Facile preparation of doxorubicin-loaded upconversion@polydopamine nanoplatforms for simultaneous in vivo multimodality imaging and chemophotothermal synergistic therapy.

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Facile Preparation of Doxorubicin-Loaded Upconversion@Polydopamine Nanoplatforms for Simultaneous In Vivo Multimodality Imaging and Chemophotothermal Synergistic Therapy Fuyao Liu, Xiuxia He, Zhen Lei, Liang Liu, Junping Zhang, Hongpeng You, Huimao Zhang,* and Zhenxin Wang* promising multifunctional nanoplatforms for both diagnosis and therapy.[1] The multiple imaging modalities (e.g., X-ray computed tomography (CT), magnetic resonance imaging (MRI), and fluorescence imaging), can provide more reliable and accurate information for detection and positioning of tumor sites than a single modality.[2] Loading chemotherapy drugs (e.g., doxorubicin (DOX)) with hyperthermia agents (e.g., gold nanoshell and graphene) can enhance therapeutic efficacy through synergistic chemotherapy and photothermal therapy (PTT).[3] In particular, imaging, drug delivery and PTT can be achieved simultaneously when the drug carriers also act as multimodal imaging agents.[3d] Based on the detailed and accurate imaging information provided by multimodal bioimaging, the drug delivery and cancer therapy processes can be well monitored noninvasively, which is expected to further optimize the efficacy of both early disease diagnosis and therapeutic treatment.[3d,4] For instance, Liu and co-workers have developed a multifunctional nanocomposite by coating magnetic iron oxide nanoclusters with a near-infrared (NIR) light-absorbing polymer polypyrrole (PPy), which after loading with DOX could be used for imaging guided, remotely controlled cancer

The development of biosafe nanoplatforms with diagnostic and therapeutic multifunction is extremely demanded for designing cancer theranostic medicines. Here, a facile methodology is developed to construct a multifunctional nanotheranostic that gathers five functions, upconversion luminescence (UCL) imaging, T1-weighted magnetic resonance imaging (MRI), X-ray computed tomography (CT) imaging, photothermal therapy (PTT), and chemotherapy, into one single nanoprobe (named as UCNP@PDA5-PEG-DOX). For generating the UCNP@PDA5-PEG-DOX, a near-infrared light (NIR)-absorbing polydopamine (PDA) shell is directly coated on oleic-acid-capped β-NaGdF4:Yb3+,Er3+@βNaGdF4 upconverting nanoparticle (UCNP) core for the first time to form monodisperse, ultrastable, and noncytotoxic core–shell-structured nanosphere via water-in-oil microemulsion approach. When combined with 808 nm NIR laser irradiation, the UCNP@PDA5-PEG-DOX shows great synergistic interaction between PTT and the enhanced chemotherapy, resulting in completely eradicated mouse-bearing SW620 tumor without regrowth. In addition, leakage study, hemolysis assay, histology analysis, and blood biochemistry assay unambiguously reveal that the UCNP@PDA5-PEG has inappreciable cytotoxicity and negligible organ toxicity. The results provide explicit strategy for fabricating multifunctional nanoplatforms from the integration of UCNP with NIR-absorbing polymers, important for developing multi-mode nanoprobes for biomedical applications.

1. Introduction Nanoparticles (NPs) can be used to integrate more than one kind of imaging or therapeutic functions, which makes them F. Y. Liu, Z Lei, Prof. Z. X. Wang State Key Laboratory of Electroanalytical Chemistry Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun 130022, P.R. China E-mail: [email protected] F. Y. Liu, Z. Lei University of Chinese Academy of Sciences Beijing 100039, P.R. China Dr. X. X. He, J. P. Zhang School of Life Science and Technology Changchun University of Science and Technology Changchun 130022, P. R. China

L. Liu, Prof. H. M. Zhang Department of Radiology The First Hospital of Jilin University Changchun 130033, P.R. China E-mail: [email protected] Prof. H. P. You State Key laboratory of Rare Earth Resource Utilization Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun 130022, P.R. China

DOI: 10.1002/adhm.201400676

Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201400676

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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chemophotothermal combination therapy.[4] Remarkable progress has been achieved in developing nanoplatforms for early cancer diagnosis or therapy, but the integration of two or more strategies in one system is still at its tentative stage and has become a representative challenge in bionanomedicine. Upconversion nanoparticle (UCNP), particularly lanthanidedoped rare-earth NP, which is able to emit shorter wavelength photons under excitation by NIR light, is a new generation of optical probes with great potential in biomedical imaging.[5] Compared with traditional downconversion fluorescent probes such as quantum dots and organic dyes, UCNP has prominent advantages including narrow emission peaks, large stokes shifts, low toxicity, good photostability as well as the autofluorescencefree nature that offers significantly improved signal-to-noise ratios during imaging.[6] For example, because of the unique MR, strong X-ray attenuation and high atomic number properties of Gd, NaGdF4-based UCNP has been developed as multimodal imaging (UCL/MRI/CT) nanoprobes, and applied to animal studies.[7] In order to obtain high-quality UCNP-based contrast and therapeutic agents, nobel metal (e.g., gold) or mesoporous silica shell are normally coated on UCNP to serve as PTT unit or drug carrier, respectively.[8] Although the gold/silica-coated UCNP exhibits high efficiency on the cancer treatment, their thermal and colloidal stability still remain to be further improved.[9] Due to its bioinspired nature, polydopamine (PDA) has served as a universal coating to NPs for various biomedical applications.[10] In particular, PDA shell can be further surface modified to improve the stability and functionality of NPs. For instance, Ji and co-workers have demonstrated that PDA-coated gold NPs are stable in vivo for a period of at least 6 weeks.[10b] Although there is rapidly growing on the applications of PDA to prepare hydrophilic NP-based core@shell nanomaterials for biomedical applications, few of examples on synthesis of hydrophobic NP@PDA NPs have been reported. The hydrophobic NP, specific UCNP, shows more attractive properties including high monodispersity and exceptional optical property than those of their hydrophilic counterpart.[6,7] Here, oleic-acid-capped UCNP is coated with NIR lightabsorbing PDA polymer by water-in-oil microemulsion method, obtaining UCNP@PDA core–shell nanocomposites, which are then modified with amino-terminated polyethylene glycol (mPEG-NH2) for improving the stability of UCNP@PDA in physiological conditions. To our best knowledge, it is the first time to directly coat PDA onto the hydrophobic NPs. The PDA shell not only exhibits strong photothermal effect, but also provides active surface for loading the aromatic chemotherapy drugs (e.g., DOX) via π–π stacking and hydrogen bonding interactions. Owing to the high UCL emission, T1 relaxivity value and CT contrast enhancement of UCNP cores, tri-modal imaging (UCL/MRI/CT) of mouse-bearing colorectal (SW620) tumor has been achieved by PEGylated UCNP@PDA with 5 nm thickness PDA shell (UCNP@PDA5-PEG). Using DOXloaded UCNP@PDA5-PEG (UCNP@PDA5-PEG-DOX) as the model system, excellent synergistic therapeutic efficacy is demonstrated in both in vitro cell culture and in vivo animal experiments. The results suggest that the drug-loaded UCNP@PDA core–shell nanocomposite can be employed as an efficient nanoplatform for biomedical applications including multimodality imaging and chemophotothermal therapy.

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2. Results and Discussion 2.1. Synthesis and Characterization of UCNP@PDAn-PEG Using 15 nm Yb and Er co-doped NaGdF4: Yb3+, Er3+ (Gd: Yb: Er = 80%: 18%: 2%) UCNP as seed, NaGdF4: Yb3+, Er3+@NaGdF4 UCNP was synthesized by a seedmediated regrowth method.[7a,11] The average diameter of NaGdF4:Yb,Er@NaGdF4 UCNP has been increased to approximately 20 nm in diameter, which suggests the growth of the NaGdF4 shell on the NaGdF4: Yb3+, Er3+ core (as shown in Figure 1c). The crystalline nature of the core and core/shell was further confirmed by the corresponding X-ray diffraction (XRD) pattern (as shown in Figure S1, Supporting Information), which were in agreement with the pure hexagonal phase (JCPDS No. 27–0699). Two bands at 1464 cm−1 and 1567 cm−1 are observed at FT-IR spectrum of the as-prepared NaGdF4 :Yb,Er@NaGdF4 UCNP (as shown in Figure S2, Supporting Information), which are assigned to the COOH stretching vibration. These bands revealed that oleic acid molecules are attached on the NaGdF4: Yb3+, Er3+@NaGdF4 UCNP surface during NPs synthesis. In the presence of oxygen, dopamine can easily form reactive quinone through spontaneous oxidation, which are further polymerized and formed an adherent PDA shell on the substrate.[10a] The polymerization reaction of dopamine has been widely used in hydrophilic NPs modification since the polar groups of PDA, such as hydroxyl and amine groups, endow the NPs with improved hydrophilicity and stability.[12] In this study, the UCNP with different thickness of PDA shell (named as UCNP@PDAn, n = 3, 5, 8) are prepared in water-in-oil microemulsion method because the oxidative polymerization of dopamine in a water-in-oil microemulsion is much easier to control than that in bulk aqueous solution because of the relative low reaction rate in microemulsion.[13] The water nanodroplets exist in the bulk cyclohexane phase supply a nanoreactor for the synthesis of UCNP@PDAn. When dopamine monomer is introduced into the alkaline microemulsion, the oxidative polymerization is immediately triggered. This spontaneous multistep intramolecular chemical reaction produces an important intermediate, named 5,6-dihydroxyindole, which further leads to form PDA through an intermolecular reaction. The thickness of PDA shell could be easily modulated by changing the amount of dopamine in the reaction mixture. For instance, the thickness of the PDA shell is increased from 3 to 8 nm by increasing the amount of dopamine hydrochloride from 8.3 to 33.3 mg (as shown in Figure 1d and S3, Supporting Information). PDA thickness-dependent on weight loss behavior of UCNP@PDAn is observed on TGA analysis (as shown in Figure S4, Supporting Information), demonstrating that UCNP@PDAn with different shell thicknesses have been synthesized. The XPS and EDS measurements clearly show the element of N in UCNP@PDA5 (as shown in Figure 1f and S5, Supporting Information). Absorption bands, 3385 cm−1 (NH stretching), 1588 cm−1 (C C aromatic ring stretching vibration), and 1511 cm−1 (NH bending) in the FT-IR spectrum of UCNP@PDA5 (as shown in Figure S2, Supporting Information) and 1350 cm−1 and 1580 cm−1 in Raman spectrum of UCNP@PDA5 (as shown in Figure 1g) further confirm the presence of PDA on UCNP.




FULL PAPER Figure 1. a) Schematic illustration of the synthesis of UCNP@PDA5-PEG and the following DOX loading. TEM images of b) NaGdF4: Yb3+, Er3+ UCNP, c) NaGdF4: Yb3+, Er3+@NaGdF4 UCNP, d) UCNP@PDA5, and e) UCNP@PDA5-PEG. f) XPS spectra and g) Raman spectra of NaGdF4: Yb3+, Er3+@ NaGdF4 UCNP and UCNP@PDA5.

The mPEG-NH2 was conjugated on UCNP@PDA surface through a reaction between terminal amine group of mPEGNH2 and PDA because PEG2000–OCH3 favors the accumulation of NPs in tumor by the enhanced permeability and retention (EPR) effect.[14] In this case, the catechol group of PDA shell is firstly oxidized to quinone under basic condition. The quinone then reacts with the nucleophilic amine group of mPEG-NH2 by a Schiff base reaction and/or a Michael-type addition pathway (as shown in Figure S6, Supporting Information). As typically example, the IR characteristic peaks of PEG at 2924 cm−1 (alkyl C H stretching) and 1092 cm−1 (C O C stretching) are clearly observed in the FI-TR spectrum of UCNP@PDA5PEG, indicating that mPEG-NH2 has been conjugated on the UCNP@PDA5 (as shown in Figure S2, Supporting Information). In addition, the zeta potential (−0.85 mV) of UCNP@PDA5-PEG is higher than that (−15.6 mV) of UCNP@ PDA5 (as shown in Figure S7, Supporting Information). The UCNP@PDA5-PEG exhibits excellent colloidal stability in different dispersants including H2O, PBS, TB, 0.9% NaCl solution, and 10% serum (as shown in Figure S8, Supporting Information) and high NIR absorption from 700 to 900 nm (as shown in Figure S8 and S9, Supporting Information), making it as a potential PTT agent. The absorbance of UCNP@PDAn-PEG at 808 nm is increased by increasing the thickness of PDA shell (as shown in Figure S9, Supporting Information). In contrast with NIR absorption, the UCL emission of UCNP@PDAn-PEG


is decreased with increasing the thickness of PDA shell (as shown in Figure S10, Supporting Information). In addition, the room temperature magnetization of NaGdF4: Yb3+, Er3+@NaGdF4 UCNP, UCNP@PDA3-PEG, UCNP@PDA5-PEG, and UCNP@ PDA8-PEG are determined to be 2.4, 2.0, 1.7, and 1.1 emu g−1 at 30 KOe, respectively (as shown in Figure 2a). The decreased magnetic saturation of UCNP@PDAn-PEG could be attributed to the increasing mass of PDA shell on the surface of the UCNP. In order to obtain relatively high photothermal conversion efficiency, UCL intensity and T1-MRI contrast effect, the UCNP@PDA5-PEG (the mass ratio of UCNP to PDA in the UCNP@PDA5-PEG is about 1:0.429) is used in the following in vitro and in vivo experiments. The mass and molar extinction co-efficients of UCNP@PDA5-PEG at 808 nm are 3.1 × 103 cm2 g−1 and 5.1 × 108 M−1 cm−1, respectively. The molar extinction co-efficient of UCNP@PDA5-PEG is comparable to that of polyaniline NPs (8.95 × 108 M−1 cm−1), or much higher than those of other reported PTT coupling agents, such as CdX NPs (X = S, Se, Te) (2–5 × 105 M−1 cm−1), copper selenide NPs (7.7 × 107 M−1 cm−1) and carbon nanotubes (7.9 × 106 M−1 cm−1).[15] The UCNP@PDA5-PEG displays three major UCL peaks at 526, 546, and 660 nm, which are attributed to the energy transitions from 2H11/2, 4S3/2, and 4F9/2 to 4I15/2 of Er3+ ions, respectively (as shown in Figure 2b).[16] The UCL intensity of UCNP@PDA5-PEG is equal to that of UCNP@SiO2, which



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Figure 2. a) Magnetization-applied magnetic field curves of NaGdF4: Yb3+, Er3+@NaGdF4 UCNP and UCNP@PDAn-PEG, b) UCL spectrum of UCNP@PDA5-PEG in water (Gd content: 5 ppm), c) T1-weighted MR images and R1 relaxivity of aqueous solution of UCNP@PDA5-PEG as a function of the molar concentration of Gd3+ in the solution, and d) CT images and HU value of UCNP@PDA5-PEG aqueous solution as a function of the mass concentration of Gd3+ in the solution, respectively.

has been widely used as UCL probe in bioanalysis (as shown in Figure S11, Supporting Information).[17] The MRI contrast capability of UCNP@PDA5-PEG is tested by a 1.5T clinical MRI unit with the spin-echo method. The molar relaxivity (r1) (which corresponds to the slope of the line in Figure 2c) is estimated to be 3.0 mM−1 s−1, which is higher than the literature reported values of UCNP@SiO2.[17] Figure 2d shows the CT phantom images of UCNP@PDA5-PEG with varying concentrations. The Hounsfield unit (HU) value (c.a. 200) of Gd (4.0 mg mL−1) in UCNP@PDA5-PEG is equivalent to iodine (8 mg mL−1) in Omnipaque, a popular iodine-based CT contrast agent currently used in clinic, showing strong enhancement of CT signal. These results indicate that UCNP@PDA5-PEG can be applied as efficient multi-modality contrast agents for UCL, MR, and CT imaging.

2.2. Photothermal Performance of UCNP@PDA5-PEG Prior to construct the multifunctional drug system, the photothermal conversion capability of UCNP@PDA5-PEG is evaluated by irradiating UCNP@PDA5-PEG aqueous solution with an 808 nm NIR laser at 1.3 W cm−2 for 600 s. The UCNP@PDA5-PEG shows effective photothermal heating of the solutions (as shown in Figure 3a). The photothermal heating effect increased monotonically with the concentration of UCNP@PDA5-PEG. In the presence of UCNP@PDA5-PEG (Gd content: 100 ppm), the solution temperature is increased to 50 °C (the temperature of efficient killing of cancerous cells)[18] 4


within 200 s irradiation. The experimental result demonstrates that UCNP@PDA5-PEG has high photothermal conversion capability. Furthermore, there is no significant absorption change of UCNP@PDA5-PEG at 808 nm when UCNP@PDA5PEG is irradiated by 808 nm NIR laser for over 100 min (as shown in Figure S12, Supporting Information). The phenomenon indicates that UCNP@PDA5-PEG has high photostability. In addition, negligible free Gd ions could be detected after one month dialysis of UCNP@PDA5-PEG under physiological conditions. The dialysis experimental result indicates that the PDA shell can effectively prevent Gd3+ leaking from UCNP core for a long time.

2.3. NIR Light-Triggered DOX Release DOX, a commonly used aromatic chemotherapy drug, is mixed with UCNP@PDA5-PEG to construct multifunctional drug system. As expected, the DOX can be effectively loaded on the PDA shell after incubated with UCNP@PDA5-PEG. The red-shifted absorption peak of DOX (from 480 to 486 nm) in the UV–vis spectrum of the UCNP@PDA5-PEG-DOX not only confirms the loading of DOX onto UCNP@PDA5-PEG, but also shows π–π stacking interaction between DOX and PDA shell (as shown in Figure 3b).[19] The saturated maximal DOX loading efficiency of UCNP@PDA5-PEG is estimated to be about 0.97 mg DOX/mg UCNP@PDA5-PEG, which is much higher than DOX loading efficiencies of other organic– inorganic nanocomposites.[8c,20] The high loading capacity




FULL PAPER Figure 3. a) Temperature elevation of water and UCNP@PDA5-PEG solution of different concentrations (Gd content: 0–100 ppm) over a period of 10 min under exposure of 808 nm NIR light (1.3 W cm−2) measured every 10 s using a thermometer, b) UV–vis spectra of DOX, UCNP@PDA5-PEG, and UCNP@PDA5-PEG-DOX, c) DOX release from UCNP@PDA5-PEG-DOX over time in PBS with two different pH values indicated, and d) NIR-triggered release of DOX from UCNP@PDA5-PEG-DOX, which were irradiated with an 808 nm NIR laser (1.3 W cm−2) for 6 min at different time points indicated by arrows under different pH values indicated. Error bars mean standard deviations (n = 5).

(ca. 100 wt%) could be contributed to the strong π–π stacking and hydrogen bond interactions of PDA shell with DOX because PDA has abundant phenyls, amino, and hydroxyl groups on its surface.[12] In the presence of DOX, the green upconversion emission is quenched partially through resonance energy transfer from UCNP to DOX, which gives further evidence on the formation of UCNP@PDA5-PEG-DOX (as shown in Figure S13, Supporting Information). The drugreleasing behaviors of UCNP@PDA5-PEG-DOX under pH 5.0 and 7.4 were investigated, respectively. Within 24 h, about 20% of DOX is released from the UCNP@PDA5-PEG-DOX at pH 5.0 (as shown in Figure 3c). The slowly released DOX would favor the long and continued therapy of tumor without frequent dosing, and subsequently improve the patients’ compliance.[3c] The DOX releasing rate at pH 5.0 is about threefold faster than that at pH 7.4. The phenomenon may due to the protonation of the amino group in the DOX molecule that offers DOX a positive charge, thus facilitating drug release under acidic pH. In addition, sustained release property of our nanocomposite is consistent with many previously reported π–π stacking-based drug controlled systems.[21] After irradiated under an 808 nm NIR laser (1.3 W cm−2, 6 min for each pulse), a burst release of DOX was observed from UCNP@PDA5-PEG-DOX at pH 5.0. In comparison, rather limited release of DOX was observed at pH 7.4 under the same condition (as shown in Figure 3d). The environmental pH-dependent NIR-responsive release processes could be used to fine-tune the amount of intracellular drug release and minimize side effect of drug.


2.4. Cell Uptake and In Vitro Cytotoxicity In order to investigate the internalization process and intracellular release behavior of UCNP@PDA5-PEG-DOX, the SW620 cells were incubated with UCNP@PDA5-PEG-DOX for 0.5, 2, 4, 6, and 12 h, respectively. As the prolonging of incubation time, the UCL signals inside cells are increased dramatically and the red fluorescence of DOX is evenly distributed in the whole intracellular region (as shown in the Figure 4). The experimental result indicates that the UCNP@PDA5-PEGDOX is internalized by SW620 cells and DOX molecules are released from cellular internalized UCNP@PDA5-PEG-DOX. In addition, the DOX shows slowly nuclear accumulating behavior since the NP-loaded DOX is firstly released within the cytoplasm, followed by accessing into the nucleus. The phenomenon is consistent with other reported drug controlled systems. 20a,[22] MTT assay of SW620 cell is employed to evaluate the cytotoxicities of UCNP@PDA5-PEG and UCNP@PDA5-PEG-DOX. The cell viability of UCNP@PDA5-PEG stained SW620 cells is still remained above 90% when the concentration of the UCNP@PDA5-PEG is as high as 100 ppm (Gd content) in culturing medium (as shown in Figure 5). The result confirms the noncytotoxicity of UCNP@PDA5-PEG. Although the cumulative release of DOX is less than 10% and cellular internalization of NPs is limited at the 2 h point, the UCNP@PDA5-PEG-DOX still shows apparent cytotoxicity on SW620 cells. The cytotoxicity can be attributed to the high DOX loading efficiency (ca. 100 wt%) of UCNP@PDA5-PEG. The cell viabilities of UCNP@



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Figure 4. Fluorescence images of SW620 cells after incubation with UCNP@PDA5-PEG-DOX for various times indicated. Images are taken from a) bright-field mode, b) the UCL channel (green), and c) DOX channel (red), respectively.

PDA5-PEG-DOX or UCNP@PDA5-PEG-stained SW620 cells were decreased dramatically when combined with an 808-nm NIR laser photothermal treatment (irradiated at a power density of 1.3 W cm−2 for 6 min, as shown in Figure 5). In particular, the cytotoxicity of UCNP@PDA5-PEG-DOX is stronger than that of UCNP@PDA5-PEG under the same concentration. This result suggests that UCNP@PDA5-PEG-DOX has an outstanding synergistic antitumor effect. This synergistic effect could be caused by hyperthermia effect on physiological function of cells and photothermal enhanced chemotherapeutic efficacy of DOX at elevated temperatures. To further identify the synergistic effect of UCNP@PDA5-PEG-DOX in vitro, UCNP@PDA5-PEG or UCNP@PDA5-PEG-DOX (Gd content: 100 ppm) treated SW620 cells with or without 808 nm NIR

Figure 5. In vitro cell viabilities of SW620 cells incubated with various concentrations of UCNP@PDA5-PEG and UCNP@PDA5-PEG-DOX (Gd content: 0–100 ppm) with or without 808 nm NIR laser irradiation (808 nm, 1.3 W cm−2, 6 min). The error bars mean standard deviations (n = 5, **p < 0.01, or *p < 0.05, by ANOVA with Tukey's post-test).

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laser irradiation are co-stained by Calcine AM and propidium iodide (PI) and imaged by confocal microscopy, respectively (as shown in Figure S14, Supporting Information). The confocal microscopic result is consistent with that of MTT assay. Furthermore, we investigate the effects of the laser power intensity and irradiation time on the efficiency of chemophotothermal therapy by the MTT assay (as shown in the Figure S15, Supporting Information). The experimental result suggests that 6 min of irradiation at 1.3 W cm−2 is sufficient for the cancer therapy.

2.5. In Vivo Imaging and Therapy Prior to in vivo imaging and therapy experiment, the toxicity of the UCNP@PDA5-PEG is further examined by hemolysis test. The hemolysis result indicates that UCNP@PDA5-PEG has excellent blood compatibility (as shown in Figure S16, Supporting Information). In vivo multimodality imaging capability of UCNP@PDA5-PEG was performed on BALB/C nude mouse-bearing colorectal (SW620) tumor. It is very important to realize early diagnosis and treatment of colorectal cancer (CRC) since CRC is the third most common cancer worldwide and has poor prognosis.[23] UCNP@PDA5-PEG solution (50 µL, 0.6 mg Gd mL−1) were administered subcutaneously into the tumor site of nude mouse. Clearly, UCL signal of the tumor region is observed (as shown in Figure S17, Supporting Information). The experimental result indicates that UCNP@PDA5-PEG would be used as a promising probe for in vivo UCL imaging with high sensitivity. Desired amounts (1.5 mg Gd mL−1 for MRI experiments and 15 mg Gd mL−1 for CT experiments) of UCNP@PDA5-PEG were injected intravenously into SW620 tumor-bearing nude mice by the tail vein, respectively. A detectible T1-weighted contrast enhancement (12%) is observed in the tumor area (marked by red circles in the figure) after 2 h injection (as shown in Figure 6a). The




FULL PAPER Figure 6. In vivo a) MR and b) CT images c) 3D volume rendering CT images of nude mice after intravenous injection of UCNP@PDA5-PEG at different timed intervals (pre-injection, 0.5, 2, and 24 h post-injection), respectively. The tumor site was marked by red circle. Corresponding data analysis of d) MR and e) CT measurements, respectively. Error bars mean standard deviations (n = 5).

signal intensities in tumor are increased 1.27 times (from 545 to 693) at 24 h post-injection (as shown Figure 6d). Figure 6b shows the in vivo whole-body CT images of nude mouse with UCNP@PDA5-PEG. Clearly positive enhancement of signal of the tumor region (marked by red circles in the Figure 6b) can be observed after injection of UCNP@PDA5-PEG. Remarkably, the signal (HU values) of tumor has been gradually increased for over 24 h (as shown in Figure 6e), suggesting the increased uptake of UCNP@PDA5-PEG by EPR effect at the prolonged post-injection. Volume-rendered CT images also confirm the signal enhancement of tumor region after 24 h injection (as shown in Figure 6c). The long-lasting blood circulation in vessels can help to improve the diagnostic accuracy of tumor.[24] Furthermore, accumulation UCNP@PDA5-PEG in tumor is estimated to be 10% ID g−1 (as shown in Figure S18, Supporting Information). The high tumor accumulation of UCNP@PDAPEG NPs could due to the EPR effect in cancerous tumors with tortuous and leaky vasculature.[25] These investigations have demonstrated that the UCNP@PDA5-PEG can be employed as contrast agents for in vivo UCL/MRI/CT imaging. After MRI


experiment, the mouse-bearing SW620 tumors were irradiated by 808 nm NIR laser for 10 min. Hematoxylin and eosin (H&E) staining of tumor slices show that the tumor cellularity exhibits typical nuclear damage characteristics (as shown in Figure S19, Supporting Information). The result suggests that UCNP@PDA5-PEG has significant PTT therapeutic efficacy on cancer treatment. We next performed a pilot chemophotothermal therapy on SW620 tumor-bearing nude mice. The mice were divided into six randomized groups including five control groups (group I to V) and one treatment group (group VI) when the sizes of tumors were reached to 3–4 mm in diameter. The mice were first treated through intravenous injection of different materials. After treatment, the tumor dimensions were tracked every 2 days with a caliper for 14 days. The nude mice were sacrificed and tumors were excised at 14th days. There is no statistically significant difference in tumor size among Group I to Group III (as shown in Figure 7 and Figure S20, Supporting Information), indicating that the administration of UCNP@ PDA5-PEG only or 808 nm NIR laser irradiation alone cannot



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Information). And there are no significant evidences of tissue damage and adverse effect of the UCNP@PDA5-PEG to various organs as shown in the Figure 8. For hematology analysis and blood biochemical assays, there is nearly no difference between treated group and control group after one month post-injection of UCNP@PDA5-PEG (as shown in Table S1, Supporting Information), which further confirmed the low toxicity of UCNP@PDA5-PEG.

3. Conclusion

Figure 7. a) Digital photographs of the tumors collected from different groups of mice at the end of intravenous treatments (day 14), and b) corresponding tumor growth curves of different groups of mice after intravenous treatments. Error bars mean standard deviations (n = 5, *p < 0.05, by ANOVA with Tukey’s post-test).

significantly regressing the growth of SW620 tumor. Although enhanced inhibitions of tumor growth are observed in Group IV and Group V, the tumors cannot be eliminated completely. Complete eradication of tumor without regrowth is noted in the treatment group (Group VI) over a course of 14 d. These results demonstrate that the UCNP@PDA5-PEG-DOX can provide remarkable chemophotothermal synergistic effect for cancer therapy. Furthermore, the treatment was also administered by intratumor injection to the SW620 tumor-bearing nude mice. Compared with the control groups, the chemophotothermal treatment resulted in complete tumor destruction without recurrence (as shown in Figure S21 and S22, Supporting Information). The synergistic antitumor effect achieved here may be attributed to the several reasons including local hyperthermia effect on cancer cells, photothermal enhancing drug delivery into cancer cells, and NIR-triggered release of DOX from cellular internalized UCNP@PDA5-PEG-DOX.

In summary, multifunctional UCNP@PDA core@shell nanocomposites have been successfully prepared by coating bioinspired PDA shell on oleic acid capped β-NaGdF4:Yb3+,Er3+@β-NaGdF4 UCNP. Our preparing strategy has several advantages including 1) it is simple since dopamine monomers are directly polymerized on hydrophobic UCNP surface via water-in-oil microemulsion method and the thicknesses of PDA shell can be easily adjusted through varying the amount of dopamine monomers in the reaction mixture; 2) the as-prepared UCNP@PDA can be further functionalized by reacting with thiol and amino-terminated molecules through Michael addition or Schiff base reaction; 3) the UCNP core can serve as an efficient nanoprobe for UCL/MRI/CT multimodality imaging and 4) the PDA shell is employed as both high photothermal conversion agent and aromatic anticancer drug carrier, resulting in chemophotothermal synergistic therapy of tumors. Furthermore, the proposed strategy is highly simple and versatile, which can be easily extended to prepare other types of hydrophobic NP@PDA nanocomposites for biomedical applications. This research paves a straightforward route to develop NIR-absorbing organic–inorganic nanocomposite-based multifunctional theranostic agents for imaging-guided chemophotothermal synergistic therapy of cancer.

4. Experimental Section Synthesis of UCNP@PDAn-PEG (n = 3, 5, 8): The NaGdF4: Yb3+, Er3+@ NaGdF4 UCNP was synthesized according to a previously reported method with slight modifications (see Supporting Information for details).[7a,11] The as-prepared NaGdF4: Yb3+, Er3+@NaGdF4 UCNP was redispersed in cyclohexane. For synthesizing PDA-coated UCNP with

2.6. In Vivo Toxicology Investigation Finally, the long-term in vivo toxicity of UCNP@PDA5-PEG was investigated by histochemical analysis, hematology analysis, and blood biochemical assays. Neither death nor significant body weight drop is noted in all test groups (as shown Figure S23, Supporting

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Figure 8. Histological changes of a) healthy mouse without injection of UCNP@PDA5-PEG and b) the mouse after 30 d post-injection of a single dose of UCNP@PDA5-PEG (Gd content: 5 mg kg−1) in NaCl solution (0.9 wt%), respectively.





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5 nm PDA shell (named as UCNP@PDA5), Igepal CO-520 (0.65 mL) was added in cyclohexane (10 mL) containing oleic-acid-stabilized NaGdF4: Yb3+, Er3+ @NaGdF4 UCNP (10 mg). After stirred for 20 min, ammonium hydroxide (75 µL, 28 wt% in water) was added into mixture followed by ultrasonic treatment for 15 min. After stirred for another 30 min, dopamine hydrochloride aqueous solution (50 µL, 25 wt%) was injected into the above reaction mixture at a rate of 3 µL min−1. After stirred for 24 h, the NPs were precipitated by adding ethanol, collected by centrifugation (10 000 rpm for 10 min) and washed with ethanol and water (10 mL, three times). Finally, the UCNP@PDA5 were redispersed in water and dried by vacuum evaporation. The UCNP with 3 nm PDA shell (named as UCNP@PDA3) or UCNP with 8 nm PDA shell (named as UCNP@PDA8) were synthesized by the same synthesis procedure of UCNP@PDA5 except dopamine hydrochloride solution (25 µL or 100 µL) was used, respectively. Finally, the as-prepared UCNP@PDAn (100 mg) was reacted with mPEG-NH2 (300 mg) in TB (Tris buffer, 50 mL, 10 × 10−3M, pH 8.5) under vigorous stirring for 12 h. Then, PEGylated UCNP@PDAn (named as UCNP@PDAn-PEG) were purified by centrifugation (10 000 rpm, 50 mL H2O, three times), and redispersed in water. CT and MR Imaging of Phantom: UCNP@PDA5-PEG aqueous solutions with various concentrations were prepared in 1.5 mL centrifuge tubes, respectively. CT or T1-weighted MR images were obtained using clinical 64-detector row CT unit or GE Signa 1.5-T MR unit (General Electric, Milwaukee, WI, see Supporting Information for detailed imaging parameters), respectively. In the CT and MR measurement, blank PBS (10 × 10−3 M PB containing 137 × 10−3 M NaCl, pH 7.4) was set as control sample. Measurement of Photothermal Performance: A series of UCNP@PDA5-PEG aqueous solution with different concentrations (Gd content: 0, 12.5, 25, 50, and 100 ppm) was irradiated with an 808 nm NIR laser at a power density of 1.3 W cm−2 for different time periods, respectively. The solution temperature was monitored by a thermometer with a thermocouple probe. Loading DOX on UCNP@PDA5-PEG: UCNP@PDA5-PEG (7 mg) were dispersed in 10× TB (1.089 mL), and mixed with DOX hydrochloride aqueous solution (9.8 mL, 1 mg mL−1). After vigorous stirring for 24 h in the dark, the nanocomposites were purified by centrifugation, washed with H2O (10 mL, three times), redispersed in PBS (10 mL, pH 7.4), respectively. The final product was named as UCNP@PDA5PEG-DOX. The loading weight of DOX was calculated as following: W = Woriginal DOX −WDOX in supernatant. The amount of DOX was analyzed by using calibration curve of DOX at the wavelength of 480 nm. The gadolinium contents of UCNP@PDAn-PEG and UCNP@PDA5-PEG-DOX were determined by inductively coupled plasma mass spectrometry (ICP-MS). DOX Releasing Study: UCNP@PDA5-PEG-DOX was sealed in a dialysis bag (molecular weight cutoff = 8000) and submerged into PBS solution (3 mL) at pH 5.0 or 7.4 at 37 °C with gentle shaking, respectively. At determined time points, the DOX containing dialysis buffer was collected and replaced with an equal volume of fresh PBS. For 808 nm NIR laser-triggered DOX release, UCNP@PDA5-PEG-DOX was sealed in a dialysis bag (molecular weight cutoff = 8000) and immersed in PBS solution (3 mL) at different pH values (5.0 and 7.4) at 37 °C with gentle shaking, respectively. At predetermined time intervals, the samples were irradiated with an 808 nm NIR laser (1.3 W cm−2) for 6 min, respectively. Dialysis buffer (0.15 mL) was collected for detection releasing amount of DOX before and after 808 nm NIR laser stimulation, respectively. Leakage Evaluation of Gd3+ Ions from UCNP@PDA5-PEG: UCNP@ PDA5-PEG (5 mL, Gd content: 5 mg mL−1) was sealed in a dialysis bag (molecular weight cutoff = 8000), submerged into NaCl solution (30 mL, 0.9 wt%) with 10% fetal bovine serum or PBS (30 mL, pH 7.4) with 10% fetal bovine serum under gentle shaking for one month. Then, the released Gd element was quantified by ICP-MS. Cell Uptake and Cell Viability Assay: The SW620 human colon cancer cells were obtained from Shanghai Cell Bank, Chinese Academy of Sciences. The cells were incubated in fresh Leibovitz's L-15 culture medium (L-15, 2 mL) containing UCNP@PDA5-PEG-DOX (Gd content: 50 ppm) for a specific time (i.e., 0.5, 2, 4, 6, and 12 h), respectively.

Finally, the UCNP@PDA5-PEG-DOX stained cells were washed with PBS (pH 7.4, three times), and directly subjected to fluorescence imaging with two excitation wavelength (λex = 980 nm for UCNP and λex = 488 nm for DOX), respectively. To quantitatively evaluate the chemophotothermal cytotoxicity of UCNP@PDA5-PEG-DOX, SW620 cells were incubated with desired amounts of UCNP@PDA5-PEG and UCNP@PDA5-PEG-DOX, respectively. Then, the UCNP@PDA5-PEG and UCNP@PDA5-PEG-DOX-stained cells were irradiated by an 808 nm NIR laser (1.3 W cm−2) for 6 min, washed with fresh L-15 (100 µL, three times) and cultured with fresh L-15 (100 µL) for another 24 h, respectively. Finally, the viabilities of SW620 cells were evaluated by an MTT assay. The relative cell viabilities (%) were calculated by using the optical densities with respect to the control value. The untreated SW620 cells were used as control sample. The cell viabilities (%) of the UCNP@PDA5-PEG and UCNP@PDA5-PEG-DOX stained cells were also measured by MTT assay as previously described except 808 nm NIR laser irradiation. In Vivo Multimodality Imaging and Chemophotothermal Therapy: Nude mice with average weight of 20 g were purchased from Vital River Company (Beijing, China). Animal procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments established by Jilin University Institutional Animal Care and Use. SW620 cells (5 × 106) suspended in serum-free L-15 (100 µL) were inoculated subcutaneously in female nude mice. For in vivo UCL imaging, nude mice were first anesthetized by chloral hydrate (10 wt%). And then NaCl solutions (50 µL, 0.9 wt%) containing UCNP@PDA5-PEG (0.6 mg Gd mL−1) was intratumorally injected into the nude mice. After injection, excited with a 980 nm CW NIR laser in a darkroom, the UCL imaging was observed and recorded by a CCD-based digital camera with a suitable filter. For in vivo CT and MRI imaging, nude mice were anesthetized using chloral hydrate (10 wt%). NaCl solutions (200 µL, 0.9 wt%) containing desired amounts (1.5 mg Gd mL−1 for MRI experiments and 15 mg Gd mL−1 for CT experiments) of UCNP@PDA5-PEG were injected intravenously into the mouse through the tail vein, respectively. In vivo dual-modal imaging was performed at desired time points after injection. CT images were acquired as previously described except 129 mm field of view was used. T1-weighted MR images were acquired using a GE Signa 1.5-T (General Electric, Milwaukee, WI) unit. Imaging parameters were as follows: TR, 240 ms; TE, 15.9 ms; field of view, 120 mm × 72 mm and slice thickness, 2.0 mm, respectively. For chemophotothermal therapy, when the tumor size reached to 3–4 mm in diameter, the nude mice were divided into six randomized groups, which were PBS treated (Group I), 808 nm NIR laser treated (Group II), UCNP@PDA5-PEG treated (Group III), UCNP@PDA5-PEG-DOX treated (Group IV), UCNP@PDA5-PEG with 808 nm NIR laser treated (Group V), and UCNP@PDA5-PEG-DOX with 808 nm NIR laser treated (Group VI), respectively. Then, for intratumor injection model, the nude mice were intratumorally injected with PBS, UCNP@PDA5-PEG, and UCNP@PDA5-PEG-DOX aqueous dispersion (150 µL, Gd content: 50 ppm). After injection, the tumors were irradiated with or without an 808 nm laser at 1.3 W cm−2 for 6 min. The tumor dimensions were tracked with a caliper for 14 d. The tumor volume was calculated according to the formula: Tumor volume = (tumor length) × (tumor width)2/2. The relative tumor volumes were calculated as V/V0 (V0 was the tumor volume when the treatment was initiated). For intravenous injection model, all the procedures were as same as previous intratumor injection model except UCNP@PDA5-PEG and UCNP@PDA5-PEG-DOX (Gd content: 500 ppm) were used, respectively. Toxicology Analysis: Hemolysis assay and in vivo toxicity study were employed to evaluate the safety of UCNP@PDA5-PEG (see Supporting Information for details). And in vivo biodistribution of UCNP@PDA5-PEG was quantified by ICP-MS (see Supporting Information for details).

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.



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