Folate-bovine serum albumin functionalized polymeric micelles loaded with superparamagnetic iron oxide nanoparticles for tumor targeting and magnetic resonance imaging.

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Folate-bovine serum albumin functionalized polymeric micelles loaded with superparamagnetic iron oxide nanoparticles for tumor targeting and magnetic resonance imaging Huan Li a,1, Kai Yan b,1, Yalei Shang a, Lochan Shrestha a, Rufang Liao a, Fang Liu a, Penghui Li c, Haibo Xu a,⇑, Zushun Xu b,c,⇑, Paul K. Chu c,⇑ a

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Department of Radiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Hubei University, Wuhan, Hubei 430062, China c Department of Physics & Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China b

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Article history: Received 19 June 2014 Received in revised form 28 November 2014 Accepted 7 January 2015 Available online xxxx

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Keywords: Folate Amphiphilic copolymers Magnetic micelles Magnetic resonance imaging Tumor

a b s t r a c t Polymeric micelles functionalized with folate conjugated bovine serum albumin (FA-BSA) and loaded with superparamagnetic iron oxide nanoparticles (SPIONs) are investigated as a specific contrast agent for tumor targeting and magnetic resonance imaging (MRI) in vitro and in vivo. The SPIONs-loaded polymeric micelles are produced by self-assembly of amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers and oleic acid modified Fe3O4 nanoparticles and functionalized with FA-BSA by electrostatic interaction. The FA-BSA modified magnetic micelles have a hydrodynamic diameter of 196.1 nm, saturation magnetization of 5.5 emu/g, and transverse relaxivity of 167.0 mM1 S1. In vitro MR imaging, Prussian blue staining, and intracellular iron determination studies demonstrate that the folatefunctionalized magnetic micelles have larger cellular uptake against the folate-receptor positive hepatoma cells Bel-7402 than the unmodified magnetic micelles. In vivo MR imaging conducted on nude mice bearing the Bel-7402 xenografts after bolus intravenous administration reveals excellent tumor targeting and MR imaging capabilities, especially at 24 h post-injection. These findings suggest the potential of FABSA modified magnetic micelles as targeting MRI probe in tumor detection. Ó 2015 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

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1. Introduction

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Morbidity and mortality caused by cancer is increasing and early detection is the key to effective treatment [1,2]. As one of the powerful techniques in cancer diagnosis, magnetic resonance imaging (MRI) offers the advantages of non-invasive, multiparametric imaging as well as deep soft tissue penetration [3]. Tumor-specific targeted MR imaging [4–6] thus has large potential and nanoparticle encapsulated contrast agents can enhance the contrast between tumors and normal tissues [7]. In this respect, superparamagnetic iron oxide nanoparticles (SPIONs) are sensitive and negative MRI probes possessing the ability to noninvasively monitor events occurring on the cellular and even molecular levels

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⇑ Corresponding authors at: Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Hubei University, Wuhan, Hubei 430062, China. Tel.: +86 27 85726410; fax: +86 27 85726919 (H. Xu). Tel.: +86 27 88661879; fax: +86 27 88665610 (Z. Xu). Tel.: +852 34427724; fax: +852 34420542 (P.K. Chu). E-mail addresses: [email protected] (H. Xu), [email protected] (Z. Xu), [email protected] (P.K. Chu). 1 These two authors contributed equally to this project.

in vivo [8,9]. However, biological applications of SPIONs are limited because of the high surface hydrophobicity making them prone to being engulfed by macrophages and rapidly removed from circulation [10]. In order to prolong the circulation time, it is essential to modify the surface of these magnetic iron oxide nanoparticles. Several methods have been explored to convert hydrophobic SPIONs into hydrophilic ones, for instance, ligand exchange [11] and amphiphilic copolymer encapsulation [12]. Amphiphilic copolymers have drawn much interest because of their self-assembling properties [13]. Amphiphilic copolymers consisting of both hydrophobic and hydrophilic segments can self-assemble into hydrophobic core–hydrophilic shell structures in an aqueous medium [14]. The hydrophobic SPIONs can form small clusters on the hydrophobic core of the polymeric micelle to produce high MRI T2 contrast [15], whereas the hydrophilic segments of the polymer derivatized with a ligand endows them with targeting ability [16]. For example, self-assembled fluorine-containing amphiphilic poly(HFMAg-PEGMA) copolymeric micelles loaded with SPIONs have an organized core–shell structure and show excellent stability and loading efficiency [17] and the cationic monomer methacryloxyethyl

http://dx.doi.org/10.1016/j.actbio.2015.01.006 1742-7061/Ó 2015 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

Please cite this article in press as: Li H et al. Folate-bovine serum albumin functionalized polymeric micelles loaded with superparamagnetic iron oxide

Q1 nanoparticles for tumor targeting and magnetic resonance imaging. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.01.006

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trimethyl ammonium chloride (MOTAC) serves as a binding site due to the positive charge [18]. Bovine serum albumin (BSA), a negatively charged plasma protein, offers advantages such as non-toxicity, good biocompatibility, and excellent biodegradability [19]. It has been used as a carrier for targeting agents such as folate [20,21] to improve the water solubility and prolong circulation in the blood. Moreover, the negatively charged BSA can serve as a stabilizing agent to bind cationic particles [22] since a polyelectrolyte complex can be formed by the electrostatic attraction between the cationic polymeric micelles and negatively charged BSA in a solution. However, in vivo application of water-soluble SPIONs-loaded polymeric micelles has been hampered by lack of specificity toward a pathological site [23]. The folate receptor (FR) is a specific tumor marker and overexpressed in many forms of cancer [24,25]. The folate receptor also has a high binding affinity to folic acid (Kd 190 pM) [26] and is thus an attractive target for site-specific delivery of folate modified contrast agents into proliferating cells. In fact, folate has been conjugated with nanoprobes to improve the sensitivity and specificity of tumor diagnosis [27]. When cationic magnetic polymeric micelles are functionalized with folic acid using BSA as the carrier and stabilizing agent, the encapsulated SPIONs can be taken up by the cancer cells via a folate receptor mediated endocytic pathway. In this study, FA-BSA modified and SPIONs-loaded polymeric micelles are prepared and the use of the materials in folate-receptor overexpressed cancer targeting and MR imaging are investigated in vitro and in vivo.

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2. Materials and methods

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2.1. Materials

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2,2,3,4,4,4-hexafluorobutyl methacrylate (HFMA) purchased from Xeogia Fluorine-Silicon Chemical Company (Harbin, China) was distilled at reduced pressure before use and methoxy poly (ethylene glycol) monomethacrylate (PEGMA) (average molecular weight of 950 g/mol), 75 wt.% methacryloxyethyl trimethyl ammonium chloride (MOTAC) solutions, and folic acid were obtained from Aldrich. 2,20 -azobisisobutyronitrile (AIBN) was purified by recrystallization in ethanol and oleic acid, iron (III) chloride hexahydrate (FeCl36H2O), iron (II) chloride tetrahydrate (FeCl24H2O), ammonium hydroxide (NH3H2O, 25–28%), dimethyl sulfoxide (DMSO), ethanol, hexane, hydrochloric acid (HCl), 1-ethyl-3-(30 dimethylaminopropyl) carbodiimide (EDC), and tetrahydrofuran (THF) were purchased from Sinopharm Chemical Reagent Co. Ltd., China. Bovine serum albumin (BSA) and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma–Aldrich.

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2.2. Sample preparation

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2.2.1. Preparation of cationic SPIONs-loaded polymeric micelles Mono-dispersed SPIONs were synthesized by chemical coprecipitation and modified with oleic acid according to the procedures described previously [28]. The cationic amphiphilic poly (HFMA-co-MOTAC)-g-PEGMA copolymers were synthesized by free radical polymerization [17,29]. Briefly, 1.02 g of PEGMA, 0.91 g of HFMA, and 0.12 g of MOTAC were dissolved in 15 mL of THF in a 50 mL round bottomed flask with a magnetic stirrer. After adding 0.068 g of AIBN as a radical initiator, the mixture was deoxygenated under vacuum and backfilled with nitrogen several times in an ice bath. Polymerization proceeded at 75 °C for 24 h and the cationic amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers were collected by precipitation in hexane. Cationic SPIONloaded polymeric micelles denoted as unmodified magnetic

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micelles were prepared by self-assembly. 0.20 g of SPIONs were dissolved in 5 mL of hexane and manually mixed with 25 mL of distilled water containing 0.21 g of the cationic amphiphilic poly (HFMA-co-MOTAC)-g-PEGMA copolymers prior to sonication for 15 min. Hexane was evaporated at 70 °C in a water bath during sonication and the solution containing the cationic magnetic micelles was purified and separated from the large particles and free copolymers by centrifugation and filtration, respectively.

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2.2.2. Preparation of FA-BSA modified magnetic micelles FA-BSA modified magnetic micelles were synthesized by functionalizing the cationic magnetic micelles with FA-BSA by electrostatic complexation [30]. Conjugation of folate with the bovine serum albumin was carried out according to the previously reported method [20,31]. 10 mg of folic acid and 10 mg of EDC were mixed in 10 mL of DMSO under stirring at room temperature for 2 h to modify the terminal carboxylate group. 50.0 mg of the BSA dissolved in 10 mL of distilled water was added to the above solution and stirred at room temperature in the dark for 4 h. The folate and other reactants in excess were removed from the conjugated protein using Sephadex G-25. 1 mL of FA-BSA and 2 mL of purified cationic magnetic micelle solutions were mixed and reacted for 8 h at room temperature under continuous agitation. The solution was dialyzed against ultrapure water for 3 days. The FA-BSA modified magnetic micelles were re-dispersed and stored at 4 °C for further studies.

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2.3. Characterization

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H NMR was conducted to investigate the chemical structure of the amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers using the UNITY INVOA 600 MHz spectrometer (Varian, USA) with CDCl3 containing 0.03% v/v tetramethylsilane (TMS) as the solvent. The structures of the SPIONs, amphiphilic poly(HFMA-co-MOTAC)g-PEGMA copolymers, and folate-modified magnetic micelles were assessed by Fourier transform infrared spectroscopy (FTIR, PerkinElmer Spectrum One, USA). The morphology of the samples was examined by transmission electron microscopy (TEM, Tecnai G20, FEI Corp., USA) at 200 kV. The hydrodynamic size and size distribution were measured using a dynamic light scattering instrument (DLS, Zetasizer NanoZS90, Malvern Instruments Ltd., Worcestershire, UK) at 25 °C at a scattering angle of 90°. The zeta potential of the particles was determined by DLS. The total iron concentration was determined by fast sequential atomic absorption spectroscopy (SpectrAA240FS, Varian, Palo Alto, USA) and the thermogravimetric analysis was performed on the Perkin Elmer TGA-7. The magnetic properties were studied on a vibrating sample magnetometer (VSM, HH-15, China) at 298 K under an applied magnetic field. The transverse relaxivity (r2) of the magnetic micelles was determined using a 3.0-T whole body MR scanner (MAGNETOM Trio, A Tim System 3 T, Siemens, Munich, Germany) in combination with an 8-channel wrist joint coil. The particles were diluted by 300 lL 0.5% agarose gel on a 96-well plate with iron concentrations in the range of 0–0.10 mmol/L and were tested by T2-weighted multi-echo spin echo sequence. The parameters were as follows: field of view (FOV) = 120 mm, base resolution = 384 384, slice thickness = 1.5 mm, multiple echo time (TE) = 20, 40, 60, 80, 100, 120, and 140 ms, repetition time (TR) = 2000 ms, and scanning time = 13–14 min. The transverse relaxation time (T2) of each suspension was quantified using the in-house software. The transverse relaxation rates (1/T2) were plotted versus iron concentrations and the transverse relaxivity (r2) was computed based on linear regression (Origin 7.5).



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2.4. In vitro cytotoxicity test

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The cytotoxicity of the magnetic micelles was estimated by means of the MTT assay. The human normal liver cell line HL-7702 was obtained from Shanghai Institute of Life Science Cell Culture Center (Shanghai, China). The cells were seeded on 96-well plates (8000 cells per well) and incubated with the FA-BSA modified or unmodified magnetic micelles dissolved in DMEM at different iron concentrations (0, 25, 50, 75, and 100 lg/mL). Each sample concentration was repeated 5 times. After incubation for 24 and 48 h, 20 lL of MTT (5 mg/mL) was added to each well and the cells were cultured for another 4 h. Afterward, the medium was removed and DMSO (100 lL/well) was added to dissolve the formazan crystals. The absorbance of each well was monitored by a microplate reader (1420 multilabel counter, Perkin Elmer, MA, USA) at 490 nm. The relative cell viability (RCV) relative to the control wells was calculated by the following equation: RCV = (ODtest/ ODcontrol) 100%, where ODtest and ODcontrol were obtained in the presence and absence of the magnetic micelles, respectively.

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2.5. In vitro cell labeling

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2.5.1. Cell MRI and intracellular iron content determination The human liver cancer cell line Hep3B and human liver cancer cell line Bel-7402 were obtained from ATCC (VA, USA) and the Shanghai Institute of Life Science Cell Culture Center (Shanghai, China). The liver cancer cells were incubated with the FA-BSA modified magnetic micelles or unmodified magnetic micelles dissolved in DMEM at different iron concentrations (0, 5, 10, 15, 20, and 25 lg/mL) for 2 h. 5 sets of each sample concentration were prepared. The cells were washed 3 times with PBS and centrifuged for collection. Each sample was re-suspended in 300 lL 0.5% agarose gel on a 96-well plate. A 3.0 T whole-body MR scanner in combination with a 8-channel wrist joint coil and the T2-weighted spin echo sequence were employed to test the prepared tubes using the following parameters: FOV = 120 mm, base resolution = 384 384, slice thickness = 1.5 mm, TE = 40 ms, TR = 2000 ms, and scanning time = 13–14 min. The FA-BSA modified and unmodified magnetic micelles were dissolved in DMEM at different iron concentrations (0, 5, 10, 15, 20 and 25 lg/mL) and incubated with hepatoma cells on 12-well plates (5 105 cell1 well1, 1 ml medium) at 37 °C for 2 h, respectively. The cells were washed, collected, and transferred to conical flasks. 0.5 mL of perchloric acid and 2 mL of nitric acid were added to each conical flask and the solution was heated to 240 °C for 1 h and cooled down to room temperature. 5 mL of distilled water was added to the conical flask and shaken thoroughly for the measurement. The intracellular iron content was determined by fast sequential atomic absorption spectrometry (Spectr AA240FS, Varian, Palo Alto, USA). The instrumental parameters were as follows: lamp current = 5 mA, wavelength = 248.3 nm, slit = 0.2 nm, and flame gas = air–acetylene.

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2.5.2. Iron-specific prussian blue staining Prussian blue staining was performed to evaluate the presence of iron cations. The untreated hepatoma cells were seeded on 6-well plates at a density of 2 105 cell well1. After incubation for 24 h, the tumor cells were incubated with either the FA-BSA modified micelles or unmodified magnetic micelles (dissolved in DMEM) at the Fe concentration of 10 lg/mL for 2 h and the cells treated with PBS served as the blank control. After labeling, the cells were washed thrice with the cell culture medium and PBS. The cells were then soaked with 4% paraformaldehyde for 1 h and washed with PBS three times, followed by staining with the Prussian blue solution (equal volume of 2% hydrochloric acid and 2% potassium ferrocyanide) for 30 min. Afterward, the cells were

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washed several times with distilled water and counterstained with the nuclear fast red solution (Sigma–Aldrich) for 3 min. Iron staining was examined by optical microscopy (IX71, Olympus, Japan).

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2.6. In vivo tumor targeting and imaging

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2.6.1. Establishment of human hepatoma xenografts in nude mice The animal protocol was approved by the Institutional Animal Care and Use Committee of Tongji Medical College at the Huazhong University of Science and Technology. The SFP athymic nude male BALB/c mice, 4–6 weeks old and weighing 18–22 g, were obtained from Beijing HFK Bioscience Co. Ltd, China. The hepatoma xenograft models were established according to the previously reported method [32]. Sterile PBS (0.1 ml) containing 3 106 human hepatoma Bel-7402 cells were injected subcutaneously to the lower back of each nude BALB/c mouse. After the tumor volume reached about 400 mm3, the mice bearing the Bel-7402 xenografts (n = 20) were randomly divided into four groups for in vivo studies.

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2.6.2. In vivo MRI The mice bearing tumors were anesthetized with 10% chloral hydrate (40 lL/10 g body weight) by intraperitoneal injection. Different contrast agents including the T1 contrast agent Gd-DTPA (Omniscan, at the dose of 2 ml /kg body weight), T2 contrast agents Feridex (Feridex I.V., Advanced Magnetics, Inc., USA), unmodified magnetic micelles, and FA-BSA modified magnetic micelles (at the dose of 5 mg Fe/kg body weight) were used. Before and after administration of the contrast agents through the tail vein, T1and T2-weighted images were acquired by using spin echo sequence from the mice placed in the 8-channel wrist joint coil. The T1-weighted sequence parameters were as follows: FOV = 80 mm, base resolution = 192 192, slice thickness = 3 mm, TE = 4.71 ms, TR = 106 ms, and scanning time = 1–2 min. The T2weighted sequence parameters were as follows: FOV = 80 mm, base resolution = 192 192, slice thickness = 1.2 mm, TE = 62 ms, TR = 3000 ms, and scanning time = 3 min. The data were analyzed in terms of tumor/muscle signal ratios at each time point and the signal intensity of the tissue was obtained from the region of interest (ROI) with 44 pixels and area of 0.12 cm2. We chose 5 ROIs of different slices in coronal and axial MR images for each tumor and the data were expressed as mean ± standard deviation.

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2.6.3. Histochemistry analysis After 24 h post-injection, the liver and tumor tissues of the aforementioned tumor-bearing nude mice were excised and stored in 4% paraformaldehyde for 24 h. Paraffin blocks were prepared from the fixed tissue samples and sectioned to a thickness of 8 lm for tissue staining. Prussian blue staining was carried out to visualize accumulation of magnetic particles in the tissues. The sections were deparaffinized for 1 h and serially hydrated by immersing twice in 100% xylene, twice in 100% ethanol, and once in 95%, 90%, 80%, and 70% ethanol. The hydrated slides were washed with tap water for 5 min and stained with the Prussian blue solution for 50 min. The slides were then counterstained with the nuclear fast red solution for 5 min and iron staining was observed by optical microscopy.

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3. Results

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3.1. Characterization

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The structure of the poly (HFMA-co-MOTAC)-g-PEGMA copolymers is determined by 1H NMR and the spectrum is shown in Fig. 1A. The NMR spectrum shows characteristic signals of the PEG graft chains ACH2CH2OA giving rise to the broad peak at

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Fig. 1. (A) 1H NMR spectrum of the amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymer. (B) FTIR spectra of (a) SPIONs, (b) amphiphilic poly(HFMA-co-MOTAC)-gPEGMA copolymers, and (c) FA-BSA modified magnetic micelles.

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d = 4.13 ppm and d = 3.68 ppm (labeled e and f in the NMR spectrum). The signals at d = 3.41 ppm (g and j) are assigned to AOCH3 and ACH3 of MOTAC. The characteristic signals of the fluorinated HFMA segments include the peaks at d = 5.01 ppm (d) assigned to ACH2CF2A and d = 4.37 ppm (c) corresponding to ACHFCF3 of the HFMA segment. The characteristic signals of the MOTAC at d = 3.85 ppm (h) and d = 3.58 ppm (i) arise from ACH2CH2 and the signals at 1.99 ppm (b) and 1.16 ppm (a) belong to ACH2A and ACHA of the copolymer backbone, respectively. FTIR is used to further determine the structure and formation of the functional magnetic micelles (Fig. 1B). The FTIR spectrum of the SPIONs is shown in Fig. 1Ba in which the peak at 578 cm1 corresponds to FeAO vibration in Fe3O4 Fig. 1Bb depicts the FTIR spectrum of the amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers. The characteristic absorption by the double bond (C@C) at 1620 cm1 disappears, indicating that the monomers have polymerized. The strong absorption of the ester carbonyl (C@O) bands in HFMA, PEGMA, and MOTAC appears at 1744 cm1. The peaks at 1189 cm1 and 1037 cm1 are attributed to absorption by the CAF groups in HFMA. The absorption peaks at 1000–1200 cm1 are wider and flatter than those of the copolymers without fluorocarbon bonds [33]. The strong peak at 1102 cm1 is attributed to the ether bonds (CAO) in PEGMA and the characteristic [AN–(CH3)3] peak of the MOTAC units appears at 1453 cm1. The results corroborate the participation of HFMA, PEGMA, and MOTAC in polymerization. Compared to the FTIR results acquired from the amphiphilic copolymers, a broader peak at 494–614 cm1 is observed from the FA-BSA modified magnetic micelles (Fig. 1Bc). The new intense peaks at 1630 cm1 and 680 cm1 are ascribed to the benzene ring in the folate and lysine in BSA, respectively. The results reveal that SPIONs are incorporated into the polymeric micelles and FA-BSA adheres to the magnetic micelles successfully. The morphology of the nanoparticles (SPIONs, unmodified magnetic micelles, and FA-BSA modified magnetic micelles) is depicted in Fig. 2A together with the corresponding size distribution diagrams in Fig. 2B. The unmodified magnetic micelles are spherical core–shell structures (Fig. 2Ab) with an average diameter of 161.5 nm and polydispersity index (PDI) of 0.21 (Fig. 2Ba). The morphology of the FA-BSA modified magnetic micelles observed by TEM is shown in Fig. 2Ac. Although the SPION content in the micelles is reduced, the magnetic micelles modified with BSA-FA still have a well-organized core–shell structure. The average hydrodynamic particle size of the FA-BSA modified magnetic micelles is about 196.1 nm (PDI = 0.26) (Fig. 2Bb), which is larger than that of the unmodified magnetic micelles because the FABSA contributes to the hydrodynamic radius of the micelle. The unmodified magnetic micelles have a positive zeta potential of

24.6 ± 6.4 mV due to the cationic monomer MOTAC in the micelles [18]. A zeta potential of 15.7 ± 4.6 mV measured from the FA-BSA indicates that the FA-BSA is negatively charged, and the zeta potential of the unmodified magnetic micelles decreases from 24.6 ± 6.4 to 20.1 ± 6.1 mV (Table 1). The results reveal that SPIONs are incorporated into the polymeric micelles and FA-BSA adheres successfully to the magnetic micelles electrostatically. The TGA thermograms of SPIONs and FA-BSA modified magnetic micelles are shown in Fig. 2C. The nanoparticles after each stage of modification give distinctive TGA curves related to the composition of the magnetic micelles. For SPIONs (Fig. 2Ca), the degradation of oleic acid on the Fe3O4 nanoparticles causes 19.7% weight loss in the temperature range of 200–400 °C. For the FA-BSA modified magnetic micelles (Fig. 2Cb), the first stage of weight loss at 160–300 °C corresponds to degradation of FA-BSA and HFMA, whereas the second stage at 300–420 °C stems from decomposition of PEGMA and MOTAC. The Fe3O4 content in the FA-BSA modified magnetic micelles is calculated to be 19.2 wt.% based on the TGA results. The saturation magnetization values of the unmodified and modified magnetic micelles are 13.9 and 5.5 emu/g, respectively (Fig. 2D). The magnetization curves do not pass directly through the origin, indicating that the prepared magnetic micelles may contain a few ferromagnetic components in addition to superparamagnetic nanoparticles. This may be because the special self-assembled pattern and crystal structure of the prepared magnetic micelles span both the superparamagnetic and ferrimagnetic regimes [34]. The loss of magnetization may be due to the presence of copolymers surrounding the magnetic cores. Fig. 3a shows sensitive and concentration-dependent dark areas in the MR images obtained from the aqueous medium with the magnetic micelles. The significantly reduced intensities even at low iron concentrations indicate that the magnetic micelles produce appreciable negative contrast enhancement in MRI. The effectiveness of an MRI negative contrast agent is commonly evaluated in terms of its transverse relaxivity (r2), which represents the efficiency of the magnetic nanoparticles to shorten the proton relaxation time. Fig. 3b shows that the relaxation rates (1/T2) of the micelles vary linearly with iron concentrations. The transverse relaxivity (r2) of the unmodified and FA-BSA modified magnetic micelles calculated from the slope of the graph are 243.9 and 179.5 mM1 s1, respectively (Fig. 3b).

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Cell viability of over 80% is maintained from the human normal liver cell line HL-7702 after incubation for 24 h and 48 h by increasing the iron concentration from 25 to 100 lg/mL (Fig. 4),

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Fig. 2. (A) TEM images of (a) SPIONs, (b) unmodified magnetic micelles, and (c) FA-BSA modified magnetic micelles. (B) Size distributions of (a) unmodified magnetic micelles and (b) FA-BSA modified magnetic micelles. (C) TGA curves of (a) SPIONs and (b) FA-BSA modified magnetic micelles. (D) Magnetic hysteresis loops of (a) unmodified magnetic micelles and (b) FA-BSA modified magnetic micelles.

Table 1 Zeta potential of nanoparticles. Nanoparticles

FA-BSA

Unmodified magnetic micelles

FA-BSA modified magnetic micelles

Zeta potential (mV)

15.7 ± 4.6

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Fig. 3. (a) T2-weighted MR images of the unmodified and FA-BSA modified magnetic micelles for different iron concentrations; (b) T2 relaxivity (r2) of unmodified and FA-BSA modified magnetic micelles. 1 mM Fe is equivalent to 55.845 lg Fe/mL.

indicating that both the FA-BSA modified and unmodified magnetic micelles have no obvious cytotoxicity and are suitable nanocarriers or probes in biomedical applications. The uptake of both the FA-BSA modified and unmodified magnetic micelles is evaluated using the human hepatoma cell lines Hep3B and Bel-7402 by different methods including MR imaging, intracellular iron content determination, and iron-specific Prussian blue staining (Fig. 5). On MR maps, the cells treated with the FA-BSA modified magnetic micelles show a more significant negative contrast enhancement compared to that with the unmodified magnetic micelles at each concentration (Fig. 5A). The hepatoma cells incubated with the targeted magnetic micelles showed an increasing iron adsorption curve dose-dependently and is higher than that of the unmodified magnetic micelles (Fig. 5B). Iron-specific Prussian blue staining results are consistent with the cell MRI and cell uptake studies. FA-BSA targeted magnetic micelles accumulate more extensively in the cytoplasmic region, in comparison, the tumor cells treated with unmodified magnetic micelles show only a few internalized blue particles (Fig. 5C). These findings demonstrate the high specificity and efficiency of the folate functionalized magnetic micelles for hepatic carcinoma cells. The iron uptake percentages of the prepared magnetic micelles by the Hep3B and Bel-7402 cells are displayed in Table S1. There is no difference between the iron uptake percentages of unmodified magnetic micelles by Hep3B and Bel-7402 cells. However, the average uptake rate of FA-BSA modified particles in Bel-7402 cells is 12.07%, which is larger than 8.83% in the Hep3B cells. The difference between Bel-7402 and Hep3B cells is due to folic acid modification.

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The feasibility to use the FA-BSA modified magnetic micelles in specific MR imaging of tumors is further explored by in vivo imaging (Fig. 6). The T2 signal intensity in the group treated with the

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Fig. 4. MTT assay results for HL-7702 cells incubated with (a) unmodified magnetic micelles and (b) FA-BSA modified magnetic micelles at different iron concentrations for 24 h and 48 h. Data are expressed as mean ± SD (n = 5).

Fig. 5. (A) Cell MRI, T2-weighted imaging of the Hep3B cells (left) and Bel-7402 cells (right) suspension collected after incubation for 2 h with unmodified micelles (upper row) and FA-BSA modified micelles (lower row) at final Fe concentrations of 0, 5, 10, 15, 20, and 25 lg/mL. (B) Iron uptake curves of the Hep3B cells (left) and Bel-7402 cells (right) after incubation for 2 h with the unmodified and FA-BSA modified magnetic micelles at final Fe concentrations of 0, 5, 10, 15, 20, and 25 lg/mL. Data are expressed as mean ± SD (n = 5). (C) Prussian blue staining results. Upper row: Prussian blue staining images of the Hep3B cells after incubation for 2 h with (b) unmodified and (c) FA-BSA modified magnetic micelles and (a) without micelles as the control. Bottom row: Prussian blue staining images of the Bel-7402 cells after incubation for 2 h with (e) unmodified and (f) FA-BSA modified magnetic micelles and (d) without micelles as the control.



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Fig. 6. (a) In vivo MR images of Bel-7402 subcutaneous xenograft in nude mice acquired after administration of Gd-DTPA (top row), Feridex (the second row), unmodified (the third row), and FA-BSA modified magnetic micelles (bottom row) at different time points (10 min, 1, 24, 48, and 72 h). (b) Tumor-to-muscle signal ratios observed on T1-images of the groups treated with Gd-DTPA at different time points different time points (10 min, 1, 24, 48, and 72 h). (c) Tumor-to-muscle signal ratios observed on T2-images of the groups treated with Feridex, unmodified magnetic micelles, and FA-BSA modified magnetic micelles at different time points (10 min, 1, 24, 48, and 72 h). TMR: tumor-to-muscle signal ratios. Tumor volume: 400–700 mm3, n = 3–5 for each group.

Fig. 7. Histochemical analysis: Prussian blue staining images of the tissue sections after injection of contrast agent for 24 h. Top row: Liver tissue sections with (a) Gd-DTPA, (b) Feridex, (c) unmodified and (d) FA-BSA modified magnetic micelles. Bottom row: Tumor tissue sections with (e) Gd-DTPA, (f) Feridex, (g) unmodified and (h) FA-BSA modified magnetic micelles.

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FA-BSA modified magnetic micelles decreases most significantly between 1 h and 24 h, while the tumors with Feridex and unmodified magnetic micelles show minimal contrast change in the same Q4 period. For comparison, the T1 signal intensity of the tumor increases significantly within 10 min after injection with Gd-DTPA. The distributions of the four different contrast agents in the normal liver at 24 h post-injection of contrast agents are measured to investigate magnetic micelle disposition (Fig. 7). Prussian blue staining performed on the group with Feridex shows large accumulation of magnetic particles in the liver (Fig. 7b black arrow), whereas the group with unmodified magnetic micelles shows less blue spots (Fig. 7c black arrow). In contrast, the FA-BSA modified magnetic micelles do not accumulate much in the liver (Fig. 7d). Prussian blue staining of the tumor tissues is performed at 24 h post-injection of contrast agents. Accumulation of blue spots in

the tumor tissues observed from the folate-targeted groups (Fig. 7h, black arrow) is obviously larger than that of untargeted groups (Fig. 7g, black arrow). There are no visible blue spots in the groups of Gd-DTPA (Fig. 7e) and a small number of blue spots trapped in the tumor vascularity in the groups of Feridex (Fig. 7f, black arrow). The distribution behavior of the FA-BSA modified magnetic micelles suggests that PEGMA and FA-BSA modification of the magnetic micelles enables the particles to escape from the reticuloendothelial system and deliver to the tumor tissue.

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The process to synthesize the FA-BSA modified and SPIONsloaded polymeric micelles is illustrated in Scheme 1. The cationic

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Scheme 1. Schematic illustration of the synthesis of the FA-BSA modified SPIONs-loaded polymeric micelles by self-assembly and electrostatic complexation.

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amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers are synthesized by polymerization of the hydrophobic HFMA monomer, hydrophilic PEGMA macromonomer, and cationic MATOC monomer. Generally, amphiphilic polymers, which form self-assembled micelles in water, can encapsulate the hydrophobic guest molecules [35]. By this approach, the hydrophobic SPIONs are incorporated into the core of polymeric micelles with the hydrophilic parts on the surface forming the water-soluble cationic magnetic micelles. In order to enhance the targeting property, the negatively charged FA-BSA is attached to the cationic magnetic micelles via electrostatic interaction to form the functional magnetic micelles. The chemical structure determined by 1H NMR and FTIR, morphology shown in the TEM images, and zeta potentials in Table 1 are in accordance with our design objective (Scheme 1). Particle size is known to be intrinsically related to the accumulation kinetics in the solid tumor. The extravasation and accumulation of larger particles in the tumor interstitium is slower compared to smaller ones, but they are retained for a longer time within the tumor tissue than smaller molecules, which rapidly clear by diffusion [36]. The pore cutoff size of porous blood vessels in majority of tumors is known to be 380–780 nm [37], and the maximum size of nanoparticles allowing penetration through cell membranes is known to be 500 nm [38]. Therefore, although the average hydrodynamic particle size of the prepared FA-BSA modified magnetic micelles is about 196.1 nm (Fig. 2B), which is larger than commercialized nanodrugs or contrast agents, it still can extravasate into the tumor interstitium and accumulate tumor tissue. And due to the large size, FA-BSA modified magnetic micelles are retained in the tumor tissue for a long time, which is useful for biomedical applications. Furthermore, the transverse relaxivity in MRI correlates positively with the size of the magnetic nanocluster. Increasing the size of the magnetic nanoclusters can increase the transverse relaxivity [39]. The transverse relaxivity of FA-BSA modified magnetic micelles is 179.5 mM1 s1, which is much higher than that of commercially available MRI contrast agents used in clinical studies such as carboxydextran-coated ResovistÒ (151.0 mM1 s1) [40], Feridex IV (98.3 mM1 s1) [41], and other tumor-targeted MRI contrast agents reported in the literature such as FA-PEG-modified Fe3O4 NPs (99.6 mM1 s1) [5]. The higher relaxivity means that administration of lower doses still achieves accurate differentiation of tissues in MRI. Therefore, the FA-BSA modified magnetic micelles have superior negative contrast effect and can be used as a negative contrast agent in T2-weighted MR imaging. The MR T2-weighted images of the hepatoma cells incubated with magnetic micelles show lower signal intensity in the MR images, indicating higher intracellular uptake of the magnetic micelles into the hepatoma cells. With increasing concentrations, the signal intensity decreases more obviously. The results are in

agreement with previous studies demonstrating the in vitro concentration dependence of the magnetic nanoparticles absorbed by hepatoma cells [25]. Both intracellular iron determination and Prussian blue staining provide more evidences of more cellular uptake of folate-targeted micelles than the non-targeted counterparts into tumor cells. Moreover, the uptake rate of the folate-targeted micelles by the Bel-7402 cells is larger than that by the Hep3B cells (Table S1). The two cell lines, Bel-7402 and Hep3B, express different levels of folate receptors. Bel-7402 is FR-positive cells [42] whereas the Hep3B cells show negligible expression which is used as the FR-negative control [43]. Therefore, the in vitro cell uptake study confirms that folic acid enhances uptake of the magnetic micelles by the over-expressed tumor cells via ligand–receptor interactions. In fact, folate is frequently used as the target conjugated with nanoprobes for site-specific delivery to tumors [27,42]. In addition, enhanced iron uptake and signal reduction are observed from the groups with unmodified magnetic micelles due to nonspecific phagocytosis. The positively charged micelles are electrostatically attracted to the negatively charged cell membrane leading to cellular uptake via clathrin-mediated endocytosis [44]. The T2 signal intensity of the tumor decreases significantly after injection with FA-BSA modified magnetic micelles, demonstrating the efficient use as negative nanoprobes in MR imaging of xenografted hepatoma. The distribution of the four different contrast agents in the normal liver and tumor tissue at 24 h post-injection provide further evidences. Prussian blue staining performed on the group with Feridex and unmodified magnetic micelles show accumulation of magnetic particles in the liver, whereas the FABSA modified magnetic micelles do not obviously accumulate in the liver (Fig. 7d). BSA is a natural polymer possessing many advantages such as low immunogenicity and good biocompatibility. BSA is not captured by macrophages in the liver nor expelled by normal kidney excretion [45], thereby prolonging the blood circulation lifetime. Moreover, the hydrophilic polymer PEGMA is frequently used to modify the surface of contrast agents. The modification reduces the uptake by the reticuloendothelial system and increases its circulation time without causing immune reactions [46]. A longer circulation time boosts the chance of contact between the FA-BSA modified magnetic micelles and folate receptor on the tumor. Prussian blue staining of the tumor tissues at 24 h post-injection of contrast agents confirms that the MR signal intensity change of the tumor after bolus intravenous administration of the folate-functionalized magnetic micelles is due to accumulation of micelles in the tumor tissue instead of being trapped in the tumor vascularity. The tumor uptake characteristics of the FA-BSA modified magnetic micelles are compared to those of Gd-DTPA, Feridex, and unmodified magnetic micelles as controls. There are no visible blue spots in the groups of Gd-DTPA, and a



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small number of blue spots are trapped in the tumor vascularity in the groups of Feridex. Accumulation of blue spots in the tumor tissues observed from the folate targeted groups is obviously larger than that of untargeted groups (Fig. 7). It confirms that the magnetic micelles enhance the T2-weighted MR images of the subcutaneous tumor as shown in Fig. 6. In fact, the uptake percentages of the unmodified and FA-BSA modified magnetic micelles by the tumor are (1.54 ± 0.041)% and (2.91 ± 0.035)%, respectively (Supporting information). The uptake percentage of the FA-BSA modified magnetic micelles by the tumor is larger than that of the unmodified magnetic micelles. The results are in agreement with those obtained by MR imaging and Prussian blue staining showing that only a small amount of the FA-BSA modified magnetic micelles is uptaken by the tumor. The possible reason is that most of magnetic micelles are excreted by way of excrement in the intestine and through urine in the kidney [47]. However, such a small quantity of magnetic micelles can produce significant MR effect, indicating that MR imaging is very sensitive to the detection of FA-BSA modified magnetic micelles. Accumulation of nanoparticles in tumors is based on blood circulation and extravasation [38]. Both PEGMA and BSA in the developed magnetic micelles can extend the circulation time, thereby increasing the fraction of micelles reaching the tumor. Moreover, folate mediated active targeting can enhance the uptake of magnetic micelles by tumor via ligand–receptor interactions. Therefore, enhanced tumor targeting can be achieved at 24 h due to the relatively long circulation lifetime of FA-BSA modified magnetic micelles and folate mediated active targeting. The unmodified magnetic micelles without folic acid only minimally accumulate in the tumor region after intravenous injection because of the enhanced permeability and retention (EPR) effect. MRI and Prussian blue staining further demonstrate that the FA-BSA functionalized magnetic micelles can be specifically assembled into the tumor tissues in vivo and also suggest a limited contribution of passive mechanisms to tumor tissue retention of the magnetic micelles.

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Tumor-targeted magnetic micelles are prepared by self-assembling of SPIONs-loaded amphiphilic poly(HFMA-co-MOTAC)-gPEGMA copolymeric micelles and FA-BSA via electrostatic interactions. In vitro studies demonstrate the high T2 relaxivity indicating potential use as a negative MRI contrast agent for folate-receptor overexpressing cancer. In vitro cellular uptake studies with hepatoma cells reveal that the FA-BSA functionalized magnetic micelles are internalized to a higher concentration than the unmodified magnetic micelles. In vivo tumor specific MR imaging and Prussian blue staining reveal the high selectivity and sensitivity to hepatoma, especially at 24 h post-injection. PEGMA and FA-BSA modification of the magnetic micelles prolong the circulation time and enable the particles to be delivered to the tumor tissue. Furthermore, folate mediated active targeting can improve the uptake of magnetic micelles by the tumor. Therefore, the FA-BSA modified magnetic micelles are potentially useful as a tumor-targeting contrast agent in cancer imaging as well as preoperative and postoperative delineation of tumors to impart important information with respect to active drug targeting and adjuvant therapy.

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Acknowledgments

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Q5 This work was financially supported by the National Natural Q6 Science Foundation of China (NSFC, 81372369, 81171386, 81101042), National Basic Research Program of China (973 Program, 2011CB933103), Hong Kong Research Grants Council (RGC)

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General Research Funds (GRF) No. CityU 112212, and City University of Hong Kong Applied Research Grant (ARG) Nos. 9667085. This work was partially supported by the National Natural Science Foundation of China (NSFC, 51273058).

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Appendix A. Figures with essential colour discrimination

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Certain figures in this article, particularly Figs. 1 and 5–7 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2015.01.006.

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Appendix B. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.01. 006.

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