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Multifunctional Core@Shell Magnetic Nanoprobes for Enhancing Targeted Magnetic Resonance Imaging and Fluorescent Labeling in Vitro and in Vivo Qian Zhang,† Ting Yin,† Guo Gao,*,† Joseph G. Shapter,‡ Weien Lai,§ Peng Huang,† Wen Qi,† Jie Song,† and Daxiang Cui*,† †

Institute of Nano Biomedicine and Engineering, Key Laboratory for Thin Film and Microfabrication Technology of the Ministry of Education, Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China ‡ School of Chemical and Physical Sciences, Flinders University, Bedford Park, Adelaide 5042, Australia § Academy of Photoelectric Technology, HeFei University of Technology, HeFei 230009, China S Supporting Information *

ABSTRACT: Core@shell magnetic nanoparticles (core@ shell MNPs) are attracting widespread attention due to their enhancement properties for potential applications in hyperthermia treatment, magnetic resonance imaging (MRI), diagnostics, and so forth. Herein, we developed a facile thermal decomposition method for controllable synthesis of a superparamagnetic, monodispersed core@shell structure (Co@Mn = CoFe2O4@MnFe2O4) with uniform size distribution (σ < 5%, dc ≈ 15 nm). The CoFe2O4 core could enhance magnetic anisotropy, and the MnFe2O4 shell could improve the magnetization value. The Co@Mn MNPs were transferred into aqueous solution with an amphiphilic polymer (labeled 2% TAMRA) and functionalized with PEG2k and target molecules (folic acid, FA) to fabricate multifunctional PMATAMRA-Co@Mn-PEG2k-FA nanoprobes. The obtained PMATAMRACo@Mn-PEG2k-FA nanoprobes exhibit good biocompatibility, high T2 relaxation values, and long-term fluorescence stability (at least 6 months). Our results demonstrate that the synthesized PMATAMRA-Co@Mn-PEG2k-FA nanoprobes can effectively enhance the targeted MRI and fluorescent labeling in vitro and in vivo. The research outcomes will contribute to the rational design of new nanoprobes and provide a promising pathway to promote core@shell nanoprobes for further clinical contrast MRI and photodynamic therapy in the near future. KEYWORDS: core@shell, magnetic nanoprobes, MRI, fluorescent labeling, biodistribution

1. INTRODUCTION

excellent superparamagnetic behavior and maximal hysteretic loss (SLP) under an alternating magnetic field, and they have been recognized as new types of magnetic nanomaterials for hyperthermia applications.13,16−18 The properties of these core@shell MNPs are controlled via the core/shell ratio and Co-ferrite content, and CoFe2O4@MnFe2O4 MNPs have been shown to have an optimal anisotropy as Co content reaches around 15%.13 Additionally, CoFe2O4@MnFe2O4 MNPs exhibit high T2 relaxation values, meaning they could be potential materials for MRI. Hence, CoFe2O4@MnFe2O4 MNPs could become one of the most promising nanomaterials for tumor diagnosis and therapy in the near future. However, in vivo mode, the majority of the injected nanomaterials will

In recent decades, magnetic nanoparticles (MNPs) have exhibited great potential for medical applications,1 including targeted drug delivery,2,3 molecular detection and separation,4−6 magnetic resonance imaging (MRI),1,7 in vitro testing,8 magnetic fluid hyperthermia treatment,9 and so forth. These applications mainly take advantage of their unique, sizedependent magnetic properties as well as their stability, biodegradability, and nontoxic properties.10 Recently, with precision medicine becoming increasingly important, mulltifunctional MNPs with controllable shape, structure, and size, as well as enhanced magnetic properties have become significant in various fields.11−15 Core@shell architectures with combinations of CoFe2O4 and MnFe2O4 are novel representative examples, especially for CoFe2O4@MnFe2O4 (hard phase CoFe2O4 as core and soft phase MnFe2O4 as shell). The CoFe2O4@MnFe2O4 presents © 2017 American Chemical Society

Received: March 27, 2017 Accepted: May 10, 2017 Published: May 10, 2017 17777

DOI: 10.1021/acsami.7b04288 ACS Appl. Mater. Interfaces 2017, 9, 17777−17785

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of Fabricated PMATAMRA-Co@Mn-PEG2k-FA Nanoprobes and Their Multifunctional Labeling in Vitro and in Vivo

2. EXPERIMENTAL SECTION

aggregate in organs after the blood circulates and induce lesions,19 and only a small amount of the nanomaterial will be localized in the target tumor site. To overcome these drawbacks and improve the specific targeting efficiency, designing multifunctional nanoprobes with a low dosage and excellent magnetic properties for fluorescence labeling and MRI in vivo will be very valuable. Many researchers have reported the applications of MNPs for multimodal imaging and tumor therapy based on magnetic hyperthermia or photodynamic therapy.9,20,21 However, the poor quality of the MNPs (e.g., stability, dispersion, and surface functional groups) always induces severe agglomeration during blood circulation and consequently affects the targeting efficiency of tumor labeling and therapy.22 Therefore, the rational design and synthesis of high-quality magnetic nanoprobes for imaging and therapy of tumors are of great interest. In this study, we synthesized monodisperse CoFe2O4@ MnFe2O4 MNPs (Co@Mn MNPs) with a highly uniform size (dc = 15.1 ± 1.2 nm). The hydrophobic Co@Mn MNPs were then transferred into aqueous solution using a TAMRA-labeled polymer, followed by functionalization with a targeting species (folic acid (FA)) using a PEGylation approach, which yields a new nanoprobe, PMATAMRA-Co@Mn-PEG2k-FA. The synthesized Co@Mn MNPs and their corresponding nanoprobes exhibit excellent superparamagnetic properties and outstanding MRI capacity. The TAMRA-labeled amphiphilic polymer can achieve the phase transfer of hydrophobic Co@Mn MNPs into aqueous solution, where highly stable yellow fluorescence on the NP surface is observed. PEGylation is a routine strategy to reduce the cell uptake to minimize cytotoxicity as well as prolong the half-life of nanoprobes in the blood by extending their circulation time.23,24 Moreover, FA molecules are specific targeting molecules for MGC 803 cell lines of gastric tumors. Attaching FA on the terminal COO− groups of NPs via conjugation of N2H-PEG-NH2 molecules can enhance the cell uptake of cultured MGC 803 cell lines in vitro and improve the specific targeting efficiency into MGC 803 tumors in vivo.25,26 Scheme 1 illustrates the fabrication process of core@shell magnetic nanoprobes and their multifunctional labeling in vitro and in vivo.

2.1. Chemicals and Materials. Iron(III) acetylacetonate (Fe(acac) 3 , 98%), cobalt(II) acetylacetonate (Co(acac)2 , 97%), manganese(II) acetylacetonate (Mn(acac)2, 97%), oleic acid (OLA, 90%), oleylamine (OLAM, 80−90%), benzyl ether (95%), dodecylamine (DoCA, 98%) and poly(ethylene glycol) (NH2-PEG-NH2, PEG2k, Mw: 2 kDa) were purchased from Aladdin. 1,2-Hexadecanediol (90%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), poly(isobutylene-alt-maleic anhydride)(PMA, Mw ∼ 6 kDa), and an In Vitro Toxicology Assay Kit, Resazurin based (Resazurin) were obtained from Sigma-Aldrich. FA, hexane (98%), chloroform (99%), and tetrahydrofurane (THF, 99.8%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Tetramethylrhodamine-5-carboxamide cadaverine (TAMRA) was ordered from Anaspec, Inc. and Invitrogen Corporation. Centrifuge filters were obtained from Millipore Corporation. All chemicals were used without further purification. 2.2. Synthesis of Monodisperse Co@Mn MNPs. The synthesis of Co@Mn MNPs was carried out by a thermal decomposition method, as described previously.13 To obtain the core@shell structure, the synthesis of Co@Mn MNPs was separated into producing CoFe2O4 seeds and MnFe2O4 shell growth. CoFe2O4 seeds were synthesized via a one-pot synthesis process.27 In detail, 0.71 g of Fe(acac)3, 0.26 g of Co(acac)2, and 2.58 g of 1,2hexadecanediol were weighed in a 100 mL three-neck flask, followed by the addition of 1.69 g of OLA, 1.6 g of OLAM, and 20 mL of benzyl ether. Under magnetic stirring for various times, the chemicals were degassed for 20 min at 100 °C with vacuum. Then, with a flow of N2 protection, the mixture was slowly heated up to 200 °C and maintained at this temperature for 2 h then heated again to 300 °C and left for 1 h. During this time, the mixture solution changed color from brown to black. Finally, the reaction was stopped by removing the heating mantle. The precipitates from the black solution were separated using the addition of 40 mL of ethanol followed by centrifugation (2800 rcf, 10 min). The recovered black precipitates were dispersed in hexane and washed with ethanol again. Finally, the CoFe2O4 seeds were dissolved in hexane and kept at a concentration of 20 mg/mL. Co@Mn MNPs were synthesized by growing a MnFe2O4 shell on the CoFe2O4 seeds.13 Briefly, 0.71 g of Fe(acac)3, 0.25 g of Mn(acac)2, and 2.58 g of 1,2-hexadecanediol were weighed in a 100 mL three-neck flask followed by the injection of 0.56 g of OLA, 0.53 g of OLAM, and 20 mL of benzyl ether, and 2 mL of CoFe2O4 seed solution. From this mixture, the hexane was removed by vacuum, and then under N2 protection, the temperature of the mixture was increased to 200 °C for 1 h, and kept heated under reflux for 0.5 h. At last, the reaction was 17778

DOI: 10.1021/acsami.7b04288 ACS Appl. Mater. Interfaces 2017, 9, 17777−17785

Research Article

ACS Applied Materials & Interfaces stopped by removing the heating mantle, and the same cleaning steps described earlier were repeated. After cleaning, the obtained Co@Mn MNPs were dissolved in chloroform. 2.3. Synthesis of TAMRA-Labeled Amphiphilic Polymer. The 2% TAMRA-labeled amphiphilic polymer (PMATAMRA) was synthesized by reacting PMA with DoCA and TAMRA.28 As per the typical synthesis process, as a hydrophilic back bone, 75% of the anhydride rings from PMA were linked with amino groups of DoCA as hydrophobic side chains, as well as 2% of TAMRA (structure of polymer shown in Figure S1). Briefly, 79.5 μg of PMA (∼0.0795 mmol) was mixed with 69.5 μg of DoCA (0.0695 mmol) in 20 mL of anhydrous THF in a 100 mL round-bottomed flask. After sonication for several seconds, 5 mg of TAMRA was added into the mixture. Afterwards, the solution was kept at 65 °C overnight with magnetic stirring. After 24 h, the pink color solution was completely evaporated by rotavapor and redispered in 5 mL of chloroform to obtain a final concentration of 0.1 M. 2.4. Polymer Coating of Co@Mn MNPs. The polymer coating process used a previously reported method.28 In this work, the amphiphilic polymer was mixed with hydrophobic Co@Mn MNPs in a round-bottomed flask with a ratio of Rp/Area = 300 monomers/nm2, where Rp/Area was described by the ratio of polymer per particle surface. Then, the solvent was evaporated slowly until the sample completely dried. Afterwards, sodium borate buffer (SBB) 12 buffer (50 mM, pH 12.0) was added to dissolve the polymer-coated samples. During the polymer coating process, hydrophobic side chains were intercalated with aliphatic ligands on the particle surface, and maleic anhydrides of the hydrophilic backbone were opened into carboxy groups, which supplied negative charges for stabilization in aqueous solution. After the reaction, the PMATAMRA-Co@Mn MNPs were washed three times to remove free polymer with centrifugation (17 000 rpm, 1 h). 2.5. Fabrication of PMATAMRA-Co@Mn-PEG2k-FA Nanoprobes. PMATAMRA-Co@Mn MNPs were gradually functionalized by PEG2k and FA via EDC chemistry.21,29 First, PMATAMRA-Co@Mn MNPs (1 μM, 1 mL) were mixed with 100 mg of PEG dissolved in 5 mL of SBB 9.0 buffer (50 mM, pH 9.0), then 57 mg of EDC in SBB9.0 was added. The mixture was kept at room temperature overnight to achieve the reaction of NPs with PEG. After 24 h, a centrifuge filter (Millipore, 100 kDa) was applied to remove free EDC and PEG2k to obtain concentrated PMATAMRA-Co@Mn-PEG2k. The activated FA dispersed in SBB9.0 buffer (3 mg/mL, 3 mL) was sequentially added to the reaction mixture and left overnight. Finally, free FA was washed away with a centrifuge filter via centrifugation (3000 rpm, 10 min) to obtain the cleaned nanoprobes, and the sample was kept in Milli-Q water. 2.6. Characterization. The morphology of the synthesized MNPs was imaged by transmission electron microscopy (TEM, JEM-2100f, Japan). The UV−vis spectra and fluorescence spectra of all samples were recorded by a UV−vis spectrophotometer (Varian Inc., Palo Alto, CA) and fluorescence measurements were taken using a fluorescence spectrophotometer (Hitachi FL-4600, Japan). The hydrodynamic diameters (dh) and zeta potential (ζ) values dispersed in H2O of all of the samples were measured by dynamic light scattering (DLS) using a Zetasizer Nano (Malvern Instruments Ltd., U.K.). Hysteresis curves of the MNPs and nanoprobes at 300 K were obtained by using a superconducting quantum interference device magnetometer (SQUID) with a magnetic applied field of 3 T, and in vitro MRI and r2 values were recorded by low-field nuclear magnetic resonance (NMR, 0.5 T, Niumag, Shanghai, China). 2.7. Cell Culture and Cell Viability Study by Resazurin Assay. The MGC-803 cell line was acquired from the Cell Bank (Chinese Academy of Sciences, China). The cells were regularly incubated under 37 °C and 5% CO2, in Dulbecco’s modified Eagle’s medium (HyClone) media (10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 0.1 mg/mL streptomycin). Viability studies of cells treated with nanoprobes (PMATAMRA-Co@Mn-PEG2k-FA) at serial concentrations were measured by resazurin assay according to the changes of fluorescence intensity (λex: 560 nm, λem: 587 nm).30 To identify the specific targeting of MGC-803 cells by FA, nanoprobes in the absence of FA (termed as the no target control) were studied as a

parallel experiment. Briefly, MGC-803 cells at the logarithmic phase of cell growth were seeded into a 96-well plate (cell density/well: 1 × 104) and incubated overnight at 37 °C (5% CO2). After 24 h, the growth medium solution from each well was aspirated and rewashed by phosphate-buffered saline (PBS) buffer. Then, 100 μL of fresh media containing various concentrations of nanoprobes and the no target sample from 7.5 to 180 nM was added, whereas for other cells, just the media with PBS was added as the negative control. After incubation for 24 h, the cells were rinsed by PBS and fresh media containing 10% resazurin solution was added. After incubation for another 4 h, the cell viability was analyzed by microplate reader, and the changes of the fluorescence intensity were measured on the basis of the following equation

cell viability (%) =

I587 nm (experimental group) × 100% I587 nm (negative control)

where I is the fluorescence intensity at 587 nm, the cell viability without added MNPs was considered as 100%. 2.8. Cellular Uptake Analysis in Vitro. Cell uptake analysis of the nanoprobes was conducted by confocal laser scanning microscopy (CLSM, Leica, Germany) and flow cytometry (FCM, BD FACS Calibur). During the CLSM study, MGC-803 cells were first plated at a density of 4.0 × 104 cells/well overnight. Then, nanoprobes and the equivalent no target control (30 nM, 100 μL) were added separately into the cells and incubated for 6, 12, and 24 h. Afterward, the cells were rinsed by PBS and fixed by adding 2.5% paraformaldehyde for 0.5 h. The nuclei of cells were stained by Hoechst 33342 for 5 min. Thus, fluorescence signals from Hoechst 33342 and TAMRA were separately collected through the 420−460 and 560−600 nm barrier filters under laser irradiation of CLSM. In addition, the uptake efficiency of nanoprobes can be measured by FCM through analysis of the yellow fluorescence of TAMRA on the nanoprobes. The MGC-803 cell line was cultured in 24-well plates (cell density: 7.0 × 104 cells/well) overnight, and then, the cells were treated with the same amount of nanoprobes and no target control for various times. All of the cells were separately rinsed with PBS, trypsinized, and resuspended in 500 μL of PBS for FCM measurements, collecting signals using the FL2 channel. 2.9. In Vivo Fluorescence Imaging and Biodistribution Analysis. Female BALB/c-nude mice (age: 6−8 weeks, weight: ∼20 g, purchased from Shanghai Slac Lab Animal Co., Ltd.) were raised under compliance of the Institutional Animal Care and Use Committee from Shanghai Jiao Tong University (SCXK-2012-0002). The mice were anesthetized using 8% chloral hydrate and MGC-803 tumors (size: ∼30 mm3) were embedded into the right flank of the mice for establishing gastric tumor-bearing mice. After the tumor size reached 100−150 mm3, all of the tumor-bearing mice were randomly divided into three groups and separately injected with 200 μL of different samples through the caudal vein: (1) 1× PBS solution as negative control, (2) nanoprobes (200 nM, 200 μL) in 1× PBS solution, and (3) the samples of the no target control (PMA-Co@MnPEG2k, 200 nM, 200 μL) in 1× PBS solution. All of the tumor-bearing mice were dissected after 24 h, the major organs (heart, liver, spleen, lung, and kidney) and tumors were separated from the body. Fluorescence signals from the organs were analyzed and quantified by using Bruker Molecular Imaging Software version 7.1. All of the organs and tumors were digested by aqua regia (HCl/HNO3, 3:1 v/v), and the elements of Co, Fe, and Mn were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS). 2.10. In Vitro and in Vivo MR Imaging. In vitro MR imaging was carried out with low-field nuclear magnetic resonance. Spin−spin relaxation time (T2) and the r2 value of PMATAMRA-Co@Mn-PEG2kFA nanoprobes were measured by changing the Fe concentration (0.035, 0.07, 0.14, and 0.56 mM). To investigate the MR imaging in vivo, T2-weighted images of all of the nude mice after intravenous injection of nanoprobes were recorded by using a Magnetom Trio medical MR system (3.0 T, Siemens) from the Sixth People’s Hospital (Radiology department, Shanghai). During the study, anesthetized mice were placed in an animal-specific body 17779

DOI: 10.1021/acsami.7b04288 ACS Appl. Mater. Interfaces 2017, 9, 17777−17785

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ACS Applied Materials & Interfaces

Figure 1. Characterizations of PMATAMRA-Co@Mn-PEG2k-FA nanoprobes. (a) TEM micrograph of Co@Mn MNPs evaporated from CHCl3 with a diameter of 15.1 ± 1.2 nm; (b) negatively staining TEM image of PMATAMRA-Co@Mn MNPs, which exhibit a homogeneous polymer shell around the MNPs with a thickness of 2.72 ± 0.67 nm; (c) HRTEM micrograph of one single Co@Mn MNP; (d) scheme of amphiphilic polymer coated with one particle; (e, f) diameter values (dh) and ζ values measured by DLS of Co@Mn MNPs in CHCl3 (I), PMATAMRA-Co@Mn MNPs in H2O (II), PMATAMRA-Co@Mn MNPs-PEG2k in H2O (III), and PMATAMRA-Co@Mn MNPs-PEG2k-FA nanoprobes in H2O (IV). The scale bar for the HRTEM image is 10 nm.

Figure 2. (a) M−H curves of PMATAMRA-Co@Mn MNPs (MNPs, black) and PMATAMRA-Co@Mn-PEG2k-FA nanoprobes (nanoprobes, red) measured at 300 K by SQUID. The inset in figure (a) shows the M−H curves in a low magnetic field; (b) T2-weighted MRI images of PMATAMRACo@Mn MNPs (upper) and nanoprobes (lower) dispersed in H2O with various Fe concentrations measured at 25 °C under 0.5 T magnetic field; (c) T2 relaxation rates (1/T2) of PMATAMRA-Co@Mn MNPs (black) and PMATAMRA-Co@Mn-PEG2k−FA nanoprobes (red). coil and MRI images were recorded over various time intervals (preinjection, 0.5, 12, and 24 h). Finally, the obtained image data were analyzed by MR imaging software.

TAMRA) was synthesized, and the PMA-coated Co@Mn MNPs to achieve phase transfer were designed. During the polymer coating (scheme is presented in Figure 1d), the hydrophobic sidechains intercalated with the aliphatic ligands on the NPs’ surface, whereas the hydrophilic backbone containing −COO− groups facilitates the phase transfer. Thus, PMATAMRA-Co@Mn MNPs can be successfully transferred into aqueous solution. The large number of carboxylic groups, applied for high stability, coupled with a labeled dye can give the NPs a yellow fluorescence under laser irradiation, which has long-term stability for at least 6 months without quenching.28 Furthermore, the negative-staining TEM image of PMATAMRA-Co@Mn MNPs demonstrate that the MNPs are well dispersed in H2O with a polymer shell thickness of 2.72 ± 0.67 nm (cf. Figures S3b and 1b).31 Subsequently, PEG2k and FA was gradually functionalized on the surface of NPs via EDC

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization. Co@Mn MNPs presented a core@shell structure formed by growing a MnFe2O4 shell on the surface of CoFe2O4 seeds. The crystal phase and elemental composition of the core@shell architecture were investigated by STEM imaging and EELS spectra, respectively (cf. Figure S2). TEM (Figures S3a and 1a) of CoFe2O4 seeds and Co@Mn MNPs demonstrates that they exhibit a highly uniform size distribution (σ < 5%). The highresolution TEM (HRTEM) in Figure 1c shows the Co@Mn MNP exhibits a spherical structure with fringe distances of 0.25 and 0.16 nm. The amphiphilic polymer (labeled with 2% 17780

DOI: 10.1021/acsami.7b04288 ACS Appl. Mater. Interfaces 2017, 9, 17777−17785

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ACS Applied Materials & Interfaces

Figure 3. (a) Viability of MGC-803 cells upon incubation by nanoprobes and the no target control for over 24 h at 37 °C with concentration ranging from 7.5 to 180 nM (the converse concentration ranges from 42 to 1000 μg/mL); (b) thin-section cell TEM images of nanoprobes with concentration of 30 nM taken into MGC 803 cells incubated for 24 h. The black dots demonstrate the enrichment of nanoprobes in the lysosome; (c) the zoomed-in image of aggregated nanoprobes in the black box in b.

Figure 4. (a) Confocal microscopy images of PMATAMRA-Co@Mn-PEG2k-FA nanoprobes and no target control in cell lines of MGC-803 for 6, 12, and 24 h. The Hoechst 33342 (nuclei) was excited with a 405 nm laser diode, and TAMRA molecules were excited with a 545 nm laser diode. The emission of Hoechst 33342 was filtered with a 440−470 nm band-pass, whereas TAMRA-labeled nanoprobes were imaged with a 576 nm long-pass filter. (b) FCM images display the cellular uptake of nanoprobes and no target control based on the fluorescence of TAMRA. Red and blue histograms represent the cellular uptake of nanoprobes and no target control, respectively. The scale bars are 100 nm.

to zero, −35.8 ± 1.2, −25.4 ± 1.6, and −28.5 ± 2.0 mV, respectively. dh values of nanoprobes at different NaCl concentrations and pH values are summarized in Tables S1 and S2, respectively. Figure S3c shows the UV−vis absorption spectra of PMATAMRA-Co@Mn MNPs and nanoprobes. Compared to that in the MNPs, after functionalization, there is evident absorption at around 350 nm in the spectra of the nanoprobes due to FA, demonstrating the successful attach-

chemistry to fabricate the PMATAMRA-Co@Mn-PEG2k-FA nanoprobes. According to the hydrodynamic diameters (dh) and ζ value of all of the samples using a Malvern Zetasizer (Figure 1e,f), the sizes of the four samples (Co@Mn MNPs in CHCl3, PMATAMRA-Co@Mn MNPs, PMATAMRA-Co@MnPEG2k, and PMATAMRA-Co@Mn-PEG2k-FA nanoprobes) are measured to be 13.1 ± 0.25, 16.5 ± 0.4, 25.3 ± 0.7, 25.7 ± 1.1 nm, respectively. However, the ζ values of the samples are close 17781

DOI: 10.1021/acsami.7b04288 ACS Appl. Mater. Interfaces 2017, 9, 17777−17785

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ACS Applied Materials & Interfaces ment of the targeting molecule. The fluorescence emission spectra of the nanoprobes (cf. Figure S3f) presents a fluorescent peak at 575 nm under excitation at 544 nm. The concentrations of nanoprobes are determined by ICP-MS to analyze the elements of cobalt (Co), manganese (Mn), and iron (Fe), and the concentration calculation is exhibited in Table S3. 3.2. Magnetic Properties and T 2-Weighted MR Relaxometry. Iron oxide NPs are generally used as T2weighted MR contrast agents due to their excellent magnetic properties, especially for Co@Mn MNPs, which exhibit excellent superparamagnetic behavior and ideal spin−spin relaxation times (T2). From Figure 2a, we can see that the M−H curves of PMATAMRA-Co@Mn MNPs present smooth curves without any hysteresis at 300 K, with saturated magnetization values (Ms) of 82 emu g−1, and the fabricated PMATAMRA-Co@Mn-PEG2k-FA nanoprobes also provide excellent M−H curves with Ms of 68 emu g−1. Moreover, the slope for the nanoprobes is even higher than that of the Co@ Mn MNPs in a low magnetic field (cf. inset image of Figure 2a), which means the fabricated nanoprobes still exhibit outstanding Ms values after functionalization with biomolecules. The results show that the nanoprobes present excellent superparamagnetic behavior and are suitable for further application in MR imaging in vitro and in vivo. The magnetic properties were further studied by measuring the spin−spin relaxation time (T2) of PMATAMRA-Co@Mn MNPs and nanoprobes via varying Fe concentrations from 0.035 to 0.56 mM using a 0.5 T MRI system (cf. Figure 2b,c). Both of them exhibit significant MRI contrast enhancement with increasing Fe concentration, and the T2 relaxation rate (1/T2) increases linearly with varying Fe concentrations, with slopes, r2, of 255.7 and 167.8 mM−1 s−1. The high contrast property of the nanoprobes highlights their potential ability for MRI in vivo. 3.3. Cellular Uptake Assay. The cytotoxicity study of PMATAMRA-Co@Mn-PEG2k-FA nanoprobes was performed using a resazurin assay with the MGC-803 cell line. Various concentrations of nanoprobes and no target control (PMATAMRA-Co@Mn-PEG2k), from 7.5 to 180 nM (the converse concentration is from 42 to 1000 μg/mL), were incubated with MGC-803 cells for 24 h at 37 °C (PBS solution in the absence of nanomaterials as the negative control). After treatment with resazurin and cell viability analysis, as shown in Figure 3a, there is no evident toxicity in the cells incubated with nanomaterials below a concentration of 80 nM (converse concentration: 444 μg/mL) in which the cell viability is over 90%, and cell damage appeared only when the concentration of nanomaterials reached over 180 nM (converse concentration: 1000 μg/mL) and the cell viability decreased to 55%; this result indicated that the nanoprobes have excellent biocompatibility in the cellular assay, except at an extremely high dosage. The cell uptake and pathway of PMATAMRA-Co@Mn-PEG2k-FA nanoprobes was elucidated by thin-section cell TEM images (Figure 3b). After cell uptake, most of the nanoprobes have been enriched into the lysosome, which is clearly visible from the cell (Figure 3c). We further investigated the cellular uptake and quantification analysis of nanoprobes in the MGC-803 cell line by CLSM experiments and the FCM system. As shown in Figure 4a, there is no significant yellow fluorescence after 6 h of incubation compared to that in the no target control, suggesting a low uptake of MNPs by cells. In contrast, bright yellow fluorescence can be clearly observed with incubation of equal amounts of

nanoprobes after 6 h incubation. This result is confirmed by the average fluorescence intensity measured by FCM. From these results, we can conclude that as a specific target molecule for MGC-803 cell lines, FA can significantly enhance the cellular uptake after being functionalized onto the surface of nanoprobes. Meanwhile, an increase of yellow fluorescence has been detected after increasing the cell incubation time from 6 to 24 h, which clearly demonstrated that more MNPs are taken into the cells with longer incubation time. 3.4. In Vivo MR Imaging. To evaluate the efficiency of PMATAMRA-Co@Mn-PEG2k-FA nanoprobes as the T2 contrast agent in vivo, nanoprobes (200 nM, 200 μL, dispersed in 1× PBS solution) and equal amounts of PMATAMRA-Co@Mn MNPs were intravenously injected into MGC-803 tumorbearing mice. MR images (cf. Figure 5a) were recorded at

Figure 5. In vivo MR images. (a) MR images of the MGC-803 tumorbearing mice (white dashed circle positions) before and after intravenous injection of PMATAMRA-Co@Mn MNPs and PMATAMRACo@Mn-PEG2k-FA nanoprobes at a time range from 0.5 to 24 h; (b) the corresponding value of quantification of tumor contrast collected at a time range from the untreated time until 24 h.

different time intervals, and MRI values around the tumors, shown in the white dashed circle area, were collected and analyzed for the MRI efficiency of the nanoprobes (cf. Figure 5b); the intensity (I) of the tumor site, from the MRI image, was found to be inversely proportional to the amount of nanoprobes in the relevant area. As we can see, due to the enhanced permeability and retention effect on the tumor, both PMATAMRA-Co@Mn MNPs and PMATAMRA-Co@Mn-PEG2kFA nanoprobes injected through the tail vein could enrich the tumor site during blood circulation over time, with the resulting decrease of MRI intensity. Meanwhile, FA molecules can further enhance the specific targeting to the MGC-803 tumor. Therefore, there is an obvious darkening effect at the tumor site after the injection of nanoprobes compared to those of PMATAMRA-Co@Mn MNPs. Besides, both samples decreased the T2 signal value to the lowest value at 12 h, which means the injected nanomaterials gradually enriched the tumor sites 17782

DOI: 10.1021/acsami.7b04288 ACS Appl. Mater. Interfaces 2017, 9, 17777−17785

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ACS Applied Materials & Interfaces

Figure 6. (a, b) Ex vivo fluorescence images and fluorescence intensity analysis of major organs and tumors of MGC-803 tumor bearing mice after injection of PMATAMRA-Co@Mn-PEG2k-FA nanoprobes and the no target control (PMATAMRA-Co@Mn-PEG2k) for 24 h; (c−e) quantitative biodistributions over 24 h of three elements (Fe, Co, and Mn) from nanomaterials in dissected organs and tumors based on the ICP-MS analysis (three mice from each group), mice treated with PBS solution were used as the negative control.

capability.32 After analyzing the biodistribution of Co and Mn, both of these elements have consistent ratios in various organs and tumors treated by nanomaterials, whereas they are rare in mice treated with PBS. For the groups, after injection of nanomaterials, large amounts of Co and Mn are enriched the liver, which indicates that the injected nanoparticles are metabolized by the liver. Additionally, due to the enhancement of targeting efficiency based on FA molecules, all of these elements have significant enhancement in the tumor area treated with nanoprobes compared to that in the other control groups. In addition, due to the under-control dosage of the intravenously injected nanomaterials (Fe: 150 μg in total), the mice in this study were kept in a healthy state without weight loss, and there is no evidence of inflammation from the dissected organs and tumor, which shows that the nanoprobes exhibit good biocompatibility in vivo.

during the blood circulation. Afterward, the amount of nanomaterials in the tumor site decreased during metabolism under blood circulation over time, with the result that the T2 signal value rose again at 24 h. In comparison, the nanoprobes acquire a significant enhancement of the T2 signal value compared to that of the PMATAMRA-Co@Mn MNPs due to the specific target molecules to the MGC-803 tumor. 3.5. Distribution Analysis of Nanoprobes in Vivo. We further evaluated the tumor-targeting property and biodistribution of PMATAMRA-Co@Mn-PEG2k-FA nanoprobes in the tumor-bearing mice. After the tumor sizes of gastric tumorbearing mice reached 100−150 mm3, nanoprobes (200 nM, 200 μL, dispersed in 1× PBS solution) and an equal amount of the no target control (PMATAMRA-Co@Mn-PEG2k) were intravenously injected into the nude mice, 200 μL of PBS solution was injected into the nude mice as the negative control. Figure 6a shows the ex vivo fluorescence images of major organs and tumors 24 h after injection. We can see that both of the groups present strong fluorescence in the liver possibly because the liver is the main organ for the metabolic pathways of these kinds of nanomaterials. Owing to the enhancement of the targeting efficiency based on FA molecules, the fluorescence signal in the tumor sites after injection of nanoprobes was stronger than that of the no target sample. The quantification of the fluorescence intensity of major organs and tumors is presented separately in Figure 6b. Additionally, biodistributions of nanoprobes in major organs and tumors were studied by measuring the concentrations of the three constituent elements (Fe, Mn, and Co) by ICP-MS. Equal injection amounts of the no target sample and PBS solution was used in the control group. Figure 6c presents the Fe distribution of the dissected organs and tumors 24 h after injection, which shows that all of the mice have the highest Fe concentration in the spleen because of its iron storage

4. CONCLUSIONS In summary, we have successfully designed a novel tumor targeting nanoprobe based on the core@shell magnetic nanoparticle. This core@shell structure has hard phase CoFe2O4 as the core, which could enhance the magnetic anisotropy, and soft phase MnFe2O4 as the shell, which could improve the magnetization value. This kind of structure not only can be applied for hyperthermia treatment, from previous studies, but also can be used for targeted fluorescence/magneto imaging of tumor regions, similar to monodispersed Co@Mn MNPs, after further modification. Co@Mn MNPs from our studies are synthesized by a thermal decomposition method with a uniform size distribution (σ < 5%, dc ≈ 15 nm). With the covalent modification of TAMRA molecules to PMA, Co@Mn MNPs show long-term fluorescence stability after polymer coating. PEG molecules were coupled between Co@Mn MNPs with FA, and with the guidance of a targeting FA molecule, the 17783

DOI: 10.1021/acsami.7b04288 ACS Appl. Mater. Interfaces 2017, 9, 17777−17785

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fabricated PMATAMRA-Co@Mn-PEG2k-FA nanoprobes can precisely target gastric tumor cells and specifically enhance cell uptake in the tumor region. The synthesized PMATAMRACo@Mn-PEG2k-FA nanoprobes could be applied as a T2weighted MR contrast agent to trace the variations of the tumor regions for tumor-targeted MR imaging. Besides, on the basis of the fluorescence of nanoprobes and element analysis in the major organs and tumors, it is easy to recognize the cell uptake and tumor targeting pathway in vitro and in vivo.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b04288. Structure of polymer, characterization of the core@shell MNPs and nanoprobes, ICP-MS analysis, supporting tables (PDF)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (G.G.). *E-mail: [email protected]. Tel: +86-21-34206886. Fax: +8621-34206886 (D.C.). ORCID

Guo Gao: 0000-0002-8698-5423 Joseph G. Shapter: 0000-0002-4000-2751 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank the 863 High-Tech project of China (2014AA020701), the National Natural Science Foundation of China (Nos. 81671737, 81225010, and 91634108) and the 973 Project (2015CB931802) for financial support.



ABBREVIATIONS MNPs, magnetic nanoparticles; Co@Mn MNPs, CoFe2O4@ MnFe 2 O 4 MNPs; ζ, zeta potential; d h, hydrodynamic diameters; AMF, alternating magnetic field; MRI, magnetic resonance imaging



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