Activatable Multifunctional Persistent Luminescence Nanoparticle

Nov 9, 2016 - Research Center for Analytical Sciences, Tianjin Key Laboratory of Molecular Recognition and Biosensing, College of Chemistry, State Key...
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Activatable Multifunctional Persistent Luminescence Nanoparticle/ Copper Sulfide Nanoprobe for in Vivo Luminescence ImagingGuided Photothermal Therapy Li-Jian Chen,† Shao-Kai Sun,§ Yong Wang,∥ Cheng-Xiong Yang,† Shu-Qi Wu,† and Xiu-Ping Yan*,†,‡ †

Research Center for Analytical Sciences, Tianjin Key Laboratory of Molecular Recognition and Biosensing, College of Chemistry, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China ‡ Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300071, China § School of Medical Imaging, Tianjin Medical University, Tianjin 300203, China ∥ School for Radiological and Interdisciplinary Sciences (RAD-X), Center for Molecular Imaging and Nuclear Medicine, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China S Supporting Information *

ABSTRACT: Multifunctional nanoprobes that provide diagnosis and treatment features have attracted great interest in precision medicine. Near-infrared (NIR) persistent luminescence nanoparticles (PLNPs) are optimal materials due to no in situ excitation needed, deep tissue penetration, and high signal-to-noise ratio, while activatable optical probes can further enhance signal-to-noise ratio for the signal turn-on nature. Here, we show the design of an activatable multifunctional PLNP/copper sulfide (CuS)-based nanoprobe for luminescence imagingguided photothermal therapy in vivo. Matrix metalloproteinases (MMPs)-specific peptide substrate (H2N−GPLGVRGC−SH) was used to connect PLNP and CuS to build a MMP activatable system. The nanoprobe not only possesses ultralow-background for in vivo luminescence imaging due to the absence of autofluorescence and optical activatable nature but also offers effective photothermal therapy from CuS nanoparticles. Further bioconjugation of c(RGDyK) enables the nanoprobe for cancer-targeted luminescence imaging-guided photothermal therapy. The good biocompatibility and the multiple functions of highly sensitive tumor-targeting luminescence imaging and effective photothermal therapy make the nanoprobe promising for theranostic application. KEYWORDS: multifunctional nanoprobe, activatable imaging, near-infrared persistent luminescence nanoparticles, CuS nanoparticles, photothermal therapy

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INTRODUCTION

enzymes are significant targets for cancer imaging as they can hydrolyze some specific peptide substrates.5,11 Recently, several kinds of nanoparticles have been reported as optical imaging materials, including metal nanoclusters,12 semiconductor quantum dots,13 and upconversion nanoparticles14 with their own specific advantages for fluorescence imaging. However, metal nanoclusters and quantum dots often suffer from high background signal due to the autofluorescence in irradiated tissues. Though upconversion nanoparticles exhibit little autofluorescence background, they are limited in applications for low quantum yield and still requiring of continuous light excitation.15 Persistent luminescence nanoparticles (PLNPs) can store excitation energy and subsequently release persistent luminescence slowly, which lasts for hours or even days after excitation stops.16 Near-infrared (NIR)-emitting PLNPs have become a newly emerging material for in vivo optical imaging

Multifunctional nanoprobes with theranostic features have drawn great attention in cancer diagnosis and therapy.1−3 Accurate cancer detection and targeted therapy are the goals of multifunctional theranostic probes. Activatable fluorescent probes are attractive due to highly sensitive turn-on imaging signals originating from specific recognition or interaction.4,5 Activatable probes keep an “off” state before reaching the target, improving the resolution and target-to-background ratio. The quenching process of these probes occurs when acceptor and donor are in a close range, inducing physical energy transfer processes, such as Förster resonance energy transfer (FRET).6,7 These probes can be activated when specific reaction or stimuli occurs, producing a restored optical signal. Protease-activatable bioimaging probes are the most noteworthy activatable probes in cancer imaging.8,9 Matrix metalloproteinases (MMPs), a class of zinc-dependent endopeptidases, possess vital functions in tumor metastasis, invasiveness, and angiogenesis.10 The majority of MMPs are overexpressed in the focus of infection, such as the area of cancer. These © 2016 American Chemical Society

Received: August 25, 2016 Accepted: November 9, 2016 Published: November 9, 2016 32667

DOI: 10.1021/acsami.6b10702 ACS Appl. Mater. Interfaces 2016, 8, 32667−32674

Research Article

ACS Applied Materials & Interfaces with merits of no in situ excitation, deep tissue penetration, and high signal-to-noise ratio.17−19 More importantly, Cr3+-doped PLNPs can emit renewable persistent luminescence under a tissue-penetrating LED light, which means the PLNP bioimaging is not limited by the luminescence decay lifetime anymore.20 Up to now, PLNP-based multifunctional nanoprobes dedicated to multimodal imaging or theranostic applications have been reported.21−26 Multimodal imaging nanoprobes combining optical imaging with magnetic resonance imaging (MRI) were developed, such as gadolinium complexes functionalized PLNPs,21 superparamagnetic persistent luminescence nanohybrids,22 and gadolinium-doped persistent nanophosphors.23 A Zn2.94Ga1.96Ge2O10:Cr3+,Pr3+@ TaOx@SiO2 core/shell nanostructure was fabricated for computed tomography and luminescence imaging.24 In addition, novel core/shell structures using mesoporous silica shell as drug carrier and integrating persistent luminescence and magnetism were reported.25,26 Therefore, PLNPs are promising for developing multifunctional nanoplatforms in various applications. As a minimally invasive treatment, photothermal therapy has received great attention for high selectivity to kill tumorous cells.27,28 This therapeutic approach converts optical energy to heat with the aid of light-absorbing agents to kill cells by hyperthermia. Photothermal therapy is an attractive choice due to its fewer side effects and easier manipulation than chemotherapy and radiotherapy.29 Though PLNPs and photothermal therapy show remarkable merits, to our best knowledge, multifunctional probes integrating PLNPs and photothermal therapy for theranostic application have rarely been reported to date. Herein, we show the design of an activatable multifunctional PLNP/copper sulfide (CuS)-based nanoprobe for in vivo luminescence imaging-guided photothermal therapy. The PLNP Zn1.1Ga1.8Ge0.1O4:Cr3+ with renewable persistent luminescence under red LED illumination is used as the NIR luminescence source.20 Meanwhile, CuS nanoparticles are employed as both the photothermal agent and the quencher for the high photothermal conversion efficiency and strong NIR absorption.30,31 The MMP-specific peptide substrate (H2N− GPLGVRGC−SH) serves as a bridge between the PLNP and CuS nanoparticles to build a MMP activatable optical probe. Further bioconjugation of succinimidyl carbonate−poly(ethylene glycol)thiol (SC−PEG−SH) and peptide c[ArgGly-Asp-(D-Tyr)-Lys] (c(RGDyK)) renders the probe good biocompatibility and tumor-targeting ability. The prepared nanoprobe (PLNP−CuS−RGD) offers multiple functions of highly sensitive tumor-targeting optical imaging and effective photothermal therapy and shows great potential for theranostic application.

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Scheme 1. (a) Schematic for the Design, Synthesis, and Activation of PLNP−CuS−RGD. (b) Illustration for the Utilization of PLNP−CuS−RGD as the Activatable Nanoprobe for in Vivo Imaging-Guided Photothermal Therapy

and Sigma-Aldrich Co. (Shanghai, China), respectively. Ultrapure water was obtained from Wahaha Group Co. (Hangzhou, China). Characterization. Transmission electron microscopy (TEM) images and energy dispersive spectra (EDS) were acquired on a Tecnai G2 F20 field emission transmission electron microscope (FEI, U.S.A.) at 220 kV accelerating voltage. X-ray diffraction (XRD) experiments were performed on a D/max-2500 diffractometer with Cu Kα radiation (Rigaku, Japan). Luminescence spectra were acquired on an F-4500 spectrofluorometer (Hitachi, Japan). Thermogravimetric analysis (TGA) was carried out on a PTC-10A TG-DTA thermoanalyzer (Rigaku, Japan). Zeta potential and size distribution were recorded on a Nano-ZS Zetasizer (Malvern, U.K.). Absorption spectra were obtained on a UV-3600 spectrophotometer (Shimadzu, Japan). Fourier transform infrared (FT-IR) spectra were obtained on a Nicolet 6700 spectrometer (Thermo Fisher Scientific, U.S.A.). Cell imaging experiments were carried out on a TCS SP8 confocal laser scanning microscope (Leica, Germany). NIR persistence photographs were recorded on a Berthold NightOWL LB 983 Imaging System (Bad Wildbad, Germany). The concentration of Cu and Zn was obtained on an X7 inductively coupled plasma mass spectrometer (Thermo Electron, U.S.A.). Preparation and Functionalization of PLNP. The PLNP (Zn1.1Ga1.8Ge0.1O4:Cr3+) was synthesized according to the previous publications with minor modification.19,32 The aqueous solutions of Zn(II), Ga(III), Ge(IV), and Cr(III) were mixed under vigorous stirring for 3 h after the pH was adjusted to 7.5 with ammonia hydroxide. The mixture was hydrothermally reacted at 160 °C for 24 h and then centrifuged. The obtained solid was sintered in air at 800 °C for 3 h and wet ground in 5 mM NaOH solution. The resulting hydroxylfunctionalized PLNP (PLNP−OH) was collected by 5 min centrifugation at 3500 rpm.

EXPERIMENTAL SECTION

Materials. Cr(NO3)3·9H2O (99.99%), Zn(NO3)2·6H2O (99.99%), Ga2O3 (99.99%), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC·HCl), 1,10-phenanthroline monohydrate (99%), N-hydroxysuccinimide (NHS), and (3-aminopropyl)triethoxysilane (APTES, 99%) were acquired from Aladdin (Shanghai, China). GeO2 was bought from Sinopharm Chemical Reagent Beijing Co. (China). Diglycolic anhydride (DGA, 97%) and p-aminophenol mercuric acid were obtained from J&K Scientific Ltd. (Beijing, China). SC−PEG−SH (MW 5000) was obtained from Seebio Biotech Inc. (Shanghai, China). H2N−GPLGVRGC−SH peptide (MW 757.91) and c(RGDyK) (MW 619.70) were obtained from China Peptides Co. (Shanghai, China). Dimethylformamide (DMF) and matrix metalloproteinase-2 (MMP-2) were obtained from Tianjin Concord Technology Co. (Tianjin, China) 32668

DOI: 10.1021/acsami.6b10702 ACS Appl. Mater. Interfaces 2016, 8, 32667−32674

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ACS Applied Materials & Interfaces

Figure 1. (a) TEM and high-resolution TEM images of PLNP-COOH. (b) TEM and high-resolution TEM images of CuS nanoparticles. (c) TEM image of PLNP−CuS−RGD. (d) TGA analysis of PLNP−COOH and PLNP−SH. (e) Size distribution analysis of PLNP−COOH, PLNP−SH, and PLNP−CuS−RGD.

Figure 2. (a) Luminescence emission spectra of aqueous solution of PLNP−COOH, PLNP−SH, and PLNP−CuS−RGD (1 mg mL−1 as PLNP), and the absorption spectra of 0.4 mg mL−1 CuS nanoparticle solution. (b) NIR persistent luminescence decay curves of PLNP−COOH and PLNP− SH (1 mg mL−1). (c) DLS measurement of PLNP−CuS−RGD, showing the stability in PBS. (d) DLS measurement of PLNP−CuS−RGD, showing the stability in serum. peptide were used in the above functionalization steps to maximize the product yield. Preparation of PLNP−CuS−RGD. The citrate-coated CuS nanoparticles were synthesized according to Zhou et al.30 To prepare PLNP−CuS−RGD (1 mg mL−1 as PLNP), PLNP−SH (3 mg) was dispersed in PBS (1 mL, 10 mM, pH 7.4) and mixed with the as-prepared CuS nanoparticle dispersion (9 mL, 1.2 mg mL−1) in the dark under 3 h stirring. The resulting composite was centrifuged and washed with PBS. Then, the precipitate was redispersed in PBS (2 mL) to obtain PLNP−CuS dispersion. Moreover, c(RGDyK) (2 mg) and SC-PEG-SH (12 mg) in PBS (5 mL) were reacted under stirring for 6 h. The preprepared SH−PEG−RGD was added into the PLNP−CuS dispersion. After stirring in the dark for another 12 h, the

The carboxyl-functionalized PLNP (PLNP−COOH) and aminefunctionalized PLNP (PLNP−NH2) were obtained as in the previous reports.22 Acarbodiimide method was used to conjugate the peptide onto PLNP. Briefly, PLNP−COOH (10 mg) was dispersed into phosphate buffer saline (PBS) (5 mL, 10 mM, pH 6.0). NHS (30 mg) and EDC (12 mg) were quickly added to the above suspension. The mixture was stirred for 2 h and then centrifuged for 5 min at 6000 rpm. The precipitant was redispersed in PBS (4.5 mL, 10 mM, pH 7.4). Then, H2N−GPLGVRGC−SH peptide (10 mg) in PBS (0.5 mL) was added for another 5 h reaction under stirring at room temperature. The resulting H2N−GPLGVRGC−SH peptide-modified PLNP (PLNP− SH) was collected, washed with PBS, freeze-dried, and kept at 4 °C for further use. Excessive APTES, DGA, and H2N−GPLGVRGC−SH 32669

DOI: 10.1021/acsami.6b10702 ACS Appl. Mater. Interfaces 2016, 8, 32667−32674

Research Article

ACS Applied Materials & Interfaces RGD-modified PLNP−CuS−RGD was obtained. The final functionalization step was carried out with excessive PEG and RGD under vigorous stirring overnight to maximize product yield. The final PLNP−CuS−RGD product was collected, washed with PBS, and redispersed in PBS (3 mL). MMP-2 Enzymatic Activation of PLNP−CuS−RGD. The NIR luminescence recovery of PLNP−CuS−RGD was examined at various concentrations of MMP-2 enzyme. MMP-2 was activated by incubation with p-aminophenol mercuric acid (2.5 mM) in the TCNB buffer (0.05% Brij 35, 100 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.8) at 37 °C for 2 h. SB-3CT (MMP-2/9 inhibitor, 1 μM) was added as control. The same amount of PLNP−CuS−RGD (0.5 mg mL−1 as PLNP) was mixed with the above solution and incubated at 37 °C for 2 h. The phosphorescence spectra were monitored on F-4500 fluorescence spectrophotometer under excitation at 254 nm. Incubation of PLNP−CuS−RGD with Cells. SCC-7 cells and 293T cells were incubated in DMEM and RPMI 1640 medium with 10% FBS and 1% penicillin−streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C, respectively. Cells were seeded in 24-well plates to adhere for 24 h. The cell culture supernatants were mixed with PLNP−CuS−RGD at 37 °C for 2 h. MMP inhibitor (SB-3CT, 1 μM) was added as negative control. In Vitro Cytotoxicity Assay. To test cytotoxicity or photothermal cytotoxicity, SCC-7 and 293T cells were seeded in the 96-well plates (1 × 104 cells/well) and incubated for 24 h. Further incubation of the cells was then performed with different concentrations of PLNP− CuS−RGD probe (calculated as PLNP) for another 24 h with or without 10 min irradiation (808 nm, 1 W cm−2). The cell viability was obtained by MTT assay. In Vivo Luminescence Imaging of SCC-7 Tumor. Animal experiments were done following the instructions of the Tianjin Committee of Use and Care of Laboratory Animals. SCC-7 cells (5 × 106 cells/mouse) were subcutaneously injected into the thigh of athymic nude mice (Balb/c, 5−6 weeks old, female) to produce tumors 5 mm in size for optical imaging. Intravenous injection of PLNP−CuS−RGD (200 μL, 4 mg mL−1 as PLNP) into anesthetized tumor-bearing mice was performed after 254 nm UV light excitation for 10 min. Excitation with a 650 nm LED light (5000 lm) was then applied on the mice for 2 min before acquiring luminescence images. In the MMP-2 inhibition experiment, SB-3CT (10 μM, 75 μL) as inhibitor was injected into tumor 30 min before injection. In Vivo Photothermal Therapy. Four groups of balb/c mice bearing a SCC-7 tumor (tumor diameter ∼5−7 mm) were obtained randomly (n = 3). Two hundred microliters of PLNP−CuS−RGD (4 mg mL−1 as PLNP) were injected into the mice of groups A and C intravenously. After 2 h, 15 min irradiation with an 808 nm NIR laser at 1.5 W cm−2 was applied to the mice tumors of groups A and B. The thermographic pictures were taken using a FLIR E50 thermal camera. A digital caliper was used to measure the tumor length and width every 2 days for 14 days after different treatments. The tumor volume was defined as length × width2/2. Histological Staining. The major organs were dissected from above four mice groups after 15 days, fixed in a 4% formaldehyde solution at room temperature for 24 h. Finally, the sections of 10 μm thickness were made in a cryostat for H&E staining.

Figure 3. (a) Activation of PLNP−CuS−RGD at different concentrations of MMP-2 with or without inhibitor. Inset: Plot of the luminescence intensity of the probe against the concentration of MMP-2 in the absence of inhibitor. (b) Change of size distribution with different concentrations of MMP-2. (c) NIR persistent luminescence and bright images in 24-well plate of PLNP−CuS−RGD (0.3 mg mL−1 of PLNP) incubated with SCC-7 cell supernatants with or without inhibitor (2 h, 37 °C). Fresh cell medium was used as control.

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RESULTS AND DISCUSSION The design, preparation, and activation of the activatable multifunctional PLNP−CuS−RGD nanoprobe are illustrated in Scheme 1a. To achieve autofluorescence-free bioimaging, we prepared the Zn1.1Ga1.8Ge0.1O4:Cr3+ PLNPs as the NIR luminescence source because of its superlong persistent luminescence and red light renewability.19,32 A luminescence activatable platform was designed to further improve the sensitivity and the signal-to-noise ratio. The activatable platform was built from the PLNP and CuS nanoparticles. Here, CuS nanoparticles were used as both the photothermal agent and the quencher for

Figure 4. Luminescence images of 293T and SCC-7 cells after incubation with PLNP−CuS−RGD on a confocal laser scanning microscope. The red color resulted from the activated PLNP, showing its intracellular location, while the blue color came from DAPI for cellular nuclei staining. Scale bar = 50 μm.

strong NIR absorption and high photothermal conversion efficiency.30 To make the persistent luminescence of the nanoprobe be activated with overexpressed MMP in tumor, we utilized MMP-specific peptide substrate (H2N−GPLGVRGC−SH) as 32670

DOI: 10.1021/acsami.6b10702 ACS Appl. Mater. Interfaces 2016, 8, 32667−32674

Research Article

ACS Applied Materials & Interfaces

Figure 5. (a) In vivo luminescence images of SCC-7 tumor-bearing mice after intravenous injection of PLNP−CuS−RGD with or without inhibitor (10 min illumination with a 254 nm UV light before injection, 2 min illumination with a 650 nm LED light before each acquisition). (b) Biodistribution of PLNP and CuS in SCC-7 tumor-bearing mice (n = 3) at 2 and 24 h after intravenous injection of PLNP−CuS−RGD.

displayed the hydrodynamic diameter of 78.82 nm. The conjugation of H2N−GPLGVRGC−SH peptide with PLNP−COOH and CuS nanoparticles and subsequent RGD immobilization made the average hydrodynamic diameter of the resulting PLNP−CuS−RGD increase to 141.0 nm. The prepared PLNP gave NIR emission at 695 nm from the 2 E → 4A2 transition of distorted Cr3+ ions (Figure S5a)33 and exhibited excellent NIR-persistent luminescence (Figure S5b). Moreover, the persistent luminescence signal can be repeatedly renewed with minimal loss under red LED light excitation (Figure S5c, S5d), which was mainly due to the excitation band originating from the 4A2 → 4T2 (t2e) transition (Figure S6).16 Functionalization with H2N−GPLGVRGC−SH peptide gave no remarkable influence on the luminescence property of the PLNP (Figure 2a, b). Conjugation of CuS nanoparticles to PLNP−SH resulted in significant luminescence quenching of the PLNP due to FRET effect resulting from the overlap of the luminescence emission of the PLNP and the wide absorption band of CuS nanoparticles (Figure 2a). The luminescence of PLNP−SH decreased as the amount of CuS increased and then remained unchanged when CuS was excessive (>10.8 mg) (Figure S8b). For RGD functionalization, excess RGD was added to the reaction to ensure the high targeting property. The PLNP− CuS−RGD nanoprobe prepared gave nearly 94% luminescence quenching to keep minimal background signal of the activatable probe (Figure 2a). No obvious change in the size distribution of PLNP−CuS−RGD in PBS and serum for 24 h shows the good stability of PLNP−CuS−RGD in physiological environment (Figure 2c, 2d). To activate the luminescence of PLNP−CuS−RGD, we tested the capability of MMP-2 to cleave the H2N− GPLGVRGC−SH peptide which bridged between the PLNP and CuS. The luminescence of PLNP−CuS−RGD nanoprobe increased with the concentration of MMP-2, but only slightly increased when inhibitor existed (SB-3CT, 1 μM) (Figure 3a). The change of size distribution of PLNP−CuS−RGD with the concentration of MMP-2 suggests that the PLNP−CuS−RGD probe was successfully cleaved by MMP-2 (Figure 3b). To further study the luminescence recovery of PLNP−CuS−RGD in extracellular environment, PLNP−CuS−RGD was also incubated with SCC-7 cell supernatant at 37 °C for 2 h. Significant luminescence recovery was observed due to the secreted MMP-2, whereas unapparent recovery was found when inhibitor existed. Additionally, the luminescence intensity increased with the cell

the bridge to link the PLNP and CuS nanoparticles. H2N− GPLGVRGC−SH peptide-functionalized PLNP was synthesized via a condensation reaction of the −NH2 group in the peptide and the −COOH group in the PLNP. The prepared peptide-functionalized PLNP gave terminal-SH group to conjugate the CuS nanoparticles via Cu−S bond to build an activatable nanoprobe. To improve the biocompatibility and target ability, the nanoprobe was further modified with PEG and c(RGDyK) targeting ligand. SC−PEG−SH was first covalently bonded to the −NH2 group of c(RGDyK) and then linked to CuS nanoparticles via Cu−S bond as well. As a result, an activatable multifunctional PLNP/CuS based nanoprobe was designed for in vivo luminescence imaging-guided photothermal therapy (Scheme 1b). The NIR-emitting PLNP and CuS nanoparticles were synthesized according to previous publications.19,30,32 The PLNP− COOH had a diameter of 56.8 ± 9.2 nm based on 100 randomly selected nanoparticles (Figure 1a) and a 4.8 Å uniform lattice fringe of d-spacing for (111) lattice plane of the PLNP (Figure S1a). The XRD pattern of PLNP shows the spinel phase of ZnGa2O4 (JCPDS 38-1240) and Zn2GeO4 (JCPDS 25-1018). The prepared CuS nanoparticles gave relatively uniform structure with a size of 11.7 ± 0.2 nm (Figure 1b). The d-spacing with a 3.3 Å lattice fringe for (100) lattice plane could be indexed as diffraction peak of covellite-phase CuS (JCPDS 79-2321) (Figure S1b). The PLNP−CuS−RGD showed core/satellite-like morphology (Figure 1c). To verify a success synthesis of PLNP−CuS−RGD, the element mapping was also analyzed (Figure S2). The elements Zn, Ga, and O are mainly distributed in the core, and the elements Cu and S are distributed in the whole nanostructures, suggesting the PLNP core is surrounded with CuS nanoparticles. The average number of CuS nanoparticles per PLNP was estimated to be 17.30 The functionalization of PLNP with H2N−GPLGVRGC− SH peptide was confirmed by TGA (Figure 1d). The TGA curve of PLNP−COOH shows the loss of absorbed water before 325 °C and the decomposition of organic layer on nanoparticles after 325 °C. The TGA curve of PLNP−SH shows a greater weight loss than PLNP−COOH before 227 °C due to the absorbed water and pyrolysis of amino acids, which corresponds to the sharp drop of weight in the temperature range of 187−227 °C for pure peptide (Figure S4). The weight loss percentage of H2N−GPLGVRGC−SH peptide was 1.04%. The average number of peptide per PLNP was 4859. The change of average hydrodynamic diameters further reveals the surface modification of PLNP (Figure 1e). The PLNP−COOH 32671

DOI: 10.1021/acsami.6b10702 ACS Appl. Mater. Interfaces 2016, 8, 32667−32674

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ACS Applied Materials & Interfaces

Figure 7. (a) Representative thermal images of SCC-7 tumor-bearing mice exposed to 808 nm laser (1.5 W cm−2) after 2 h of intravenous injection of PLNP−CuS−RGD. (b) SCC-7 tumor growth curves in each mice groups after different treatments. Tumor volumes were normalized to the initial size (n = 3 for each group). For therapeutic group, mice were intravenously treated with PLNP−CuS−RGD and exposed to 808 nm laser illumination (1.5 W cm−2, 15 min) at 2 h postinjection. P value: < 0.05. (c) Representative photographs of tumors collected from various mice groups after 14 day treatment.

athymic nude mice model. The PLNP−CuS−RGD was preirradiated with 254 nm UV light for 10 min and injected to the mice intravenously. Two minute LED light illumination was applied on mice before obtaining the NIR afterglow photographs each time (Figure 5a). No autofluorescence background was observed because the imaging was performed with no in situ excitation. Since large amounts of nanoprobes were accumulated in the liver, one of the major reticuloendothelial system organs, the uncompleted quenched luminescence of the probe produced observable signal in the liver at 0.5 h after injection, but drastically decreased after 2 h to offer low probe background signal in activatable optical imaging (Figure 5a). However, significant luminescence was always observed in the tumor area of SCC-7 tumor-bearing mice for at least 2 h, indicating effective MMP activation and excellent tumortargeting performance of the probe. In contrast, the control tumor-bearing mice group with inhibitor gave dramatically reduced signal intensity in the tumor. The above results indicate that the MMP in tumor area specifically cleaved the linked peptide to activate the luminescence of the PLNP, leading to sensitive optical imaging with high specificity and signal-tonoise ratio. Furthermore, the element Zn (from PLNP) and Cu (from CuS) concentrations within the organs were analyzed to investigate the PLNP−CuS−RGD distribution in mice. Figure 5b confirms that the uptake of PLNP−CuS−RGD by SCC-7 tumor increased with time. The photothermal property of the PLNP−CuS−RGD probe with different concentrations was investigated under irradiation of 808 nm laser (2 W cm−2) to evaluate its potential for

Figure 6. (a) Real-time thermal images and (b) temperature-varying curves of PLNP−CuS−RGD at various concentrations under laser irradiation with 2 W cm−2. (c) Cell viability of 293T and SCC-7 cells against PLNP−CuS−RGD probe (in the presence or absence of 808 nm laser irradiation with 1 W cm−2 for 10 min). P values: * < 0.05, ** < 0.01.

density (Figure S9). The above results indicate that PLNP− CuS−RGD was successfully activated with MMP-2 in vitro. To investigate the application of the prepared activatable PLNP−CuS−RGD nanoprobe for imaging cancer cells, MMP2 enzyme positive (SCC-7) and negative (293T) cell lines were incubated with PLNP−CuS−RGD. Obvious luminescence signal in SCC-7 cell was observed (Figure 4). However, no luminescence signal was found in 293T cells. Meanwhile, low luminescence signal was found in the inhibitor pretreated SCC-7 cells. These results suggest that the activated PLNP successfully internalized into the SCC-7 cells and offered the ability for imaging cancer cells. The inspiring results of the in vitro enzymatic activation and cell luminescence imaging encouraged us to evaluate the in vivo activation of PLNP−CuS−RGD with SCC-7 tumor-bearing 32672

DOI: 10.1021/acsami.6b10702 ACS Appl. Mater. Interfaces 2016, 8, 32667−32674

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ACS Applied Materials & Interfaces ORCID

photothermal therapy (Figure 6a). The temperature of the probe (2 mg mL−1 of PLNP) under laser irradiation incremented from 25 to 44 °C in 8 min (Figure 6b). Furthermore, the photothermal effect was primarily demonstrated via the MTT assay. The cell viabilities of SCC-7 and 293T cells were all over 80% when no laser light irradiated (Figure 6c), suggesting low cytotoxicity of PLNP−CuS−RGD. In contrast, the cell viability decreased regardless of cell line imparity along with the increase of PLNP−CuS−RGD concentration when 10 min irradiation with 808 nm laser was applied to the cells. The results show that PLNP−CuS−RGD offers good photothermal therapeutic effect. SCC-7 tumor-bearing mice were used to demonstrate the performance of PLNP−CuS−RGD for photothermal therapy in vivo. For this purpose, the mice contained one experimental group (mice treated with PLNP−CuS−RGD with laser irradiation) and three control groups (mice treated with laser irradiation alone, mice treated with PLNP−CuS−RGD alone, and mice without any treatment). The tumors of the experimental group were irradiated under 808 nm laser at 2 h postinjection; the temperature rapidly increased and approached 60 °C in the tumor area (Figure 7a). Tumor volumes were monitored every other day during the subsequent 14 days. The tumor volumes had hardly changed compared with primary volumes (Figure 7b). However, the control groups with treatment of nanoprobe alone, with laser irradiation alone, and with no treatment showed 25-fold, 25-fold, and 37-fold increase in tumor volume, respectively, compared with the original volume (Figure 7b). This result indicates the effective photothermal therapeutic effect of PLNP−CuS−RGD. To further demonstrate the low system toxicity in photothermal therapy, H&E staining was performed (Figure S10). No remarkable abnormality was found in the major organs, such as spleen, liver, lung, heart, or kidney. This result shows that PLNP−CuS− RGD caused minimal lesion to organs and had no side effect in SCC-7 tumor-bearing mice.

Xiu-Ping Yan: 0000-0001-9953-7681 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly appreciate the financially support from the National Natural Science Foundation of China (Nos 21435001, 21275079).

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(1) Bardhan, R.; Lal, S.; Joshi, A.; Halas, N. J. Theranostic Nanoshells: From Probe Design to Imaging and Treatment of Cancer. Acc. Chem. Res. 2011, 44, 936−946. (2) Lee, D. E.; Koo, H.; Sun, I. C.; Ryu, J. H.; Kim, K.; Kwon, I. C. Multifunctional Nanoparticles for Multimodal Imaging and Theranosis. Chem. Soc. Rev. 2012, 41, 2656−2672. (3) Kim, J.; Piao, Y.; Hyeon, T. Multifunctional Nanostructured Materials for Multimodal Imaging, and Simultaneous Imaging and Therapy. Chem. Soc. Rev. 2009, 38, 372−390. (4) Jiang, T.; Olson, E. S.; Nguyen, Q. T.; Roy, M.; Jennings, P. A.; Tsien, R. Y. Tumor Imaging by Means of Proteolytic Activation of Cell-Penetrating Peptides. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 17867−17872. (5) Lee, S.; Cha, E.-J.; Park, K.; Lee, S.-Y.; Hong, J.-K.; Sun, I.-C.; Kim, S. Y.; Choi, K.; Kwon, I. C.; Kim, K.; Ahn, C.-H. A Near-InfraredFluorescence-Quenched Gold-Nanoparticle Imaging Probe for in Vivo Drug Screening and Protease Activity Determination. Angew. Chem., Int. Ed. 2008, 47, 2804−2807. (6) Jares-Erijman, E. A.; Jovin, T. M. FRET Imaging. Nat. Biotechnol. 2003, 21, 1387−1395. (7) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. Self-Assembled Nanoscale Biosensors Based on Quantum Dot FRET Donors. Nat. Mater. 2003, 2, 630−638. (8) Wang, S.; Huang, P.; Chen, X. Stimuli-Responsive Programmed Specific Targeting in Nanomedicine. ACS Nano 2016, 10, 2991−2994. (9) Mo, R.; Gu, Z. Tumor Microenvironment and Intracellular Signal-Activated Nanomaterials for Anticancer Drug Delivery. Mater. Today 2016, 19, 274−283. (10) Egeblad, M.; Werb, Z. New Functions for the Matrix Metalloproteinases in Cancer Progression. Nat. Rev. Cancer 2002, 2, 161−174. (11) Lin, X.; Xie, J.; Zhu, L.; Lee, S.; Niu, G.; Ma, Y.; Kim, K.; Chen, X. Hybrid Ferritin Nanoparticles as Activatable Probes for Tumor Imaging. Angew. Chem., Int. Ed. 2011, 50, 1569−1572. (12) Zhang, L.; Wang, E. Metal Nanoclusters: New Fluorescent Probes for Sensors and Bioimaging. Nano Today 2014, 9, 132−157. (13) Wegner, K. D.; Hildebrandt, N. Quantum Dots: Bright and Versatile in Vitro and in Vivo Fluorescence Imaging Biosensors. Chem. Soc. Rev. 2015, 44, 4792−4834. (14) Sun, L. D.; Wang, Y. F.; Yan, C. H. Paradigms and Challenges for Bioapplication of Rare Earth Upconversion Luminescent Nanoparticles: Small Size and Tunable Emission/Excitation Spectra. Acc. Chem. Res. 2014, 47, 1001−1009. (15) Zhong, Y.; Tian, G.; Gu, Z.; Yang, Y.; Gu, L.; Zhao, Y.; Ma, Y.; Yao, J. Elimination of Photon Quenching by A Transition Layer to Fabricate A Quenching-Shield Sandwich Structure for 800 nm Excited Upconversion Luminescence of Nd3+-Sensitized Nanoparticles. Adv. Mater. 2014, 26, 2831−2837. (16) Pan, Z.; Lu, Y.-Y.; Liu, F. Sunlight-Activated Long-Persistent Luminescence in the Near-Infrared from Cr3+-Doped Zinc Gallogermanates. Nat. Mater. 2012, 11, 58−63. (17) le Masne de Chermont, Q.; Chaneac, C.; Seguin, J.; Pelle, F.; Maitrejean, S.; Jolivet, J. P.; Gourier, D.; Bessodes, M.; Scherman, D. Nanoprobes with Near-Infrared Persistent Luminescence for in Vivo Imaging. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 9266−9271. (18) Abdukayum, A.; Chen, J. T.; Zhao, Q.; Yan, X. P. Functional Near Infrared-Emitting Cr3+/Pr3+ Co-Doped Zinc Gallogermanate

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CONCLUSIONS We have shown the design, characterization, and application of an activatable multifunctional PLNP/CuS-based nanoprobe for effective persistent luminescence imaging-guided photothermal therapy. The synergistic effect of the renewable long persistent luminescence, the MMP activatable nature, the tumor-targeting ability, and the photothermal therapeutic effect makes the PLNP−CuS−RGD probe promising for luminescence imagingguided photothermal therapy with high sensitivity and excellent specificity. Our results demonstrate that the strategy for designing activatable multifunctional nanoplatforms has great potential for theranostics.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b10702. Supplementary method, figures, and additional characterizations of PLNP, CuS nanoparticles, and PLNP−CuS− RGD probe, such as XRD patterns, optical properties, FT-IR spectra, and zeta potential (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Fax: +86-22-23506075. E-mail: [email protected]. 32673

DOI: 10.1021/acsami.6b10702 ACS Appl. Mater. Interfaces 2016, 8, 32667−32674

Research Article

ACS Applied Materials & Interfaces Persistent Luminescent Nanoparticles with Superlong Afterglow for in VivoTargeted Bioimaging. J. Am. Chem. Soc. 2013, 135, 14125−14133. (19) Maldiney, T.; Bessière, A.; Seguin, J.; Teston, E.; Sharma, S. K.; Viana, B.; Bos, A. J. J.; Dorenbos, P.; Bessodes, M.; Gourier, D.; Scherman, D.; Richard, C. The in Vivo Activation of Persistent Nanophosphors for Optical Imaging of Vascularization, Tumours, and Grafted Cells. Nat. Mater. 2014, 13, 418−426. (20) Li, Z.; Zhang, Y.; Wu, X.; Huang, L.; Li, D.; Fan, W.; Han, G. Direct Aqueous-Phase Synthesis of Sub-10 nm “Luminous Pearls” with Enhanced in Vivo Renewable Near-Infrared Persistent Luminescence. J. Am. Chem. Soc. 2015, 137, 5304−5307. (21) Abdukayum, A.; Yang, C. X.; Zhao, Q.; Chen, J. T.; Dong, L. X.; Yan, X. P. Gadolinium Complexes Functionalized Persistent Luminescent Nanoparticles as a Multimodal Probe for Near-Infrared Luminescence and Magnetic Resonance Imaging in Vivo. Anal. Chem. 2014, 86, 4096−4101. (22) Teston, E.; Lalatonne, Y.; Elgrabli, D.; Autret, G.; Motte, L.; Gazeau, F.; Scherman, D.; Clement, O.; Richard, C.; Maldiney, T. Design, Properties, and in Vivo Behavior of Super-Paramagnetic Persistent Luminescence Nanohybrids. Small 2015, 11, 2696−2704. (23) Maldiney, T.; Doan, B.-T.; Alloyeau, D.; Bessodes, M.; Scherman, D.; Richard, C. Gadolinium-Doped Persistent Nanophosphors as Versatile Tool for Multimodal in Vivo Imaging. Adv. Funct. Mater. 2015, 25, 331−338. (24) Lu, Y. C.; Yang, C. X.; Yan, X. P. Radiopaque Tantalum Oxide Coated Persistent Luminescent Nanoparticles as Multimodal Probes for in Vivo Near-Infrared Luminescence and Computed Tomography Bioimaging. Nanoscale 2015, 7, 17929−17937. (25) Shi, J.; Sun, X.; Li, J.; Man, H.; Shen, J.; Yu, Y.; Zhang, H. Multifunctional Near Infrared-Emitting Long-Persistence Luminescent Nanoprobes for Drug Delivery and Targeted Tumor Imaging. Biomaterials 2015, 37, 260−270. (26) Shi, J.; Fu, H.; Sun, X.; Shen, J.; Zhang, H. Magnetic, Long Persistent Luminescent, and Mesoporous Nanoparticles as Trackable Transport Drug Carriers. J. Mater. Chem. B 2015, 3, 635−641. (27) McGrath, A. J.; Chien, Y.-H.; Cheong, S.; Herman, D. A. J.; Watt, J.; Henning, A. M.; Gloag, L.; Yeh, C.-S.; Tilley, R. D. Gold over Branched Palladium Nanostructures for Photothermal Cancer Therapy. ACS Nano 2015, 9, 12283−12291. (28) Chen, M. C.; Lin, Z. W.; Ling, M. H. Near-Infrared LightActivatable Microneedle System for Treating Superficial Tumors by Combination of Chemotherapy and Photothermal Therapy. ACS Nano 2016, 10, 93−101. (29) Xiao, Q.; Zheng, X.; Bu, W.; Ge, W.; Zhang, S.; Chen, F.; Xing, H.; Ren, Q.; Fan, W.; Zhao, K.; Hua, Y.; Shi, J. A Core/Satellite Multifunctional Nanotheranostic for in Vivo Imaging and Tumor Eradication by Radiation/Photothermal Synergistic Therapy. J. Am. Chem. Soc. 2013, 135, 13041−13048. (30) Zhou, M.; Zhang, R.; Huang, M.; Lu, W.; Song, S.; Melancon, M. P.; Tian, M.; Liang, D.; Li, C. A Chelator-Free Multifunctional [64Cu]CuS Nanoparticle Platform for Simultaneous Micro-PET/CT Imaging and Photothermal Ablation Therapy. J. Am. Chem. Soc. 2010, 132, 15351−15358. (31) Zhang, L.; Gao, S.; Zhang, F.; Yang, K.; Ma, Q.; Zhu, L. Activatable Hyaluronic Acid Nanoparticle as a Theranostic Agent for Optical/Photoacoustic Image-Guided Photothermal Therapy. ACS Nano 2014, 8, 12250−12258. (32) Allix, M.; Chenu, S.; Véron, E.; Poumeyrol, T.; KouadriBoudjelthia, E. A.; Alahraché, S.; Porcher, F.; Massiot, D.; Fayon, F. Considerable Improvement of Long-Persistent Luminescence in Germanium and Tin Substituted ZnGa2O4. Chem. Mater. 2013, 25, 1600−1606. (33) Bessière, A.; Jacquart, S.; Priolkar, K.; Lecointre, A.; Viana, B.; Gourier, D. ZnGa2O4:Cr3+: A New Red Long-Lasting Phosphor with High Brightness. Opt. Express 2011, 19, 10131−10137.

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DOI: 10.1021/acsami.6b10702 ACS Appl. Mater. Interfaces 2016, 8, 32667−32674