Biomaterials 35 (2014) 2915e2923

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Protein modified upconversion nanoparticles for imaging-guided combined photothermal and photodynamic therapy Qian Chen a, Chao Wang a, Liang Cheng a, Weiwei He b, Zhengping Cheng b, Zhuang Liu a, * a Institute of Functional Nano & Soft Materials (FUNSOM) & Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou, Jiangsu 215123, China b Department of Polymer Science and Engineering, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, Jiangsu 215006, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 October 2013 Accepted 19 December 2013 Available online 10 January 2014

In this work, we develop a multifunctional nano-platform by coating upconversion nanoparticles (UCNPs) with bovine serum albumin (BSA), obtaining UCNP@BSA nanoparticles with great solubility and stability in physiological environments. Two types of dye molecules, including a photosensitizer, Rose Bengal (RB), and an NIR-absorbing dye, IR825, can be simultaneously loaded into the BSA layer of the UCNP@BSA nanoparticles. In this carefully designed UCNP@BSA-RB&; IR825 system, RB absorbs green light emitted from UCNPs under 980-nm excitation to induce photodynamic cancer cell killing, while IR825 whose absorbance shows no overlap with upconversion excitation and emission wavelengths, offers nanoparticles a strong photothermal perform under 808-nm laser irradiation. Without showing noticeable dark toxicity, the obtained dual-dye loaded nanoparticles are able to kill cancer via combined photothermal and photodynamic therapies, both of which are induced by NIR light with high tissue penetration, by a synergetic manner both in vitro and in vivo. In addition, the intrinsic paramagnetic and optical properties of Gd3þ-doped UCNPs can further be utilized for in vivo dual modal imaging. Our study suggests that UCNPs with well-designed surface engineering could serve as a multifunctional nanoplatform promising in cancer theranostics. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Upconversion nanoparticles Toxicity Photothermal therapy Photodynamic therapy Combination therapy

1. Introduction Conventional cancer therapies including chemotherapy and radiotherapy have many limitations such as toxic side effects and drug resistance, and often fail to completely eradicate the tumor. Phototherapy is a class of non-invasive therapeutic techniques with many advantages such as remote controllability, improved selectivity, and low systemic toxicity [1]. Photothermal therapy (PTT) and photodynamic therapy (PDT) are two different types of phototherapy methods. PTT involves optical absorbing agents, such as gold nanostructures (nanoshells, nanorods, nanostars and nanocages) [2e5], carbon nanomaterials (carbon nanotube and graphene) [6,7], and various other inorganic [8] and organic nanoparticles [9,10] with strong near infrared (NIR) absorbance, to effectively convert the photo energy into heat to kill cancer cells

* Corresponding author. Institute of Functional Nano & Soft Materials (FUNSOM) & Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou, Jiangsu 215123, China. E-mail address: [email protected] (Z. Liu). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.12.046

under light irradiation [10e12]. On the other hand, PDT includes three key components: light, photosensitizer molecules and oxygen [13,14]. Under the appropriate light irradiation, the photosensitizers will transfer the absorbed optical energy to surrounding oxygen molecules, generating cytotoxic singlet oxygen (1O2) or reactive oxygen species (ROS) to kill cancer cells. Ideal phototherapy agents should exhibit minimal dark toxicity and are effective in cancer destruction under light exposure, via either photothermal or photodynamic mechanisms. Light penetration is one of major challenges in phototherapy. The NIR window in the range of 700e1000 nm, in which biological tissues have the minimal light absorption, is ideal for optical imaging [15,16] and phototherapy. Although NIR light is commonly used in PTT, unfortunately, most photosensitizers in current PDT are excited by UV or visible light, which has limited tissue penetration depth, limiting the therapeutic efficacy of PDT for large or deep tumors [17e20]. Upconversion nanoparticles (UCNPs) usually containing rare-earth elements under the NIR excitation can emit UV-visible light [21], which can in turn active photosensitizers absorbed on their surface via resonance energy transfer, generating

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reactive 1O2 or ROS to kill cancer cells [22e28]. Hitherto, we and others have used UCNPs coupled with photosensitizers for photodynamic therapy, such as using Chlorin e6 (Ce6) adsorbed/conjugated UCNPs to realize highly effective NIR-induced in vitro and in vivo PDT therapy [29e32]. Moreover, by coating UCNPs with gold or silver shells or graphene oxide, the obtained nanocomposites could also serve as photothermal agents for cancer cell ablation in vitro and in vivo [33,34]. However, the combination of NIRinduced photodynamic and photothermal therapy based on UCNPs to realize synergistic in vivo anti-tumor effect has not yet been reported to our best knowledge. In this work, we use a protein, bovine serum albumin (BSA), to modify NaGdY4-based UCNPs. The obtained UCNP@BSA nanoparticles show excellent solubility in water as well as physiological solutions. Utilizing the hydrophobic domains in the BSA protein, two different dye molecules, including a photosensitizer, Rose Bengal (RB) [27], and an NIR-absorbing dye, IR825, can be effectively loaded onto UCNP@BSA nanoparticles. The obtained dual-dye loaded nanoparticles on one hand could serve as a dual modal magnetic resonance (MR) and upconversion optical imaging probe, on the other hand are effective in both photodynamic and photothermal therapy, which if combined together could result in excellent synergetic cancer killing effects both in vitro and in vivo. This is the first demonstration of using UCNPs for in vivo imagingguided combined photodynamic and photothermal therapies, both of which are induced by NIR light with high tissue penetration. 2. Materials and methods 2.1. Materials All chemicals were analytical grade and used without further purification. Gd2O3, Yb2O3, Er2O3 and trifluoroacetic acid (CF3COOH) were purchased from shanghai chemical industrial Co. All the trifluoroacetates were prepared by dissolving the respective rare-earth oxides in trifluoroacetic acid (CF3COOH). Sodium trifluoroacetate, oleic acid (OA, 90%), 1-octadecene (ODE >90%), poly(acrylic acid) (PAA MW ¼ 1800) and Rose Bengal were purchased from SigmaeAldrich. Bovine serum albumin (BSA) was purchased from J&C chemical CO. Triethylamine (TEA), diethylene glycol (DEG) were purchased from Sinopharm Chemical Reagent Co. IR825 dye was synthesized following our previously reported protocol [35].

their characteristic absorbance peak at 540 nm and 825 nm, respectively, after subtracting the corresponsive absorbance contribution from UCNP@BSA before drug loading at the same nanoparticle concentration. The release of RB and IR825 from UCNP@BSA was studied by dispersing the sample under 37  C in PBS and water for different periods of time. The released RB and IR825 from the nanocomplex were collected and determined by the UVeVise NIR spectroscopy. 2.5. Determination of singlet oxygen Singlet oxygen sensor green (SOSG), which was highly sensitive to singlet oxygen, was employed here during the detection process. Different samples were mixed with 2.5 mM SOSG, and then irradiated by a 980-nm laser (0.5 W/cm2) for different periods of time. The generation of singlet oxygen was determined by measuring recovered fluorescence of SOSG (excitation ¼ 494 nm). 2.6. In vitro cell experiments Murine breast 4T1 cancer cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin at 37  C under 5% CO2. For confocal fluorescence imaging, 4T1 cells (1  105 cells) were cultured in 35 mm culture dishes containing different concentrations of UCNP@BSA-RB&; IR825 for 4 h. After washing with PBS (pH ¼ 7.4) for three times, cells were labeled with 40 , 6-diamidino-2phenylindole (DAPI) and then imaged by a laser scanning confocal fluorescence microscope (Leica SP5) equipped with an external 980-nm excitation laser, to determine the cellular uptake of UCNP@BSA-RB& IR825. The in vitro cytotoxicity was measured using a standard methyl thiazolyl tetrazolium (MTT, SigmaeAldrich) assay. 4T1 cells were seeded into 96-well cell culture plate at 1  104/well until adherent and then incubated with various concentrations of UCNP@BSA, UCNP@BSA-RB, UCNP@BSA-IR825, and UCNP@BSA-RB& IR825 for 24 h. The standard MTT assay was carried out to determine the cell viabilities relative to the control untreated cells. For in vitro PDT experiments, 4T1 cells (1  104 cells per well) seeded in 96-well plate were incubated with various concentrations of nanoparticles for 4 h, and then irradiated by the 980-nm laser at a power density of 0.4 W/cm2 for 10 min, with 1min interval for every 1 min of light exposure to avoid heating. Whereas for in vitro PTT experiments, cells after the nanoparticles incubation were exposed to the 808nm laser at a power density of 0.5 W/cm2 for 5 min. The cells were then incubated at 37  C for additional 24 h before MTT assay to determine the cell viabilities relative to the control untreated cells. For in vitro combined PDT/PTT experiment, 4T1 cells (1  104 cells per well) seeded in 96-well plate were incubated with 0.1 mg/ml UCNP@BSA-RB& IR825, and then irradiated by the 808-nm laser (0.5 W/cm2, 5 min) and then 980-nm laser (0.4 W/cm2, 1-min interval, 10 min). The cells were then re-incubated at 37  C for additional 24 h before MTT assay to determine the relative cell viabilities.

2.2. Synthesis of NaGdF4:Yb:Er nanoparticles

2.7. Targeted cancer cell imaging

NaGdF4:Yb:Er nanocrystals were synthesized using a thermal decomposition method. 1 mmol of Re (CF3COO)3 (Gd:Yb:Er ¼ 78%:20%:2%), 2 mmol of CF3COONa, and 20 ml solvent (10 ml OA/10 ml ODE) were added into a 100 ml three-necked flask simultaneously and degassed at 120  C for 0.5 h under vacuum. In the presence of nitrogen, the mixture was rapidly heated to 320  C and kept at this temperature for 1 h under vigorous magnetic stirring. After cooling down to the room temperature, the product was precipitated by addition of ethanol, separated by centrifugation, washed by cyclohexane, and then washed three times with ethanol. The yielded nanoparticles could be re-dispersed in various non-polar organic solvents.

In order to increase the specificity, RGD was covalently linked to UCNP@BSA. Sulfosuccinimidyl 4-N-maleimidomethy cyclohexane-1-carboxylate (Sulfo-SMCC) was mixed with UCNP@BSA-RB& IR825 solutions at 1:10 M ratios at pH 7.4 for 2 h. Upon removal of excess reagents by centrifuging at 14,800 rpm for 5 min, the activated nanoparticles were reacted overnight with thiolated RGD at 1:5 M ratios at pH 7.4. The resulting UCNP@BSA-RGD nanoconjugates were collected by centrifu gation and washed with PBS three times, re-dispersed in PBS, and stored at 4 C for further applications. For confocal imaging, U87 cells over-expressing integrin avb3 receptor were seeded in 35 mm culture dishes and treated with 0.2 mg/ml of UCNP@BSA or  UCNP@BSA-RGD for 2 h at 4 C. After washing with PBS (pH ¼ 7.4) for three times, the cells were labeled with DAPI and then imaged by the confocal fluorescence microscope.

2.3. Preparation of the UCNP@BSA UCNPs were firstly coated with polyacrylic acid (PAA) following a literature methods [33]. The obtained PAA-modified UCNPs were soluble in water. To conjugate BSA to PAA-coated UCNPs, 100 mg BSA was mixed with 5 ml aqueous solution of PAA-coated UCNPs (2 mg/ml) and then added with 5 mg of 1-Ethyl-3- (3dimethylaminopropyl) carbodimide (EDC). The solution was then stirred at room temperature for 2 h, added with another 5 mg of EDC, and then further stirred for 6 h at room temperature. The obtained UCNP@BSA nanoparticles were collected by centrifuging at 14,800 rpm for 5 min, with the precipitation re-dispersed in deionized water and stored at 4  C.

2.8. Tumor model Nude mice weighing 18 w 20 g were purchased from Suzhou Belda BioPharmaceutical Co., Ltd. and used in accordance with regulations provided by Soochow University Laboratory Animal Center. 4T1 tumors were inoculated by subcutaneous injection of 5  106 cells in ～30 mL of serum-free RMPI-1640 medium onto the back of each nude mouse. The mice were treated when the tumor volumes approached 50 mm3.

2.4. Loading of RB and IR825 molecules on UCNP@BSA

2.9. In vivo PDT/PTT treatment

In a typical experiment, loading of RB and IR825 into UCNP@BSA nanocomposite was accomplished by mixing 80 mM Rose Bengal and 0.4 mM IR825 with UCNP@BSA solution (0.2 mg/ml). The mixture was placed at 4  C in the dark overnight. Excess RB and IR825 were removed by centrifugation and washing with deionized water for several times. The obtained UCNP@BSA-RB&; IR825 nanocomplex was stored at 4  C in the dark. The UVeViseNIR absorbance spectra of UCNP@BSA-RB&; IR825 were measured by using UV765 (Shanghai Precision & Scientific Instrument Co. Ltd). The concentrations of Rose Bengal and IR825 loaded onto UCNPs were determined by

4T1 tumor-bearing mice were divided into 6 groups (n ¼ 5 per group): (a) untreated; (b) irradiated by 980-nm þ 808-nm laser; (c) intratumorally (i.t.) injected with 20 ml UCNP@BSA-RB&IR825; (d) i.t. injected with 20 ml UCNP@BSA-RB&IR825 and irradiated by the 980-nm laser; (e) i.t. injected with 20 ml UCNP@BSA-RB& IR825 and irradiated by the 808-nm laser; (f) i.t. injected with 20 ml UCNP@BSA-RB& IR825 and irradiated by both 980-nm and 808-nm lasers. The power density of 980 nm laser was 0.4 W/cm2 (30 min, 1 min interval after each minute of irradiation), where as that of 808-nm laser was 0.5 W/cm2 (5 min, continuous irradiation). An IR

Q. Chen et al. / Biomaterials 35 (2014) 2915e2923 thermal camera was used to monitor the temperature change on mice during laser irradiation. The tumor sizes were recorded every 2 days for 2 weeks, with their lengths and widths measured by a digital caliper. The tumor volume was calculated according to the following formula: width2  length/2. Relative tumor volumes were calculated as V/V0 (V0 was the tumor volume when the treatment was initiated).

3. Results and discussion The strategy for construction of BSA-coated theranostic UCNPs is shown in Fig. 1a. NaGdF4:Yb:Er (Gd:Yb:Er ¼ 78%:20%:2%) UCNPs were synthesized by a high temperature thermal decomposition method and then coated with polyacrylic acid (PAA) according to our published protocol [33], yielding PAA-coated UCNPs with carboxyl groups on their surface. Then the BSA protein was covalently conjugated to PAA-coated UCNPs via the formation of amide bonds between carboxylic groups in the PAA layer and amino groups of BSA with the help of 1-Ethyl-3-(3-dimethylaminopropyl) carbodimide (EDC). Transmission electron microscopy (TEM) images (Fig. 1b) showed that those UCNPs were mono-dispersed with

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an average diameter of approximately 60 nm. Compared to PAAcoated UCNPs, a thin layer (w2 nm in thickness) of BSA coating on the nanoparticle surface was clearly observed in the TEM images of UCNP@BSA nanoparticles, evidencing the successful BSA coating on UCNPs. Although PAA-coated UCNPs were soluble in water, they would rapidly aggregate in the presence of salts when phosphate buffered saline (PBS) was added, owing to the electron screening effect [36]. In marked contrast, UCNP@BSA nanoparticles were well dispersed in both water and PBS without any noticeable agglomeration (Fig. 1c), suggesting the remarkably enhanced physiological stability of those nanoparticles after BSA protein coating. Owing to the existence of Gd3þ in UCNPs, those nanoparticles could serve as a T1 MR contrasting agent [37]. With a concentration-dependent brightening effect in T1-weighted MR images, The T1 relaxivity (r1) of UCNP@BSA was measured to be 2.55 mM1 S1 (Fig. 1d). Albumin, the majority component of serum proteins, has been widely used as a non-cytotoxic nano-carrier for drug delivery [38] .For example, human serum albumin (HAS) is able to bind paclitaxel

Fig. 1. Preparation and characterization of UCNP@BSA-RB& IR825 nanocomplex. (a) A schematic illustration to show the synthesis of UCNP@BSA-RB& IR825 nanocomplex, as well as the mechanism of both PDT and PTT therapies based on this system. (b) TEM images of UCNP-PAA (left) and UCNP@BSA (right) nanoparticles. Insert is higher-resolution images of single nanoparticles. (c) Photos of UCNP-PAA and UCNP@BSA nanoparticles dispersed in water and PBS. Although both soluble in water, UCNP-PAA but not UCNP@BSA would precipitate in the presence of salts. (d) T1-weighted MR images of UCNP@BSA at different Gd concentrations and relative relaxation rate r1.

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Fig. 2. Characterization of dye loading nano-complexes. (a) Photos of free RB, free IR825, UCNP@BSA-RB, UCNP@BSA-IR825, UCNP@BSA-RB& IR825 dispersed in water before (upper) and after centrifugation (bottom). Both RB and IR825 could be adsorbed by UCNP@BSA, which were precipitated after centrifugation. (b) UVeViseNIR absorbance spectra of UCNP@BSA, UCNP@BSA-RB, UCNP@BSA-IR825, and UCNP@BSA-RB& IR825 solutions at the same UCNP concentration (0.2 mg/ml). (c) Fluorescence spectra of UCNP@BSA, UCNP@BSA-RB, UCNP@BSA-IR825, and UCNP@BSA-RB& IR825 solutions at the same UCNP concentration (0.2 mg/ml). Insert: photos of UCNP@BSA and UCNP@BSA-RB& IR825 under the 980 nm laser excitation. (d) The generation of singlet oxygen by measuring the fluorescence intensity changes of SOSG as the functional of 980-nm light radiation time (0.5 W/cm2). The increase of SOSG fluorescence was a result of SO generation. (e) Temperature curves of different solutions over a period of 5 min under exposure to the 808-nm light (0.5 W/cm2). Insert: IR thermal photos of water, UCNP@BSA, UCNP@BSA-IR825, UCNP@BSA-RB& IR825 after 5 min of 808-nm laser irradiation.

via the hydrophobic interaction between the drug molecule and the hydrophobic domain of the protein, forming paclitaxel-loaded HSA nanoparticles, or namely AbraxaneÒ, which has already been approved for clinical use as an anti-cancer drug [39]. We thus wondered whether our UCNP@BSA nanoparticles could serve as a loading and delivery platform for hydrophobic molecules with photo-therapeutic functions. Rose Bengal is widely used photosensitizer molecule for photodynamic therapy [40]. On the other hand, IR825 is a non-fluorescent heptamethine indocyanine dye with strong NIR absorption, and has been demonstrated to be highly effective for in vitro and in vivo photothermal therapy of cancer [35]. In our experiment, RB, IR825, or both RB and IR825, were mixed with UCNP@BSA overnight in the dark. Excess dye molecules were removed by centrifugation at 14,800 rpm for 5 min

and washed with water for several times, obtaining UCNP@BSA-RB, UCNP@BSA-IR825, and UCNP@BSA-RB& IR825 nano-complexes well dispersed in water, respectively (Fig. 2a). As a straightforward evidence, aqueous solutions of UCNP@BSA, UCNP@BSA-RB, UCNP@BSA-IR825, UCNP@BSA-RB& IR825, as well as two types of free dyes at pH 7.4, were centrifuged for 5 min. Colored precipitate and nearly colorless supernatant were observed for dye-loaded nanoparticles (Fig. 2a), suggesting that the RB and IR825 molecules were indeed complexed with UCNP@BSA nanoparticles. Since no coupling agent has been induced during loading of those dye molecules, the binding of RB and IR825 on UCNP@BSA is likely by simple hydrophobic interactions. The UVeViseNIR absorption spectra and upconversion luminescence (UCL) spectra (excitation at 980 nm) of different dye-

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Fig. 3. Cell uptake and in vitro combined cancer therapy. (a) Relative viabilities of 4T1 cells after being incubated with various concentrations of UCNP@BSA, UCNP@BSA-RB, UCNP@BSA-IR825, and UCNP@BSA-RB& IR825 for 24 h. (b) Confocal UCL/fluorescence images of 4T1 cells incubated with different concentrations of UCNP@BSA-RB& IR825 after 4 h. Green and blue colors represented UCL emissions and DAPI-stained cell nuclei. (c) Relative viabilities of 4T1 cells treated with various concentrations of UCNP@BSARB under 980 nm laser irradiation (0.4 W/cm2, 10 min). (d) Relative viabilities of 4T1 cells treated with various concentrations of UCNP@BSA-IR825 under 808 nm laser irradiation (0.5 W/cm2, 5 min). (e) In vitro cytotoxicity effect induced by PDT, PTT, or combined PTT-PDT treatment. Combined PTT þ PDT offered significantly higher cancer cell killing effect than mono-therapy by PDT only or PTT only. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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loaded nanoparticles were then recorded (Fig. 2b and c). Both RB and IR825 remained their characterized absorption peaks after being loaded on UCNP@BSA nanoparticles (Fig. 2b). Importantly, UCNP@BSA-RB& IR825 showed the RB characteristic peak at w540 nm, which well matched the green emission peak of UCNPs to allow resonance energy transfer from UCNPs to RB. Therefore, the loading of RB on UCNP@BSA resulted in w30% of decrease in the green emission (the UCL peak at 540 nm) of UCNPs (Fig. 2c). On the other hand, the IR825 adsorbed on nanoparticles exhibited the characteristic peak at w825 nm, which showed no overlap with either the red emission peak (w660 nm) or the excitation wavelength (980 nm) of UCNPs. Therefore, the loading of IR825 would not affect the upconversion luminescence (UCL) emission of UCNPs (Fig. 2c), a unique advantage over Au or Ag shelled UCNPs which usually showed partially quenched UCL emissions [33,41]. Overall, compared with UCNP@BSA without dye loading, UCNP@BSA-RB& IR825 with dual-dye loading showed reduced green emission at 540 nm and essentially unchanged red emission peaked at 660 nm. The loading capacities of RB and IR825 on UCNP@BSA were evaluated, showing the saturated RB and IR825 loading ratios of w7.6% (w/w) and w22% (w/w), at the feeding RB, and IR825 concentrations of 80 mM and 0.4 mM, respectively (Supporting Figs, S1 and S2). UCNP@BSA-RB& IR825 prepared under this condition was chosen for further experiments. The release of RB and IR825 on UCNP@BSA-RB& IR825 was measured in water and PBS, showing that the majority of dye molecules retained being loaded on nanoparticles after 24 h incubation in PBS (pH 7.4) at room temperature (Supporting Fig. S3). Since the generation of cytotoxic singlet oxygen (SO) is critical in PDT, we then assessed the production of SO by UCNP@BSA-RB& IR825 under the 980-nm laser irradiation using the singlet oxygen sensor green (SOSG), whose quenched fluorescence in the aqueous solution would be recovered in the presence of SO [42]. In our experiment, we found obvious production of SO when 1 mg/ml UCNP@BSA-RB or UCNP@BSA-RB& IR825 was exposed to a 980-nm laser at the power density of 0.5 W/cm2 (Fig. 2d). In contrast, UCNP@BSA-IR825 without RB loading, or free RB under 980-nm excitation, both showed no detectable SO generation. The effective generation of SO by UCNP@BSA-RB and UCNP@BSA-RB& IR825 under NIR light would enable NIR-induced PDT using our nanoparticles. Then we studied the photothermal effect of UCNP@BSA-RB& IR825. Different solutions were irradiated by an 808 nm NIR laser for 5 min at the power density of 0.5 W/cm2. The temperature rises of UCNP@BSA-IR825 and UCNP@BSA-RB& IR825 were found to be much higher than that of other samples without IR825 loading (Fig. 2e). In contrast, under irradiation by a 980-nm NIR laser for 5 min at the power density of 0.5 W/cm2, the temperature increases for all the tested solutions were not significant (Supporting Fig. S4), suggesting that the 980-nm excitation at such a power density would mainly introduce the photodynamic instead of photothermal effect. Before testing the photo-therapeutic efficacy of our UCNP@BSA-RB& IR825 nanocomplex in cell culture experiments, we firstly measured the dark cytotoxicity of UCNP@BSA before and after drug loading. Cell viability test based on the standard thiazolyl blue tetrazolium bromide (MTT) assay was carried out on 4T1 murine breast cancer cells, revealing no obvious cytotoxicity to cells even at high nanoparticle concentrations up to 0.4 mg/ml (Fig. 3a). We next studied the cell uptake of UCNP@BSA-RB& IR825 by confocal fluorescence microscopy. Strong green UCL signals from UCNPs in cytoplasm of 4T1 cells showed up after they were incubated with different concentrations of UCNP@BSA-RB& IR825 for 4 h, indicating the efficient cellular uptake of dye-loaded UCNPs (Fig. 3b).

Next, in vitro phototherapy efficacies of PDT, PTT, and combined PDT-PTT, were evaluated with 4T1 cells. Under irradiation of the 980-nm laser at the power density of 0.4 W/cm2 for 10 min, the cell viability gradually decreased with the increase of UCNP@BSA-RB concentrations, demonstrating the NIR-induced photodynamic killing of cancer cells (Fig. 3c). The PTT effect caused by the hyperthermia induced by UCNP@BSA-IR825 and the 808-nm laser (0.5 W/cm2, 5 min) was also assessed, showing slightly more effective cell destruction ability than that of the PDT effect (Fig. 3d). After demonstrating individual PDT and PTT treatment, we then carried out combined PDT and PTT using the UCNP@BSA-RB& IR825 nanocomplex. To our expectation, cells incubated with UCNP@BSARB& IR825 after 808-nm and 980-nm lasers irradiation were nearly completely destructed by such combined PTT þ PDT therapy, resulting in much lower remaining cell viability compared to those after mono-therapy with only 808-nm laser or only 980-nm laser irradiation (Fig. 3e). To further study the potential of UCNP@BSA-RB& IR825 nanocomplex for in vivo imaging and cancer combination therapy, mice bearing 4T1 tumors were intratumorally (i.t.) injected with UCNP@BSA-RB& IR825 (20 ml, 10 mg/ml). Upon injection with the nanoparticles, obvious UCL and T1-weighted MR signals were observed from tumors of mice, demonstrating the ability of GdUCNPs for multimodal imaging (Fig. 4a and b). We then used an IR thermal camera to measure the real-time temperature change of the tumor upon laser irradiation (Fig. 4c and d). After i.t. injection with UCNPs@BSA-RB& IR825, rapid temperature rise from w30  C to w45  C was observed for tumors under the 808-nm laser irradiation (0.35 W/cm2) within 5 min, but not for those under 980-nm laser irradiation (0.4 W/cm2) for the same period of time. In contrast, saline injected tumors showed no significant temperature increase under either 808-nm or 980-nm laser irradiation under the same conditions. The therapeutic efficacies of PTT, PDT and combined PTT þ PDT based on our UCNP@BSA-RB& IR825 theranostic nanocomplex were next evaluated in animal experiments. A total of 30 mice bearing 4T1 tumors were divided into six groups with 5 mice per group: untreated, laser only (808 nm þ 980 nm), injection only, PTT only, PDT only and PTT þ PDT. For the laser only group, the tumors were exposed with both 808-nm (0.5 W/cm2, 5 min) and then 980nm laser (0.4 W/cm2, 30 min, 1 min interval for every 2e3 min). For the later four groups, a solution of UCNP@BSA-RB& IR825 (40 ml, 10 mg/ml) was i.t. injected into each tumor. For PDT, those tumors were exposed to a 980-nm laser for 30 min at the power density of 0.4 W/cm2 (1 min interval for every 2e3 min to avoid any heating) to induce singlet oxygen production for cancer cell killing. In the case of PTT, an 808-nm laser was introduced for tumor irradiation with a power density of 0.5 W/cm2 for 5 min. The tumor sizes were then measured by a caliper every the other day to determine the tumor growth (Fig. 4e). The mice were then sacrificed at day 14 after the treatment was initiated for tumor collection (Fig. 4f). It was found that either PDT or PTT by itself could only slightly delay the tumor growth. In marked contrast, the tumor development after combined PTT þ PDT treatment was remarkably inhibited, without showing significant growth within 14 days (Supporting Fig. S5). HematoxylineEosin (H&E) staining of tumor slices collected from different groups of mice showed that the tumor cells after combined PDT-PTT treatment were severely damaged, while those after mono-therapy (PDT only or PTT only) were only partially destructed, confirming the tumor growth data and further demonstrating the superior efficacy of combination therapy (Fig. 4g). In this UCNP@BSA-RB& IR825 complex, the optical properties of each component are precisely controlled to achieve the optimized theranostic outcomes. While the green UCL emission from UCNPs

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Fig. 4. In vivo cancer therapy in 4T1 tumor-bearing nude mice. (a & b) In vivo T1-weighted MR images (a) and UCL images (b) of mice before and after intratumoral injection with UCNP@BSA-RB& IR825. (c) IR thermal images of 4T1 tumor-bearing mice i.t. injected with either PBS or UCNP@BSA-RB& IR825 and the exposed to the 808-nm or 980-nm laser irradiation at the power density of 0.5 W/cm2. (d) The tumor temperature changes based on IR thermal imaging data in (c). (e) In vivo tumor growth in different groups of mice after various treatments indicated. Six groups of mice (n ¼ 5 per group) were used in our experiment: 1: untreated, 2: laser only (808 nm þ 980 nm), 3: injection only, 4: PDT, 5: PTT, 6: PTT þ PDT. 808-nm (0.5W/cm2, 5 min) and 980-nm (0.4 W/cm2, 30 min) were used to separately trigger PTT and PDT, respectively. (f) Photographs of tumors collected from different groups of mice 14 day after the treatment was initiated. (g) Micrographs of H&E-stained tumor slices harvested from mice with different treatments indicated.

are utilized to excite RB molecules to trigger the PDT effect, the red emission of UCNPs with better tissue penetration are not affected and could be fully used in optical imaging. Moreover, the NIR absorption of IR825 peaked at w825 nm perfectly locates inside the ‘window’ of the UCL anti-Stokes shift of UCNPs, without affect their excitation (980 nm) and emission (660 nm). Thus, unlikely the previously developed UCNP-based photothermal agents by coating

UCNPs with noble metal shells [33,43], the UCL emission in our system would not be compromised, ideal for applications in imaging-guided therapy. However, although encouraging synergistic therapeutic efficacies of the combined photothermal and photodynamic therapy are realized both in vitro and in vivo using our UCNP@BSA-RB& IR825 theranostic nanoparticles, there is still much room for the

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further improvement of this multifunctional nano-platform. The recent development of Nd3þ containing UCNPs which could be effective excited under w800 nm laser would be promising for applications in UCNP-based photodynamic therapy to avoid water heating under 980 nm light [44,45]. Moreover, further modification of the BSA shell, either by polyethylene glycol (PEG) to enable long blood circulation and passive tumor targeting, or conjugated with targeting molecules to achieve active tumor targeting, may allow tumor imaging and combined therapy upon systemic administration of those nano-agents. In fact we have already demonstrated in our preliminary work (Supporting Fig. S6) that by conjugating ArgGly-Asp (RGD) peptide on the BSA shell, specific cancer cell targeting and selective cell killing could become possible. 4. Conclusion In summary, we develop a surface functionalization strategy for UCNPs by coating those nanoparticles with BSA protein. In such a UCNP@BSA system, the Gd-based UCNPs, which are useful contrast agents for dual modal optical/MR imaging, in the mean time are able to trigger photodynamic therapy under NIR light by resonance energy transfer if coupled with photosensitizers. On the other hand, the BSA coating not only improves the physiological stability of UCNPs, but also could serve as a delivery platform to load various molecules with therapeutic functions. RB, a photodynamic agent, together with IR825, a photothermal agent, are simultaneously loaded on UCNP@BSA for combined phototherapy of cancer, demonstrating outstanding synergistic anti-tumor effect in our animal experiments. Although further studies are still ongoing in our laboratory aiming at achieving combination therapy of cancer upon systemic administration (e.g. intravenous injection) of such UCNP-based theranostic agent, our work presents a new design of a simple nano-platform, in which multiple imaging and therapy functions can be integrated together for imaging-guided cancer combination therapy. Acknowledgment This work was partially supported by the National Basic Research Programs of China (973 Program) (2012CB932600, 2011CB911002), the National Natural Science Foundation of China (51222203, 51132006, 51302180), and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. Liang Cheng was supported by a Postdoctoral science foundation of China (2013M531400). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2013.12.046. References [1] Sahu A, Choi WI, Lee JH, Tae G. Graphene oxide mediated delivery of methylene blue for combined photodynamic and photothermal therapy. Biomaterials 2013;34:6239e48. [2] Chen J, Yang M, Zhang Q, Cho EC, Cobley CM, Kim C, et al. Gold nanocages: a novel class of multifunctional nanomaterials for theranostic applications. Adv Funct Mater 2010;20:3684e94. [3] Ma Y, Liang X, Tong S, Bao G, Ren Q, Dai Z. Gold nanoshell nanomicelles for potential magnetic resonance imaging, light-triggered drug release, and photothermal therapy. Adv Funct Mater 2012;23:815e22. [4] Zhang Z, Wang L, Wang J, Jiang X, Li X, Hu Z, et al. Mesoporous silica-coated gold nanorods as a light-mediated multifunctional theranostic platform for cancer treatment. Adv Mater 2012;24:1418e23. [5] Wang S, Huang P, Nie L, Xing R, Liu D, Wang Z, et al. Single continuous wave laser induced photodynamic/plasmonic photothermal therapy using photosensitizer-functionalized gold nanostars. Adv Mater 2013;25(22):3055e61.

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