temperature-responsive drug release

Accepted Manuscript An imaging-guided platform for synergistic photodynamic/photothermal/chemotherapy with pH/temperature-responsive drug release Ruichan Lv, Piaoping Yang, Fei He, Shili Gai, Guixin Yang, Yunlu Dai, Zhiyao Hou, Jun Lin PII:

S0142-9612(15)00466-4

DOI:

10.1016/j.biomaterials.2015.05.016

Reference:

JBMT 16853

To appear in:

Biomaterials

Received Date: 31 January 2015 Revised Date:

1 May 2015

Accepted Date: 14 May 2015

Please cite this article as: Lv R, Yang P, He F, Gai S, Yang G, Dai Y, Hou Z, Lin J, An imaging-guided platform for synergistic photodynamic/photothermal/chemo-therapy with pH/temperature-responsive drug release, Biomaterials (2015), doi: 10.1016/j.biomaterials.2015.05.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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An imaging-guided platform for synergistic photodynamic/photothermal/chemo- therapy with pH/temperature-responsive drug release Ruichan Lv a, Piaoping Yang a,*, Fei He a, Shili Gai a, Guixin Yang a, Yunlu Dai a,

a

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Zhiyao Hou b, Jun Lin b,*1

Key Laboratory of Superlight Materials and Surface Technology, Ministry of

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Education, College of Material Sciences and Chemical Engineering, Harbin Engineering University, Harbin, 150001, P. R. China b

State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of

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Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China

*

Corresponding author: Fax: +86 431 86598041.

E-mail: [email protected] (P. Yang), [email protected] (J. Lin) 1

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ABSTRACT To integrate biological imaging and multimodal therapies into one platform for enhanced anti-cancer efficacy, we have designed a novel core/shell structured

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nano-theranostic by conjugating photosensitive Au25(SR)18– (SR refers to thiolate) clusters, pH/temperature-responsive polymer P(NIPAm-MAA), and anti-cancer drug (doxorubicin, DOX) onto the surface of mesoporous silica coated core-shell

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up-conversion nanoparticles (UCNPs). It is found that the photodynamic therapy

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(PDT) derived from the generated reactive oxygen species and the photothermal therapy (PTT) arising from the photothermal effect can be simultaneously triggered by a single 980 nm near infrared (NIR) light. Furthermore, the thermal effect can also stimulate the pH/temperature sensitive polymer in the cancer sites, thus realizing the

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targeted and controllable DOX release. The combined PDT, PTT and pH/temperature responsive chemo-therapy can markedly improve the therapeutic efficacy, which has been confirmed by both in intro and in vivo assays. Moreover, the doped rare earths

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endow the platform with dual-modal up-conversion luminescent (UCL) and computer

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tomography (CT) imaging properties, thus achieving the target of imaging-guided synergistic therapy under by a single NIR light.

Keywords: Up-conversion, photodynamic, photothermal, pH/temperature-responsive

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1. Introduction For the drug delivery systems (DDSs), if referred to imaging-guided photodynamic therapy (PDT) and photothermal therapy (PTT), two irrelevant lights

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were usually utilized to achieve the diagnosis and therapy which made it hard for real-time assessment. Meanwhile, the loaded drug molecules are confronted with the leak problem before arriving at the tumor focus, which may introduce serious side

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damage to the normal organs and body [1–7]. The up-conversion luminescent (UCL)

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emission generated upon NIR excitation is potential to track the treatment, and the UCL emissions in visible regions could be used as the donors for transferring energy to photosensitizers [8–11]. Moreover, NIR light located in the optical transmission window of biological specimens has the merits of high penetration depth, high

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detection sensitivity, increased image contrast, and low damage to cells [12–16]. The traditional organic dyes as photosensitizers have some disadvantages, such as poor water solubility, high skin toxicity, low selectivity, instable physical/chemical

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property, and other ambiguous problems [17–19]. Thus, developing novel inorganic

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materials as the photosensitizers is of great importance. Recent research progress on the solution-phase atomically precise thiolate-protected Aun(SR)m– clusters have taken up part of photosensitizers field [20–22]. Especially, Au25(SR)18– clusters with higher body clearance and lower kidney toxicity have been proposed to produce 1O2 production owing to its high stability, long lifetime of the electronic excited states, presence of triplet excited states with which the adsorbed 3O2 could be transferred to 1

O2 [23,24]. Meanwhile, Au25(SR)18– clusters may also generate the photothermal 3

ACCEPTED MANUSCRIPT effect owing to the strong electromagnetic fields inside particles caused by their closely positioned and coupled sharp features [25,26]. When the PDT agent is used for in vivo clinical application, short irradiation time, high 1O2 quantum yield and low

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laser pump power are required. The luminescent spectral overlap between the donor (phosphor) and acceptor (photosensitizer), the distance between the two counterparts, and the quantity of loaded photosensitizers all play important role in the 1O2 yield.

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Thus, the structure design of the UCL host/dopant and material structure is essential

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[27–31]. The silica coated functional materials have been especially used as effective drug carriers to reduce the toxicity of the inorganic and organic materials [32–36]. By controlling the shell thickness of mesoporous silica, the BET surface which decides the quantity of loaded nanoparticles and the distance between donors and acceptors

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could be adjusted. Particularly, the large surface area, porous structure, adjustable pore channel, and easily modified surface of mesoporous silica make it possible to store more drug molecules.

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For the conventionally synthesized multi-functional nanoparticles with silica or

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hollow structure for the DDSs, the anticancer drug molecules in these systems always cannot distinguish the normal cells from the cancer ones. Besides, they may show an early leak and uncontrollable release under the normal body fluid environment (37 °C, neutral) [37,38]. To resolve this formidable challenge, the gatekeepers are needed to regulate the release and prevent the early leak before entering the tumor cells [39,40]. In recent years, pore blocking caps have been proposed such as polymer brushes and macromolecule chains which could be self-changed under various conditions. 4

ACCEPTED MANUSCRIPT Poly(N-isopropyl acrylamide) (P(NIPAm)), as one of the most important temperature-sensitive polymer, could translate its phase in aqueous environment under changed temperature which is suitable for blocking pores as a gatekeeper.

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Especially, by conjugating co-monomer or tuning the environment pH value, the low critical solution temperature (LCST) of the P(NIPAm) polymer can be changed which is above normal body temperature (even higher than 50 °C under pH value of 7.4)

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[41–44]. In the tumor-bear body, the tumor focus always has higher temperature and

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lower pH value than the normal ones due to the inflammation and cancer cell immortalization [45]. Thus, the positive pH/temperature-sensitive “on-off” drug release is attractive to apply in the anti-cancer therapy DDSs because photothermal effect could be generated due to the photosensitive agent irradiated by NIR laser and

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they can intelligently distinguish the normal cells from the tumor ones due to the characteristic property of cancer focus.

In this study, we proposed a facile and mass-production method to construct

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core/shell structured Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-P(NIPAm-MAA) (YSAP)

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up-conversion nanoparticles with adjustable shell thickness. An inert Y2O3:Yb shell was employed to enhance to the UC emission intensity of Y2O3:Yb,Er core. The silica shell thickness is controlled by adjusting the amount of added TEOS. Meanwhile, Au25(SR)18– clusters with size of 2.5 nm were conjugated and well dispersed in the silica mesopores to produce PDT and PTT effect by receiving energy from the UCNPs. Meanwhile, the pH/temperature-responsive polymer NIPAm-MAA is introduced to realize the targeted and controllable release triggered by the high 5

ACCEPTED MANUSCRIPT temperature arising from the system under NIR irradiation and low pH condition in the cancer cells. The biocompatibility, dual-modal imaging (CT and UCL), especially in vitro and in vivo toxicity of Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-P(NIPAm-MAA)

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have also been investigated in detail.

2. Experimental section

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2.1. Reagents and materials

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All the chemical reagents in this experiment are of analytical grade and used without any further purification, including urea, hydrochloric acid (HCl), nitric acid (HNO3), HAuCl4·3H2O, methanol, dimethyl sulfoxide (DMSO) (Beijing Chemical Corporation), Y2O3, Yb2O3, and Er2O3 (Sinopharm Chemical Reagent Co., Ltd.),

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cetyltrimethyl ammonium Bromide (CTAB), NaOH, tetraethoxysilane (TEOS), ammonium nitrate (NH4NO3), 1,4-dioxane, phosphate buffered saline (PBS), potassium hydrogen phthalate (PHP), glutaraldehyde (Tianjin Kermel Chemical Co.,

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Ltd.), diphenyl (2, 4, 6-trimethylbenzoyl)-phosphine oxide (TPO) (Energy Chemical),

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and aminopropyltrimethoxysilane (APTES), tetraoctylammonium bromide (TOAB), NaBH4, N-isopropyl acrylamide (NIPAm), methacrylic acid (MAA), DOX, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride

(EDC),

nhydroxysuccinimide (NHS), folic acid (FA), 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT), 4’,6-diamidino-2-phenylindole (DAPI), calcein AM and propidium iodide (PI), trypan blue (Sigma-Aldrich). 2.2. Synthesis 6

ACCEPTED MANUSCRIPT 2.2.1. Synthesis of Y(OH)CO3:Yb,Er Briefly, by dissolving corresponding Ln2O3 (Ln = Y, Yb, and Er) into HNO3 under heating, 1 mmol of Ln(NO3)3 solutions were obtained. In the co-precipitation process,

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1 mL of Ln(NO3)3 (94%Y, 5%Yb, and 1%Er) were mixed with 3 g of urea in a beaker containing 50 mL of deionized water. The solution was heated to 90 °C and kept for 3

2.2.2. Synthesis of Y(OH)CO3:Yb,Er@Y(OH)CO3:Yb

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h. After washed several times, Y(OH)CO3:Yb,Er precursor was prepared.

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The coating process of the Y(OH)CO3:Yb was similar to the precursor. Firstly, the as-synthesized Y(OH)CO3:Yb,Er was dispersed into 50 ml deionized water and ultrasonic treated, and then 1 mmol of Ln(NO3)3 (95%Y and 5%Yb) and 3 g of urea were mixed and dissolved into the solution. After stirring for another 5 min, the

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mixture was kept stable with water bath at 90 °C for 3 h. The resulting precipitate was centrifuged and dried at 60 °C for 12 h, and the Y(OH)CO3:Yb,Er@Y(OH)CO3:Yb spheres were obtained.

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2.2.3. Synthesis of core/shell Y2O3:Yb,Er@Y2O3:Yb@mSiO2

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Before the coating process, the Y(OH)CO3:Yb,Er@Y(OH)CO3:Yb spheres were firstly calcinated at 600 °C for 3 h in order to obtain Y2O3:Yb,Er@Y2O3:Yb spheres. Here, NaOH solution instead of conventional NH3.H2O solution was used to give rise to the facile and fast coating process. Typically, 0.15 g of Y2O3:Yb,Er@Y2O3:Yb and 0.1 g of CTAB was mixed with 10 mL ethanol and 40 mL of deionized water with continuous stirring, and then 3 mL of NaOH (1 mol L–1) was added. Then, the mixture was heated up to 70 °C, and then 150 µL of TEOS was added slowly. After kept for 10 7

ACCEPTED MANUSCRIPT min, the mixture was centrifuged and washed with ethanol and water for several times. To remove CTAB surfactant, the as-synthesized silica-coated spheres were mixed with 100 mL of ethanol with 1 g of NH4NO3, and then kept at 70 °C for 2 h. After that,

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the mixture was centrifuged and washed with ethanol several times. Finally, the Y2O3:Yb,Er@Y2O3:Yb@mSiO2 composite was obtained after drying at 60 °C for 12 h. The samples with different silica shell thickness were synthesized by adjusting the

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amount of added TEOS.

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2.2.4. Synthesis of Au25(SR)18− clusters

In a typical process, 78.7 mg of HAuCl4·3H2O and 126.8 mg of TOAB were dissolved in 10 mL of methanol solvent and stirred for 20 min vigorously. After that, 1 mmol of captopril dissolved in 5 mL of methanol was rapidly added into the solution,

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which was further stirred for another 30 min. Then, 2 mmol of NaBH4 dissolved in 5 mL of ice-cold deionized water was rapidly injected in the solution with vigorous stirring, which was continued for another 8 h. After that, the sample was centrifuged

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to remove the excessive Au(I) polymer. The supernatant was recovered and further

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treated by rotary evaporation and then precipitated by ethanol. After extracting several times with little amount of methanol, the extracted clusters were precipitated by ethanol and dried in vacuum. The TEM image and the size distribution of the as-synthesized Au25(SR)18− clusters are shown in Fig. S1. 2.2.5. Preparation of Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25 In order to make negative Y2O3:Yb,Er@Y2O3:Yb@mSiO2 and negative Au25(SR)18− clusters conjugate, the positive –NH2 group was firstly modified. Briefly, 8

ACCEPTED MANUSCRIPT the as-synthesized Y2O3:Yb,Er@Y2O3:Yb@mSiO2 was dispersed in 20 mL deionized water with continuous stirring. After that, 2 mL of APTES (50 g L–1) was added into the solution and reacted for another 12 h. Then, the product was centrifuged to

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remove the excess APTES. The obtained sample was dispersed in 20 mL of ethanol containing 5 mL of as-prepared Au25(SR)18− clusters with continuous string for 4 h. At last, the solution was centrifuged and dried at 60 °C for 12 h. The obtained product

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was denoted as Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25.

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2.2.6. Preparation of Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-P(NIPAm-MAA) Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-P(NIPAm-MAA) was achieved by a facial photo-induced polymerization. Firstly, 20 mg of Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25 powders were dispersed in 1 mL of 1,4-dioxane, and then 0.125 g of NIPAm, 0.003 g

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TPO, and 7.6 µL of MAA were added. The solution was ultrasonic treated to obtain the mixed gel. After the photo-crosslinking process, the resulting mixture was then exposed to the UV lamp (200 W cm–2, PHILIPS) for 5 min to complete the

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photo-curving process. Subsequently, the product was washed with deionized water

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and ethanol for several times to remove the unreacted monomers, then dried in vacuum at 50 °C to obtain Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-P(NIPAm-MAA) (denoted as YSAP). Before the in vivo administration, the as-synthesized sample was further modified by FA molecules. Typically, 20 mg of YSAP was dissolved into 20 mL of deionized water. 1 mL of FA (10 mg mL–1),1 mL of NHS (2 mg mL–1), 1 mL of EDC (6 mg mL–1) were kept stirring for 2 h and then added into the YSAP solution with continuous stirring for another 12 h in dark. Finally, the product was recovered 9

ACCEPTED MANUSCRIPT by centrifugation and washed twice to remove the free FA. 2.3. Characterization Powder X-ray diffraction (XRD) measurements were performed on a Rigaku

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D/max TTR-III diffractometer with graphite monochromatized Cu Kα radiation (λ = 0.15405 nm), and the scanning rate is 15° min–1 within the 2θ range from 20° to 80°. Images were obtained digitally on transmission electron microscopy (TEM, FEI

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Tecnai G2 S-Twin). Fourier transform infrared spectroscopy (FT-IR) spectra were

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measured on a Perkin-Elmer 580B IR spectrophotometer using KBr pellet technique. N2 adsorption/desorption isotherms were acquired on a Micromeritics ASAP Tristar II 3020 apparatus, the pore size distribution was obtained by the Barrete-Jonere-Halenda (BJH) method. UCL emission spectra were obtained on Edinburgh FLS980 apparatus

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using a 980 nm laser diode Module (K98D08M-30W) as the irradiation source and recorded in the visible light region. DOX concentration was calculated by UV-1601 ultraviolet visible (UV-vis) spectrophotometer at the wavelength of 480 nm.

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2.4. DOX loading and release test of YSAP

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Typically, 30 mg of YSAP was ultrasonically dispersed into 5 mL of PBS (pH = 7.4), and then 2.5 mL of DOX (1 mg mL–1) was added into the YSAP solution with slow stirring for 24 h at room temperature. The as-prepared mixture was centrifugally separated, and the supernatant solution was retained for further UV-vis analysis. The mixture was kept for further release process. Typically, 10 mL of fresh PBS was replenished and set in water bath kettle at 37 °C with magnetic stirring, and then the supernatant solution was kept for further analysis. The obtained sample was rinsed 10

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different temperature was carried out by adjusting the water bath temperature. The loading efficiency and concentration of DOX in the solution were determined by UV-vis measurement with the typical absorbance wavelength at 480 nm.

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2.5. In vitro viability of YSAP

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6000-7000 well–1 of L929 fibroblast cells were put in a 96-well plate and incubated with 5% CO2 at 37 °C. Among them, 8 wells were left with culture only for blank control, and then incubated for 24 h to confirm the cells to attach the wells. The YSAP UCNPS were dispersed with culture and diluted into diverse concentrations of

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500, 250, 125, 62.5, 31.3, and 15.63 µg mL–1, and then the solutions with different concentrations were added to the wells and further incubated with L929 cells for 24 h. After that, 20 µL of MTT solution (5 mg mL–1) was added to every well and the cells

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were incubated at 37 °C for another 4 h. Note that only the viable cells could make

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MTT reduce into formazan. At last, DMSO (150 µL) was added to each well. The absorbance at 490 nm was measured using a micro-plate reader for calculation. 2.6. In vitro cellular uptake and UCL microscopy (UCLM) observation Cellular uptake process was performed by A549 cancer cell lines using a confocal

laser scanning microscope (CLSM). In a 6-well culture plate, the A549 cells were seeded and allowed to grow overnight to obtain monolayer cells. After that, the wells were added with as-prepared YSAP (1 mg mL–1) at 37 °C with incubation time of 30 11

ACCEPTED MANUSCRIPT min, 1 h, and 3 h, respectively. And then the cells were rinsed with PBS several times, fixed with 1 mL of glutaraldehyde (2.5%) at 37 °C for 10 min, then further washed with PBS three times. 1 mL of DAPI solution (20 µg mL–1) was added and kept for 10

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min. After rinsed three times, the cells were detected by Leica TCS SP8 instrument. For the UCL microscopy observation, the slide was executed with the same process except that the cells were detected by inverted fluorescence microscopy (Nikon Ti-S).

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

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The in vitro CT imaging experiments were performed on a Philips 64-slice CT scanner with the voltage of 120 kV. The YSAP UCNPs were dispersed in saline and diluted to a series of concentrations of 16, 8, 4, 2, 0.5, and 0.25 mg mL–1 and then placed in a line for CT imaging. The mice were first anesthetized with 10% chloral

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hydrate (0.03 mL g–1 of mouse) by intra-peritoneal injection to perform in vivo CT imaging. Then, 100 µL of YSAP UCNPs were injected intratumorally into the tumor-bearing mice in situ for scanning.

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2.8. In vitro cytotoxicity of YSAP

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A549 cells were seeded in a 6-well culture plate and grown overnight as a monolayer. After that, the cells were incubated with different treatments: with culture only, with NIR irradiation, with YSAP UCNPs, with YSAP UCNPs under NIR irradiation, with YSAP-DOX, with YSAP-DOX under NIR irradiation. The pump power of the NIR irradiation is 0.72 W cm–2. The materials were added and incubated for 6 h to complete the cell uptake, and then the irradiation was carried on. After the treatment, the wells were washed with PBS both dyed with calcine AM and PI, and 12

ACCEPTED MANUSCRIPT visualized using CLSM. 2.9. In vivo toxicity of YSAP The H22 tumors were generated in the left axilla of each female mouse through

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injection of murine hepatocarcinoma cell lines (20–25 g) subcutaneously. After grown for the next 7 days, the tumor size is about 6-8 mm. The tumor-bearing mice were randomly divided into five groups (n = 5 per group) and injected intravenously with

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YSAP UCNPs, with NIR irradiation, YSAP-DOX, and YSAP-DOX with NIR

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irradiation, respectively. One group was used for blank control without any treatment. The mouse was injected every two days with YSAP amount of 100 µL (1 mg mL–1). The tumor site was irradiated by the NIR light for 10 min every time (pump power is 0.72 W cm–2). After treatment for two days, the body weight and tumor size were

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detected and recorded. 2.10. Histology examination

After treatment for two weeks, the histology analysis was carried out. The

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representative organs of kidney, liver, heart, lung, spleen, and the tumor tissues of the

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mice in the control group and treatment group were isolated to less than 3 cm × 3 cm for each tissue. And then the isolated tissues were successively dehydrated using buffered formalin, ethanol of different concentrations, and xylene. After that, the dehydrated tissues were embedded with liquid paraffin, and sliced to 3-5 mm for hematoxylin and eosin (H&E) staining. The final stained slices were examined using an optical microscope.

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3. Results and discussion 3.1. Formation, phase, structure, shape, and luminescent properties The schematic illustration for the formation of YSAP UCNPs and the

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imaging-guided synergistic multimodal anticancer therapy application are depicted in Scheme 1. As shown, there are four important points in the synthesis: the coated inert shell is beneficial to obtain the higher UCL emission intensity which is good to the

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final diagnosis and the FRET process. The further coated mesoporous silica shell (the

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shell thickness can be adjusted) is utilized to attach photosensitizers and store more drug molecules. The attached Au25(SR)18– clusters are photothermal/photodynamic agents, and P(NIPAm-MAA) is the gatekeeper to obtain pH/temperature-responsive drug release. Through the master design, the imaging-guided synergistic anti-cancer

precursor,

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therapy effect is obtained. Fig. 1 presents the XRD patterns of Y(OH)CO3:Yb,Er Y(OH)CO3:Yb,Er@Y(OH)CO3:Yb,

Y2O3:Yb,Er@Y2O3:Yb,

and

Y2O3:Yb,Er@Y2O3:Yb@mSiO2 spheres with adjusted silica thickness. There are no

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clear diffraction peaks in Y(OH)CO3:Yb,Er and Y(OH)CO3:Yb,Er@Y(OH)CO3:Yb,

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which indicate the two samples are amorphous. After calcination, the diffraction peaks of the product fit well with the cubic-phased Y2O3 (JCPDS No. 43–1036). The sharp and strong peaks indicate the high crystallinity. When the mesoporous silica is coated, the XRD pattern of Y2O3:Yb,Er@Y2O3:Yb@SiO2 has no obvious change due to the high crystallinity of the sample which covers the peaks of silica. The cell lattice parameter of Y2O3:Yb,Er@Y2O3:Yb is 10.442 Å which is well consistent with that of Y2O3 (10.604 Å). There is a tiny decrease of the unit cell because the larger Y3+ ions 14

ACCEPTED MANUSCRIPT are replaced by the doped Yb3+ ions with smaller ionic radius (10.435 Å). TEM images of Y(OH)CO3:Yb,Er and Y(OH)CO3:Yb,Er@Y(OH)CO3:Yb are given in Fig. S2. It is obvious that the two samples are uniform with smooth surface.

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As expected, the Y(OH)CO3:Yb,Er@Y(OH)CO3:Yb has lager particle size (160 nm) than that (140 nm) of Y(OH)CO3:Yb,Er. Meanwhile, there is an ambiguous boundary between Y(OH)CO3:Yb,Er core and the Y(OH)CO3:Yb shell, and the thickness is

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about 20 nm. The coating of an inert shell will increase the UCL intensity due to the

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decreased surface defects and cross-relaxation caused by the shorter Yb-Yb distance for the increased Yb content in the host. The UC emission spectra of Y2O3:Yb,Er and Y2O3:Yb,Er@Y2O3:Yb are given in Fig. 2A. It is obvious Y2O3:Yb,Er@Y2O3:Yb has much higher emission intensity than that of Y2O3:Yb,Er and the emission colour

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changes from yellow/green to yellow in the CIE chromaticity image (Fig. 2B). Even under the NIR laser irradiation with low pump power (0.72 W cm–2), Y2O3:Yb,Er@Y2O3:Yb emits brighter green colour than that of Y2O3:Yb,Er (Fig. 2C,

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D). Usually, this enhancement effect arising from the coated inert shell has been only

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achieved in oleic acid-capped nanoparticles (about 50 nm) obtained from hightemperature solvent method [46–52], and this facile co-precipitation process derived UC emission enhancement has rarely been reported. After calcination, uniform microspheres with particle size of 140-170 nm are

obtained (Fig. 3A1-A3). After coating a layer of mesoporous silica, the as-obtained product (Y2O3:Yb,Er@Y2O3:Yb@mSiO2) is still uniform. In Fig. 3B1-B3, the thickness of silica shell is 5 nm when adding 50 µL of TEOS during the coating 15

ACCEPTED MANUSCRIPT process, whereas the thickness is increased to 20 nm when 150 µL of TEOS was introduced (Fig. 3C1-C3). Note that the thickness of the silica layer can be adjusted to 0, 5, 10, 20, and 30 nm by controlling the amount of added TEOS (Fig. S3). Thus, the

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controllable sizes can provide the opportunity to further optimize the performance of the core/shell material because the shape and size are important when applied in the biological field..

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After modification of APTES, positive-charged –NH2 groups are conjugated on

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the surface of Y2O3:Yb,Er@Y2O3:Yb@mSiO2, and then the negative Au25(SR)18− clusters are easily attached on the surface by electrostatic interaction. Finally, after modifying P(NIPAm-MAA), the multi-functional YSAP UCNPs are achieved. Fig. 3D shows the TEM image of the final YSAP UCNPs. As seen, the final product is

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uniform with size of 160-190 nm, and the core/shell structure can be clearly seen due to the different electron penetrability of the core and shell. The average thickness of silica shell and P(NIPAm-MAA) shell is about 20 nm and 40 nm, respectively. EDS,

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STEM element mapping and the corresponding cross-section compositional line

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profiles of YSAP are presented in Fig. 3E, F. The elements of Y, Yb, Si, O, and Au are well distributed in the spheres. Meanwhile, the line profiles indicate the core of Y2O3:Yb,Er@Y2O3:Yb and the shell of mesoporous silica, which further confirms the formation of core/shell structure. N2 adsorption/desorption isotherms of Y2O3:Yb,Er@Y2O3:Yb@mSiO2 with various silica thickness of 0, 5, and 20 nm are exhibited in Fig. S4. The three samples all exhibit typical IV-type isotherms, indicating the mesoporous channels. The BET 16

ACCEPTED MANUSCRIPT surface areas of the three samples are calculated to be 13, 86 and 251 m2 g–1, respectively. The N2 adsorption/desorption isotherm of pure Y2O3:Yb,Er@Y2O3:Yb (Fig. S4A) has the hysteresis loop in the high relative pressure (0.9-1.0) region

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corresponding to H4 hysteresis loops, which is the typical character of layer structure. For comparison, other two silica coated samples (Fig. S4C, E) have the extra H1 hysteresis loops in the medium relative pressure (0.3-0.8), revealing the formation of

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small pores. As shown in Fig. S4D, F, both samples have two main pores located at

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3.34 and 3.17 nm, respectively. This core/shell structure with large surface area and suitable pore size will be beneficial to store more drug molecules and conjugate functional nanoparticles. FT-IR

spectra

of

Y2O3:Yb,Er@Y2O3:Yb,

Y2O3:Yb,Er@Y2O3:Yb@mSiO2,

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Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25, YSAP, and YSAP-DOX are shown in Fig. S5. Similar bands at 3400 and 564 cm–1 in the three spectra are assigned to the hydroxyl group stretching of water and –Y–O–. For Y2O3:Yb,Er@Y2O3:Yb@mSiO2, the

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characteristic absorption bands of –Si–O–Si (1083 and 801 cm–1) indicate the

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existence of silica [53]. For the APTES modified and further Au25– clusters conjugated sample, two new peaks at 2924 cm–1 and 1407 cm–1 are ascribed to –CH3 and –NH2, respectively [54]. The typical peaks at 1387, 1457, 1658, 2928, and 2972 cm–1 are assigned to the conjunction of the P(NIPAm-MAA) [55]. Furthermore, for the DOX loaded sample, several new peaks at 1000-1800 cm–1 (1171, 1245, 1546, and 1720 cm–1) and 3290 cm–1 can be attributed to the load DOX molecules [56]. 3.2. In vitro compatibility, imaging, drug release, and toxicity 17

ACCEPTED MANUSCRIPT It is essential to detect the biocompatibility of YSAP UCNPs for potential biological application. Standard MTT assay was employed on L929 cell lines to detect the short-term viability. Fig. S6 demonstrates the cell viability of the cells

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incubated with different concentrations of the particle varying from 15.63 to 500 µg mL–1 for 24 h. The cell viability of the as-prepared sample in all dosages is 99.7%– 112.3%, indicating the good biocompatibility in vitro. In the optical microscopy

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images of the cells grown with culture only (Fig. S5B) and in the presence of 1 mg

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mL–1 YSAP UCNPs (Fig. S5C) dyed by trypan blue, the L929 cells in both groups spread and begin to proliferate similarly. In addition, almost no blue cells are found which indicates the non-toxicity of the UCNPs.

Fig. S7 presents the CLSM photographs of A549 cancer cells incubated with

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YSAP-DOX for 0.5 h, 1 h, and 3 h in order to verify the cell uptaken process. The blue fluorescence in the nuclei is attributed to the DAPI, and the red emission is from the YSAP-DOX which is used to track the carrier. The overlay of the two channels is

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also shown correspondingly. In the first 0.5 h, little red emission is found, indicating

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only a few of YSAP-DOX particles have been taken up. Stronger red fluorescence emission is found in both cytoplasm and cell nuclei with enhanced incubation time, which suggests more particles are localized in the cells gradually. The in vitro results reveal that YSAP UCNPs with loaded DOX can be taken up by cancer cells effectively. Computed X-ray tomography (CT) imaging is one of the most common clinic diagnostic techniques due to the deep tissue penetration and high-resolution 3D 18

ACCEPTED MANUSCRIPT structure details, and Yb doped particles can be used as contrast agents for CT imaging. Here, CT contrast imaging of YSAP UCNPs in vitro and in vivo were performed. As shown in Fig. 4A, the CT signal increases obviously with enhanced

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Y/Yb concentration, and the values show high positive contrast enhancement as a function of the concentration with a large slope of 13.38 (Fig. 4B). The in vivo CT imaging of mice was presented in Fig. 4C1-D2. The CT value is up to 1922.5 HU

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(Hounsfield units) after injection, which is markedly higher that without injection

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(53.4 HU). The high CT value of YSAP UCNPs may be due to the component of Au, which possess longer imaging time and higher absorption coefficient than traditional contrast agents [57]. Although the high resolution of CT imaging is important, there is a limitation to the single-modal image due to the low sensitivity, especially when it is

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used to tumor-imaging with small density differences [58–60]. Therefore, the UCL emission of YSAP UCNPs is more important for real-time anticancer diagnosis. Fig. 4E1-E4 present the inverted florescence microscope images of A549 cells incubated

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with YSAP UCNPs (1 mg mL–1) for 1 h marked with DAPI. It is obvious the YSAP

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UCNPs in the cells emit bright red emissions upon 980 nm NIR irradiation. Meanwhile, most of the UCL emissions are focused on the cytoplasm instead of in the nuclei compared with the YSAP-DOX during the cell-uptaken process shown by CLSM, which indicate the drug carriers are uptaken by endocytosis through inside endosomes and lysosome instead of passive adsorption. The results reveal that the as-prepared YSAP UCNPs are effective contrast agent for CT and UCL imaging. Fig. 5A (blue line) shows the absorbance spectrum of Au25(SR)18– clusters 19

ACCEPTED MANUSCRIPT solution and the UCL spectra of Y2O3:Yb,Er@Y2O3:Yb@mSiO2 (black line) and YSAP UCNPs (red line). The Au25(SR)18– clusters have obvious absorption band at the visible region. As shown, three main emission peaks of YSAP UCNPs (red line)

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are all decreased compared to Y2O3:Yb,Er@Y2O3:Yb@mSiO2 (black line). According to the Förster theory, the emission intensity of UCL donor would be decreased with the acceptor of PDT/PTT agent (photosensitizer) combined. In Fig. 5A, there is an

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obvious whole overlap between the UCL Y2O3:Yb,Er@Y2O3:Yb@mSiO2 host and

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Au25(SR)18– clusters which can produce singlet oxygen and thermal effect irradiated by the visible light [61]. Fig. 5B shows the absorbance of DPBF solution mixed with YSAP UCNPs under NIR laser irradiation for different irradiation times (0, 1, and 5 min). The absorption peaks of the DPBF solutions in the visible region (especially at

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the wavelength of 350-470 nm) decrease gradually when irradiated by NIR light, which indicates singlet oxygen 1O2 has been generated. Fig. 5C shows the in vivo infrared thermal images of tumor-bearing mice after injection of saline and YSAP

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UCNPs with different exposure time under NIR laser irradiation, and the enhanced

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temperature curve versus the irradiation time is also displayed in Fig. S8. The group injected with YSAP shows an obvious enhanced temperature from 36.1 °C to 52.3 °C, which can be used to kill the targeted tumor cells effectively. DOX was usually selected as model anti-cancer drug to evaluate the loading and

release behavior of the drug carriers. As shown in Fig. 6A, the loading amount of DOX on Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25 with silica thickness of 0, 5, 20 nm (without P(NIPAm-MAA) modified) are 0.54 mg, 0.95 mg, and 1.57 mg, respectively. 20

ACCEPTED MANUSCRIPT When the corresponding Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-P(NIPAm-MAA) are utilized as carriers with other conditions unchanged, the loading amounts are not decreased but enhanced to 0.98 mg, 1.33 mg, and 2.22 mg, respectively. There are

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several factors attributed to the loading efficiency: physical attraction and chemical bonding due to the different functional groups. The physical mesopores and channels are beneficial to load most of DOX molecules. After modification of Au25(SR)18–

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clusters, the BET surface areas of the samples decreas which is disadvantaged to the

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loading of DOX, while the attached negative Au25(SR)18– clusters could link more positive DOX molecules. Meanwhile, the conjugated P(NIPAm-MAA) under the room-temperature is stretched which could store more DOX molecules inside the pores.

Thus,

YSAP

has

higher

loading

amount

than

that

of

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Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25.

When referred to the release efficiency among the three samples with different shell thickness, they may have the different release processes (Fig. 6B). Obviously,

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the samples with mesoporous silica shell show a fast release process followed by a

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modest release, while Y2O3:Yb,Er@Y2O3:Yb-Au25-DOX without silica shell almost do not have the slow release process due to the low loading efficiency and lack of mesoporous channels. The result indicates it is important to coat a layer of mesoporous silica on the UCL particles to store/adsorb more drug molecules and control the release. For an effective DDSs, two release processes are necessary: the initial quick and booming release could kill most of the cancer cells and inhibit the rapid growth, and the following modest platform is beneficial to extinguish the 21

ACCEPTED MANUSCRIPT survived few cells. The pH- and temperature-sensitive release properties were investigated through comparison of the release process for Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-DOX and

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YSAP-DOX. As shown in Fig. S9, Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-DOX shows higher and faster release at lower pH value and higher temperature. Note that, when the pH value is 7.4, 37.1% of DOX is released at 37 °C (Fig. S9A). When the

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temperature is 25 °C at pH value of 7.4, 26.3% of DOX has been released (Fig. S9B).

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After conjugation of P(NIPAm-MAA), an interesting and important release behavior has been achieved. In Fig. 6C, when the temperature is 37 °C and the pH value is 7.4, the release efficiency within 36 h is only 12.5%, which is much lower than that of Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-DOX under the same condition, and the release

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efficiency is as high as 73.6% at pH value of 4.0, which indicates there is an obvious pH-responsive release process. This pH-sensitive result may be attributed to the protonation of the MAA and DOX under lower pH value which induce the increased

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electrostatic forces. The increased release rate and efficiency under lower pH value is

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beneficial for the anti-cancer clinical therapy, because the tumors own a weakly acidic extracellular environment with the pH value of 6.0-7.0 than that of normal body fluid (7.4), and the inside endosomes (pH = 5.0-6.0) and lysosome (pH = 4.0-5.0) in the tumor cells also have lower pH values [62]. The temperature-responsive release result is depicted in Fig. 6D. When the pH value of the release environment is 7.4, the respective release efficiency at different temperatures within 36 h is 8.1% (25 °C) and 51.4% (50 °C) which suggest an excellent temperature-responsive release property. It 22

ACCEPTED MANUSCRIPT is proposed that P(NIPAm-MAA) is served as a temperature-sensitive gatekeeper. When it is placed in the cold environment, the polymer brushes stretch a lot which play a role as a bolt in preventing the leak of the DOX. When the temperature is

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higher than the LCST of the polymer, the brushes shrink which makes the channels open and thus promote the release.

On basis of above photothermal effect and temperature-responsive results, we

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carried out the NIR light triggered drug release test under the condition of pH value of

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7.4 and temperature of 37 °C, which is shown in Fig. 6E. It is apparent that the release of DOX almost terminates at the 4th hour. It is noted that the DOX release is activated when there is an NIR irradiation which induces photothermal effect to increase the release temperature. The release efficiency within 2 hours under the laser irradiation is

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20.9% which is much higher than that without laser irradiation (4.4%). Fig. 8F shows the UCL intensity of the YSAP-DOX system as a function of the release time and the corresponding UCL spectra are given in Fig. S10. The gradually increased intensity is

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due to the decreased quenching effect caused by the DOX. UCL image of a mouse

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injected with YSAP-DOX under the NIR irradiation is shown in inset of Fig. 6F, a clear red spot further indicates the potential application as the real-time tracked diagnosis material. In conclusion, because of the high drug loading efficiency, pH-responsive and thermo-responsive release, the as-synthesized YSAP UCNPs with UCL emission can be used as potential candidates for multifunctional drug carrier and simultaneous tracking with targeted therapy. Calcein AM (dyed living cells with green color) and propidium iodide (PI) (dyed 23

ACCEPTED MANUSCRIPT dead cells with red color) were used to distinguish the live/dead state of A549 cancer cells under different treatment conditions detected by CLSM. All the added YSAP UCNPs concentration is 1 mg mL–1 and the corresponding DOX concentration is 74

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µg mL–1. Fig. 7A and 7B show the CLSM images of A549 cells incubated with culture only and YSAP UCNPs. It is clear that almost all of cells are green, indicating the YSAP UCNPs has no negative effect to the cells. In Fig. 7C for A549 cells

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incubated with culture under NIR irradiation, the green color implies that the pure

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culture has no obvious absorbance of NIR irradiation. After the YSAP UCNPs were incubated and irradiated by NIR light, a lot of A549 cells are killed with the viability of about 40% (Fig. 7D) due to the photo-thermal and photodynamic effect caused by the Au25(SR)18− clusters which are responsive to the NIR irradiation. The pure

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chemo-effect of YSAP-DOX was also detected and shown in Fig. 7E of which the cell viability is about 60%. When A549 cells were incubated with YSAP-DOX and irradiated under the NIR light, almost no cells survive (Fig. 7F), suggesting a clear

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synergistic chemo-, photothermal, and photodynamic anti-cancer effect.

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3.3. In vivo anticancer therapy

The good biocompatibility and anticancer effect in vitro inspired us to further

study the effect in vivo. Folate receptor (FR) is well known as cancer cell marker that up-regulated in different cancer cell lines. Before the in vivo administration, FA was firstly

conjugated

to

realize

the

stronger

targeted

effect

[63–66].

The

receptor-mediated endocytosis of FA-conjugated YSAP UCNPs has been confirmed by the CLSM images of the HeLa tumor cells after incubation of FA and FA-free 24

ACCEPTED MANUSCRIPT samples, as depicted in Fig. S11. Besides HeLa cells (FR positive), A549 cell lines (FR negative) and SKOV3 cell lines (FR positive) were also chosen to study the selectivity of this YSAP material for comparison. As shown in Fig. S12, there is a

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receptor-mediated endocytosis process due to the conjunction of FA to the FR positive cell lines. As a principle experiments proof, the H22 tumor-bearing mice were intravenously injected with YSAP UCNPs, DOX, and YSAP-DOX after tumor grew

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to 6-8 mm in size (about one week). Fig. 10A reveals that the results are much

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different after diverse treatments, the corresponding tumor photographs are presented in Fig. S13. It is found that the tumor size is very large (about 28.6 mm) in the first group without any treatment. When the tumor is directly exposed to NIR irradiation without any injection, the tumor size is inhibited a little bit, which may be due to the

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thermal arising from the absorbance of hemoglobin itself. The third group of the tumor-bearing mice injected with YSAP-DOX has a tumor size of 18.0 mm which should be ascribed to the released DOX from the system. The tumor size in the fourth

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group after injection with YSAP UCNPs and irradiation by NIR light is inhibited to

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16 mm which is derived from the photothermal and photodynamic effect. The final group was injected with YSAP-DOX under NIR irradiation, and the tumor is dramatically inhibited with size of 6.7 mm which keeps the same even lower than the beginning size. Meanwhile, the histological analysis of the tumors indicates the tumors are almost killed and become normal in the best treated group (Fig. 8B). Fig. 8C shows there is no decrease of body weight in all of the groups indicating there is no adverse drug reaction in all of the five groups. Fig. 8D, E show the T-test results of 25

ACCEPTED MANUSCRIPT the normalized tumor sizes in the five groups. There is an extremely significant discrepancy between the best treated group and the control group (P = 0.0056). When there is only chemo- effect, the P value is 0.0091, and the P value is 0.0083 when

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there is only photothermal/photodynamic effect. That means there is a synergistic anti-tumor effect among the chemo-, photothermal, and photodynamic effects partly due to the pH/temperature-responsive of drug molecules.

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Histological analysis of the typical heart, lung, kidney, liver, and spleen of the

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mice is given in Fig. S14. In the best treated group, hepatocytes in the liver samples are found normal, and there is no pulmonary fibrosis detected in the lung samples. Meanwhile, the glomerulus structure in the kidney section is clearly observed and there is no concentration tendency. Necrosis is not found in any of the histological

4. Conclusion

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samples analyzed.

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In summary, a multifunctional nano-platform combined dual-modal imaging and

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multimodal therapies for improved cancer ablation has been constructed. The UCL emission intensity of the Y2O3:Yb,Er core as the donor is greatly enhanced by coating an inert shell, and the thickness of the silica shell can be controllably tuned for loading DOX (chemo-therapy agent) and Au25(SR)18– clusters (PDT/PTT agents). The conjugated pH/temperature-responsive P(NIPAm-MAA) gives rise to a much faster DOX release in the tumor sites when irradiated by NIR light, achieving the target of releasing DOX molecules inside of cancer cells after the endocytosis process under 26

ACCEPTED MANUSCRIPT NIR irradiation. The in vitro and in vivo results reveal that the as-synthesized nanoplatform not only exhibits excellent synergistic (PDT/PTT/chemo-) anti-cancer therapeutic effect but shows dual-modal imaging properties (CT and UCL) when

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irradiated by 980 nm NIR light, demonstrating the feasible application of imaging guided therapy.

Appendix

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Supplementary material associated with this article can be found, in the online

Acknowledgments

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version.

We greatly acknowledged the financial supports from the National Natural Science Foundation of China (NSFC 21271053, 21401032, 51472058, and 51332008),

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Natural Science Foundation of Heilongjiang Province (B201403), and Harbin

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Sci.-Tech. Innovation Foundation (2014RFQXJ019).

27

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delivery system based on luminescent CaF2:Ce3+/Tb3+-poly(acrylic acid) hybrid

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[57] Xing H, Bu W, Zhang S, Zheng X, Li M, Chen F, et al. Multifunctional nanoprobes for upconversion fluorescence, MR and CT trimodal imaging. Biomaterials 2012;33:1079-89.

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BaGdF5:Yb,Er nanoprobes for multi-modal upconversion fluorescent, in vivo computed

2012;33:9232-8.

tomography

and

biomagnetic

imaging.

Biomaterials

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[59] Xing H, Bu W, Ren Q, Zheng X, Li M, Zhang S, et al. A NaYbF4:Tm3+

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nanoprobe for CT and NIR-to-NIR fluorescent bimodal imaging. Biomaterials 2012;33:5384-93.

[60] Jin S, Zhou L, Gu Z, Tian G, Yan L, Ren W, et al. A new near infrared photosensitizing

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nanoparticles and hypocrellin A for photodynamic therapy of cancer cells. Nanoscale 2013;5:11910-8. [61] Kawasaki H, Kumar S, Li G, Zeng C, Kauffman DR, Yoshimoto J, et al. 35

ACCEPTED MANUSCRIPT Generation of singlet oxygen by photoexcited Au25(SR)18 clusters. Chem Mater 2014;26:2777-88. [62] Schmaljohann D. Thermo- and pH-responsive polymers in drug delivery. Adv

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Drug Deliver Rev 2006;58:1655-70. [63] Lee KY, Seow E, Zhang Y, Lim YC. Targeting CCL21-folic acid-upconversion nanoparticles conjugates to folate receptor-alpha expressing tumor cells in an

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endothelial-tumor cell bilayer model. Biomaterials 2013;34:4860-71.

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[64] Lee RJ, Low PS. Folate-mediated tumor cell targeting of liposome-entrapped doxorubicin in vitro. Biochim Biophys Acta 1995;1233:134-44. [65] Reddy JA, Allagadda VM, Leamon CP. Targeting therapeutic and imaging agents to folate receptor positive tumors. Curr Pharm Biotechno 2005;6:131-50.

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[66] Liu S-Q, Wiradharma N, Gao S-J, Tong YW, Yang Y-Y. Bio-functional micelles self-assembled from a folate-conjugated block copolymer for targeted

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intracellular delivery of anticancer drugs. Biomaterials 2007;28:1423-33.

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Scheme

1.

Schematic

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illustration

for

Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-P(NIPAm-MAA)

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synergistic multimodal anticancer therapy.

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the

and

formation the

of

imaging-guided

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Fig. 1. XRD patterns of the two precursors and as-synthesized Y2O3:Yb,Er@Y2O3:Yb

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@mSiO2 with different coated silica thickness.

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Fig. 2. (A) The UC emission spectra of Y2O3:Yb,Er and Y2O3:Yb,Er@Y2O3:Yb under

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980 nm irradiation, (B) the corresponding CIE chromaticity image, the digital photographs of (C) Y2O3:Yb,Er and (D) Y2O3:Yb,Er@Y2O3:Yb under daylight and

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980 nm NIR irradiation with the pump power of 0.72 W cm–2.

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Fig. 3. TEM images with different magnification of (A1-A3) Y2O3:Yb,Er@Y2O3:Yb, (B1-B3)

Y2O3:Yb,Er@Y2O3:Yb@mSiO2

(5

nm),

and

(C1-C3)

Y2O3:Yb,Er@Y2O3:Yb@mSiO2 (20 nm); (D) TEM image, (E) EDS spectrum, (F) STEM image and the corresponding cross-section compositional line profiles of Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-P(NIPAm-MAA).

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Fig. 4. (A) In vitro CT images of with different Y/Yb concentrations, (B) CT value of YSAP aqueous solutions versus the Y/Yb concentration. CT images of tumor-bearing Balb/c mice (C1, C2) before and (D1, D2) after injection. Inverted fluorescence microscope images of A549 cells incubated with YSAP UCNPs for 1 h (E1) under bright field, (E2) dyed by DAPI, (E3) irradiated under 980 nm laser, and (E4) overlay of the channels. 41

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Fig. 5. (A) Absorption spectrum of Au25(SR)18– clusters (blue line), emission spectra

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of pure Y2O3:Yb,Er@Y2O3:Yb@mSiO2 (black line), and YSAP UCNPs (red line) under 980 nm irradiation. (B) Absorption spectra of YSAP UCNPs after adding DPBF under different irradiation times. (C) In vivo infrared thermal images of a

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tumor-bearing mouse after injection of YSAP UCNPs with different irradiation times

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under the 980 nm NIR irradiation (0.72 W cm–2).

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Fig. 6. (A) DOX loading efficiency of pure Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25 and YSAP

with

different

silica

thickness

and

(B)

release

efficiency

of

Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25 with different silica thickness. The release efficiency of YSAP-DOX (C) at different pH values at 37 °C and (D) at different release temperatures at pH = 7.4. (E) The release efficiency of YSAP-DOX triggered by NIR laser with the pump power of 0.72 W cm–2 (pH = 7.4, 37 °C) and (F) the corresponding luminescence intensity of YSAP-DOX as function of the release time. 43

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Fig. 7. CLSM image of A549 cancer cells incubated (A) with culture only, (B) with UCNPs only, (C) with culture under NIR irradiation, (D) with YSAP under NIR irradiation, (E) with YSAP-DOX, (F) with YSAP-DOX under NIR irradiation. All the

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cells were dyed with Calcein AM and PI.

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Fig. 8. (A) Photographs of tumor-bearing mice after different treatments: without treatment, with NIR irradiation, with YSAP-DOX, with YSAP under NIR irradiation,

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and with YSAP-DOX under NIR irradiation. (B) H&E stained images of tumor

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tissues obtained after 14 days of treatment. (C) The normalized body weight and (D) tumor size of H22 tumor in different groups after treatment. (E) The T-test assay of the final results at 14th day in different groups.

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An imaging-guided platform for synergistic photodynamic/photothermal/chemo- therapy with pH/temperature-responsive drug release Ruichan Lv a, Piaoping Yang a,*, Fei He a, Shili Gai a, Guixin Yang a, Yunlu Dai a,

Key Laboratory of Superlight Materials and Surface Technology, Ministry of

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Zhiyao Hou b, Jun Lin b,*

Education, College of Material Sciences and Chemical Engineering, Harbin Engineering University, Harbin, 150001, P. R. China b

State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of

Corresponding author: Fax: +86 431 86598041.

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Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China

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E-mail: [email protected] (P. Yang), [email protected] (J. Lin)

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Fig. S1. TEM image and particle size distribution (inset) of as-prepared Au25(SR)18–

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clusters.

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Fig.

S2.

TEM

images

of

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(A,

B)

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Y(OH)CO3:Yb,Er@Y(OH)CO3:Yb.

3

Y(OH)CO3:Yb,Er

and

(C,D)

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Fig. S3. TEM images of Y2O3:Yb,Er@Y2O3:Yb@mSiO2 spheres with silica thickness

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of (A) 0 nm, (B) 5 nm, (C) 10 nm, (D) 20 nm, (E) 30 nm.

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Fig. S4. N2 adsorption/desorption isotherms and corresponding pore size distribution curves of Y2O3:Yb/Er@Y2O3:Yb@mSiO2 spheres with different silica thickness of (A, B) 0 nm, (C, D) 5 nm, and (E, F) 20 nm.

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Fig. S5. FT-IR spectra of Y2O3:Yb,Er@Y2O3:Yb, Y2O3:Yb,Er@Y2O3:Yb@mSiO2, Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25,

Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-

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P(NIPAm-MAA), and Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25-P(NIPAm-MAA)-DOX.

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Fig. S6. (A) Cell viability of L929 cell lines incubated with YSAP UCNPs with

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different concentrations, and the optical microscope images of L929 cells incubated (B) with culture only for 24 h, and (C) with YSAP UCNPs (1 mg mL–1) for another 24

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h dyed by trypan blue.

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Fig. S7. Confocal laser scanning microscopy (CLSM) images of A549 cancer cells

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incubated with YSAP-DOX at 37 °C for different times: 0.5 h, 1 h, and 3 h.

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Fig. S8. Temperature curve of tumor-bearing mice after injection of saline and YSAP

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UCNPs versus exposure time under the 980 nm NIR irradiation (0.72 W cm–2).

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Fig. S9. The release efficiency of Y2O3:Yb,Er@Y2O3:Yb@mSiO2-Au25 (A) at different pH values at 37 °C and (B) at different release temperatures at pH = 7.4 as a function of the release time.

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Fig. S10. The UC emission spectra of YSAP-DOX under 980 nm irradiation as a

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function of the release time.

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Fig. S11. Confocal laser scanning microscopy (CLSM) images of HeLa cells incubated with YSAP-DOX and FA-YSAP-DOX for 1 h. All the scale bars are 50 µm.

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Folate receptor (FR) is well known as cancer cell marker that up-regulated in many types of cancer cell lines. HeLa cells were seeded in the 6-well plate and incubated with YSAP-DOX and FA-YSAP-DOX for 1 h in the same condition at

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37 °C with 5% CO2. The FA-YSAP-DOX presents stronger red luminescence than the cells incubated with YSAP-DOX without FA, suggesting more nanoparticles could be

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Fig. S12. Confocal laser scanning microscopy images of A549 cells and SKOV3 cells

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incubated with YSAP-DOX and FA-YSAP-DOX for 1 h. All the scale bars are 50 µm.

Here, besides HeLa cells (FR positive) we have utilized, A549 cell lines (FR

negative) and SKOV3 cell lines (FR positive) were chosen as the comparison. Typically, A549 cells and SKOV3 cells seeded in 6-well plate separately and incubated with YSAP-DOX and FA-YSAP-DOX for 1 h in the same condition at 37 °C with 5% CO2. As shown, A549 cells incubated with the FA-YSAP-DOX present no obvious change compared with those incubated with YSAP-DOX due to the negligible non-specific binding of FA with the FR negative cell membrane. While to SKOV3 cell lines, cells incubated with the FA-YSAP-DOX present stronger red 13

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positive cell lines.

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Fig. S13. The photographs of mice tumor with different treatments at 14th day.

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groups at the 14th day.

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Fig. S14. H&E stained images of heart, liver, spleen, lung, and kidney from different

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