Yb3+@SiO2

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Biological and Medical Applications of Materials and Interfaces

NIR-to-NIR Deep Penetrating Nanoplatforms Y2O3:Nd3+/ Yb3+@SiO2@Cu2S towards Highly Efficient Photothermal Ablation Zhiyu Zhang, Hao Suo, Xiaoqi Zhao, Dan Sun, Li Fan, and Chongfeng Guo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03239 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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ACS Applied Materials & Interfaces

NIR-to-NIR

Deep

Penetrating

Y2O3:Nd3+/Yb3+@SiO2@Cu2S

towards

Nanoplatforms Highly

Efficient

Photothermal Ablation Zhiyu Zhang†, Hao Suo†, Xiaoqi Zhao†, Dan Sun†, Li Fan*‡ and Chongfeng Guo*† † National Key Laboratory of Photoelectric Technology, Functional Materials (Culture Base) in Shaanxi Province, National Photoelectric Technology, Functional Materials & Application of Science and Technology International Cooperation Base, Institute of Photonics & Photon-Technology, Northwest University, Xi’an, 710069, China. ‡ Department of Pharmaceutical analysis, School of Pharmacy, and The State Key Laboratory of Cancer Biology (CBSKL), The Fourth Military Medical University, 169th Changle West Road, Xi’an, Shaanxi, 710032, China. ------------------------------------------------------------------------------------------------------KEYWORDS: NIR-to-NIR; Core-shell structure; Penetration depth; Photo-thermal conversion; Ablation. ABSTRACT:

A

di-functional

nano-photo-thermal

therapy

platform

with

near-infrared excitation to near-infrared emission (NIR-to-NIR) were constructed through core-shell structures Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC), in which the core Y2O3:Nd3+/Yb3+ and shell Cu2S play the role of bio-imaging and photo-thermal conversion function, respectively. The structure and composition of the present photo-thermal therapy agents (PTAs) were characterized by powder x-ray diffraction (XRD), field emission scanning electron microscope (FE-SEM), transmission electron

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microscope (TEM) and X-ray photoelectron spectra (XPS). The NIR emissions of samples in biological window area (BWA) were measured by photoluminescence (PL) spectra under the excitation of 808 nm laser, further the penetration depth of NIR emission at different wavelength in biological tissue were also demonstrated by comparing with visible (Vis) emission from Y2O3:Yb3+/Er3+@SiO2@Cu2S and NIR emission from Y2O3:Nd3+/Yb3+@SiO2@Cu2S through injecting different depths in pork muscle tissues. The photo-thermal conversion effects were achieved through the outer ultra-small Cu2S nano-particles efficiently simultaneously absorb the 808 nm excitation source and NIR light emission from the core Y2O3:Nd3+/Yb3+ to generate heat. Further, the heating effect of YRSC nano-particles was confirmed by thermal imaging and ablation of YRSC to E. coli and human hepatoma (HepG-2) cells. Results indicate that the YRSC has potential applications in photo-thermal therapy and NIR imaging in biological tissue.

1. INTRODUCTION

As a new developed and promising cancer treatment, photo-thermal therapy (PTT) offers high specificity and minimal side effects in comparison conventional surgery, chemotherapy and radiotherapy treatments though significant effects have been gotten on traditional cancer treatment in past decades.1-4 PTT could convert external near infrared (NIR) photon energy into massive heat through efficient photo-thermal therapeutic agents (PTAs), in which the fast conversion speed is conductive to reduce the pain and duration of treatment.5, 6 For ideal PTAs, the small nano-scaled particles size is not only easy to accumulate in tumor sites by enhanced permeability but also to ACS Paragon Plus Environment

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provide more opportunity to be modified with more functional molecules in a small volume due to its larger surface area.7,

8

Some conventional gold nanostructures,

carbon dots or nano-tubes, copper-based nano-particles have been developed for PTAs due to their strong absorption in the NIR region and efficient photo-thermal conversion efficiency.9, 10 Amongst them, copper-based nanoparticles are considered as prospective PTAs because they offer high stability, low cost and low cytotoxicity.11, 12

To achieve the precise treatment for the tumor tissue, it is necessary to accurately control the position of the nano-PTAs in the lesion region, which will become powerful tools for detection and treatment of cancer if endowing the PTAs with the function of optical imaging.13 Compared with the visible imaging, NIR imaging is more valuable for biological application because the NIR light located in the optical biological window area (BWA, 600-1350nm) provides deep tissue penetration and excellent bio-security.14-16 An ideal biological imaging luminescent particle had better posses strong absorption and emission in biological window area, which could provide clear and high quality imaging through avoiding the in vivo auto-fluorescence and exhibit deeper tissue.17-19 Typically, NIR-to-NIR emissions used for highly penetrating fluorescence bio-imaging were realized by two-photon excited up-conversion nano-particles (UCNPs) in Yb3+-Tm3+ co-doped system,20 in which Yb3+ ion is typical sensitizers owing to its large absorption cross-section at 980 nm and efficient Yb3+ to Tm3+ energy transfer.21, 22 However, the maximum absorption of water molecules at around



980 nm in biological tissue greatly hinder their potential applications, resulting in overheat effect to damage the normal tissues and cells with the continuous irradiation of 980 nm laser. Nd3+ not only has broad and strong absorption cross section at around 808 nm, but also efficiently transfers the absorbed energy to Yb3+;23 moreover, the emissions of Nd3+ and Yb3+, ranging from 850 to 1200 nm under the excitation of 808 nm laser, are benefit to fluorescence bio-imaging and successfully avoid absorption of water.24, 25 Thus, a new photo-thermal therapy nano-platform with NIR imaging and photo-thermal conversion capacity was designed through coating traditional PTAs cuprous sulfide on the surface of Y2O3:Nd3+/Yb3+@SiO2 nano-spheres, in which PTAs cuprous sulfide not only absorbs energy of 808 nm from the excitation light source to generate heat but also absorbs part of the Nd3+-Yb3+ NIR emission to further enhance the photo-thermal effect. Here, a core-shell structured di-functional photo-thermal therapy nano-platform were constructed, in which the core with NIR-to-NIR single photon excited Y2O3:Nd3+/Yb3+ nano-sphere plays the role of imaging and the absorbed ultra-small cuprous sulfide offer the function of photo-thermal conversion. The new PTAs could be expressed as Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC), the penetration depth of NIR light and visible light in biological tissues were investigated in a comparable method, and the photo-thermal ablation of Escherichia coli (E. coli) and human hepatoma cells (HepG-2) were also evaluated to demonstrate the potential application of nano-platform.


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2. EXPERIMENTAL SECTION Reagents and materials. All chemicals are used without further purification in present experiments, including high purity RE2O3 (RE=Y, Nd and Yb) (99.99%) and analytical grade reagents (A. R.) nitric acid (HNO3), urea, tetraethoxysilane (TEOS), ammonium hydroxide (NH3.H2O), copper chloride dehydrate (CuCl2·2H2O), sodium citrate (Na3Cit), sodium sulfide (Na2S), aminopropyltrimethoxysilane (APTMS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium

bromide

(MTT)

and

dimethyl sulfoxide (DMSO). In addition, phosphate buffered saline (PBS), nutrient broth and agar medium, Escherchia coli (E. coli) (reference number: ATCC25922), HepG-2 cells and live/dead cell imaging kit were also used. Synthesis of Y2O3:0.01Nd3+/0.1Yb3+@SiO2 and Y2O3@SiO2 nano-spheres. At the beginning,

mono-dispersed

precursor

nano-spheres

of

Y2 O3

and

Y1.89O3:0.01Nd3+/0.1Yb3+ were prepared through a co-precipitation technique, in which a stoichiometric amount of raw materials Y2O3, Yb2O3, Nd2O3 and Er2O3 were first dissolved in nitric acid with stirring and heating to get hydro-soluble rare earth nitrate. Then, 12.4 g urea and 50 mL deionized (DI) water were added to form transparent solution and kept at 95 ℃ for 30 min. After centrifuging and washing the precursor precipitations several times with water and alcohol, the precursors were obtained after drying at 80 ℃ for 6 hours. Finally, the dried precursors were calcined in air at 700℃ for 3 h with 3 ℃ min-1 heating rate to get Y2O3:Nd3+/Yb3+ or Y2O3 nano-spheres. To improve the biocompatibility of the samples, coating a layer of SiO2 on the



surface of above obtained nano-spheres is necessary, which is achieved via a modified Stӧber process. Above obtained Y2O3:Nd3+/Yb3+ or Y2O3 nano-spheres (0.1g) were ultrasonically dispersed in the mixture of 80 mL ethanol, 20 mL DI water and 2.0 mL ammonia aqueous solution (25 wt%) to get a high dispersion solution, the following step is to introduce 0.4 mL TEOS drop by drop with continuous stirring. After reacting and stirring for 5 h at room temperature, the obtained particles were separated and washed with ethanol and DI water to dry at 80 ℃ to obtain core-shell structured Y2O3:Nd3+/Yb3+@SiO2 (named as YRS) and Y2O3@SiO2 (named as YS) nano-spheres. Synthesis of Y2O3:Nd3+/Yb3+@SiO2@Cu2S and Y2O3@SiO2@Cu2S. To better connect ultra-small Cu2S particles with the out-layer SiO2 of Y2O3:Nd3+/Yb3+@SiO2 (YRS) or Y2O3@SiO2 (YS), as-prepared YRS or YS nano-spheres (0.06 g) were first ultrasonically dispersed in 40 mL ethanol for 10 minutes and followed adding APTMS (0.12 mL) drop by drop with constant stirring for 12 h to formed -NH2 on the surface of YRS and YS. Cu2S nano-particles were synthesized according to the following procedures. 0.014 g CuCl2·2H2O and 0.02 g sodium citrate were dissolved in 100 mL DI water and then adding 2 mL of Na2S·9H2O (0.04 M) with stirring 5 min to form the brown solution. Subsequently, the brown solution was kept at 90 °C for 30 min to get a dark-green solution, and this solution was preserved at 4 °C. After that, 0.03 g as-prepared -NH2 modified Y2O3:Nd3+/Yb3+@SiO2-NH2 or Y2O3@SiO2-NH2 was dispersed in the 40 mL dark-green Cu2S nano-particles solution, respectively.


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Followed

reacting

and

stirring

for

2

h,

the

final

samples

Y2O3:Nd3+/Yb3+@SiO2@Cu2S (named YRSC) or Y2O3@SiO2@Cu2S (named YSC) were obtained after centrifuging, washing and drying at 70 °C for 2h. Sample Y2O3:Yb3+/Er3+@SiO2@Cu2S was also synthesized as contrast group using the same method. Photothermal ablation of bacteria. The diluted Bacteria (E. coli) by sterile nutrient broth solution were adequately mingled with PBS buffer (1.0 mg/mL) containing Y2O3@SiO2 (YS) or Y2O3@SiO2@Cu2S (YSC) or Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC), respectively. The acquired solution was exposed to the irradiation of a 1.1 W cm-2 808 nm laser for 30 s. After that, the spread plate method was used to transfer the solution treated with laser in the nutrient agar medium, and cultured at 37 ℃ for 24 h. Followed that, the bacteria colony numbers were calculated. A blank group without bacteria, samples and irradiation of 808 nm laser was set as control. To reduce the experimental error, tests were repeated three times. Cytotoxicity assay and In Vitro Photothermal Ablation of cancer cells. A standard MTT assay was applied to evaluate the cytotoxicity of Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC) using HepG-2 cells. The cells were seeded in sextuplicate into 96-well plates and treated with the indicated concentrations of YRSC (0.25, 0.5, 1, 2.5, 5, 15, 30, 62.5 and 125 µg/mL) for 24 h. Subsequently, MTT was added into the medium at a final concentration 125 µg/mL and incubated at 37 °C for 4 h to allow the formation of formazan. Dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan crystals. The absorbance was measured by a microplate reader at 570 nm to



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determine the relative cell viability. Before the photo-thermal ablation experiment, the selected HepG-2 cells were seeded into 96-well plates for 24 h. For qualitative analysis, the cells were incubated in the condition with and without YRSC for 4 h, followed by an 808 nm NIR laser (1.1 W/cm2) irradiation for 5 min. Then, the cells were stained with live/dead cell imaging kit and visualized under a fluorescence microscope. Characterization. The phase purity and crystal structure of samples were carried out using powder X-ray diffraction (XRD) on a Rigaku-Dmax 3C powder diffractometer (Rigaku Corp, Tokyo, Japan) with Cu Kα radiation (λ = 1.5406 Å) in the range of 10°≤ 2θ ≤ 70° and Fourier transform infrared (FT-IR) spectra using a Bruker EQUINOX55 spectrometer. The Zeta potential test was measured using a Malvern Zen 3600 (Malvern Instruments Ltd, England). The morphology and microstructure of samples were recorded on a field emission scanning electron microscope (FE-SEM, Hitachi SU-8010) and a transmission electron microscope (TEM, JEM-2100F, Japan) equipped with an energy dispersive X-ray (EDX) spectrometer. For TEM measurements, samples were suspended in ethanol solution and a drop of the solution was placed on a carbon coated nickel grids. X-ray photoelectron spectra (XPS) were carried out on a Kratos AXIS-ULTRA DLD (The United Kingdom) instrument using a monochromatic Al Kα excitation source. The photoluminescence (PL) spectra were recorded

by

a

FLS920

fluorescence

spectrophotometer

equipped

with

a

power-controllable 808 nm semiconductor laser as excitation source, and the UV-vis-NIR absorption spectra were received by Cary 5000 UV-Vis-NIR


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spectrophotometer. The photo-thermal behaviors of the samples were assessed using an InfReC R500 infrared thermal camera (Nippon electronic company, Japan). The confocal fluorescence images of HepG-2 cells were detected by FV1000 (OLYMPUS, Japan).

3. RESULTS AND DISCUSSIONS

3.1. Structure and morphology of samples. The synthetic procedures of the core-shell structured Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC) and Y2O3@SiO2@Cu2S (YSC) could be divided into four steps: the synthesis of precursor, calcined precursor to obtain Y2O3 (Y) or Y2O3:Nd3+/Yb3+ (YR), coating silica layer on Y2O3 (or Y2O3:Nd3+/Yb3+) and the modification of core-shell structured samples by Cu2S ultra-small particles, as schematically illuminated in Fig. 1A. To identify the phase purity and structure of products at every step, corresponding XRD patterns were displayed in sequence in Fig.1B, in which no distinct diffraction peaks were found for the precursor in comparison with the well matched peaks with standard profile of Y2O3 (JCPDS No. 65-3178) for the calcined precursor. A broad band centered at 2θ = 22° characteristic peak from amorphous SiO2 (JCPDS No. 29-0085) were observed in the patterns of silica coated Y2O3:Nd3+/Yb3+, implying the successful coating of SiO2 on the surface Y2O3:Nd3+/Yb3+.26 However, no characteristic diffraction peaks of Cu2S were found in the XRD patterns of Cu2S coated sample Y2O3:Nd3+/Yb3+@SiO2, which may be attributed to the low crystallinity or less amount of Cu2S nano-particles. Furthermore, Fourier transform infrared (FTIR) spectroscopy of precursor, YR, YRS, ACS Paragon Plus Environment


YRS-NH2 and YRSC were given in Fig. S1, in which broadband absorption at about 3471 cm-1 from O-H of water were observed in all samples. The characteristic vibration from O-C-O, -OH and -CO3 appeared in the precursor, which imply that the precursor may be Y(OH)CO3. With further calcination and modification, Y-O, -Si-O-Si-, SiO4 and Si-O appeared with the disappearance of -CO3, which further prove the formation of Y2O3 and the silica coating layer. In order to determine the morphology and size distribution of products at each step, TEM images of Nd3+-Yb3+ co-doped samples were representatively displayed in Fig. 1C-F. The highly mono-dispersed precursor particles exhibit regular spherical shape with about 110 nm uniform size (Fig. 1C) and no significant changes were found in the shape and size distribution of sample with and without dopants (in Fig. S2); similarly, no obvious changes were observed in the morphology and dispersibility of Y2O3:Nd3+/Yb3+ obtained through heating precursor samples at 700 ℃ for 3 h (Fig. 1D). Subsequently, Y2O3:Nd3+/Yb3+ were coated a layer of silica with about 20 nm mean thickness, whereas the uniformity and dispersibility of coated sample Y2O3:Nd3+/Yb3+@SiO2 was preserved (Fig. 1E) except the increase to about 150 nm for average size. Finally, the above obtained products were further modified by Cu2S particles to get final core-shell structured sample Y2O3:Nd3+/Yb3+@SiO2@Cu2S, as shown in Fig. 1F, in which Y2O3:Nd3+/Yb3+@SiO2 particles were first modified with the positive charge -NH2 group from APTMS to make the connection of negative-charged ultra-small Cu2S nanoparticles with the surface of Y2O3:Nd3+/Yb3+@SiO2 more solid by electrostatic interaction (in Fig. S3).19


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Figure 1. Scheme of the synthesis of Y2O3:Nd3+/Yb3+@SiO2@Cu2S core-shell structured nano-spheres (A) and the XRD patterns of each step (B) together with their corresponding TEM images of sample particles of precursors (C), Y2O3:Nd3+/Yb3+ (D), Y2O3:Nd3+/Yb3+@SiO2 (E) and Y2O3:Nd3+/Yb3+@SiO2@Cu2S (F). All scale bars are 100 nm. To further character the microstructure and composition of the final sample Y2O3:Nd3+/Yb3+@SiO2@Cu2S, a TEM image with higher magnification was provided in Fig. 2A, in which lots of ultra-small nano-particles were detected on the surface of spherical shape sample. The high resolution transmission electron microscopy (HRTEM) images of zones a and b in Fig. 2A were shown in Fig. 2B and C, in which the clear lattice distances of the inner core (zone a) and the outer ultra-small particles (zone b) were 0.43 and 0.24 nm corresponding to the (211) plane of the cubic Y2O3 (JCPDS No. 65-3178) and (102) plane of the Cu2S (JCPDS No.26-1116), respectively.



Their fast Fourier transform (FFT) analysis of the corresponding core and the adsorbed ultra-small particles (Fig. 2D and E) also substantiates the cubic Y2O3 and hexagonal Cu2S. Furthermore, XPS analysis was performed to explore the valence state of copper. Figure S4 displays the Cu 2p spectrum of Cu2S, where the two strong peaks at 932.5 and 952.4 eV were assigned to the binding energy of Cu 2p3/2 and Cu 2p1/2, respectively.27 The inset of Fig. S4 shows the L3VV kinetic energy spectra of Cu peaked at 916.3 eV, which is a characteristic peak of mono-valent Cu+ species.28 The clear bright/dark contrast among the core, shell and the adsorbed ultra-small nano-particles in the high-angle annular dark-field scanning TEM (HAADF-STEM) image further confirm the core-shell structure of the Y2O3:Nd3+/Yb3+@SiO2@Cu2S, as presented in Fig. 2F. The cross-section element compositional line profiles in Fig. 2G and the elemental mapping analysis (Fig. 2H) clearly depict the elemental composition and distribution area, in which elements Yb, Nd, Y distributed in the interior but elements Si, Cu, S concentrated on the outer shell of sample particles. Above results further confirm the components and core-shell structure of final sample Y2O3:Nd3+/Yb3+@SiO2@Cu2S.


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Figure 2. (A) TEM image, (B,C) HR-TEM images and corresponding (D,E) FFT patterns in different zones, (F) HAADF-STEM, (G) Cross-section compositional line profiles and (H) Elemental mapping images of sample Y2O3:Nd3+/Yb3+@SiO2@Cu2S. 3.2. Optical property.



Figure 3. (A) NIR emission spectra of Y2O3:Nd3+/Yb3+(YR), Y2O3:Nd3+/Yb3+@SiO2 (YRS), Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC) together with the digital photographs of the YRSC solution irradiated by 808 NIR laser for different time; (B) UV-vis-NIR absorption spectra of PBS buffer solution and Cu2S, YR, YRS, and YRSC dispersed in PBS; (C) Measured NIR emission intensity of YRSC with irradiation of 808 nm laser and (D) visible emission (λex=980nm) intensity of Y2O3:Yb3+/Er3+@SiO2@Cu2S as a function of injection depth in pork as well as (E) the corresponding variable curves of integrated intensity. Inset of Fig.E is the intensity ratio of IYb/INd in YRSC with different injection depth. Under the excitation of 808 nm NIR light, the samples Y2O3:Nd3+/Yb3+ (YR), Y2O3:Nd3+/Yb3+@SiO2 (YRS) and Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC) exhibit similar emission in NIR optical biological window area (BWA) ranging from 850-1200 nm, as shown in Fig. 3A, in which emission peaks are composed of the two parts. The strongest peak at 975 nm comes from the 2F5/2 → 2F7/2 transition of Yb3+ due to the energy transfer from Nd3+ → Yb3+; the other peaks are attributed to the


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characteristic emissions of Nd3+. The corresponding NIR digital photographs of YRSC aqueous solution irradiated by 808 nm laser for different time (i.e. 0.1, 0.3 and 0.5 ms) were given in the inset of Fig. 3A, in which an 850 nm cutoff filter were used to avoid the effect from laser. However, the PL intensity of sample YR, YRS and YRSC gradually decreased in turn because of the high absorbency of both the SiO2 shell and Cu2S nanocrystals.29,

30

Furthermore, the Vis-NIR absorption spectra of

sample Cu2S, YR, YRS, and YRSC dispersed in PBS solution together with the blank PBS solution were provided in Fig. 3B, in which no distinct absorption appeared for blank PBS solution but a broad absorption band ranged from about 500 to 1200 nm were observed for Cu2S. In comparison with that of YRS, the absorption in the region of 750-1200 nm was greatly enhanced for the sample YRSC, which is mostly responsible for the decrease of YRSC emission intensity due to the Cu2S absorption for 808 nm laser and the NIR emission from the core YR. In order to investigate the penetrating depth of the YRSC emission, the monitored emission intensity of sample YRSC was presented in Fig. 3C with variable injection depth in pork muscle tissues; as a contrast, the depth-dependent up-conversion (UC) emission intensity of Y2O3:Yb3+/Er3+@SiO2@Cu2S in visible region was also given in Fig. 3D. In order to avoid the influence of the sample morphology and size distribution on penetration depth, Figure S5 displays the same morphology and size of Y2O3:Yb3+/Er3+@SiO2@Cu2S as YRSC. It is clear that the appreciable fluorescence signals from both samples drastically decreases with increasing the injection depth. For the sake of comparison, the normalized integrated intensity of YRSC and



Y2O3:Yb3+/Er3+@SiO2@Cu2S were depicted in Fig. 3E, in which the descent speed of the visible emission intensity from Y2O3:Yb3+/Er3+@SiO2@Cu2S is faster than that of NIR emission intensity from YRSC. NIR emission could still be detected up to over 8 mm injection depth, whereas the visible emission is completely attenuated at a much shallower depth of about 4 mm. It is important to remark that the data in Fig. 3E present the direct evidence that the penetration depth of NIR emission in tissues is substantially improved in comparison with that of visible light. The inset of Fig. 3E further presents the variable integrated intensity ratio (R=IYb/INd) of Yb3+ emission (966-984 nm) to Nd3+ (1000-1150 nm) as a function of injection depth in pork muscle tissues, in which the continuously decreased R values represent the direct measurement that the absorption of 980 nm NIR light is stronger than that of 1000-1150 nm NIR light in biological tissues.

Figure 4. Irradiation-time-dependent temperature variation curves of (A) YRS, YSC


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and YRSC powder using 1.1W/cm2 808nm laser and (B) powder-density dependent temperature of YRSC for different irradiation time together with (C) photographs and (D) bacteria viability of the colonies of E. coli under different incubated conditions. To detect the photo-thermal conversion effects of samples Y2O3:Nd3+/Yb3+@SiO2 (YRS), Y2O3@SiO2@Cu2S (YSC) and Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC), the real-time temperatures of YRS, YSC and YRSC powder collected by an infrared thermal camera under the excitation of an 808 nm (1.1W/cm2) laser, as shown in Fig. 4A. At the beginning, the temperature of sample YSC and YRSC rapidly increase, and gradually achieved the maximum upon the prolonging the exposure time to 27 s. The expectation is that the temperature increment (∆T) of YRSC (41.3℃) is higher than that of YSC (33℃) due to the enhanced absorption for 808 nm laser by Nd3+ and the re-absorption of NIR emission from Y2O3:Nd3+/Yb3+ by Cu2S, whereas only 0.9 ℃ was improved for YRS. These data included in Fig. 4A represent that Cu2S nano-particles are mostly responsible for the significantly enhanced photo-thermal effect. Furthermore, the power-density-dependent photo-thermal effect of the optimal sample YRSC were depicted in Fig. 4B as function of exposure time of 808 nm laser, in which the surface temperatures of samples rapidly heat up and arrive balance within 5 s. With power density increasing from 0.2 to 1.6 W/cm2, the heat effect is more efficient and the surface temperature reach at about 29.5, 38.9, 54.2 and 76.6 ℃ as irradiation for 12 s using 0.2, 0.3, 1.1 and 1.6 W/cm2 808 nm laser, respectively. In consideration of heat effect and he biological safety, the 1.1 W/cm2 of power density was used in the follow-on experiments.



3.3. Photo-thermal ablation. A contrast experiment to further evaluate the photo-thermal effects of samples Y2O3:Nd3+/Yb3+@SiO2 (YRS), Y2O3@SiO2@Cu2S (YSC) and Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC) was performed by assessing their ablation efficiencies on bacteria (E. coli), in which bacteria were first incubated in YRS, YSC or YRSC+PBS solution (1.0 mg/mL), and then irradiated with 808 nm laser (1.1 W/cm2) for 30 s. A blank group only including PBS buffer (pH=7.4) was treated in the same environment as control and no any bacteria colonies was observed, which indicate that all the utensils and mediums were cleaned. As shown in Fig. 4C, a larger number of bacterial colonies were observed in the YRS+808 group; by contrast, a few colonies were seen in the YSC+808 group and very few colonies were observed in the YRSC+808 group. The corresponding E. coli viability in different groups were also counted and displayed in Fig. 4D, it is found that no obvious decline in colony for the group of YRS+808, whereas about 56 and 80% bacteria were ablated in YSC+808 and YRSC+808 group, respectively. This result further confirmed that most heat effect is attributed to Cu2S nano-particles and the introduction of Yb3+/Nd3+ enhanced the photo-thermal effect, thus the best phto-thermal ablation was observed with YRSC+808 against E. coli. To further confirm the antibacterial action of YRSC plus laser group, SEM was used to compare the morphology of E. coli (Fig. S6) in YRSC+laser group with that of the untreated E. coli group. No significant bacterial morphological changes were observed in untreated E. coli, whereas nano-particles were clearly found on the surface of E. coli and ablated the E. coli through destructing the bacterial wall.


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Figure 5. (A) Cell viabilities of HepG-2 cells incubated for 24 h with different concentrations of YRSC and confocal fluorescence image of HepG-2 cells (B), HepG-2 cells with 808 (C) as well as HepG-2 cells with YRSC under 808 nm laser (1.1 W/cm2) irradiation for 5 min (D). Cells were dyed with LIVE/DEAD Cell Imaging Kit. For determining the applicability of the Y2O3:Nd3+/Yb3+@SiO2@Cu2S (YRSC) nano-particles in real photo-thermal therapy, the toxicity and cell viability of the HepG-2 cells after treatment with different contents of YRSC nano-particles were investigated

by

a

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

standard bromide)

MTT assay,

and

the

obtained results were shown in Fig. 5A. As expected, the average cell viability of HepG-2 after incubation with different concentration YRSC nano-particles for 24 h is over 90%, which indicate that no cytotoxicity of YRSC is observed. In order to



visualize the localized photo-thermal ablation of HepG-2 cells, the untreated HepG-2 cells, treated HepG-2 cells with laser of 808 nm (1.1 W/cm2) for 5 min and the incubated HepG-2 cells in YRSC for 4 h with 5 min irradiation of 808 nm (1.1 W/cm2) are stained by the LIVE/DEAD Cell Imaging Kit. Then, the stained HepG-2 cells were visualized by confocal laser scanning microscopy imaging to distinguish living or dead cells, as shown in Fig. 5 B, C, and D. A vivid green color was observed in the entire well when cells were treated without or with only 808 nm laser (Fig. 5B and C), suggesting that the exposure of cells to laser irradiation alone is safe. Whereas, a little green color was observed among red color in the well when cells were incubated with YRSC+808 nm laser due to the predominant death cells killed by huge heat from photo-thermal effect of YRSC. These results indicate that YRSC offer great promise for cancer cell treatment as an efficient photo-thermal agent.

4. CONCLUSIONS In summary, we have developed a core-shell structured difunctional photo-thermal therapy nano-spherical platform Y2O3:Nd3+/Yb3+@SiO2@Cu2S. The microstructure characterization of samples illuminate that ultra-small Cu2S particles were absorbed on the surface of Y2O3:Nd3+/Yb3+@SiO2, in which NIR-to-NIR emission from the core of Y2O3:Nd3+/Yb3+@SiO2@Cu2S has deeper penetration in biological tissue than that of Vis emission from the core of Y2O3:Yb3+/Er3+@SiO2@Cu2S. This not only endows the present sample with great promise in biological imaging applications, but also Cu2S nano-particles on the outer surface could simultaneously absorb the 808 nm excitation light and partial emission from Nd3+ and Yb3+ to generate heat. The optimal ACS Paragon Plus Environment

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heat effect was achieved in sample Y2O3: Yb3+/Er3+@SiO2@Cu2S by a contrast experiment

among

Y2O3@SiO2,

Y2O3@SiO2@Cu2S

and

Y2O3:Nd3+/Yb3+@SiO2@Cu2S, which further confirmed by the photo-thermal ablation and over 80% E. coli were killed after irradiation 30s with 808nm laser. Furthermore,

the

result

MTT

tests

indicate

that

the

sample

of

Y2O3:Nd3+/Yb3+@SiO2@Cu2S is non-toxic, and most of the HepG-2 cells were ablated through photo-thermal effect of the samples. Results confirmed that combining NIR-to-NIR imaging with efficient photothermal effects of conventional PTAs in one nano-platform is significant for further biological applications. But how to precisely detect the actual amount of heat generated by PTAs in biological tissue to ensure the safety of photo-thermal therapy is our further step. ASSOCIATED CONTENT

Supporting Information

This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (No.51672215), Research Fund for the Doctoral Program of Higher Education of China



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Yb3+@SiO2

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