Yolk-Structured Upconversion Nanoparticles with Biodegradable

Aug 21, 2017 - Fourier-transform Infrared (FT-IR) spectra were tested on a Vertex PerkinElmer 580BIR spectrophotometer (Bruker), using the KBr pellet ...
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Yolk-Structured Upconversion Nanoparticles with Biodegradable Silica Shell for FRET Sensing of Drug Release and Imaging-Guided Chemotherapy Jiating Xu,† Fei He,*,† Ziyong Cheng,‡ Ruichan Lv,§ Yunlu Dai,† Arif Gulzar,† Bin Liu,† Huiting Bi,† Dan Yang,† Shili Gai,† Piaoping Yang,*,† and Jun Lin*,‡ †

Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, Harbin Engineering University, Harbin 150001, P. R. China ‡ State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China § Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School of Life Science and Technology, Xidian University, Xi’an 710071, P. R. China S Supporting Information *

ABSTRACT: Silica related nanovehicles are being widely studied for bioapplication, while the use in vivo has been restricted due to the biodegradation reluctance. Herein, a facile Mn-doping method was used to endow the upconversion nanoparticles (UCNPs) with a biodegradable shell, simply by transforming mesoporous silica coated UCNPs (UCNPs@mSiO2) to Mn-doped upconversion nanocapsules (MnUCNCs). The yolk-structured Mn-UCNCs have huge internal space, which is greatly beneficial for DOX (a chemotherapeutic agent) storage. Furthermore, the Mn-doped nanoshell is responsive to mild reductive and acidic tumor condition, which enables the biodegradation of the silica shell in tumor sites and further accelerates the breakup of Si−O−Si bonds within the silica framework. This tumor-sensitive degradation of the shell not only facilitates DOX release in the tumor location but also allows faster nanoparticle diffusion and deeper tumor penetration, thus realizing efficient particle distribution and improved chemotherapy. Moreover, the biodegradability-enhanced DOX release brings a rapid recovery to the total emission intensity and a drastic decline to the red/green (R/G) ratio, which can be used to sense the drug release extent. The MRI effect caused by Mn release coupled with the inherent MRI/CT/UCL imaging derived from the UCNPs (NaGdF4:Yb,Er@NaGdF4:Yb) under NIR irradiation endow the nanocarrier with superior multiple imaging functions. The high biocompatibility of PEGylated Mn-UCNCs was validated, and the excellent anticancer effectiveness of the DOX loaded nanosystem was also achieved.

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INTRODUCTION

cancer cells as possible, so that a reliable tumor delineation is difficult to achieve. For safe and efficient chemotherapy, the nanocarriers should primarily meet the criteria of high drug loading and minor adverse effects, so to avoid the harmful drug release and drug resistance as much as possible.16−18 Hence, a number of mesoporous silica related nanovehicles have been developed to obtain high storage and controlled release of chemotherapy agents.19 Nevertheless, the stable physicochemical property of the −Si−O−Si− famework makes it hard to degrade in physiological condition, thus causing the incomplete drug release. Besides, the reluctant silica biodegradation may also lead to the accumulation of nanocarriers within the biobodies,

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Cancer is one of the world’s leading causes of death. The innovation of intelligent cancer therapeutics concurrently exerts accurate diagnosis, and efficacious treatment becomes a significant work.3−8 Chemotherapy is the most widely used modality for anticancer therapy.9−11 Recently, the integration of upconversion nanoparticles (UCNPs) with mesoporous silica for delivery of chemotherapeutic agents to the tumor sites has attracted a great deal of attention because of the potency in realizing multiple imaging-guided chemotherapy.12−15 However, the limited drug delivery efficacy and dull drug release are the two major obstacles in achieving ideal treatment outcomes. Besides, the traditional mesoporous silica coated UCNPs are size-unvaried at tumor focus and, thus, are usually are restrained in the interstitium of a solid tumor after extravasation. Under these circumstances, the nanoparticles cannot reach as many © 2017 American Chemical Society

Received: August 16, 2017 Revised: August 21, 2017 Published: August 21, 2017 7615

DOI: 10.1021/acs.chemmater.7b03461 Chem. Mater. 2017, 29, 7615−7628

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promote the degradation of the shell itself including the cleavage of Si−O bonds. Furthermore, the disintegration of the Mn-doped silica shell can further improve the T1-weighted MRI thanks to the Mn (II) release.60 Notably, the biodegradation of the shell brings an obvious size decrease to the Mn-UCNCs and therefore makes the exposed core particles reach more cancer cells to achieve efficient tumor distribution. Because of the fluorescence resonance energy transfer (FRET) between green UCL peaks and the DOX, the green emissions of the DOX-loaded nanocapsules are largely diminished. As a result, the biodegradability-enhanced DOX release inevitably brings a rapid decline of the R/G ratio, which can be used to sense the drug release extent when used for in vivo anticancer theranostic. The specific contributions of the DOX loaded nanocarrier are (1) PEGylation prolonged the blood circulation; (2) extravasation at the tumor sites through the EPR effect; (3) fabrication of yolk-structured Mn-UCNCs with a biodegradable shell; (4) regulation of the silica biodegradability simply by doping Mn into the shell; (5) biodegradation-enhanced DOX release and the MRI effect; (6) FRET sensing of the drug release, and (7) tumor-responsive chemotherapy with multiple imaging guidance.

resulting in potential biosafety issues. However, the biodegradation of silica has not been efficiently achieved so far, which is the most critical hindrance in their practical application.20,21 Hence, it is still a formidable challenge to manage the biodegradability of the silica shell portion on iorganic nanoparticles when applied as chemothrapeutic drug carriers. As known, a reliable lesion delineation is greatly significant before treatment.22−24 Recently, a growing number of pieces of research have indicated that rare-earth upconversion nanomaterials are promising in combining multiple bioimaging into one system,25−28 which will extremely offset the defects of singlemodality imaging.29−31 Upconversion, known as an anti-Stokes process, is a special multiphoton emission, which transduces more than one long-wavelength excitation photon to a shortwavelength emission photon.32−41 This unique feature is especially beneficial for optical imaging because the longwavelength photons have deeper penetration, allowing a longer imaging distance.42−44 And the upconverted emission can be easily distinguished from the autofluorescence of biotissues, thus avoiding the background interference during the visualization processes.42 Moreover, the UCNPs doped with Gd3+ and Yb3+ ions are promising nanoprobes for magnetic resonance imaging (MRI) and X-ray computed tomography (CT) imaging,45,46 which unites with the UCL imaging to offer whole-body visualization at high spatial resolutions as well as the real-time observation of nanocarriers in tumor tissues. It is known of that PEGylation usually endows the UCNPs-based nanotheranostics with large particle size, which makes them selectively accumulate in tumor lesions through the enhanced permeability and retention (EPR) effect.47,48 However, the large size for particles is detrimental for nanoagents to penetrate into the tumor parenchyma deeply. After extravasating from the tumor vessels, the agents are mainly restricted to the adjacent zones of tumor vasculatures due to the dense extracellular matrix and high interstitial fluid pressure, thus greatly limiting their penetration depth.49−52 These two contradictory aspects of improved tumor extravasation and inferior tumor penetration are intractable to be compromised when using UCNPs-based nanocarriers for anticancer theranostic. Actually, the dilemma also reveals that it is significant to develop a size-variable nanosystem that could maintain large size for longer blood circulation and selective tumor extravasation, while evolving into small-sized particles at lesions for deep tissue penetration and effective distribution. Here, a facile Mn-doping strategy was used to concurrently enable tumor-triggered biodegradation of a Mn-doped silica shell and tumor-improved diagnostics of a nanosystem. Via this method, the biodegradation property of a silica shell was intrinsically altered. As accepted, Mn is an important element in human bodies for regulating metabolism; its uptake and excretion can be efficiently controlled by a biological sysytem.53 In addition, the paramagnetic Mn ions in the silica nanoshell can act as a T1-weighted magnetic resonance imaging (MRI) contrast agent, which couples with the inherent CT, MRI, and UCL imaging effects of UCNPs to perform tumor-improved multiple imaging.54−56 Moreover, the −Mn−O− bonds can respond to acidic or reductive condition.57−59 Hence the Mndoped silica shell of the fabricated nanocapsules can quickly biodegrade in the mild acidic and reductive tumor microenvironment due to the inherent breakup nature of the Mn−O bonds. The breakage of the Mn−O bonds and the subsequent release of Mn ions from the silica shell can produce a large amount of defects within the silica framework and further

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EXPERIMENTAL SECTION

Chemicals and Reagents. Sodium fluoride (NaF), Gd 2O3 (99.99%), Er2O3 (99.99%), Yb2O3 (99.99%) and hydrochloric acid (HCl) (from Sinopharm Chemical Reagent Co., Ltd.); oleic acide (OA), glutathione (GSH), 1-octadecene (ODE), disodium maleate, propidium iodide (PI), doxorubicin (DOX), 4′,6-diamidino-2-phenylindole (DAPI), methoxy PEG silane (Mw = 2000) and 3−4,5dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) (from Sigma-Aldrich); ammonium nitrate (NH4NO3), tetraethyl orthosilicate (TEOS) and cetyltrimethylammonium bromide (CTAB) (from Tianjin Kermel Chemical Co., Ltd.); manganese(II) sulfate monohydrate (MnSO4·H2O), trifluoroacetic acid (CF3COOH), and sodium trifluoroacetate (CF3COONa) (from Beijing Chemical Regent Co.). All the reagents used in this work are of analytical grade without further purification. Synthesis of OA-Stabilized NaGdF4:Yb,Er (OA-NaGdF4:Yb,Er) Nanoparticles. Briefly, 1 mmol of lanthanide oleates (Er/Yb/Gd = 2:18:80) and 5 mmol of NaF were added to a three-neck flask having ODE (15 mL) and OA (15 mL). The mixture was heated to 110 °C and degassed at this temperature for 0.5 h. After flushed with N2, the system was heated to 300 °C and maintained for 1 h. The solution was then cooled down to about 40 °C naturally. The ethanol and cyclohexane were used to centrifuge the product and the resulting particles were dispersed in 5 mL cyclohexane for further use. Synthesis of OA-Capped NaGdF4:Yb,Er@NaGdF4:Yb Nanoparticles (designated as OA-UCNPs). The above cyclohexane solution of core nanoparticles was added to a three-neck flask having OA (15 mL) and ODE (15 mL). Then 0.1 mmol of Yb(CF3COO)3, 0.4 mmol of Gd(CF3COO)3, and 1 mmol of CF3COONa were added. The mixture was degassed at 120 °C for 1 h. After flushed with N2, the system was heated to 310 °C and maintained for 1 h. The resulting particles were dispersed in cyclohexane (∼10 mg mL−1) for further use. Synthesis of UCNPs@mSiO2. The silica coating procedure was carried out according to a previous report.63 Typically, a beaker with CTAB (0.1 g) dispersed in deionized water (20 mL) was ultrasonically treated to obtain a transparent solution. Then 2 mL of OA-UCNPs in cyclohexane were added, and the mixed solution was stirred overnight to obtain a homogeneous solution. For coating mesoporous silica onto water dispersible UCNPs, the obtained aqueous solution was added to a mixture of deionized water (40 mL), ethanol (6 mL) and NaOH solution (0.3 mL, 2 M). The mixed solution was transferred to a water bath and heated to 70 °C under vigorous stirring. Several minutes later, TEOS (0.15 mL) was slowly added into the mixture and stirred 7616

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injection. Then the sample (0.1 mL, 1 mg mL−1) was injected intratumorally into the tumor-bearing mice for scanning. In Vitro Cellular Uptake and UCL Microscopy (UCLM) Observation. HeLa cells were seeded n a 6-well culture plate and cultured overnight to obtain monolayer cells. After that, PEG/MnUCNCs-DOX (1 mL, 1 mg mL−1) was added to the wells and incubated for 0.5, 1, and 3 h, respectively. Then, the cells were washed with PBS three times and stained by DAPI for 10 min. One mL of glutaraldehyde (2.5%) was used to fix the cells for 10 min, and then further washed with PBS three times. At last, the fluorescence images of cells were recorded using a Leica TCS SP8 instrument. For the UCLM observation, the slides were prepared using the same process except that the images were recorded using an inverted fluorescence microscope (Nikon Ti−S), and an external continuous wave of 980 nm laser was used to irradiate the samples. In Vitro Cytotoxicity. MTT method was used to evaluate the in vitro cytotoxicity of the samples. Typically, HeLa cells (6000−7000 well−1) were seeded in a 96-well plate and cultured in a humid incubator (37 °C, 5% CO2) for 24 h. Free DOX, PEG/UCNPs@ mSiO2-DOX, and PEG/Mn-UCNCs-DOX were dispersed into the culture medium at the equivalent DOX contents, and the DOX concentrations are 0, 0.2, 0.5, 1, 2, 4, 8, 16, and 30 μg mL−1. Then the culture was removed and MTT solution (20 μL, 5 mg mL−1) was added into each well. After incubation for another 4 h, DMSO (150 μL) was added to the wells and the absorbance at 490 nm was recorded for calculation. The proportion of viable cells in experiment group to the control group was used to express the cytotoxicity. The in vitro viability of PEG/Mn-UCNCs to L929 fibroblast cells was also assessed by MTT assay. The sample concentrations are 18.8, 37.5, 75, 150, 300, and 600 μg mL−1. In Vivo Antitumor Efficiency of PEG/Mn-UCNCs-DOX. The tumor xenograft was first established in the left axilla of each female mice (15−20 g). The mice were purchased from Second Affiliated Hospital Harbin Medical University, and all the mouse experiments were performed in compliance with the criteria of The National Regulation of China for Care and Use of Laboratory Animals. When the tumor size is about 6−8 mm, the mice were randomly divided into three groups (n = 5 group−1) and intravenously injected with saline (control group), PEG/UCNPs@mSiO2-DOX and PEG/Mn-UCNCsDOX solutions at the DOX dose of 2 mg kg−1. The tumor size and body weight were recorded every day after the therapy. Tumor volume (mm3) was calculated as V = lw2/2, in which l and w represent the length and width of the tumor. Histological Examination. After 14 days treatment, the histological analysis was carried out. Less than 1 cm × 1 cm of tissues on the organs of liver, lung, kidney, heart, spleen and tumor of the representative mice in three groups were excised. Then, the excised tissues were successively dehydrated using buffered formalin, ethanol at varied concentrations, and xylene. Finally, the above dehydrated tissues were embedded by liquid paraffin, and sliced for hematoxylin and eosin (H&E) staining. The stained slices were examined using an optical microscope.

vigorously for 10 min. The resultant nanospheres were collected by centrifugation and washed with ethanol three times. To extract CTAB template, the obtained nanoparticles were transferred to ethanol (50 mL) containing NH4NO3 (0.3 g) and refluxed at 60 °C for 2 h. Synthesis of Mn-Doped Upconversion Nanocapsules (MnUCNCs). The as-prepared UCNPs@mSiO2 nanospheres were initially dispersed in deionized water (10 mL). Then, an aqueous solution (10 mL) of MnSO4·H2O (8 mg mL−1) and disodium maleate (10 mg mL−1) was dropwise added into UCNPs@mSiO2 solution under stirring, then the mixture was maintained in a hydrothermal condition (180 °C, 3 h). Finally, the resulting Mn-UCNCs were collected by centrifugation and washed washed with ethanol and deionized water three times to remove the residual reactants. Synthesis of PEGylated Mn-UCNCs (PEG/Mn-UCNCs). Ethanol (50 mL) with PEG (50 mg) and Mn-UCNCs (20 mg) dissolved in was magnetically stirred at 60 °C for 24 h. After that, PEGylated MnUCNCs were centrifuged, and the product was washed with deionized water and ethanol several times to remove the unreacted PEG. The similar procedure was used to prepare PEGylated UCNPs@mSiO2 (PEG/UCNPs@mSiO2). DOX Loading and Release from DOX Loaded PEG/MnUCNCs (labeled as PEG/Mn-UCNCs-DOX). In a dark room, PEG/ Mn-UCNCs (20 mg) were dispersed into PBS containing DOX (20 mL, 0.5 mg mL−1) under magnetic stirring for 24 h. Thereafter, the PEG/Mn-UCNCs-DOX was collected by centrifugation, and the supernatant was collected for UV−vis measurement. The DOX content in the supernatant was determined by UV−vis spectra at 480 nm. Similar process was used to prepare PEG/UCNPs@mSiO2-DOX and calculate the corresponding DOX loading rate. PBS (10 mL) was added to the precipitated mixture and set in a water bath at 37 °C with magnetic stirring, and then the supernatant was kept for further UV− vis analysis. At varied time intervals, this release process was repeated in PBS at different GSH contents (0, 5.0 mM, and 10.0 mM) or PBS with different pH values (7.4 and 5.5). Characterization. X-ray diffraction (XRD) patterns of the samples were achieved on a D8 Focus diffractometer (Bruker) using CuKa radiation (λ = 0.15405 nm). Transmission electron microscopy (TEM) micrographs were obtained from a FEI Tecnai G2 S-twin transmission electron microscope with a field emission gun operating at 200 kV. Upconversion emission spectra were measured on an Edinburgh FLS 980 apparatus, from 400 to 800 nm, using 980 nm laser diode module (K98D08M-30W, China) as the irradiation source. The N2 adsorption−desorption isotherm and corresponding pore-size distribution were tested to characterize the mesoporous structure of Mn-UCNCs on a Micrometitics Tristar 3000 system. UV−vis absorption spectra were acquired by a TU-1901 dual beam UV−vis spectrometer. Fourier-transform Infrared (FT-IR) spectra were tested on a Vertex PerkinElmer 580BIR spectrophotometer (Bruker), using the KBr pellet technique. In Vitro Degradation Experiment. Briefly, Mn-UCNCs were added into PBS solutions with different pH values (pH = 7.4 and 5.5) without or with GSH (5.0 mM and 10.0 mM). All of the testing solutions were put into a water bath at 37 °C under magnetic stirring and the sample concentrations are 0.1 mg mL−1. At given time intervals, a small amount of solution was taken out for ICP-OES test. In Vitro T1-Weighted MR Imaging. The PEG/Mn-UCNCs were dissolved into different concentrations. T1 was acquired using an inversion recovery sequence. T1 measurements were conducted using a nonlinear fit to changes in the mean signal intensity within each well as a function of repetition time using a Huantong 1.5 T MR scanner. Finally, the r1 relaxivity values were determined through the curve fitting of 1/T1 relaxation time (s−1) versus the sample concentration (mg mL−1). In Vitro and in Vivo X-ray CT Imaging. The in vitro CT imaging experiments were performed on a Philips 64-slice CT scanner (120 kV). Then the PEG/Mn-UCNCs were dissolved into varied concentrations and placed in a line for CT imaging measurements. For in vivo CT imaging test, the female mice were anesthetized with 10% chloral hydrate (0.03 mL g−1 of mouse) by intraperitoneal

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RESULTS AND DISCUSSION The schematic illustration for the synthesis of DOX-loaded PEG/Mn-UCNCs is depicted in Scheme 1A. As shown, there are several important steps in the synthetic process. The coated active-shell (NaGdF4:Yb) is beneficial to achieve superbright upconversion of NaGdF4:Yb,Er core nanoparticles, which is significant for the UCL imaging. The OA-UCNPs were then coated with mSiO2, and then a gas−liquid template method was used to enable the formation of Mn-doped upconversion nanocapsules (Mn-UCNCs). After the Mn-doped silica shell was PEGylated, the resulting PEG/Mn-UCNCs were used to store DOX for chemotherapeutic application. The addition of disodium maleate enables the formation of a mild alkalescent solution (Scheme 1B, reaction 1). Under hydrothermal condition, a small portion of silica from the inner shell was 7617

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Mn-doped silica shell can facilate the DOX release and improve T1-weighted MRI imaging. The DOX release also triggers the R/G sensor on the nanosystem for FRET sensing of DOX release extent. In other words, this PEG/Mn-UCNCs-DOX nanosystem is highly promising in achieving biodegradationenhanced chemotherapy and tumor diagnosis. Figure 1A−C respectively display the TEM images of the OA-NaGdF4:Yb,Er core nanoparticles, core−shell OA-UCNPs, and Mn-UCNCs. The core and core−shell nanoparticles were fabricated by a thermal decomposition method according to a previous report.63 In the TEM image of OA-NaGdF4:Yb,Er nanoparticles (Figure 1A), the sample consists of uniform and monodispersed particles (mean diameter: 11.4 nm). To obtain the superior upconversion of the core nanoparticles under NIR laser excitation, an active shell of NaGdF4:0.2Yb was coated on the core portion via an epitaxial growth method.64 The TEM image in Figure 1B reveals that the dispersity and uniformity of the OA-UCNPs have been kept well, and the mean size increased to 20.6 nm. The OA-UCNPs were transferred into an aqueous phase with CTAB before the mesoporous silica shell coating. The TEM image given in Figure S1A indicates that the UCNPs@mSiO2 consists of dispersible nanoparticles with a mean size of 40.7 nm. Besides, close observation reveals the wormlike channels on the silica shell. The energy-dispersive spectrum (EDS) in Figure S1B depicts the elementary composition of the UCNPs@mSiO2. The yolk-like nanostructure was generated after the UCNPs@mSiO2 spheres were hydrothermally treated in an aqueous solution with MnSO4· H2O and maleic disodium. To find out the most appropriate processing time for yolk-structure formation, the mesoporous nanospheres have been treated for various durations (0.5, 1, 2, 3, and 4 h). The TEM images in Figure 1C together with that in Figure S2 indicate that 3 h is the most suitable time duration for yolk-structure formation. When the treatment time is less than 3 h, the inner volume of the yolk structure is not maximal (Figure S2A−C). However, when the treatment time reaches 4 h, the silica shell of the yolk structure cannot be maintained perfectly (Figure S2E). As shown in Figure 1C, the hydrothermal treatment brings an increase to the diameter of UCNPs@mSiO2 of about 10 nm; that is, to say the particle size of the yolk-structured Mn-UCNCs is about 50 nm. The X-ray photoelectron spectroscopy (XPS) measurement in Figure S3 verifies that the Mn-UCNCs are composed of Na, Gd, F, Yb, Er, Si, C, O, and Mn elements. Moreover, the EDS, scanning TEM (STEM) image, and corresponding cross-sectional compositional line profiles of Mn-UCNCs are displayed in Figure 1D and E. The corresponding element testing result is in accordance with that in Figure S3. Note here, the line profiles indicate the UCNPs core and the Mn-doped silica shell, which validates the formation of core−shell structure. The XRD spectra of the OA-UCNPs, Mn-UCNCs, PEG/ Mn-UCNCs, and the standard line of β-NaGdF4 (JCPDS No. 27-0699) are exhibited in Figure 2A. Simultaneously, we supplied the XRD spectra of the as-synthesized NaGdF4:Yb,Er core nanoparticles and the UCNPs@mSiO2 in Figure S4. As shown, the diffraction peaks of pure β-NaGdF4 kept well in these samples. In comparison with UCNPs, there is a new peak for amorphous materials at 2θ = 22° for UCNPs@mSiO2, which indicates the successful silica coating.63 Note here, the XRD patterns of Mn-UCNCs and the later obtained sample exhibit a broadened peak of Mnx(SiO4)y at about 2θ = 33°, implying the existence of covalent bonds between Mn species and the silica shell.65

Scheme 1. Schematic Illustration for the Synthesis of PEG/ Mn-UCNCs-DOX (A); the Reactions between Disodium Maleate and Water Result in the Formation of Mn-UCNCs: (1) Causing Gentle Alkalescent Solution Environment and Further Promoting Hydrolysis of the Mesoporous Silica Shell and (2) Generating CO2 as Gas-Liquid Template (B)

hydrolyzed to H4SiO4.61,62 Simultaneously, the active sites produced on the mSiO2 shell adsorb carboxylate species and Mn2+ ions from decomposition of disodium maleate (Scheme 1B, reaction 2). Thereafter, the carboxylate decomposed into gaseous species including CO2 under hydrothermal condition. Notably, the reaction between H4SiO4 and Mn2+ ions results in the deposition of Mn-doped silica on the liquid−gas interface, forming solid spheres on the shell. The same growth processes and deposition on the shell continue to produce unique yolkstructured nanocapsules with a nanospheres-stacked shell. Through these elaborate designs, a multiple imaging-guided chemotherapeutic nanosystem with tumor-responsiveness is obtained. As depicted in Scheme 2, the nanosized PEG/MnUCNCs were transported within blood vessels and accumulated at tumor tissues via the EPR effect. The biodegradation of the silica shell can be easily triggered by either a mild acidic or reductive microenvironment of tumor issue and then release Mn ions. Then the Mn release-triggered biodegradation of the Scheme 2. Schematic Illustration of Transport of PEG/MnUCNCs-DOX in Blood Vessel, EPR-Mediated Tumor Accumulation, Tumor-Enhanced Chemotherapy, and Multiple Imaging Functions

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Figure 1. TEM images of OA-NaGdF4:Yb,Er core nanoparticles (A), core−shell OA-UCNPs (B), and Mn-UCNCs (C). EDS spectrum (D), STEM image (E), and the corresponding cross-sectional compositional line profiles of Mn-UCNCs.

Figure 2. XRD patterns (the standard pattern of β-NaGdF4 is supplied for comparison) (A) and FT-IR spectra of (B) OA-UCNPs, Mn-UCNCs, and PEG/Mn-UCNCs. N2 absorption−desorption isotherm (C) and corresponding pore-size distribution of Mn-UCNCs (D).

peaks at 802 and 1088 cm−1 are derived from the vibration of Si−O−Si bands. The FT-IR spectrum of Mn-UCNCs (Figure 2B) poses a similar profile compared with UCNPs@mSiO2 due to the same functional groups on the sample surface. When the Mn-UCNCs were modified with PEG, a strong C−O stretching vibration of the PEG unit is detected when compared with the vibration behavior of Mn-UCNCs. As for the final product PEG/Mn-UCNCs-DOX, the new peaks appearing at 1000− 1800 cm−1 are attributed to the DOX molecules (Figure S5).18 In Figure 2C and D, the N2 adsorption/desorption isotherm and the corresponding pore-size distribution of Mn-UCNCs is displayed. As shown, the sample represents typical type IV isotherms, implying the mesoporous structure of silica channels.62 This yolk-structured sample possesses a relatively

FT-IR spectra of the OA-UCNPs, Mn-UCNCs, and PEG/ Mn-UCNCs (Figure 2B) were measured to detect the functional groups on the surface of samples and provide additional evidence for the successful modification. As shown, the OA-UCNPs exhibit bands at 1463 and 1564 cm−1 associated with the vibrations of the carboxylic groups, and the broad band at around 3450 cm−1 originates from the stretching vibration of O−H. The strong transmission bands at 2854 and 2924 cm−1 are attributed to the asymmetric and symmetric stretching vibrations of methylene (−CH2). As For UCNPs@mSiO2 (Figure S5), the peaks at 3432 and 947 cm−1 suggest that a large number of OH (Si−OH and −OH groups) exist on the surfaces of the UCNPs@mSiO2, which is beneficial for adsorbing drug molecules through hydrogen bonds. The 7619

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Figure 3. Upconversion emission spectra of the NaGdF4:Yb,Er core nanoparticles, UCNPs, Mn-UCNCs, and the final PEG/Mn-UCNCs-DOX (A). UV−vis absorption spectra of the DOX molecules and the final PEG/Mn-UCNCs-DOX (B). The standard curve for DOX solutions detected at 480 nm (C). The absorbance spectra of the initial DOX solution and the supernatant obtained after the drug loading process with PEG/Mn-UCNCs (D).

Figure 4. Accumulated releasing profiles of biodegraded Mn and Si in PBS at various pH values (A, B), at various GSH contents under neutral condition (C, D), and at various GSH contents under acidic and neutral conditions (E, F).

and a pore volume of 0.25 cm3 g−1 (Figure S6), which is due to DOX loading in the silica channels. Figure 3A exhibits the upconversion emission spectra of NaGdF4:Yb,Er core nanoparticles, UCNPs, Mn-UCNCs, and the final obtained PEG/Mn-UCNCs-DOX. When excited with a 980 nm laser, there are three characteristic emission peaks of

high Brunauer−Emmett−Teller (BET) surface area of 367.3 m2 g−1 with an average pore size of 5.7 nm, and a high pore volume of 0.43 cm3 g−1. The mesopores and the large surface area are ideal to save cargos. The final PEG/Mn-UCNCs-DOX still has mesoporous structure, while the BET surface area is decreased to 170.3 m2 g−1 with an average pore size of 5.3 nm, 7620

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Chemistry of Materials Er3+ at 630−680 nm (4F9/2 → 4I15/2), 530−570 nm (4S3/2 → 4 I15/2), and 510−530 nm (2H11/2 → 4I15/2). To enable the superbright upconversion under low excitation power, an active shell of NaGdF4:Yb was coated on the core component. In Figure 3A, the emission intensity of the core−shell UCNPs is obviously higher than that of core nanoparticles. The activeshell here serves two roles: (1) to transfer absorbed NIR photons to the luminescent core zone and (2) to protect the luminescent Er3+ ions from the nonradiative decay.64 In comparison with the core−shell UCNPs, the emission intensity of the Mn-UCNCs was decreased obviously perhaps due to the quenching effect caused by Mn-doped silica shell. Notably, the red-emissive peaks of Mn-UCNCs are stronger than green ones because the red light falls into the optical transmission window31 and, thus, has deeper penetration, making it easier to be detected. As for PEG/Mn-UCNCs-DOX, it has lower emission intensity than Mn-UCNCs especially in the green region. This is caused by the strong absorption of DOX in the green range (Figure 3B). The UV−vis absorption spectrum of PEG/Mn-UCNCs-DOX also has an obvious absorption peak during 450 to 550 nm, which is originated from DOX. Figure 3C and Figure 3D respectively show the standard curve for DOX detected at 480 nm and the absorption spectra of the DOX solution before and after the loading process using PEG/ Mn-UCNCs. According to the Lambert−Beer Law, the DOX loading rate of PEG/Mn-UCNCs was calculated to be 75.4%. Similarly, the DOX loading rate of the PEG/UCNPs@mSiO2 is calculated to be 66.6% (Figure S7), which is obviously lower than that of PEG/Mn-UCNCs. As accepted, the Mn−O bonds are sensitive to reductive and mild acidic tumor microenvironment.65 Thus, we envisage that the introduced Mn−O bonds within the silica framework of silica can be disintegrated in a tumor-like condition, which can further accelerate the shell degradation. To simulate the tumor microenvironment, the PBS with various pH values and GSH contents were used for detailed biodegradation assays. The MnUCNCs were dissolved in various PBS solutions, and the degradation processes were monitored by ICP tests. The pH of the tumor extracellular microenvironment is about 7.2−6.5 depending on the tumor stage and type while that of intracellular early endosome and lysosome reaches 6.2−5.0.1 The PBS solutions with low pH (5.5) and GSH (5.0 mM and 10.0 mM) were used to mimic the acidic and reductive tumor microenvironment, respectively. Herein, the PBS with pH = 5.5 was adopted to study the acidity-sensitive biodegradation and theranostic of Mn-UCNCs. In comparison with that in neutral PBS, the release of Mn is obviously accelerated under mild acidic condition (Figure 4A). Moreover, the fast Mn release from the shell of Mn-UCNCs has accelerated the release of Si (Figure 4B). The Si/Mn release profiles indicate that the breakup of Mn−O bonds in acidic condition induces the fast breakage of the −Si−O−Si− framework afterward. According to a previous report, tumor cytosol and tumor tissues are reductive with GSH content at least 4-fold higher than that in normal tissues.56 Herein, the biodegradation behaviors of a Mndoped shell on Mn-UCNCs in different reductive PBS solutions were investigated. As expected, GSH can also accelerate the release of Mn and Si. In other words, the biodegradation of the Mn-doped silica shell is GSH dependent (Figure 4C, D). This result also validates the GSH-dependent biodegradation of the Mn-doped shell. Furthermore, the degradation behaviors of Mn-UCNCs under concurrent reductive (GSH = 5.0 mM and 10.0 mM) and acidic (pH =

5.5) conditions were also assessed. It can be seen that the release of Mn (Figure 4E) and Si (Figure 4F) under a combined condition are much higher than the biodegradation in either acidic or reductive condition. The additional evidence in Figure S8 validates that the silica shell of Mn-UCNCs collapses quickly in the combinational condition. The dynamic light scattering (DLS) test of the PEG/Mn-UCNCs after the biodegradation at the GSH content of 10 mM, pH = 5.5 for 24 h reveals that the diameter of the nanoparticles is about 32.7 nm (Figure S9). To study the intracellular biodegradation of Mn-UCNCs, HeLa cancerous cells were incubated with MnUCNCs for different time durations (3, 6, 12, and 24 h). As displayed in Figure 5, PEG/Mn-UCNCs can be easily

Figure 5. Bio-TEM images of HeLa cells incubated with PEG/MnUCNCs for 3 h (A), 6 h (B), 12 h (C), and 24 h (D) at 37 °C.

endocytosized into the cell cytoplasm. Notably, the shell kept well at the initial stage (Figure 5A), while the biodegradation of the shell portion can be clearly found in the bio-TEM image at the 6 h intracellular uptake (Figure 5B). With the incubation prolonged, the shell portion becomes ambiguous (Figure 5C), and when the incubation time reaches 24 h, nearly no obvious shell component can be observed (Figure 5D), validating the intracellular biodegradation of silica shell on the Mn-UCNCs. The bio-TEM observation results are consistent with the degradation experiments in different PBS solutions (Figure 4), thus a conclusion that the intracellular condition of cancerous cells can efficiently trigger the breakup of Mn−O bonds and the subsequent shell biodegradation can be drawn. Inspired by the biodegradable behavior of Mn-UCNCs, the drug release profiles of PEG/Mn-UCNCs-DOX were studied. As shown in Figure 6A, the PEG/Mn-UCNCs-DOX show a pH-responsive drug release in PBS where acidic PBS enables the quicker drug release. DOX release was also studied by dispersing PEG/Mn-UCNCs-DOX into PBS with different GSH contents (Figure 6B). It can be seen that the DOX release in pure PBS at 48 h is as low as 9.6%, but the release rate increases to 29.2% and 37.8% at GSH contents of 5.0 and 10.0 mM, respectively. Besides, the drug release shows much 7621

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Figure 6. DOX release profiles from PEG/Mn-UCNCs-DOX in PBS at varied pH values (A) and GSH contents (B), and combined acidic and reductive conditions (C). Emission intensity of PEG/Mn-UCNCs-DOX as a function of release time at pH = 5.5 and GSH = 10 mM PBS buffer (D). The integrated upconversion emission intensity and the R/G ratio as a function of the release time (E). Schematic illustration of DOX release from PEG/Mn-UCNCs-DOX along with the biodegradation of the silica shell (F).

enhanced efficacy in concurrent acidic and reductive condition (Figure 6C), which is due to the fast shell biodegradation. The upconversion emission spectra of the nanosystem in mild acidic and reductive PBS buffer versus release time are shown in Figure 6D. Because of the FRET between the nanocarrier and the DOX, the green-emissive peaks of the nanocarriers are greatly inhibited. With the release time boosted, the FRET effect is reduced because of the decreased DOX loading. As accepted, the FRET effect will disappear when the distance between the donor and the acceptor is more than 10 nm. Figure 6D depicts the variations of integrated emission intensity and the R/G ratio versus release time. It can be easily concluded that the efficient biodegradability of the silica shell brings a rapid enhancement of the integrated emission intensity. In particular, the biodegradation-enhanced DOX release leads to a faster recovery of the green-emissive peaks than red, further leading to a rapid decline of the R/G ratio. In Figure 6E, the R/G ratio decreases from 8.39 to 1.21 during a release time of 24 h. In conclusion, the UCL performance of the yolk-structured PEG/Mn-UCNCs-DOX can be used to reflect the drug release extent of the nanocarrier when used for in vivo antitumor therapy. This biodegradation-enhanced DOX release is very ideal for chemotherapeutic applications (Figure 6F). On one hand, the shell biodegradation-enhanced DOX release could enable the superior chemotherapy outcome. On the other hand, the adopted method is much easier than the construction of nanovalves into the mesopores of a shell for ondemand drug release.

To investigate the cellular uptake behavior of the resultant nanomedicine, the HeLa cancerous cells were incubated with PEG/Mn-UCNCs-DOX for 0.5, 1, and 3 h at 37 °C, and the corresponding CLSM images were exhibited in Figure 7. The blue-emissive DAPI was used to mark the cell nuclei. When excited with a 488 nm light, the loaded DOX can radiate red fluorescence. Correspondently, the merged channels are given. As shown, in the first 0.5 h, there is only feeble red emissions, which indicates that only slight PEG/Mn-UCNCs-DOX have been engulfed by HeLa cells. When incubation time increased, the red signal becomes stronger, implying that more particles are internalized in the cells. These results validate that the developed nanomedicine can be efficiently taken up by cancer cells. It should be noted that the uptake of the nanomedicine brings no morphology change to the cells, which proves the good biocompatibility of the developed nanosystem. The accumulation of PEG/Mn-UCNCs-DOX in cells was further proved by 3D fluorescence reconstruction of the Z-axis-scanned CLSM image (Figure S10) where the sample could be clearly found in the cell cytoplasm. According to knowledge, both the Mn2+- and Gd3+-doped nanomaterials have positive enhancing abilities of the T1 MRI signal,3,66 so we envisage that the combination of the NaGdF4based UCNPs and the Mn-doped silica shell deserve superior T1 MRI imaging performance. Here, the T1-weighted MRI imaging property of PEG/Mn-UCNCs after incubation for 24 h in PBS (pH = 7.4, GSH = 0 mM) and PBS (pH = 5.5, GSH = 10.0 mM) were investigated. As expected in Figure S11, due to 7622

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Figure 7. CLSM images of HeLa cells incubated with PEG/Mn-UCNCs-DOX for 0.5, 1, and 3 h at 37 °C. Scale bars, 50 μm.

the degraded paramagnetic Mn centers, the initial longitudinal relaxivity r1 of PEG/Mn-UCNCs in neutral and nonreductive PBS is as low as 0.55 mg−1 s−1 but markedly increased to 0.66 mg−1 s−1 at reductive and acidic PBS. Such a GSH- and pHresponsive MRI performance is caused by the shell biodegradation of PEG/Mn-UCNCs, which facilitates the interaction probability of water molecules with paramagnetic Mn centers. Besides, the MRI effect derived from the exposed UCNPs together with that caused by Mn release, performing an improved MRI performance. Therefore, PEG/Mn-UCNCs can play a role of tumor-responsive MRI contrast agent. As known, the CT imaging is reliable because it affords high-resolution three dimension structure details and deep tissue penetration. The Yb-doped materials have been studied extensively as CT imaging contrast agents.67,68 Herein, the in vitro and in vivo CT imaging effect of PEG/Mn-UCNCs was investigated. As displayed in Figure 8A, with the sample concentrations boosted, the CT signal increases obviously. Besides, the CT values show positive enhancement versus the concentrations with a high slope of 40.89 (Figure 8B). The in vivo CT imaging is displayed in Figure 8C and D. As shown, after injection with sample saline, the tumor site has a CT value of 214.8 Hounsfield units (HU), which is obviously higher than that without injection (45.2 HU). To demonstrate that the obtained sample can be used as an UCL imaging contrast agent and a FRET sensor of DOX release, HeLa cells were incubated with DOX-loaded PEG/MnUCNCs-DOX for 1 h and then further incubated with pure culture medium for 0.5, 1, and 3 h, respectively. As depicted in Figure 9, it is obvious that the particles in the cells emit upconverted emission (green and red) upon 980 nm laser excitation and the emissions become brighter with the incubation time prolonged. Note here, the green emission

Figure 8. In vitro CT images of PEG/Mn-UCNCs at different concentrations (A). The CT values of PEG/Mn-UCNCs versus concentrations (B). In vivo CT images of a tumor-bearing mouse before (C) and after injection (D).

markedly becomes stronger and stronger, while the intensity of red color only has slight enhancement, which reveals that the nanocarrier can be utilized for R/G-based FRET sensing of drug release (Scheme 2). Besides, no fluorescence signal was detected outside of the cells, whereas the signal located at the intracellular region implies that the as-prepared sample has been internalized into the cells instead of merely stained on the surface of the membrane. Moreover, the UCL signal is mainly located at the cell cytoplasm, which validates that the nanoparticles are engulfed by endocytosis through endosomes and lysosomes into the cells rather than passive adsorption. 7623

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Figure 9. UCL microscopy images of HeLa cells incubated with PEG/Mn-UCNCs-DOX at 37 °C for 1 h and then further incubated with pure culture medium for 0.5, 1, and 3 h. Scale bars, 20 μm.

Figure 10. Cell viabilities of L929 fibroblast cells with incubation of PEG/Mn-UCNCs for 24 and 48 h (A). Cytotoxicity of free DOX, PEG/ UCNPs@mSiO2-DOX, and PEG/Mn-UCNCs-DOX against HeLa cells (B). CLSM images of HeLa cells after various treatments, dyed with PI. Scale bars, 50 μm (C).

concentration range even at 600 μg mL−1 after 48 h incubation, indicating the nanosystem is low-toxic. Also, the in vitro cytotoxicity of PEG/Mn-UCNCs-DOX against HeLa cells was evaluated by MTT assay, as displayed in Figure 10B. In comparison with equivalent DOX, the cells treated with PEG/ UCNPs@mSiO2-DOX show much lower viabilities, indicating that the DOX delivered by PEG/UCNPs@mSiO2 can achieve improved therapeutic outcome of DOX against cancerous cells.

These results imply that the invented nanostructure with UCNPs loaded inside is an effective contrast agent for in vitro UCL imaging with ignorable background. As accepted, the biocompatibility of the as-prepared sample should be first assessed before practical application. Figure 10A shows the viabilities of L929 cells after incubating with the PEG/Mn-UCNCs at various concentrations for 24 and 48 h. The sample shows a high viability of 98.1−99.4% in the whole 7624

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Figure 11. Variations of body weights (A) and the relative tumor volume (B) achieved from the mice with different treatments. Photographs of representative mice and excised tumors (C). H&E stained images of tumor tissues obtained after 14 days therapy. Scale bars, 100 μm (D).

tumors collected from representative mice also confirm that the tumor volume of the PEG/Mn-UCNCs-DOX treated group is the smallest, revealing its highest antitumor efficacy. The H&E staining images in Figure 11D showed the most serious cell necrosis after the PEG/Mn-UCNCs-DOX therapy, which is consistent with Figure 11C. In Figure S12, the H&E staining images of the main organs, including the liver, lung, kidney, heart, and spleen, are shown. It can be seen that there is no evident organ damage in all groups, uncovering the excellent in vivo biocompatibility of the samples. Additionally, the major blood indexes and liver-function indexes of mice injected with PEG/Mn-UCNCs (Table S1) show no obvious abnormity and difference when compared with mice in the control group, further demonstrating the high biocompatibility of the nanoplatform.

The PEG/Mn-UCNCs-DOX exhibits much higher cell killing efficacy than PEG/UCNPs@mSiO2-DOX, which is due to the fast intracellular biodegradation of the silica shell to trigger the DOX release and enhance the therapeutic result. Moreover, PI which could dye dead cells with a red color was applied to highlight dead cells under varied conditions to demonstrate the cell killing efficacy.69 As shown in Figure 10C, it is reasonable that nearly no red cells can be observed for the cells incubated with pure culture medium. For the cells treated with medium containing free DOX, a small amount of red cells can be seen, which implies that a small number of cells have been killed by DOX. In comparison with free DOX, more red cells can be seen in the PEG/UCNPs@mSiO2-DOX treated group, which is in accordance with the results in Figure 10B. As expected, the rightmost picture for the group treated with medium having PEG/Mn-UCNCs-DOX (Figure 10C) has the largest proportion of dead cells. The CLSM observation and the in vitro cytotoxicity assay validate that PEG/Mn-UCNCs pose efficient DOX delivery into cancerous cells and exert superior anticancer effectiveness. Encouraged by the above in vitro therapy outcomes, U14 tumor xenograft was implanted on female mice for in vivo chemotherapy. The therapy effect was assessed by intravenous (i.v.) injection of nanomedicines (PEG/UCNPs@mSiO2-DOX and PEG/Mn-UCNCs-DOX) at an equivalent DOX dose of 2 mg kg−1. Figure 11A and B display the mean body weights and relative tumor volume of mice in each group during 14 days treatment. The body weights in three groups have no abnormal fluctuation over the investigation period, demonstrating low toxicity of samples to the health of mice. When compared with the control group, the tumor growth on the PEG/Mn-UCNCsDOX treated group is greatly inhibited over a course of 14 days therapy. The tumor growth on the PEG/UCNPs@mSiO2DOX injected group was slightly inhibited after 2 weeks treatment. The higher tumor-inhibition of PEG/Mn-UCNCsDOX is due to the highly selective particle accumulation at tumor tissues and the efficient DOX release caused by shell biodegradation. In Figure 11C, the digital photographs of

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CONCLUSIONS In a sum, a novel yolk-structured nanotheranostic integrating biodegradable silica shell and UCNPs were innovated for tumor microenvironment-responsive chemotherapy and bioimaging. These yolk-structured Mn-UCNCs with large inner space and mesoporous silica shell were simply constructed by treating UCNPs@mSiO2 under a hydrothermal condition. The adopted Mn-doping strategy enables the biodegradation of the silica shell on Mn-UCNCs in either mild reductive or acidic condition by disintegrating the Mn−O bonds and subsequent initializing Mn release, which further accelerates the shell degradation. This is the first invention of yolk upconversion nanostructure with mesoporous silica shell which is responsive to tumor condition. Importantly, the biodegradation of the shell not only promotes DOX release but also enhances T1weighted MRI due to the Mn release. The integrated UCNPs endow the Mn-UCNCs with CT, MRI, and UCL imaging functions upon a single NIR laser irradiation. Additionally, the biodegradability-enhanced DOX release brings a rapid decline to the R/G ratio, which can act as a sensor for FRET sensing of drug release extent. The PEG/Mn-UCNCs-DOX poses higher anticancer effectiveness than those of free DOX or DOX7625

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loaded PEG/UCNPs@mSiO2, revealing its potency in imagingguided chemotherapy fields.

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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/acs.chemmater.7b03461. Characterization of UCNPs@SiO2; XRD pattern of assynthesized NaGdF4:Yb,Er nanoparticles; TEM images of UCNPs@mSiO2 under different hydrothermal treatment durations; XPS spectrum of Mn-UCNCs; FT-IR spectrum, TEM images before and after biodegradation, and BET tests of PEG/Mn-UCNCs-DOX; the UV−vis analysis of the DOX loading rate of PEG/UCNPs@ mSiO2; DLS test of PEG/Mn-UCNCs after biodegradation; MR imaging tests of the PEG/Mn-UCNC before and after biodegradation; 3D CLSM images of HeLa cells incubated with PEG/Mn-UCNCs-DOX for 1 h; H&E stained images of the tissues in the three groups; and blood biochemistry and hematology data of PEG/MnUCNCs (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (F. H.). *E-mail: [email protected] (P. Y.). *E-mail: [email protected] (J. L.). ORCID

Ruichan Lv: 0000-0002-6360-6478 Piaoping Yang: 0000-0002-9555-1803 Jun Lin: 0000-0001-9572-2134 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (NSFC 51472058, 51502050, 51422209, 51720105015, 51628201, and 51572258), the project for science and technology development plan of Jilin province (20170414003GH), Outstanding Youth Foundation of Heilongjiang Province (JC2015003), Special Financial Grant from the China Postdoctoral Science Foundation (2015T80321), PhD Student Research and Innovation Fund of the Fundamental Research Funds for the Central Universities (HEUGIP201713), and the Fundamental Research funds for the Central Universities is greatly acknowledged.

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REFERENCES

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Chemistry of Materials

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DOI: 10.1021/acs.chemmater.7b03461 Chem. Mater. 2017, 29, 7615−7628

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DOI: 10.1021/acs.chemmater.7b03461 Chem. Mater. 2017, 29, 7615−7628