Bioresponsive upconversion nanostructure for combinatorial bioimaging and chemo-photothermal synergistic therapy

Chemical Engineering Journal 342 (2018) 446–457

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Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej

Bioresponsive upconversion nanostructure for combinatorial bioimaging and chemo-photothermal synergistic therapy

T

Jiating Xu, Wei Han, Tao Jia, Shuming Dong, Huiting Bi, Dan Yang, Fei He, Yunlu Dai, Shili Gai, ⁎ Piaoping Yang Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Material Sciences and Chemical Engineering, Harbin Engineering University, Harbin 150001, PR China

H I G H L I G H T S

G RA P H I C A L AB S T R A C T

was in situ grown on the meso• POM porous silica coated upconversion na-

DOX-loaded nanoplatform with tumor-responsive nanocluster in situ grown on the mesoporous upconversion nanoparticles was innovated for thermo-chemotherapy and multiple bioimaging.

noparticles.

POM endows the nanomedicine • The tumor-responsive photothermal therapy function.

DOX loaded system poses • The photon and tumor co-improved

NIR

thermo-chemotherapy.

NIR can excite the upconversion • The nanoparticles for UCL, CT, and MRI imaging.

self-assembly POM is helpful for • The enhancing tumor accumulation of particles.

A R T I C L E I N F O

A B S T R A C T

Keywords: Upconversion In situ growth Bioresponsiveness Chemo-photothermal Imaging

Inventing a tumor-responsive theranostic nanoconstruct shows great potential for improving the therapeutic outcome on cancer. Herein, a facile in situ growth strategy based on polyoxometalate (POM) integrated with the mesoporous silica coated upconversion nanoparticles (UCNPs@mSiO2) has been developed. The product was named as USP, and can be triggered by 808 nm light for cancer theranostic. The POM is ultra-sensitive to the intratumoral acidity and reducibility, which enables the photothermal conversion of 808 nm photon for photothermal therapy (PTT). The POM endows the nanostructure highly hydrophilic surface, which is very significant for in vivo application. Need to point out, the photothermal conversion ability of POM enhances the inner temperature of doxorubicin (DOX)-loaded USP (USP-DOX), realizing a bioresponsiveness and NIR photon co-enhanced chemo-photothermal therapy. The POM can also obviate the DOX leakage in normal tissues while accelerate DOX release in tumor tissues under NIR irradiation. Significantly, the POM endows the nanomedicine self-assemble property in acidic tumor microenvironment, which is highly beneficial for enhancing intratumoral accumulation. Highly effective anticancer therapy of the developed USP-DOX has been validated by the in vitro and in vivo assays. The Gd3+/Nd3+/Yb3+/Er3+ co-doped UCNPs ensure the nanosystem MRI/CT/UCL imaging functions, thus achieving the integration of therapy and diagnosis.

⁎

Corresponding author. E-mail address: [email protected] (P. Yang).

https://doi.org/10.1016/j.cej.2018.02.109 Received 2 January 2018; Received in revised form 23 February 2018; Accepted 25 February 2018 Available online 26 February 2018 1385-8947/ © 2018 Elsevier B.V. All rights reserved.

Chemical Engineering Journal 342 (2018) 446–457

J. Xu et al.

1. Introduction

nanoparticles by non-tumorous regions and the consequent mis-hyperthermia. A feasible strategy to prevent side effects is to differentiate the photothermal conversion nature between the agents outside and inside the tumor, namely to realize a tumor-responsive photothermal conversion, which is sensitive to the physicochemical characteristics of tumorous tissues. As known, the photothermal effect essentially depends on the resonant oscillation of delocalized electrons under an external electromagnetic field [77]. So it is urgent for researchers to find qualified photothermal conversion nanomaterials with sensitive electronic structure to achieve tumor-specific therapy. In this study, for the first time, POM cluster with self-adaptive electronic structure was modified on the surface of mesoporous silica coated UCNPs using an in-situ growth strategy. The intelligent theranostic nanoplatform can be used for tumor-targeted drug delivery, tumor-triggered photothermal conversion of NIR photon, and ultrasensitive NIR photon-enhanced drug release, so as to achieve tumor microenvironment and NIR photon co-enabled thermo-chemotherapy under multiple imaging guidance. In comparison with previously reported photothermal agents [24,36,42–44,46–48,78,79], the specific advantages of the POM modification are (1) tumor-responsiveness enables the DOX-loaded nanostructure tumor-targeted PTT and PTT-improved chemotherapy; (2) POM decoration endows the nanocarriers highly biocompatible surface property; (3) formation of larger structure in acidic tumor sites through hydrogen bonds is beneficial for enhancing intratumoral accumulation of the nanomedicine particles. Significantly, Gd3+/Yb3+/Nd3+/Er3+ co-doped UCNPs endow the nanosystem MRI/CT/UCL tri-modal imaging capabilities.

Developing nanotherapeutic responsive to tumor microenvironment has attracted considerable attention in recent years [1–6]. Such therapeutic modalities can simultaneously reduce the damage of anticancer agents to normal tissues and improve inhibiting effectiveness to tumorous tissues [7–9]. Among the various physiological parameters of the tumor microenvironment, acidity and reducibility are the characteristic status of the tumor tissues compare to normal tissues [10–12]. It has been demonstrated that the tumor pH of extracellular tumor microenvironment is ∼7.2–6.5 depending on the tumor stage and type while that of intracellular early lysosome and endosome reaches 6.2–5.0 [13–17]. Besides, glutathione (GSH) is the most abundant reductive agent in the tumorous tissues, with content at least 4-fold higher than that in normal tissues [18–21]. Innovation of nanotherapeutic poses effective therapy and diagnosis is highly desirable [22–27]. To date, chemotherapy is the most widely used approach for treatment of highly metastatic malignancies [28,29]. Nevertheless, the unspecific distribution of chemotherapy drug brings severe side effects and heavy burdens to patients [30,31]. Thus the selective accumulation of chemotherapy agents to both primary and metastatic tumor sites is urgent for improving the therapy of malignant tumor. Recent literatures showed that the integration of rare earth upconversion nanoparticles (UCNPs) with mesoporous silica for delivery of chemotherapeutic agents holds promise in realizing multimode (e.g., upconversion luminescence (UCL), magnetic resonance imaging (MRI), computed tomography (CT), and so forth) imaging-guided chemotherapy [32–35]. For superior chemotherapeutic modality, it is very important for nanocarriers to possess high drug loading and low side effects, so to avoid the harmful drug release and drug resistance as much as possible [36–38]. However, for mesoporous silica related nanocarriers, there is an issue of incomplete drug release when applied for chemotherapy due to the complex structure of silica channels [39–41]. To compromise this dilemma, the combination of chemotherapeutic agent with photothermal agent becomes popular for overcoming drug releasing inertia and achieving a synergistic photothermal therapy (PTT) [42–47]. It is important to give reliable tumor delineation before treatment [48–50]. In recent decades, growing number of literatures demonstrate that the lanthanide-doped UCNPs hold great promise in achieving multiple imaging, which will greatly offsets the flaws of single-modality imaging [51–53]. Upconversion is a multiphoton process, which transduces more than one low-energy excitation photons to a high-energy emission photon [54–60]. This unique feature is especially beneficial for optical imaging because the long-wavelength photons have deeper penetration, allowing longer imaging distance [61–64]. Besides, upconverted emission can be easily distinguished from the auto-fluorescence of tissues, thus avoiding the background interference during visualization [65,66]. Furthermore, the UCNPs with Gd3+ and Yb3+ (with unpaired electrons and high-Z number) doping are promising nanoprobes for MRI and CT imaging, which can unite with the UCL imaging to offer whole-body visualization and the real-time monitoring of nanocarriers in tumor sites [67–70]. To date, various types of nanomedicine based on UCNPs have been developed for cancer theranostic research [71–73]. However, there is still a common concern about nanomedicine for imaging-guided cancer treatment is the low accumulation in tumorous tissues due to the poor enhanced permeability and retention (EPR) effect and the nonspecific uptake of nanoparticles by mononuclear phagocyte systems (e.g., liver/spleen) [74,75]. To address this intractable dilemma, self-assembled nanomedicine should be designed to keep as small-sized particles during blood circulation and then self-assemble into larger nanostructures in the tumorous tissues, triggered by the tumor microenvironment [76]. The chemo-photothermal nano-agent with self-assembly nature responsive to tumor condition is significant in achieving satisfied therapy outcome. This feature is helpful in obviating the inevitable capture of the

2. Experimental section 2.1. Reagents and materials All the chemical reagents used here are of analytical grade. Hexaammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O), sodium dihydrogen phosphate dodecahydrate (NaH2PO4·2H2O), hydrochloric acid (HCl), Er2O3 (99.99%), Nd2O3 (99.99%), Gd2O3 (99.99%), Yb2O3 (99.99%) and sodium fluoride (NaF) (from Sinopharm Chemical Reagent Co., Ltd.); Reduced glutathione (GSH), L-ascorbic acid, 1-octadecene (ODE), Oleic acid (OA), 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT), disodium maleate, 4′,6-diamidino2-phenylindole (DAPI), doxorubicin (DOX), and propidium iodide (PI) (from Sigma-Aldrich); Cetyltrimethyl ammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES) and ammonium nitrate (NH4NO3) (from Tianjin Kermel Chemical Co., Ltd.); Trifluoroacetic acid (CF3COOH), and sodium trifluoroacetate (CF3COONa) (from Beijing Chemical Regent Co.). 2.2. Synthesis 2.2.1. Synthesis of NaGdF4:Yb,Er@NaGdF4Fd,Yb (designated as UCNPs) In a brief, 5 mmol of NaF and 1 mmol of lanthanide oleates (Er/Yb/ Gd = 2:18:80) were mixed in a three-necked bottle with 30 mL of OA/ ODE (v/v = 1:1). After the mixture was heated to 110 °C and degassed for 0.5 h, the system was flushed with N2 and heated to 300 °C for 1 h. When the solution was cooled down to about 40 °C, ethanol and cyclohexane were used to centrifuge the solution and the obtained particles were dispersed in cyclohexane. Subsequently, 0.05 mmol of Yb (CF3COO)3, 0.15 mmol of Nd(CF3COO)3, 0.3 mmol of Gd(CF3COO)3 and 1 mmol of CF3COONa were mixed with core nanoparticles in 30 mL of OA/ODE (v/v = 1:1). The mixture was heated to 120 °C and degassed for 1 h. After flushed with N2, the system was heated to 310 °C and maintained for 1 h. The obtained sample was dispersed in cyclohexane (∼10 mg mL–1) for further use. 2.2.2. Synthesis of UCNPs@mSiO2 A beaker with 0.1 g of CTAB and 20 mL of deionized water was 447

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

ultrasonically treated to obtain a transparent solution. Then 2 mL of UCNPs was added, and the mixture was stirred overnight to obtain a homogeneous solution. Subsequently, 40 mL of deionized water, 6 mL of ethanol and 0.3 mL of NaOH (2 M) were added to the above solution. Then the system was transferred to a water bath and heated to 70 °C under vigorous stirring. Several minutes later, 0.2 mL of TEOS was added slowly into the solution and stirred vigorously for 10 min. The resulting solution was centrifuged and the product was washed with ethanol for three times. To remove the CTAB template, the obtained sample was transferred to 50 mL of ethanol containing 0.3 g of NH4NO3 and refluxed at 60 °C for 2 h. To endow the sample amino groups, 0.15 mL of APTES was added to 20 mL of deionized water with UCNPs@mSiO2 dissolved in and then heated to 45 °C for 8 h under stirring. Finally, the product was collected by centrifugation and dried at 60 °C.

The in vitro CT imaging experiments were performed on a Philips 64-slice CT scanner at a voltage of 120 kV. The sample was diluted into various concentrations and then placed in a line for CT imaging test. The female mice were first anesthetized with 10% chloral hydrate (30 µL g–1 of mouse) by intra-peritoneal injection. Then, 100 µL of USP solution (1 mg mL–1) was injected intratumorally into the tumor-implanted mice for CT imaging tests. 2.7. In vitro cellular uptake and UCL microscopy (UCLM) observation To investigate the cellular uptake process on HeLa cells using a confocal laser scanning microscope (CLSM), the HeLa cells were seeded n a 6-well culture plate and cultured overnight to attach them. 1 mL of USP-DOX (1 mg mL–1) was added to the wells and incubated for 0.5, 1, and 3 h, respectively. After that, the cells were washed with PBS three times and stained by DAPI for 10 min. 1 mL of glutaraldehyde (2.5%) was used to fix the cells for 10 min, and then further washed with PBS three times. At last, the fluorescence images of cells were recorded using a Leica TCS SP8 instrument. For the UCLM observation, the slides were prepared using the same process except that the images were recorded using an inverted fluorescence microscope (Nikon Ti-S), and an external continuous wave 808 nm laser was used to radiate the samples.

2.2.3. Synthesis of UCNPs@mSiO2-POM (abbreviated as USP) 20 mg of the amino-modified UCNPs@SiO2 was firstly dissolved in 10 mL of ultrapure water, then 0.2 mmol of (NH4)6Mo7O24·4H2O was added. The mixture was ultrasonically treated and stirred continuously at 25 °C. A 2 mL solution of 0.12 mmol of NaH2PO4·4H2O was then rapidly added into the system. To obtain the USP at reduction state, 1 mL of L-ascorbic acid at the concentrations of 120 mg mL−1 was added dropwise into the system under stirring. The obtained sample at reduction degree was labeled as USP(R) in comparison to USP(O), which is the oxidation state with no addition of L-ascorbic acid. After further stirring at 25 °C for 30 min, the resultant sample was precipitated with 20 mL of ethanol, collected by centrifugation, washed with water and ethanol for three times, and finally dried in a lyophilizer. Note here, the USP(O) was used for the subsequent in vitro and in vivo biological experiments.

2.8. In vitro cytotoxicity The typical MTT assay was used to evaluate the in vitro cytotoxicity. To assess the cytotoxicity of USP-DOX against cancer cells, HeLa cells (6000–7000 well–1) were seeded in a 96-well plate and cultured in a humid incubator (37 °C, 5% CO2) for 24 h. USP cluster and USP-DOX were dispersed into the culture media at the concentrations of 0, 15.6, 31.2, 62.5, 125, 250, and 500 μg mL–1, and then the cells were treated with culture (control group), USP + NIR, USP-DOX, USP-DOX + NIR (pump power: 0.72 W cm–2), respectively. The samples were added and further incubated for 4 h to complete the cell uptake, and then laser irradiation was conducted. After that, 20 μL of MTT (5 mg mL–1) was added into each well. After incubation for 4 h, 0.15 mL of DMSO was added to the wells and the absorbance at 490 nm was recorded for calculation. The percentage of viable cell in experimental group to that in control group was used to express the cytotoxicity. The in vitro viability of USP to HeLa and L929 fibroblast cells was also evaluated by the MTT method. The concentrations of USP were 15.6, 31.2, 62.5, 125, 250 and 500 µg mL–1.

2.3. Instruments The crystal phase and particle size were tested by X-ray powder diffraction (XRD) and transmission electron microscopy (TEM). Upconversion fluorescence spectra were measured on an Edinburgh FLS 980 apparatus using 808 nm laser diode module as irradiation source. N2 adsorption-desorption isotherm and pore-size distribution, UV–vis absorption spectra, dynamic light scattering (DLS) measurements, and Fourier-transform Infrared (FT-IR) were all performed similarly with our previously reported methods [81]. 2.4. DOX loading and releasing test 20 mg of USP was dispersed into PBS solution of DOX (20 mL, 0.5 mg mL–1) under stirring for 24 h in dark room. After that, the DOXloaded USP (USP-DOX) were rinsed by PBS for three times to remove superficially adsorbed DOX molecules, collected by centrifugation and the supernatant was used to evaluate the DOX loading rate. The precipitated mixture was kept for the further DOX release process. 10 mL of PBS was replenished and set in a water bath kettle at 37 °C with magnetic stirring, and then the supernatant was kept for further UV–vis analysis. The DOX loading rate and the DOX content in solutions were measured by UV–vis instrument at the wavelength of 480 nm.

2.9. In vivo toxicity The tumor xenograft was planted in the left axilla of each female mouse (15–20 g). When the tumor size is about 6–8 mm, the mice were randomly divided into four groups (n = 5 group–1). The first group was used as control group. The residual three groups were treated USP injection and NIR irradiation (USP + NIR), USP-DOX injection (USPDOX), USP-DOX injection and NIR irradiation (USP-DOX + NIR). For the NIR irradiated process, the tumor site was irradiated with 808 nm laser for 10 min after injecting sample for 4 h. The body weights and tumor sizes were recorded every 2 days after the treatment. Tumor growth was recorded by measuring the perpendicular diameter of the tumor with calipers. Tumor volume (mm3) was calculated as V = lw2/2, in which l and w represent the tumor width and length, respectively.

2.5. In vitro T1-weighted MR imaging The in vitro MR imaging experiments were conducted in a 0.5 T MRI magnet. The USP was diluted into various concentrations. T1 was acquired using an inversion recovery sequence. T1 measurements were finished using a nonlinear fit to changes in the mean signal intensity within each well as a function of repetition time (TR) using a Huantong 1.5 T MR scanner. Finally, the r1 relaxivity values were determined through the curve fitting of 1/T1 relaxation time (s–1) versus the sample concentrations (mg mL–1).

2.10. Histological examination Histological analysis was conducted after 2 weeks therapy. The tumor, kidney, liver, lung, heart and spleen on representative mice in control and therapy groups were excised and sliced to less than 1 cm × 1 cm, then the achieved tissues were successively dehydrated 448

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Scheme 1. Schematic illustration of the synthetic procedure of the USP-DOX and the multiple imaging guided chemo-photothermal synergetic therapy.

silica layer was coated on them to obtain UCNPs@mSiO2. The TEM image in Fig. 1b indicates that UCNPs@mSiO2 is well-dispersed and has an average diameter of 68.3 nm. Besides, worm-like channels can clearly be observed on the mesoporous silica layer. After the UCNPs@ mSiO2 particles were modified with POM clusters, the UCNPs@mSiO2POM (USP) was obtained. From the TEM image in Fig. 1c, it can be seen that the POM modification on silica surface. The EDS spectra of the UCNPs@mSiO2 and USP samples are respectively displayed in Fig. S2a and b, validating the successful POM modification. The XRD patterns in Fig. S3 uncover the β-phase of the NaGdF4:Yb,Er nanoparticles, besides after coating a shell of NaGdF4:Nd,Yb, the β-phase maintained for UCNPs (Fig. 1d). The XRD spectrum of the UCNPs@mSiO2 shows a broad diffraction peak of amorphous materials at 2θ = 22° besides the characteristic sharp peaks of UCNPs, indicating the successful coating of the silica shell. In XRD spectrum of the USP, there is a broad diffraction peak during 25° to 35° caused by POM decoration. FT-IR spectrophotometer was used to detect the functional groups on the prepared samples. As depicted in Fig. 1e, for OA-capped UCNPs, the spectrum exhibits bands at 1463 cm–1 and 1564 cm–1 associate with the vibrations of the carboxylic groups, and the broad band at around 3450 cm–1 derives from the stretching vibration of O–H. The strong transmission bands at 2924 cm–1 and 2854 cm–1 are attributed to the symmetric and asymmetric stretching vibrations of–CH2. After the mesoporous silica coating, the FT-IR spectrum of the obtained UCNPs@ mSiO2 has the characteristic peaks at 3432 cm–1 and 947 cm–1, implying that the sample surface has large number of OH group, which is advantageous for adsorbing drug molecules through hydrogen bonds. Additionally, the peaks at 802 cm–1 and 1088 cm–1 derived from the vibration of Si–O–Si bands. The FT-IR spectrum of USP shows the bands during 1100–400 cm–1 originates from the asymmetric stretching vibrations P–O and Mo–O bonds in POM clusters. In the FT-IR spectrum of the USP-DOX (Fig. S4), the new peaks during 1000–1800 cm–1 are caused by vibrations of the loaded DOX molecules [80].

using buffered formalin, ethanol at varied concentrations, and xylene. After that, the tissues were embedded in liquid paraffin, and then sliced to 3 × 5 mm for hematoxylin and eosin (H&E) staining. The final stained slices were examined using an optical microscope. 3. Results and discussion 3.1. Synthesis and characterization of samples The synthetic procedure of USP-DOX is depicted in Scheme 1. As shown, there are several important steps in the synthetic procedure. The OA-stabilized NaGdF4:Yb,Er core nanoparticles and core-shell structured NaGdF4:Yb,Er@NaGdF4:Nd,Yb (noted as UCNPs) were fabricated by a thermal decomposition method. The active-shell of NaGdF4:Nd,Yb is highly beneficial for achieving superior upconversion of NaGdF4:Yb,Er nanoparticles under 808 nm laser irradiation. After that, the UCNPs were coated with a shell layer of mesoporous silica (mSiO2), and then APTES was used to enable the positively-charged surface of UCNPs@mSiO2 with amino groups. After the POM cluster was decorated on the silica surface using an in-situ growth method, the chemotherapeutic agent of DOX was housed in the USP, and the obtained USP-DOX was used for multiple (UCL, CT and MRI) imagingguided chemo-photothermal therapy. Especially, the photothermal conversion property of the USP-DOX is tumor microenvironment-responsive, and can be markedly enhanced by tumor acidity and reducibility. The TEM image in Fig. S1 reveals that the NaGdF4:Yb,Er core nanoparticles are uniform and monodisperses with a mean size of 21.2 nm. To enable efficient upconversion under 808 nm laser excitation, a Nd3+/Yb3+ incorporated shell (NaGdF4:Nd,Yb) was grew on the core portion via an epitaxial growth method. The TEM image in Fig. 1a indicates that the uniformity and dispersity of the UCNPs have been kept well, and the average size is 32.8 nm. After the hydrophobic UCNPs were transferred into hydrophilic using CTAB, a mesoporous 449

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Fig. 1. TEM images (a–c), XRD patterns (the standard pattern of β-NaGdF4 is given here for comparison) (d), and FT-IR spectra (e) of UCNPs, UCNPs@mSiO2 and USP samples.

Fig. 2. N2 absorption-desorption isotherm (a) and corresponding pore-size distribution of USP (b). UV–vis absorption spectra of USP(R) and USP(O) solutions at pH 7.4 (c), and the USP (R) solutions at varied pH values (d). Photothermal heating curves of USP(R) and USP(O) solutions at pH 5.5 and pH 7.4 (e) and the photothermal images of USP(R) solution at pH 5.5 and PBS buffer solution under 808 nm laser irradiation (0.72 W cm−2) (f).

Halenda method. Before POM modification, the BET surface area of UCNPs@mSiO2 is determined as 1018.4 m2 g–1 (Fig. S5a), and decreased to 91.2 m2 g–1 for the final prepared USP-DOX (Fig. S5b). These results verify the successful loading of POM cluster and DOX molecules. In this work, an in-situ growth strategy was used to decorate POM on the surface of silica. At first, the UCNPs@mSiO2 nanospheres were

The N2 adsorption/desorption isotherm and the pore-size distribution of the prepared USP are exhibited in Fig. 2a and b, respectively. From them we can see that USP represents typical type IV isotherm, implying the mesoporous structure of silica channels. The BET surface area of the USP is calculated to be 380.2 m2 g–1, and the mean size of the mesopore is identified to be 5.37 nm using the Barrett-Joiner450

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range. The USP-DOX has lower emission intensity than USP especially in green region is caused by the strong absorption of DOX during 450 nm to 550 nm. According to the Lambert-Beer’s law and the measuring results exhibited in Fig. S9, the encapsulation efficiency and loading content of DOX was calculated to be 55.3% and 27.6%, respectively. Moreover, the UV–vis absorption spectrum of USP-DOX in Fig. 3a also has an obvious absorption profile of POM during 800 nm to 860 nm. The DOX releasing profiles from USP-DOX were tested in different PBS buffer solutions without and with 808 nm laser irradiation (Fig. 3b). The DOX releasing rate is not more than 5% when incubated in PBS (pH 7.4, GSH 0 mM) for 48 h. When irradiated with 808 nm laser form 4 to 8 h, there is a slight increase of DOX releasing rate during 48 h incubation, which is caused by low photothermal energy generated by low-NIR-absorptive USP in PBS (pH 7.4, GSH 0 mM). When the USPDOX was incubated in the mild acidic and reductive PBS for 48 h, the DOX releasing amount reaches 33.2%, while for NIR-irradiated process the DOX releasing rate increased to 63.4%. The DOX releasing enhancement is caused by the acidity and reducibility co-enhanced photothermal conversion of 808 nm photon to thermal energy, which promotes the DOX diffusion from silica channels. Fig. 3c exhibits the in vivo infrared thermal images of two groups of tumor-implanted mice after injection of saline and USP-DOX with different exposure times under NIR irradiation (0.72 W cm−2). After irradiation with NIR light for 5 min, the temperature of the USP-DOX injected site increased to 53.7 °C, which is high enough to kill tumorous cells. As a comparison, the saline injected mouse has no obvious temperature change and its temperature is 37.1 °C after 5 min NIR irradiation.

endowed positively-charged surface using APTES. The lone pair electron of the nitrogen atom on amino groups tends to integrates with the hydrogen ions in aqueous solution, thus enabling the positively-charged surface of silica. As shown in Fig. S6, the UCNPs@mSiO2 sample has a zeta potential of −2.48 mV and increased to 8.42 mV after amino groups decorated on. After continuous stirring with (NH4)6Mo7O24·4H2O, the zeta potential decreased to −30.4 mV, and for the prepared USP at reduced state, the zeta potential was measured to be −16.8 mV. The UV–vis absorption spectra of the USP at reduced and non-reduced states were tested to investigate the reducibility-triggered absorption property. As shown in Fig. 2c, the USP(O) solution exhibits ignorable NIR absorptivity, while for the USP(R) the absorption profiles show an obvious enhancement in NIR wavelength range. The reduction of USP, actually is the reduction of POM cluster will facilitate the occupied cation sites and delocalized electron density of Mo(V) through the reversible and multiple steps of electron exchange, which will simultaneously strengthen the electron relaxation polarization, resulting in enhanced NIR absorptivity [8]. Also, the pH-improved NIR absorption of USP was observed in Fig. 2d, where the acidity improves the NIR absorptivity of USP. The blue shift toward 808 nm couples with the reducibility and acidity dependent NIR absorptivity enable the USP to be a promising photothermal agent in tumor condition, where featured with a mild acidity and reducibility. Interestingly, in the acidification process, the USP nanoparticles have a self-assemble nature (Fig. S7). The dynamic light scattering (DLS) tests reveal that these USP particles could self-assemble into larger assemblies with an average hydrodynamic size of ∼0.9 μm in the mild acidic PBS (Fig. S8). In the PBS of pH 7.4, these POM cluster is well-ionized with high charge density to repulse each other, which renders them extremely stable in small-sized particles. However, the acidification-induced protonation of POM will greatly decrease the electrostatic repulsion with the decreased charge density and, meanwhile, increase the attractive force through the hydrogen bonds. The subsequent re-balance between these opposite interactions results in the self-assembly of USP at low pH of 5.5. It is worth noting that the USP nanoparticles can self-assemble into much larger nanostructures under mild acidic conditions is highly beneficial for enhancing intratumoral accumulation. After that, the photothermal conversion property of the USP(R) and USP(O) at pH 5.5 and pH 7.4 was investigated upon 808 nm laser irradiation. As displayed in Fig. 2e, the temperature of the USP(R) solution (pH 5.5) increased to 48.4 °C within 5 min of irradiation, which is obviously higher than the USP(R) at pH 7.4. As for USP(O) solution, there is no obvious temperature change at the acidic and neutral PBS solutions. These results demonstrate that the reduced USP at acidic condition is highly efficient in converting 808 nm photon into thermal energy. The acidity and reducibility, which coincidently are characteristic features of the tumor microenvironment, can co-enhance the photothermal conversion of USP, implying the potency of USP in tumor responsive photothermal fields. In other words, this mesoporous USP can be used as a tool for delivering chemotherapeutic drugs for tumor-targeted chemo-photothermal therapy. In Fig. 2f, in vitro photothermal images of USP(R) (pH ∼5.5) and PBS buffer solution under NIR irradiation at varied time durations were exhibited. As shown, the PBS nearly has no temperature change while the temperature of the acidic and reduced USP increased to 50.6 °C, which affords the direct evidence for proving the high photothermal conversion of USP(R) at mild acidic condition. Fig. 3a exhibits the upconversion emission spectra of the UCNPs, UCNPs@mSiO2, USP and USP-DOX. Note here, the USP at reduced state was used for optical tests. The UV–vis absorption spectrum of USP-DOX is also provided. As shown, when excited with 808 nm laser, there are three characteristic emission peaks of Er3+ at 510–530 nm (2H11/2 → 4 I15/2), 530–570 nm (4S3/2 → 4I15/2), and 630–680 nm (4F9/2 → 4I15/2). In comparison with UCNPs, the emission intensity of UCNPs@mSiO2 was decreased obviously due to the quenching effect caused by silica shell. After POM modification, the USP has a slight decrease of emission intensity perhaps caused by low absorption of the POM in visible light

3.2. UCL/MR/CT imaging and cell uptake performance We next use USP-DOX for in vitro anticancer assays. HeLa cells incubated with USP-DOX for different durations were imaged by a confocal laser scanning microscopy (CLSM). As shown in Fig. 4a, the DOX loaded in the nanosystem radiates red emission when excited by a 488 nm laser, besides the DAPI which can emit blue fluorescence was used to label the cell nuclei. Correspondingly, the merged images of the above two channels were given. Obviously, the red fluorescence inside cells was significantly boosted with prolonging of incubation time. When the incubation time is 3 h, obvious accumulation of USP-DOX was found in the cells, indicating as-prepared nanomedicine can be efficiently internalized by HeLa cells. The merged CLSM image in Fig. S10 together with the 3D reconstructed image in Fig. S11 further demonstrate that the developed nanomedicine can be intracellularly accumulated in the cytoplasm of HeLa cells. As accepted, it is a significant work to make a precise diagnosis before therapy. Here, the UCL imaging property of USP-DOX has been investigated intracellularly. As shown in Fig. 4b, the HeLa cells were incubated with USP-DOX at 37 °C for 1 h. It is clearly that the nanoparticles in the cell radiate luminous green emission upon 808 nm laser excitation. Besides, no fluorescence signal was detected outside of the cells, whereas the signal locates at the intracellular region implies that the as-prepared sample has been internalized into the cells instead of merely being stained on the surface of membrane. Moreover, the UCL signal is mainly located at the cell cytoplasm, which validates that the nanoparticles are engulfed by endocytosis through lysosomes and endosomes into the cells rather than passive adsorption. These results imply that the invented nanostructure with UCNPs loaded inside is an effective contrast agent for in vitro UCL imaging with ignorable background. As well reported, the paramagnetic Gd3+ ions have positive enhancing nature of T1 MRI signal, so we envisage that the integration of the NaGdF4-based UCNPs and mesoporous silica shell posing superior T1 MRI imaging contrast performance. In this study, the T1-weighted MRI property of USP-DOX was studied. Fig. 5a displays the in vitro MRI images of USP solutions with different concentrations. It is clear of that the T1 MRI signal increased positively with the samples concentrations increased. In Fig. 5b, the longitudinal relaxivity r1 of USP-DOX is 451

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Fig. 3. Upconversion emission spectra of UCNPs, UCNPs@mSiO2 and USP, and UV–vis absorption spectrum of USP-DOX (a). DOX releasing profiles from USP-DOX in PBS (pH 7.4, GSH 0 mM and pH 5.5, GSH 8 mM) without and with NIR irradiation (b). Photothermal images of mice without and with USP-DOX injection when irradiated with 808 nm laser for various durations (c).

Fig. 4. CLSM images of HeLa cells incubated with USP-DOX for 0.5, 1, and 3 h at 37 °C (a). Bright-field, UCL and the merged images of HeLa cells incubated with USP-DOX for 1 h at 37 °C (b). Scale bar: 50 µm.

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Fig. 5. In vitro T1-weighted MR images of USP-DOX (a) and corresponding relaxation rate R1 versus sample concentrations (b). In vitro CT images of USP-DOX at different concentrations (c) and corresponding CT values versus sample concentrations (d); In vivo CT images of tumor-bearing mice before (e) and after USP-DOX injection (f).

calculated to be 0.5149 mg−1 s−1. Moreover, the CT imaging technique is reliable because it affords deep tissue penetration and high-resolution three dimension structure details, besides the lanthanide-doped nanomaterials have been studied extensively as CT imaging contrast agents due to the high atomic number of lanthanide elements. Herein, the in vitro and in vivo CT imaging contrast performance of USP-DOX was investigated. As presented in Fig. 5c, the intensity of CT signal increases markedly with the sample concentrations increased. Besides, the CT values show positive enhancement versus the sample concentration with a slope of 24.9 (Fig. 5d). The in vivo CT imaging is conducted on tumor-bearing mice without and with injection of USP-DOX. As shown in Fig. 5e and f, the post-injected tumor site has the CT value of 148.3 Hounsfield Unit (HU), which is obviously higher than that pre-injected tumor site (58.1 HU).

treatments exhibit high lethality on HeLa cells. Similarly, the chemotherapy effect of the USP-DOX was also evaluated by incubating HeLa cell with USP-DOX and calculating the relative cell viability after 24 h. From Fig. 6b it can be seen that the USP-DOX incubation shows higher cell-killing efficacy than PTT modality. The combined PTT and chemotherapy based on USP-DOX was then demonstrated by treating HeLa cells with USP-DOX plus NIR laser irradiation. Compared with PTT alone (USP plus light) or chemotherapy alone (USP-DOX in dark), the combination therapy (USP-DOX plus light) was found to be the most effective in killing HeLa cells by a synergistic manner under different drug concentrations. The synergistic effect between PTT and chemotherapy was further demonstrated by using the Student’s two-tail t test (Fig. S12). It shows that the cancer cells after incubation with USPDOX and 808 nm irradiation had a lower cell viability than the projected additive value (37.1%, ∗P < 0.05), strongly confirming the considerable synergistic effect. In addition, PI which could dye dead cells with a red color was applied to highlight dead cells under varied conditions to demonstrate the cell killing efficacy. As shown in Fig. 6c, for the cells treated with USP + NIR, portion of red cells can be observed, which implies that a small number of cells have been killed by the PTT effect of USP. In comparison with USP + NIR treated group, little more red cells can be seen in USP-DOX treated group, which is in accordance with the results in Fig. 6b. As expected, the cells treated with USP-DOX + NIR has the largest amount of red cells, which demonstrates that the NIR irradiated USP-DOX exert superior anticancer effectiveness.

3.3. In vitro toxicity study After that, we ought to study the efficacy of USP as a multifunctional drug delivery system at the in vitro level. The cytotoxicity of USP nanoparticles on L929 and HeLa cells was firstly evaluated using the standard methyl thiazolyl tetrazolium (MTT) method. The results in Fig. 6a confirm that USP posing no obvious toxicity to both of L929 and HeLa cells even at high concentrations of USP up to 500 μg mL−1. To evaluate the PTT effect of USP, HeLa cells were incubated with USP for 4 h, then the cells were treated with 808 nm light (0.72 W cm−2, 5 min). 24 h later, the relative viability were determined by the MTT method (Fig. 6b). In comparison with control group, the USP plus NIR 453

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Fig. 6. Cell viability of L929 fibroblast and HeLa cells incubated with various concentrations of USP for 24 h (a); Cytotoxicity of USP and USP-DOX against HeLa cells with or without 808 nm laser irradiation (b); CLSM images of HeLa cells after various treatments after being dyed with PI. Scale bar: 50 µm (c).

the mice treated with USP-DOX + NIR, uncovering a good consistence with above tumor growth data. The pathomorphological analysis of the main organs including heart, liver, spleen, lung and kidney in control and USP-DOX + NIR groups is provided in Fig. S13. There is no evident damage to the checked organs in two groups, indicating high in vivo biocompatibility of USP-DOX. Furthermore, the biochemical results of USP including blood urea nitrogen (BUN), total protein (TP), alanine transaminase (ALT), creatinine (CRE), and aspartate transaminase (AST), which is closely related to the liver and kidney functions, are presented in Table S1. When compared with the control group, there is no distinct injury of kidney or liver. The complete tests of blood show no obvious interference with the physiological regulation of haem or immune response. All in all, the invented nanocarrier presents huge potency for anticancer application.

3.4. In vivo toxicity study Mouse experiments were conducted to study the antitumor efficiency of USP-DOX in vivo. Here, first group of U14 tumor-bearing mouse was treated with saline injection as control group, second group was treated with USP injection and NIR irradiation, third group was injected with USP-DOX, and the fourth group was injected with USPDOX and irradiated by NIR light. The mean body weights and relative tumor volume of mice were recorded every two days after the initial therapy. All formulations were intravenously administered through the tail vein. In Fig. 7a, the body weights of these four groups are not evidently affected over the investigation period, demonstrating the sample nearly has no adverse effects to the mice. Compared with the control group, the USP + NIR group has little lower tumor growth speed (Fig. 7b), which demonstrates the PTT effect of USP under NIR irradiation. The tumor growth of USP-DOX injected group is obviously inhibited over the course of 2 weeks of treatment, which is due to chemotherapy effect of the released DOX triggered by tumor acidity. It is found that the NIR irradiated USP-DOX shows the highest tumor inhibition efficacy than other groups, which is derived from the NIR photon and tumor condition enabled chemo-photothermal therapy effect of USP-DOX. The NIR photon can be converted by modified POM cluster (adapted to NIR-absorptive in tumor microenvironment) to obvious heat, which not only promotes the DOX release, but also acts as a synergetic PTT modality. The tumor in forth group is shrunk dramatically and its size is even smaller than the initial size, confirming that the NIR irradiation plays a key role in the aspect of boosting antitumor activity (Fig. 7b). In Fig. 7c, photographs of representative mice and excised tumors also confirm that the tumor size upon USP-DOX injection plus NIR radiation has the smallest tumor size, revealing its highest antitumor effectiveness among the four investigated groups. As a further proof experiment, hematoxylin and eosin (H&E) stained tumor sections exhibited in Fig. 7d show the highest tumor damage degree for

4. Conclusions In a sum, a novel nanomedicine with tumor microenvironment responsiveness was invented for combined multiple imaging and NIRenabled chemo-photothermal therapy of cancer. The USP was fabricated by in-situ growing POM cluster on the surface of UCNPs@mSiO2. Significantly, the USP performs a smart dual-response to both the acidity and reducibility in tumor microenvironment, resulting in a tumor-triggered photothermal conversion of 808 nm photons. When excited by 808 nm laser, the USP-DOX acts as a photothermal agent to simultaneously promote DOX releasing and generate PTT effect, thus achieving a synergetic chemo-photothermal therapy. The nanomedicine particles can self-assemble into larger construct under acidic microenvironment, which is highly beneficial for improving intratumoral accumulation. Furthermore, the USP-DOX poses tri-modal (CT, MRI, and UCL) imaging capabilities and potent antitumor efficacy under a single NIR light irradiation, revealing its potency in cancer theranostic. 454

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Fig. 7. Changes in the body weights (a) and relative tumor volume (b) achieved from the mice under various treatments. Photographs of the mice and the excised tumors (c), and H&E stained images of tumor tissues obtained after 2 weeks treatment. Scale bar: 100 μm (d).

Acknowledgments

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