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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 13, 2017

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ACS Applied Materials & Interfaces

Multifunctional core@shell magnetic nanoprobes for enhancing targeted magnetic resonance imaging and fluorescent labeling in vitro and in vivo Qian Zhang1, Ting Yin1, Guo Gao1*, Joseph G. Shapter2, Weien Lai3, Peng Huang1, Wen Qi1, Jie Song1 and Daxiang Cui1*

1

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 Rd, Shanghai 200240, China.

2

School of Chemical and Physical Sciences, Flinders University, Bedford Park, Adelaide 5042, Australia.

3

Academy of Photoelectric Technology, HeFei University of Technology, HeFei, 230009, China.

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ABSTRACT: Core@shell magnetic nanoparticles (core@shell MNPs) are attracting wide attention due to the enhancement properties for potential applications in hyperthermia treatment, magnetic resonance imaging (MRI) and diagnostics, etc. 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 the 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

PMATAMRA-Co@Mn-PEG2k-FA

nanoprobes

exhibit

good

biocompatibility, high T2 relaxation value and long-term fluorescence stability (at least 6 months). Our results demonstrated that the synthesized PMATAMRA-Co@Mn-PEG2k-FA nanoprobes can effectively enhance the targeted magnetic resonance imaging and fluorescent labeling in vitro and in vivo. The research outcomes will contributed 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


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1. INTRODUCTION In recent decades, magnetic nanoparticles (MNPs) have exhibited great potential for medical applications1, including targeted drug delivery2-3, molecular detection and separation4-6, magnetic resonance imaging (MRI)1, 7, in-vitro testing8 and magnetic fluid hyperthermia treatment9, etc. These applications mainly take advantage of their unique size-dependent magnetic properties as well as their stability, biodegradability and nontoxic properties in nature.10 Recently, with precision medicine as increasing emergency needs, multi-functional MNPs with controllable shape, structure and size, as well as enhanced magnetic properties have gained more and more requirements 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 excellent superparamagnetic behavior and the maximal hysteretic loss (SLP) under an alternating magnetic field (AMF), and they have been recognized as new types of magnetic nanomaterials for hyperthermia applications.13, 16-18 The properties of those 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 magnetic resonance imaging (MRI). Hence, CoFe2O4@MnFe2O4 MNPs could become one of the most promising nanomaterials for tumor diagnosis and therapy in the near future. However, as the complex of in vivo mode, the majority of the injected nanomaterials will aggregate in organs after blood cycle, and induce lesions,19 whereas 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 ACS Paragon Plus Environment

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multifunctional nanoprobes with low dosage and excellent magnetic properties for fluorescence labeling and MRI in vivo will be very valuable. Many researchers have reported the applications of magnetic nanoparticles for multimodal imaging and tumor therapy based on magnetic hyperthermia or photodynamic therapy.9,

20-21

However, the poor quality of the magnetic

nanoparticles (e.g. stability, dispersion and surface functional groups) always induces severe agglomeration during blood circulation, and consequently effect the targeting efficiency of tumor labeling and therapy.22 Therefore, rational design and synthesis of high-quality magnetic nanoprobes for imaging and therapy of tumor is of great interest. In this study, we synthesized monodisperse CoFe2O4@MnFe2O4 MNPs (Abb. Co@Mn MNPs) with highly uniform size (dc =15.1 ± 1.2 nm). The hydrophobic Co@Mn MNPs were then transferred into aqueous solution using TAMRA labeled polymer, followed by functionalization with a targeting species (folic acid) 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 a highly stable yellow fluorescence on the NP’s 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, folic acid molecules are the specific targeting molecules for MGC 803 cell lines of gastric tumor. Attaching folic acid on the terminal COO- groups of NPs via conjugation of N2HPEG-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 illustrated the fabrication process of core@shell magnetic nanoprobes and their multifunctional labeling in vitro and in vivo.


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Scheme 1. Schematic illustration of fabricated PMATAMRA-Co@Mn-PEG2k-FA nanoprobes and their multifunctional labeling in vitro and in vivo. 2. EXPERIMENTAL SECTION 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) (NH2PEG-NH2, PEG2k, Mw: 2 kDa) were purchased from Aladdin. 1,2-hexadecanediol (90 %), N-(3Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), poly(isobutylene-alt-maleic anhydride)(PMA, Mw~6 kDa) and In Vitro Toxicology Assay Kit, Resazurin based (Resazurin) were obtained from Sigma-Aldrich. Folic acid (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.


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and Invitrogen Corporation, respectively. 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 In order to obtain core@shell structure, the synthesis of Co@Mn MNPs was separated into producing CoFe2O4 seeds and MnFe2O4 shell growth, respectively. CoFe2O4 seeds were synthesized via one-pot synthesis process.27 In detail, 0.71 g of Fe(acac)3, 0.26 g of Co(acac)2, 2.58 g of 1,2-hexadecanediol were weighed in a 100 mL three-neck flask, followed by the addition 1.69 g of OLA, 1.6 g of OLAM and 20 mL benzyl ether. Under various magnetic stirring, 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 kept this temperature for 2 h, then heated again to 300 °C and left for 1 h at 300 °C. During this time, mixture solution changed color from brown to black. At last, 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 following 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, 2.58 g of 1,2-hexadecanediol were weighed in a 100 mL three-neck flask followed by the injection 0.56 g of OLA, 0.53 g of OLAM, 20 mL of benzyl ether and 2 mL of CoFe2O4 seeds 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 heating under reflux for 0.5 h. At last, the reaction was stopped by removing the ACS Paragon Plus Environment

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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 2 % TAMRA labeled amphiphilic polymer (PMATAMRA) was synthesized by reacting PMA with DoCA and TAMRA28. Under a typical synthesis process, as a hydrophilic back bone, 75 % of 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 THF anhydrous in a 100 mL round 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 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 flask with ratio of Rp/Area=300 monomers/nm2, hereby Rp/Area was described by the ratio of polymer per particle surface. Then, the solvent was evaporated slowly until the sample was completely dried. Afterwards, SBB 12 buffer (sodium borate 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 hydrophilic backbone was opened into carboxy groups, which supplied negative charges for stabilization in aqueous solution. After reaction, the PMATAMRA-Co@Mn MNPs were washed three times to remove free polymer with centrifugation (17,000 rpm, 1 h). ACS Paragon Plus Environment

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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 Firstly, PMATAMRA-Co@Mn MNPs (1 µM, 1 mL) were mixed with 100 mg of PEG dissolved in 5 mL SBB 9.0 buffer (sodium borate 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 centrifuge filter via centrifugation (3000 rpm, 10 min) to obtain the cleaned nanoprobes, and kept the sample in Milli-Q water. 2.6 Characterization The morphology of the synthesized MNPs was imaged by transmission electron microcopy (TEM, JEM-2100f, Japan). The UV-Vis spectra and fluorescent spectra of all samples were measured by UV-Vis spectrophotometer (Varian InC., Palo Alto, CA, USA) 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 the samples were measured by dynamic light scattering using a Zetasizer Nano (Malvern Instruments Ltd., UK).

Hysteresis curves of the MNPs and nanoprobes at 300 K were obtained by using

superconducting quantum interference device magnetometer (SQUID, USA) with a magnetic applied field of 3 T; and in vitro MRI and r2 value was 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


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The MGC-803 cell line was acquired from 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 (DMEM, HyClone) media (10 % fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 0.1 mg/mL streptomycin). Viability studies of cells treated with nanoprobes (PMATAMRACo@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. In order to identify the specific target of FA to MGC-803 cells, nanoprobes in absence of FA (named as 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, growth medium solution from each well was aspirated and rewashed by PBS buffer. Then 100 µL of fresh media containing various concentrations of nanoprobes and no target sample from 7.5 nM to 180 nM was added while for other cells just the media with PBS was added as negative control. After incubation for 24 h, the cells were rinsed by PBS and fresh media contained with 10 % resazurin solution was added. After incubation for another 4 h, the cell viability was analyzed according to the changes of fluorescence intensity by microplate reader based on the following equation:

Cell viability (%) =

( ) ( )

× 100%,

Hereby 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 nanoprobes was obtained by Confocal Laser Scanning Microscopy (CLSM, Leica, Germany) and flow cytometry (FCM, BD FACS Calibur, USA), respectively. During the CLSM study, MGC-803 cells were first plated at a density of 4.0 × 104 cells/well ACS Paragon Plus Environment

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overnight. Then nanoprobes and equivalent no target control (30 nM, 100 µL) were added separately into the cells and incubated for 6 h, 12 h and 24 h, respectively. Afterwards, 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 420 ~ 460 nm and 560 ~ 600 nm barrier filter under laser irradiation of CLSM. In addition, the uptake efficiency of nanoprobes can be measured by flow cytometry through analysis of the yellow fluorescence of TAMRA on the nanoprobes. The of 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 same amount of nanoprobes and no target control for various times. All the cells were separately rinsed with PBS, trypsinized, and resuspended in 500 µL of PBS for FCM measurements collecting signal 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 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 right flank of the mice for establishing gastric tumor-bearing mice. After the tumor size reached 100 ~ 150 mm3, all the tumor-bearing mice were randomly divided into 3 groups and separately injected 200 µL of different samples from caudal vein: 1) 1 × PBS solution as negative control, 2) nanoprobes (200 nM, 200 µL) in 1 × PBS solution, and 3) the samples of no target control (PMA-Co@MnPEG2k, 200 nM, 200 µL) in 1 × PBS solution. All the tumor-bearing mice were dissected after 24 h, the major organs (heart, liver, spleen, lung and kidney) and tumor were separated from the body. Fluorescence signals from the organs were analyzed and quantified by Bruker Molecular Imaging Software Version 7.1. All the organs and tumors were digested by aqua regia ACS Paragon Plus Environment

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(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/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-PEG2k-FA 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 the nude mice after intravenous injection of nanoprobes were recorded by 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 coil and MRI images were recorded for various time intervals (pre-injection, 0.5 h, 12 h and 24 h). Finally, the obtained image data were analyzed by MR imaging software. 3. RESULTS AND DISCUSSION 3.1 Synthesis and characterization Co@Mn MNPs presented the core@shell structure formed by growing a MnFe2O4 shell on the surface of CoFe2O4 seeds, the crystal phase and elemental composition of core@shell architecture were investigated by STEM image and EELS spectra, respectively (c.f. Figure S2). Transmission electron microcopies (TEM, Figure S3a and Figure 1a) of CoFe2O4 seeds and Co@Mn MNPs demonstrate that they exhibit highly uniform size distribution (σ < 5 %). The high-resolution TEM (HRTEM) in Figure 1c shows the Co@Mn MNP exhibits a spherical structure with the fringe distance of 0.25 nm and 0.16 nm, respectively. The amphiphilic polymer (labeled with 2 % TAMRA) was synthesized and PMA coated Co@Mn MNPs to achieve phase transfer were made.


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During the polymer coating (scheme is presented in Figure 1d), the hydrophobic sidechains intercalated with the aliphatic ligands on the NP’s surface, while 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 an yellow fluorescence under laser irradiation, which the fluorescence have long-term stability at least 6 months without quenching.28 Further, 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. Figure S3b and Figure 1b).31 Subsequently, PEG2k and folic acid was gradually functionalized on the surface of NPs via EDC chemistry to fabricate the PMATAMRA-Co@MnPEG2k-FA nanoprobes. According to the hydrodynamic diameters (dh) and zeta-potential (ζ) of all the samples by Malvern Zetasizer (Figure 1e and 1f), the sizes of four samples (Co@Mn MNPs in CHCl3, PMATAMRA-Co@Mn MNPs, PMATAMRA-Co@Mn-PEG2k and PMATAMRACo@Mn-PEG2k-FA nanoprobes) are measured to be 13.1 ± 0.25 nm, 16.5 ± 0.4 nm, 25.3 ± 0.7 nm, 25.7 ± 1.1 nm, respectively. While the zeta-potential values of the samples are close zero, 35.8± 1.2 mV, -25.4± 1.6 mV, -28.5± 2.0 mV, respectively. dh values of nanoprobes at different NaCl concentrations and pH values are summarized in Table S1 and S2, respectively. Figure S3c shows the UV-Vis absorption spectra of PMATAMRA-Co@Mn MNPs and nanoprobes, respectively. Compared with the MNPs, after functionalization there is an evident absorption at around 350 nm in spectra of nanoprobes due to folic acid, demonstrating the successful attachment of the targeting molecule. Fluorescence emission spectra of 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.


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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 thickness of 2.72 ± 0.67 nm; c) HRTEM micrograph of one single Co@Mn MNPs; d) Scheme of amphiphilic polymer coated with one particle; e and f) diameter values (dh) and zeta-potential values (ζ) 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), respectively. The scale bar for the HRTEM image is 10 nm. 3.2 Magnetic properties and T2-Weighted MR Relaxometry Iron oxide NPs are generally used as T2 -weighted MR contrast agents due to their excellent magnetic properties, especially for Co@Mn MNPs, which exhibit excellent superparamagnetic behavior and ideal spin-spin relaxation time (T2). From Figure 2a we can see that, M-H curves of


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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@MnPEG2k-FA nanoprobes also provide excellent M-H curves with Ms of 68 emu g-1. Moreover, the slope of nanoprobes is even higher than that of 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 mM to 0.56 mM using a 0.5 T MRI system (cf. Figure 2b and 2c). Both of them exhibit significantly MRI contrast enhancement with increasing Fe concentration, and the T2 relaxation rate (1/ T2) increases linearly with varying Fe concentration with a slope r2 both of 255.7 mM-1 s-1 and 167.8 mM-1 s-1. The high contrast property of the nanoprobes highlight their potential ability for MRI in vivo.

Figure 2. a) M-H curves of PMATAMRA-Co@Mn MNPs (MNPs, black) and PMATAMRA-Co@MnPEG2k-FA nanoprobes (nanoprobes, red) measured at 300 K by SQUID. The inset in figure a) shows the M-H curves in low magnetic field; b) T2-weighted MRI images of PMATAMRACo@Mn MNPs (upper) and nanoprobes (lower) dispersed in H2O with various Fe concentrations


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measured at 25 °C under 0.5 T magnetic field; c) T2 relaxation rates (1/T2) of PMATAMRACo@Mn MNPs (black) and PMATAMRA-Co@Mn-PEG2k–FA nanoprobes (red). 3.3 Cellular uptake assay The cytotoxicity study of PMATAMRA-Co@Mn-PEG2k-FA nanoprobes was performed using a resazurin assay with MGC-803 cell line. Various concentrations of nanoprobes and no target control (PMATAMRA-Co@Mn-PEG2k) from 7.5 nM to 180 nM (the conversed concentration is from 42 µg/mL to 1000 µg/mL) were incubated with MGC-803 cells for 24 h at 37 °C (PBS solution in absence of nanomaterials as negative control). After treatment with resazurin and cell viability analysis in Figure 3a, there is no evident toxicity after incubated cells with nanomaterials below concentration of 80 nM (conversed concentration: 444 µg/mL) which the cell viability is over 90 %, and the cell damage was appeared until the concentration of nanomaterials reached over 180 nM (conversed concentration: 1000 µg/mL) and the cell viability decreases to 55 %, this result indicated that the nanoprobes have excellent biocompatibility in cellular assay except at extremely high dosage. The cell uptake and pathway of PMATAMRACo@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 lysosome which are clearly visible from the cell (Figure 3c).


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

We further investigated the cellular uptake and quantification analysis of nanoprobes in MGC803 cell line by CLSM experiments and FCM system. As shown in Figure 4a, there is no significant yellow fluorescence after 6 h incubation compared with no target control, suggesting a low uptake of MNPs by cells. In contrast, bright yellow fluorescence can be clearly observed with incubation of equal amount of nanoprobes after 6 h incubation. This result is confirmed by average fluorescence intensity measured by FCM. From those 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, the increase of yellow fluorescence has been detected after increasing the cell incubation time from 6 h to 24 h, which can be clearly demonstrated that, more MNPs are taken up into the cells with longer incubation time.


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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 h, 12 h and 24 h, respectively. 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 440-470 nm band-pass, while TAMRA labeled nanoprobes were imaged at a 576 nm long-pass (LP). b) FCM images related the cellular uptake of nanoprobes and no target control based on the fluorescence of TAMRA. Red and blue histograms are the cellular uptake of nanoprobes and no target control, respectively. The scale bars are 100 nm. 3.4 In Vivo MR Imaging In order to evaluate the efficiency of PMATAMRA-Co@Mn-PEG2k-FA nanoprobes as T2 contrast agent in vivo, nanoprobes (200 nM, 200 µL, dispersed in 1 × PBS solution) and equal amount of PMATAMRA-Co@Mn MNPs were intravenously injected into MGC-803 tumor-bearing mice. MR images (cf. Figure 5a) were recorded at different time intervals, and MRI values around the ACS Paragon Plus Environment

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tumors shown in the white dashed circle area was collected and analyzed for the MRI efficiency of the nanoprobes (cf. Figure 5b), hereby, the intensity (I) of tumor site from MRI image is inversely proportional to the amount of nanoprobes in the relevant area. As we can see, due to the enhanced permeability and retention (EPR) effect to tumor, both PMATAMRA-Co@Mn MNPs and PMATAMRA-Co@Mn-PEG2k-FA nanoprobes injected by tail-vein could enrich into the tumor site during the blood circulation over time, with the result of decrease of MRI intensity. Meanwhile, folic acid molecules can further enhance the specific targeting to MGC-803 tumor. Therefore, there is an obvious darkening effect at the tumor site after injection of nanoprobes compared to the one 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 to the tumor sites during the blood circulation. Afterwards, the amount of nanomaterials in the tumor site decreased during metabolism under blood circulation over time, with the result of T2 signal value raised up again at 24 h. In comparison, nanoprobes acquire a significant enhancement for T2 signal value than PMATAMRA-Co@Mn MNPs due to the specific target molecules to MGC-803 tumor.


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Figure 5. In vivo MR images. a) MR images of the MGC-803 tumor-bearing mice (white dashed circle positions) before and after intravenous injection of PMATAMRA-Co@Mn MNPs and PMATAMRA-Co@Mn-PEG2k-FA nanoprobes at the time from 0.5 h to 24 h; b) the corresponding value quantification of tumor contrast collected at the time from untreated time to 24 h, respectively. 3.5 Distribution analysis of nanoprobes in vivo We further evaluated the tumor targeting property and biodistribution of PMATAMRA-Co@MnPEG2k-FA nanoprobes in the tumor-bearing mice. After tumor sizes of gastric tumor-bearing mice reached to 100~150 mm3, nanoprobes (200 nM, 200 µL, dispersed in 1 × PBS solution) and equal amount of 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 negatively control.


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Figure 6a shows the ex vivo fluorescence images of major organs and tumors after post-injection for 24 h. We can see that both of the groups present strong fluorescence in liver possibly because liver is the main organ of metabolic pathways for these kinds of nanomaterials. Owing to the enhancement of targeting efficiency based on FA molecules, the fluorescence signal in the tumor sites after injection of nanoprobes was stronger than that of no target sample. The quantification of fluorescence intensity of major organs and tumor is separately presented in Figure 6b. Additionally, biodistributions of nanoprobes in major organs and tumor were studied by measuring the concentrations of three consisted elements (Fe, Mn and Co) by ICP-MS. An equal injection amount of no target sample and PBS solution were used as the control group. Figure 6c presents the Fe distribution of the dissected organs and tumors after injection for 24 h, which shows that all the mice have the highest Fe concentration in spleen because of its iron storage capability in nature32. After analyzing the biodistribution of Co and Mn, both of these two elements have consistent ratio in various organs and tumor treated by nanomaterials, while there are rare contents in mice treated by PBS. For the groups after injection of nanomaterials, large amounts of Co and Mn have been enriched in liver, which indicates that the injected nanoparticles are metabolized by liver. Additionally, due to the enhancement of targeting efficiency based on FA molecules, these elements all have significant enhancement in tumor area treated with nanoprobes compared to other control groups. In addition, due to the under controlled dosage of intravenous injected nanomaterials (Fe: 150 µg in total), the mice in this study were keeping in a healthy state without weight loose, and there is no evidence inflammation from the dissected organs and tumor, which the nanoprobes exhibit the good biocompatibility in vivo.


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Figure 6. a, b) Ex vivo fluorescence images and fluorescence intensity analysis of major organs and tumors of MGC-803 tumor bearing mice after post-injection of PMATAMRA-Co@Mn-PEG2kFA nanoprobes and 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 (3 mice from each group), mice treated with PBS solution were used as negative control. 4. CONCLUSIONS In summary, we have successfully designed a novel tumor targeting nanoprobe based on core@shell magnetic nanoparticle. This core@shell structure has hard phase CoFe2O4 as core which could enhance the magnetic anisotropy, and soft phase MnFe2O4 as shell which could improve the magnetization value. This kind of structure can not only applied for hyperthermia treatment from previous studies, monodispersed Co@Mn MNPs after further modification can be also used for targeted fluorescent/magneto imaging of tumor regions. Co@Mn MNPs from our


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studies are synthesized by thermal decomposition method with uniform size distribution (σ < 5 %, dc ≈ 15 nm). With the covalent modification of TAMRA molecules to PMA, Co@Mn MNPs presents 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 molecules, the fabricate PMATAMRA-Co@Mn-PEG2k-FA nanprobes can precisely target the gastric tumor cells and specific enhance the cell uptake in the tumor region. The synthesized PMATAMRA-Co@MnPEG2k-FA nanprobes could be applied as a T2-weighted MR contrast agent to trace the variations of tumor region for tumor-target MR imaging. Besides, based on fluorescence of nanoprobes and element analysis in major organs and tumor, it is easy to recognize the cell uptake and tumor targeting pathway in vitro and in vivo. ACKNOWLEDGEMENTS We thank the financial support from 863 High-Tech project of China (2014AA020701), National Natural Science Foundation of China (No. 81671737, 81225010 and 91634108) and 973 Project (2015CB931802).

ASSOCIATED CONTENT Supporting Information Structure of polymer, characterization of the core@shell MNPs and nanoprobes, ICP-MS analysis, supporting tables. The material is available free of charge via the Internet at http://pubs.acs.org. ABBREVIATIONS


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MNPs, Magnetic nanoparticles; Co@Mn MNPs, CoFe2O4@MnFe2O4 MNPs; ζ, zeta potential; dh, hydrodynamic diameters; AMF, alternating magnetic field; MRI, magnetic resonance imaging.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (G. Gao), [email protected] (D. Cui); Tel: +86-21-34206886; Fax: +86-21-34206886. ORCID Qian Zhang: 0000-0002-5242-7861

Notes The authors declare no competing financial interest.

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