CS@Ag2 Se (Ag+ concentration =10 mg/kg) were intravenous injection into the tumor-bearing mice and the tumors were irradiated with an 808-nm laser (1.3 W/cm2 ) after 2 h of intravenous administration. It can be clearly observed that the temperature of tumor site increased rapidly with NIR laser irradiation whereas the temperature of the control mice was not obvious change (Fig. 6b). From the temperature variation (Fig. 6c), the temperature of the tumor regions reached up to 59.3 ◦ C in the presence UCNPs@CS@Ag2 Se nanocomposites under NIR irradiation, which is sufficient to kill the cancer cells and effectively inhibit their malignant proliferation. In contrast, the temperature of tumor regions in the control group exhibited a minor elevation. The encouraging results indicate that UCNPs@CS@Ag2 Se nanocomposites possess

Please cite this article as: K. Du, P. Lei, L. Dong et al., In situ decorating of ultrasmall Ag2 Se on upconversion nanoparticles as novel nanotheranostic agent for multimodal imaging-guided cancer photothermal therapy, Appl. Mater. Today, https://doi.org/10.1016/j.apmt.2019.100497

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Fig. 6. Time-dependent biodistribution (heart, liver, spleen, lung, kidney, and tumor) of Ag in tumor-bearing Kunming mice after intravenous injection of UCNPs@CS@Ag2 Se nanocomposites (Ag+ concentration =10 mg/kg) at different post-injection time points. (b) Infrared thermal images of tumor site of tumor-bearing mice intravenously injected with 5 % glucose solution (control) and UCNPs@CS@Ag2 Se nanocomposites (Ag+ concentration =10 mg/kg) followed by an 808 nm laser irradiation for 6 min. (c) Corresponding temperature change curves at tumor sites based on thermal images. (d) Representative photographs of mice on different groups after various treatments. (e) Body weight of mice under different treatments. (f) Relative tumor growth curves of different groups after various treatments. (g) The digital photographs of excised tumors from representative euthanized mice and (h) mean tumor weight of each group after various treatments from the last day of the experiment (day 14). (i) Hematoxylin and eosin (H&E)-stained slices of tumor tissues collected from different groups. All scale bars stand for 100 ␮m.

excellent photothermal effect, thus could be used as the PTT agent for tumor ablation in vivo. Afterward, when the tumor volume reaches around 200 mm3 , Kunming mice bearing H22 cells were randomly divided into four groups (n = 6) as follows: 1) control group; 2) NIR laser-only group; 3) UCNPs@CS@Ag2 Se group; 4) UCNPs@CS@Ag2 Se + NIR laser group. Under the monitoring of an infrared thermal camera, the tumor sites were irradiated for 6 min by an 808-nm NIR laser (1.3 W/cm2 ) at 2 h post-injection. Representative mouse digital photos in each group during PTT process were shown in Fig. 6d. As known, the change of tumor volume and body weight during a period of 14 days are direct indicators to evaluate the therapeutic efficacy. The tumor growth curves of each group are studied and shown in Fig. 6f. For groups 1–3, the tumor volume shows rapid growth trend during the detection period, in sharp contrast, the tumor volume in terms of group 4 can be effectively suppressed and shows a downward trend. Meanwhile, the body weight of all groups maintained steady growth upon different treatments in duration (Fig. 6e), indicating no systemic side effects of the theranostic agent toward PTT. Fig. 6g and h show

the representative photographs and the average weights of the final tumors excised from mice after 14 days treatment respectively. It is found that the group 4 treated with UCNPs@CS@Ag2 Se nanocomposites upon NIR laser irradiation impose significant tumor inhibition without any regrowth, the tumors have been even completely eliminated after 14 days of PTT, which presents a sharp contrast to the other three groups. In addition, H&E staining analysis of the tumors slices after treatments was used to further evaluate the therapeutic efficacy. As shown in Fig. 6i, there was no obvious necrosis in the groups 1-3. The tumor tissue from group 4 was damaged seriously, at the same time, the irregular cell morphology and the increased intercellular spaces were observed. Taken together, the results confirmed that UCNPs@CS@Ag2 Se nanocomposites possess great potential as an ideal PTT agent for in vivo treatment of cancer. Finally, we evaluate the potential long-term toxicity of UCNPs@CS@Ag2 Se nanocomposites in vivo. The major organs (heart, liver, spleen, lung, and kidney) from control and treated mice were harvested at 45 days after intravenous injec-

Please cite this article as: K. Du, P. Lei, L. Dong et al., In situ decorating of ultrasmall Ag2 Se on upconversion nanoparticles as novel nanotheranostic agent for multimodal imaging-guided cancer photothermal therapy, Appl. Mater. Today, https://doi.org/10.1016/j.apmt.2019.100497

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Fig. 7. Hematoxylin and eosin (H&E) stained images of major organs (heart, liver, spleen, lung, and kidney) of mice 45 days post-injection of UCNPs@CS@Ag2 Se nanocomposites (Ag+ concentration =10 mg/kg). All scale bars stand for 100 ␮m.

tion of UCNPs@CS@Ag2 Se nanocomposites (Ag+ concentration =10 mg/kg). As the H&E stained images shown in Fig. 7, there was no appreciable tissue damage and abnormal inflammatory lesion in all major organs. Moreover, no obvious abnormal behavior of mice was observed during the whole observation period. The positive result confirms that UCNPs@CS@Ag2 Se nanocomposites have negligible toxicity and side effects in vivo for potential multimodal imaging and photothermal therapy. 4. Conclusion In summary, novel UCNPs@CS@Ag2 Se nanocomposites have been successfully fabricated via a facile in-situ growth routine and served for multimodal imaging guided cancer photothermal therapy. Ultrasmall Ag2 Se nanodots can be firmly grown and well distributed on the surface of UCNPs through the intermediate CS shell. UCL/DSL dual mode luminescence were simultaneously realized when such nanocomposites were excited by 808-nm laser, which allows us to choose optical probes for bioimaging selectively. UCNPs@CS@Ag2 Se nanocomposites can not only exhibit pronounced UCL imaging signals in vitro as well as DSL imaging signals in vivo but also offer contrasts in CT/PA imaging due to high X-ray attenuation coefficient and strong NIR absorbance. Moreover, UCNPs@CS@Ag2 Se nanocomposites possess favorable biocompatibility, high photothermal conversion efficiency, excellent photothermal stability and outstanding in vitro and in vivo photothermal killing effect, showing great potentials as ideal PTT agent. In vitro and in vivo experimental results were carried out to prove the potential of UCNPs@CS@Ag2 Se nanocomposites as a significant multifunctional nanoplatform for tetra-modal UCL/DSL/CT/PA imaging-guided PTT of cancer in the biomedical field. Impressively, UCNPs@CS@Ag2 Se nanocomposites are wellmetabolized from mice and have negligible toxicity or side effect in vivo. Taken together, the facile and novel synthesis, favorable biocompatibility, negligible toxicity as well as multimodal imaging guided PTT, enable the development of UCNPs@CS@Ag2 Se nanocomposites as the excellent therapeutic carrier for further biomedical applications. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors are grateful for the financial aid from the National Natural Science Foundation of China (21871248, and 21590794), the Youth Innovation Promotion Association of Chinese Academy of Sciences (2015181), the Key Research Program of Frontier Sciences,

CAS (YZDY-SSW-JSC018), K. C. Wong Education Foundation (GJTD2018-09), and Jilin Province Science and Technology Development Plan Project (20180101172JC).

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Please cite this article as: K. Du, P. Lei, L. Dong et al., In situ decorating of ultrasmall Ag2 Se on upconversion nanoparticles as novel nanotheranostic agent for multimodal imaging-guided cancer photothermal therapy, Appl. Mater. Today, https://doi.org/10.1016/j.apmt.2019.100497