Superhigh-magnetization nanocarrier as a doxorubicin delivery platform for magnetic targeting therapy

Biomaterials 32 (2011) 8999e9010

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Superhigh-magnetization nanocarrier as a doxorubicin delivery platform for magnetic targeting therapy Mu-Yi Hua a,1, *, Hung-Wei Yang a,1, Hao-Li Liu b,1, Rung-Ywan Tsai c, See-Tong Pang d, Kun-Lung Chuang d, Yu-Sun Chang e, Tsong-Long Hwang f, Ying-Hsu Chang d, Heng-Chang Chuang d, Cheng-Keng Chuang d, ** a

Molecular Medicine Research Center, Department of Chemical and Materials Engineering, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, ROC Department of Electrical Engineering, Chang-Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, ROC Electronics and Optoelectronics Research Laboratories, Industrial Technology Research Institute, 195, Sec. 4, Chung-Hsing Rd., Hsin-chu 31040, Taiwan, ROC d Department of Urology, Chang Gung University College of Medicine and Memorial Hospital, 5 Fu-shing Road, and Graduate Institute of Clinical Medical Sciences, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan ROC e Graduate Institute of Biomedical Sciences and Molecular Medicine Research Center, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan ROC f Graduate Institute of Natural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan ROC b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 June 2011 Accepted 7 August 2011 Available online 27 August 2011

The aim of this study describes the creation of superhigh-magnetization nanocarriers (SHMNCs) comprised of a magnetic Fe3O4 (SHMNPs) core and a shell of aqueous stable self-doped poly[N-(1-onebutyric acid)]aniline (SPAnH), which have a high drug loading capacity (w27.1 wt%) of doxorubicin (DOX). The SHMNCs display superparamagnetic property with a magnetization of 89.7 emu/g greater than that of Resovist (a commercial contrast agent used for magnetic resonance imaging; 73.7 emu/g). Conjugating the anticancer drug DOX to these nanocarriers enhances the drug’s thermal stability and maximizes the efficiency with which it is delivered by magnetic targeting (MT) therapy to MGH-U1 bladder cancer cells, in part by avoiding the effects of p-glycoprotein (P-gp) pumps to enhance the intracellular concentration of DOX. The high R2 relaxivity (434.7 mM
1s
1) of SHMNCs not only be a most effective MT carrier of chemotherapeutic agent but be an excellent contrast agent of MRI, allowing the assessment of the distribution and concentration of DOX in various tissues and organs. This advanced drug delivery system promises to provide more effective MT therapy and tumor treatment using lower therapeutic doses and potentially reducing the side effects of cardiotoxicity caused by DOX. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: P-glycoprotein pump Bladder cancer Superhigh-magnetization nanocarriers Magnetic targeting therapy Magnetic resonance image

1. Introduction Bladder cancer is the second most frequent urologic malignancy and the 13th most common cause of cancer death globally, although the chemotherapeutic agent has been considered to treat bladder cancer effectively with significant improvement in survival of patients [1,2]. DOX is one of the most effective drugs against a wide range of cancers. However, its clinical use is limited by severe side-effects such as cardiotoxicity and acquired drug resistance. Multidrug resistance such as that caused by the P-gp pumping effect commonly limits the effectiveness of chemotherapeutic agents in treating malignancies [3]. Consequently, new drug

* Corresponding author. Tel.: þ886 3 211 8800x5289; fax: þ886 3 211 8668. ** Corresponding author. Tel.: þ886 3 328 1200x2103; fax: þ886 3 328 5818. E-mail addresses: [email protected] (M.-Y. Hua), [email protected] (C.-K. Chuang). 1 M.-Y. Hua, H.-W. Yang, and H.-L. Liu contributed equally to this work. 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.08.014

delivery systems such as polymeric nanoparticles [4e6], polymeric micelles [7e10] and self-aggregated nanoparticles [11] are used widely to reduce the toxic side-effects of chemotherapeutic agents, enhance their effective lifespan during blood circulation, and reverse P-gperelated drug resistance [4]. However, these systems are nonspecific and the rate at which they release drugs cannot be controlled accurately. It is therefore important to combine the drug delivery system with a targeting therapy to minimize the adverse effects of chemotherapeutic agents on healthy cells. Two major approaches have been studied to target specific sites in the body: molecular targeting using specific ligands or antibodies [12,13], and magnetic targeting (MT) using magnetic drug nanocarriers [14,15]. Magnetic nanoparticles (MNPs) can be used for a variety of biomedical applications, such as contrast-enhancing agents for magnetic resonance imaging (MRI) [16e18], molecular imaging study [19], magnetically guided drug targeting [20e23], enhancing enzyme stability (which normally is limited by conformational restrictions imposed by binding of the enzyme to carriers) [24e26]

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for repeated use, and magnetic diagnosis to detect tumor cells in blood or bone marrow by modifying the surfaces with different specific proteins or antibodies [27]. Although MNPs are well-known drug carriers, they have many limiting characteristics, such as low water solubility and a lack of specificity in their ability to target their chemotherapeutic effects. To create more effective drug carriers for MT therapy, it is necessary to enhance particle magnetization and avoid their aggregation, while maintaining their original advantages (e.g., superparamagnetic properties, nano-scale size). This study presents the development of a superhighmagnetization nanocarriers (SHMNCs) comprised of a magnetic Fe3O4 (SHMNPs) core and a shell of aqueous stable self-doped poly[N(1-one-butyric acid)]aniline (SPAnH), that can conjugate DOX on its surface covalently. These drug-conjugated nanocarriers (i.e., SHMNCDOX) are small enough to penetrate tissues and be taken up effectively by tumor cells [28,29] by the pathways of endocytosis. This high magnetic drug delivery system should provide more drug accumulation at tumor site for cancer therapy using lower therapeutic doses and could potentially reduce the side effects of chemotherapy. 2. Materials and methods 2.1. Preparation of SHMNPs and SHMNCs SHMNPs were generated by dissolving 4.32 mmol FeCl3 and 6.48 mmol FeCl2∙4H2O in 400 mL deionized (DI) water and stirring for 5 min under N2 gas. The solution was heated slowly to 50  C and 30 mL of 0.576 N NaOH was added over a 70min period, after which the temperature was increased to 80  C for 20 min. The solution was then quenched rapidly in ice and 0.1 N HCl was added slowly until neutralized. The SHMNPs were separated from the solution by attracting them to the wall of a separation funnel using a strong magnet, washed with DI water several times to remove unreacted material, and dispersed uniformly in DI water by sonication at 300 W for 1 h. Further, the detailed procedures of SHMNCs formation were reported previous [22]. The SHMNCs were dispersed in DI water and analyzed by Fourier transform infrared (FT-IR) spectroscopy (TENSOR Model 27, Bruker). 2.2. Magnetic resonance relaxivity Enhancement of SHMNCs delivery via MT was also evaluated by magnetic resonance (MR) relaxivity measurement, which the spinespin relaxation rates (R2) can be utilized to calibrate the iron concentration of the particles. A series of T2weighted images (indicating susceptibility artifact-induced signal loss caused by MNPs accumulation) with different echo times (TE) were obtained to produce R2 values of gel phantoms containing 0.1e1 mM of iron (Fe) particles from SHMNCs, low-magnetization nanocarriers (LMNCs) or Resovist in 24-well plates. All magnetic resonance signals were produced from a clinical 3-T MRI scanner (Trio with Tim, Siemens, Erlangen, Germany) and a standard wrist coil with an inner diameter of 13 cm. The imaging parameters were set to be as follows: repetition time (TR) ¼ 3860 ms, TE ¼ 8/14, 28/57, and 85/228 ms, matrix size ¼ 128  256, slice thickness ¼ 1.5 mm, and field of view ¼ 38  76 mm. The R2 values were then calculated from the series of TE images by using standard procedures [30].

0.1 M MES buffer, resuspended in 0.2 mL of MES, and mixed with 0.1 mL of DOX at 25  C. The solution was mixed by vortexing for 1 h, then sonicated for another 1 h. The primary amino groups of DOX coupled with resultant active ester, and resulted in covalent conjugation of DOX on the surface of SHMNCs or LMNCs (i.e., SHMNCDOX or LMNC-DOX) then separated from the solution, washed with DI water to remove the MES buffer and unbound DOX, and then dispersed in 0.2 mL DI water. The binding capacity was analyzed by high-performance liquid chromatography (HPLC) on a SUPELCOSILÔ LC-18 column (4.6  250 mm) using an L-2130 pump and an L-2400 UV-detector (Hitachi). The mobile phase of the HPLC was a 50/15/35 (v/v/ v) mixture of DI water, acetonitrile and methanol with a flow rate of 1.5 mL/min; data were measured at 256 nm. 2.5. Synthesis of SHMNC-Cy5 and LMNC-Cy5 conjugates EDC (12 mg) and sulfo-NHS (27 mg) were dissolved in 2 mL of 0.5 M MES buffer (pH 6.3) in the dark. The solution was mixed with 0.1 mL of SHMNCs and LMNCs (10 mg/mL) at 25  C and sonicated for 30 min in the dark. After separation and washing with DI water, SHMNCs were added to 0.2 mL MES buffer, mixed with 0.1 mL of the monofunctional hydrazide cyanine 5 (Cy5) at 25  C, and vortexed for 2 h in the dark. Cy5 immobilized on the surface of SHMNCs and LMNCs (i.e., SHMNCCy5 and LMNC-Cy5) was separated from the solution and washed with DI water. 2.6. Stability test for SHMNC-DOX Degradation analysis of DOX was performed using separated supernatant by HPLC. The mobile phase consisted of a mixture of 45 volumes of acetonitrile and 55 volumes of solution containing 10 g of sodium laurisulphate, 2.5 mL of phosphoric acid (85%) and 1000 mL of DI water. The flow rate was 1.0 mL/min and UV detection was performed at 298 nm. The internal standard was a solution of papaverini hydrochloridum at a concentration of 50 mg/mL. The stabilities of free-DOX and SHMNC-DOX were analyzed for a period of 108 h at 37  C. 2.7. Determination of the SHMNCs uptake pathway The uptake of SHMNCs was examined by incubating MGH-U1 cells (obtained from Dr. C. W. Lin, Massachusetts General Hospital, USA.) with filipin III (1.5 mg/mL), amiloride (15 mg/mL) or chlorpromazine (12 mg/mL) for 1 h at 37  C to inhibit caveolae, macropinocytosis and the formation of clathrin vesicles, respectively. Cells were washed with Hank’s balanced salt solution (HBSS) and treated with SHMNCCy5 (150 mg/mL) for an additional 2 h at 37  C. Next, the cells were washed twice with ice cold phosphate-buffered saline (PBS) (pH 7.0), fixed with fresh ice ethanol for 5 min at room temperature. Cells were washed three times with HBSS and analyzed by TCS SP2 confocal spectral microscopy using Leica QWin software (Leica). 2.8. Cell uptake study Typical cellular membrane drug resistance bladder-cancer cells (MGH-U1) were incubated with SHMNCs and SHMNC-DOX for 8 h at 37  C. The cells were pretreated with three membrane entry inhibitors for 1 h, respectively, before co-culturing with SHMNC-DOX. After washing three times with PBS (pH 7.0), the cells were fixed with 3% glutaraldehyde for 2 h at 4  C, postfixed with 1% OsO4 for 1 h at 4  C, washed three times with 0.1 M cacodylate buffer (pH 7.4), and dehydrated using a graded series of ethanol and embedding medium. Cells were embedded in molds in Spurr’s resin (i.e., 1:1 alcohol:Epon [v/v]) and polymerized at 60  C for 24 h. Ultrathin sections (80 nm) were cut using a diamond knife and stained with 4% uranyl acetate and lead citrate for 2 h and 10 min, respectively. Images were acquired using an H7500 transmission electron microscope (TEM) (Hitachi) operating at an acceleration voltage of 100 kV.

2.3. In vitro magnetic attractability 2.9. DNA interstrand crosslinking The magnetic attractability of SHMNCs was tested in vitro using an external magnetic field. Thin glass tubing (0.25-mm i.d.) was positioned 4 mm below the pole of a 0.4-T magnet. Suspensions of Resovist or SHMNCs (2.5 mg Fe/mL) in 1 mL of sample solution were infused continuously using a syringe pump through one end of the tubing at a fixed flow rate of 15 cm/s and collected at the other end. After initiation of the magnetic field, serial images of the tubing segment located near the pole were acquired at 2-min intervals over a 10-min period with a digital camera. The amounts of Fe of attracted SHMNCs and Resovist were analyzed by the inductively coupled plasma optical emission spectrometry (ICP-OES, 720-ES, Varian Inc., Palo Alto, CA, USA). 2.4. Immobilization of DOX on SHMNCs and LMNCs Twenty-four mg of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 27 mg of N-hydroxysulfosuccinimide (sulfo-NHS) were dissolved in 2 mL of 0.5 M 2-(N-morpholino)ethanesulfonic acid hydrate (MES) in the dark. A 0.2 mL aliquot of this solution was mixed with 0.2 mL of SHMNCs or LMNCs (10 mg/ mL) at 25  C and sonicated for 30 min in the dark to produce the formation of amide bond between activated carboxyl groups. After separation then washed with 0.8 mL

To confirm the cytotoxic effects of DOX, an ethidium bromide fluorescence assay was used to measure the level of DNA interstrand crosslinking in MGH-U1 cells. The cells were exposed initially to different concentrations (1e5 mM) of free-DOX or SHMNC-DOX and incubated for 16 h. After incubation, w1  106 cells were collected by centrifugation at 5000 rpm for 6 min at 8  C and resuspended in PBS. Forty mL of the cell suspension were incubated for 15 min at 4  C with 200 mL of lysis buffer. After lysis, the cell pellets were separated by centrifugation at 5000 rpm for 6 min and the suspension incubated with 25 mL of heparin (500 IU/mL) for another 20 min at 37  C, followed by the addition of 1 mL of ethidium bromide solution. The mixture was heated for 5 min at 100  C to denature the DNA, then cooled in an ice bath for 6 min to renature it. Fluorescence was measured with excitation and emission wavelengths of 525 and 580 nm, respectively [31]. 2.10. Quantitative real-time PCR (Q-PCR) Q-PCR SYBR Green primers for the MGH-U1 gene were designed using Beacon Designer software (PREMIER Biosoft International). The primer TTAgACAgCCTCATATTTTg, specific for multidrug resistance gene 1 (MDR-1), was used to assess its

M.-Y. Hua et al. / Biomaterials 32 (2011) 8999e9010 expression in MGH-U1 cells relative to that of a common sequence upstream primer, TTCTggATggTggACAggCg (MDR-U) [32]. Reactions were performed using SYBR Green Supermix (Bio-Rad) and an iCycler iQ real-time detection system (Bio-Rad), and incubated for 3 min at 95  C, 15 s at 95  C and 30 s at 56  C for 55 cycles. Relative expression levels of ezrin, radixin and moesin were analyzed in real-time using the iCycle iQ system software and expressed as a ratio relative to the expression of the housekeeping gene b-actin. Each sample was replicated twice from three independent sets of RNA preparations. 2.11. In vitro cytotoxicity assay MGH-U1 cells were cultured in RPMI 1640 medium supplemented with 2.2 mg/ mL sodium carbonate, 10% fetal bovine serum, 50 mg/mL gentamycin, 50 mg/mL penicillin, and 50 mg/mL streptomycin at 37  C and 5% CO2. Approximately 10,000 cells (i.e., 150 mL of a suspension of 6.67  104 cells/mL) were placed in each well of a 96-well culture plate and incubated in a humidified chamber at 37  C and 5% CO2 for 24 h. Fifty mL of different concentrations of SHMNCs, free-DOX and SHMNC-DOX in medium were added and the culture continued. Cell cultures were performed in the presence of a 900-Gauss magnetic field applied beneath the culture plate. The MT efficiency in cell toxicity was tested by adding 50 mL different concentrations of LMNC-DOX and SHMNC-DOX co-cultured with the cells and changed the magnetic field to 300-Gauss. Cell growth was observed by counting after 24 h. Before counting, the culture medium was removed and cells were incubated in 120 mL of XTT for 3 h. After the reaction, 100 mL of XTT solution was sampled from each well and transferred to a 96-well counting dish. Cytotoxicity toward MGH-U1 cells in vitro was evaluated by measuring the optical density (OD) at 490 nm using an ELISA reader. In another series of experiments, 2 mL of MGH-U1 cells (5000 cells/ mL) were cultured in 35-mm dishes in a humidified chamber at 37  C and 5% CO2 for 48 h. One hundred mL of SHMNC-Cy5 were added and co-cultivated for another 24 h. The medium was removed, cells were washed with 1 mL of HBSS and 1 mL LIVE/ DEAD reagent was added. After 30 min, the reagent was removed and the cells washed again with HBSS. SHMNC-Cy5 uptake and cytotoxicity were observed by a TCS SP2 confocal spectral microscopy. In a separate series of experiments, 2 mL of MGH-U1 cells (10,000 cells/mL) were plated in 35-mm diameter plates and cultured in a humidified chamber at 37  C and 5% CO2 for 48 h. One hundred mL of identical concentrations of free-, LMNC- and SHMNC-DOX in RPMI 1640 medium were added and performed in the presence of 300- and/or 900-Gauss magnetic field applied beneath the culture dish, and then the incubation continued for 12 h. The medium was removed, cells were washed with 1 mL of HBSS and 1 mL LIVE/DEAD reagent (Invitrogen) was added. After 30 min, the reagent was removed and the cells washed again with HBSS. Cytotoxicity was monitored using a TCS SP2 confocal spectral microscope. 2.12. In vivo MRI image All animal experiments were approved by the Institutional Animal Care and Use Committee of Chang Gung University and performed in accordance with the guidelines. Mice were raised in a room with a thermostat at 26  C. NU/NU mice weighing w25e30 g (5e6 weeks old) were tested to confirm the efficacy of the proposed approach. Briefly, cultured MGH-U1 tumor cells (107 cells/mouse) were injected over a 2-min period into the hypoderm using a syringe, and needlewithdrawal was carried out over another period of 0.5 min. Experiments consisted of two groups of mice (n ¼ 3). One group was injected a single dose of Resovist (15 mg/kg) and another group was injected a same dose of SHMNC-DOX. Those mice were administered the drugs via the external jugular vein three weeks after they were injected with the tumor cells and a neodymiumeironeboron permanent magnet with a maximum magnetic flux density of 0.4 T was secured to the hypodermic tumors for 18 h. The amounts of drug were quantified by ICP-OES using a Varian 720-ES spectrometer. All MRI images were acquired on a 7T scanner. The mice were anesthetized with 2% isoflurane throughout the MRI process, placed in an acrylic holder and positioned in the center of the magnet. Tumor size was quantified using T2-weighted images with the following parameters: TR/TE ¼ 2500 ms/68 ms, matrix size ¼ 176  256, FOV ¼ 31  35 mm (resolution ¼ 0.18  0.14 mm). For the biodistribution of nanocarrier by in vivo imaging, multiple-TE T2-weighted images were acquired and R2 maps (where R2 is the inverse of T2) were generated using the images acquired with various TE values at each time point. A self-developed code executed in MATLAB (Mathworks Inc.; Natick, MA, USA) was used to generate the R2 values based on the equation SI ¼ A  e
TE/T2 þ B, where SI is the signal intensity, TE is the echo time, A is the amplitude and B is the offset. T2-weighted images were obtained to produce R2 maps both in gel phantom or in vivo experiments by using a double-TE spineecho sequence, and acquired three times using the following parameters: TR ¼ 2000 ms, TE ¼ 6.4/13/26/39/45/64/77/84/96 ms, matrix size ¼ 208  256, FOV ¼ 45  55 mm (resolution ¼ 0.22  0.21 mm), slice thickness ¼ 0.7 mm. Mice were sacrificed after MRI process. Slides were stained with Prussian blue (Sigma) to detect iron deposited in cells/tissue samples. Briefly, hypodermic tumor sections mounted on slides were stained in a 1:1 mixture of 2% potassium ferrocyanide and 2% hydrochloric acid for 30 min at room temperature. The slides were rinsed with DI water.

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2.13. In vivo fluorescence imaging Tumor bearing mice were intravenously injected with 100 ml of 5 mg/ml SHMNC-Cy5 and LMNC-Cy5, respectively, then a neodymiumeironeboron permanent magnet with a maximum magnetic flux density of 0.4 T was secured to the hypodermic tumors for 18 h and imaged using the in vivo fluorescence imaging system (XENOGEN IVIS 100). Visible light with a central wavelength at 649 nm was used as the excitation source. Auto-fluorescence (particularly from food residues in the stomach and intestine) was removed by using the spectral unmixing software.

3. Results 3.1. Characterizations of SHMNCs The average hydrodynamic size of SHMNCs (87.2 nm) (Fig. 1A) was greater than that measured by TEM (12.3 nm) (Fig. 1B). The diameter of the SHMNPs core as measured by TEM (and confirmed by XRD) was 10.5 nm, suggesting that the thickness of polymer shell was w0.9 nm. The magnetization of nanocarrier was measured using a superconducting quantum interference device (SQUID; Quantum Design, model MPMS7) with an applied magnetic field of 6 kOe at room temperature. The SHMNPs had a magnetization of 99.1 emu/g; this decreased to 89.7 emu/g in the SHMNCs, likely because SHMNPs represent only a portion of the per-gram weight of the SHMNCs (Fig. 1C). Both SHMNCs and SHMNPs displayed superparamagnetic properties with magnetizations greater than those of Resovist (73.7 emu/g), LMNCs (56.4 emu/g) and other reported magnetic drug carriers [33e35], likely because the single domain crystal structures of SHMNPs more perfect than those of LMNPs were successfully prepared by controlling the reaction temperature and time during the synthesis process (Fig. 1D). 3.2. Magnetic attractability and MRI R2 relaxivity The magnetic attractability of SHMNCs was tested in vitro using an external magnetic field of 0.4-T (Fig. 2A). Limited amounts of Resovist (w9.59 mg) were attracted, whereas under the same conditions, accumulation of SHMNCs (w54.78 mg) was readily observed (Fig. 2B), likely because the high-magnetization of SHMNCs could against the strength of the flow rate. The measured magnetization of SHMNCs compared to Resovist was 89.7 and 73.7 emu/g, respectively, which brought an increase of 1.22-fold magnetic attractability [36]. Thus, the performance of MT therapy might be improved significantly by using SHMNCs. Besides, SHMNCs also provided better contrast enhancement for MRI than Resovist because of its higher R2 relaxivity (434.7 mM
1s
1 vs. 95.2 mM
1s
1; Fig. 2C,D), indicating that SHMNCs could provide about a four-fold sensitivity improvement in MRI image. 3.3. Characterization and quantification of SHMNC-DOX Immobilization of DOX on SHMNCs was confirmed by FT-IR spectroscopy (Fig. 3B). In addition to the characteristic absorption peaks of Fe3O4 and SPAnH [20], four new peaks corresponding to DOX stretching vibrations (n) were obtained: n of C]O (1728 cm
1), n of CeOeC (1119 cm
1), n of CeO (1057 cm
1) and n of CeH bending overlapped with the stretching vibration of CeN (1408 cm
1). The zeta potential of SHMNCs in DI water was 44.2  0.9 mV and decreased to 32.6  0.7 mV (n ¼ 10) when DOX was bound covalently to the nanocarriers. This likely is the result of the conjugation between the carboxyl groups of SPAnH and the amino groups of DOX (Fig. 3A). Overall, these data indicated that the surfaces of the SHMNPs were covered with a shell of SPAnH and that the DOX was immobilized covalently with SPAnH. The

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outermost layer of SHMNCs presented eCOOH groups, which could be used to immobilize antibodies or drugs. As measured by HPLC, the amount of DOX immobilized per mg of SHMNCs increased as the amount of DOX added increased, reaching a saturating concentration of 270.9 mg DOX/mg SHMNCs when the amount of DOX added surpassed 0.5 mg (Fig. 4A). 3.4. Thermal stabilities of free- and SHMNC-DOX To determine if bioconjugation to SHMNCs affected the thermal stability of DOX, degradation analyses were performed by HPLC. The mobile phase consisted of 45 volumes of acetonitrile and 55 volumes of a solution containing 10 g of sodium lauryl sulfate, 2.5 mL of phosphoric acid (85%) and 1000 mL of DI water. The flow rate was 1.0 mL/min and UV detection was performed at 298 nm. Papaverine hydrochloride (50 mg/mL) was used as an internal standard. Retention times of 5.5, 11.2 and 14.9 min were measured for degraded DOX, intact DOX and the standard, respectively (Fig. 4B,C). The half-lives (i.e., the time required for the drug to be degraded to 50% of its original activity) of free- and SHMNC-DOX were 65 and 105 h, respectively (Fig. 4D). 3.5. The study of the SHMNCs uptake pathway Unlike other chemotherapeutic agents, DOX emits red fluorescence when excited at 488 nm [37]. The distributions of free- and SHMNC-DOX in MGH-U1 human bladder cancer cells were detected by confocal spectral microscopy. In contrast, SHMNC-DOX appeared to be taken up by endocytotic pathways (Fig. 5A) [28], thus avoiding the effects of P-gp pumps and enhancing the

intracellular concentration by 23% relative to free-DOX (n ¼ 20). Indeed, after 4 h of incubation, SHMNC-DOX was still detectable in the nuclei and was dispersed evenly within the cytoplasm, possibly because of its release from lysosomes and endosomes [38,39]. To investigate the specific pathway by which SHMNCs was internalized, it was co-cultivated with cells that had been preincubated separately with three membrane entry inhibitors d filipin III (Fil), amiloride (Amil), and chlorpromazine (Cpz) d used to inhibit caveolae-mediated endocytosis, macropinocytosis, and clathrin-mediated endocytosis, respectively. SHMNCs uptake was reduced by 61%, 6% and 20% by pretreatment of the cells with Fil, Amil, and Cpz respectively, relative to untreated cells (n ¼ 100) (Fig. 5B,C). Accumulation of SHMNCs in cells was lowest for cells pre-incubated with Fil, suggesting that the major uptake pathway for SHMNCs was caveolae-mediated endocytosis; the secondary pathway was clathrin-mediated endocytosis. When MGH-U1 cells were incubated with SHMNCs for 4 h, large quantities of nanoparticles passed through cell membranes via endocytotic pathways without exhibiting any cytotoxic effects (Fig. 6A). Selected-area diffraction analysis confirmed that the black nanoparticles in the cells were polycrystallines of Fe3O4 (Fig. 6C) [14]. In contrast, when SHMNC-DOX entered the cells, the micrographs exhibited the nuclei of the MGH-U1 cells undergo the buckling and folding characteristic of apoptosiss (Fig. 6B). 3.6. In vitro antitumor cell efficiency The cytotoxicity of magnetic nanocarriers toward normal cells had been reported by us [40]. In this case, we also determined the cytotoxicity of SHMNCs toward MGH-U1 cells using the XTT assay.

Fig. 1. (a) Hydrodynamic particle size distributions of SHMNCs and SHMNC-DOX. (b) TEM image of SHMNCs (bar: 92 nm). (c) SQUID spectra at room temperature of SHMNPs, SHMNCs, Resovist, and LMNCs. (d) XRD patterns of LMNCs and SHMNCs.

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Fig. 2. (A) Extraction of Resovist (top) and SHMNCs (bottom) from a stable colloidal fluid in DI water at 25  C pumped at a constant flow rate of 15 cm/s through tubing targeted with a 0.4-T magnet placed 4 mm from the tubing. (B) The attracted efficiency of magnetic targeting. (C) Spinespin relaxation rates (R2, in s
1) of LMNCs (left), Resovist (middle) and SHMNCs (right) containing different concentrations of Fe ions (1: 0.1, 2: 0.2, 3: 0.25, 4: 0.5, 5: 0.75, 6: 1.0 mM). (D) Fe ion relaxivities (mM
1s
1) are indicated by the slope of each line.

Fig. 3. (A) Illustration of the chemical formation of SHMNC-DOX. (B) FT-IR spectra of DOX, SHMNCs, and SHMNC-DOX at room temperature.

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The cells co-cultured for 24 h with SHMNCs remained viable even at concentrations up to 500 mg/mL (Fig. 7A), indicating that the carriers had low toxicity for tumor cells. Fluorescence microscopy of MGH-U1 cells co-cultured with SHMNC-Cy5 for 24 h also showed that SHMNCs were taken up into cells without influencing the fluorescent signal produced by live cells (Fig. 7B), confirming that the SHMNCs itself was not cytotoxic. Cytotoxicity and DNA expression are related to the level of DNA interstrand crosslinking. The occurrence of interstrand crosslinking was w14.6% after treatment with 5 mM of free-DOX for 16 h but increased w1.5-fold to 21.8% using the same concentration of SHMNC-DOX (Fig. 8A). This higher level of interstrand crosslinking correlated with increased cytotoxicity and reduced the DNA expression. The DNA expression was analyzed using the specific primer and Q-PCR to confirm DNA damage to MGH-U1 cells after treatment with 5 mM of free- and SHMNC-DOX. DNA expression after treatment with SHMNC-DOX for 16 h decreased w19% relative to treatment with free-DOX, which itself was decreased to 73.6% of controls (Fig. 8B). According to above results, the cytotoxicities of free- and SHMNC-DOX toward MGH-U1 cells were determined using the XTT assay. In contrast, free- and SHMNC-DOX were both toxic toward MGH-U1 cells in a dose-dependent manner (Fig. 8C). The IC50 of free-DOX was 5.85 mM, slightly higher than that of SHMNC-DOX of 5.16 mM. The IC50 value was reduced significantly to 3.23 mM of SHMNC-DOX when a 900-Gauss magnetic field was applied, presumably because more SHMNC-DOX was guided to the cells, further enhancing local drug concentration. The efficiency of MT therapy was also tested by reducing the strengths of the applied magnetic fields and the magnetizations of

the nanocarriers. When a 300-Gauss magnet field was applied (Fig. 8D), the IC50 of LMNC-DOX was 5.09 mM, which was higher than that of SHMNC-DOX (4.06 mM) presumably because the LMNCs were not as readily attracted by the magnet. Fluorescence microscopy confirmed that cytotoxicity was proportional to the dose of SHMNC-DOX and MT efficiency. SHMNCs alone had no cytotoxic effect (Fig. 9A). Treatment with free-DOX decreased the number of live cells (Fig. 9B) but was not as effective as treatment with SHMNC-DOX at the same effective dosage of DOX (Fig. 9C). Furthermore, when SHMNC-DOX was concentrated to a specific area by application of a 300-Gauss magnetic field (Fig. 9D), cytotoxicity within this region increased, whereas the cells outside the area of the magnetic field survived. The field was less effective in inducing cytotoxic effects when used in conjunction with LMNCDOX (Fig. 9E). Thus, when exposed to a magnetic field of constant strength, the efficacy of the drugs in MT therapy is dependent on the magnetization of the carriers. 3.7. In vivo contrast and targeting efficiency The presence of SHMNC-DOX in biological tissues profoundly alters the spinespin relaxation rate (R2), thus serving as an MRI contrast agent. After 18 h of the injection with 5 mg/kg of Resovist and SHMNC-DOX and MT application, the accumulations in tumor tissue were 274.2 and 630.8 ppm, respectively. The concentrations were increased to 965.5 and 1905.9 ppm while injected 15 mg/kg of Resovist and SHMNC-DOX, respectively (Fig. 10A). The R2 maps (showing changes caused by different amounts of MNPs) and T2 imaging (indicating susceptibility artifacteinduced signal loss caused by MNPs accumulation) of in vivo imaging showed that

Fig. 4. (A) Quantity of DOX bound per 1 mg of SHMNCs vs. added DOX. (inset) HPLC analysis of DOX. HPLC graphs of thermal stabilities: (B) free-DOX and (C) SHMNC-DOX after incubation for various times at 37  C (i) degraded DOX; (ii) intact DOX; (*) papaverine hydrochloride (internal standard); (D) degradation curves of free-DOX (-) and SHMNC-DOX ( ) over 108 h.

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Fig. 5. (A) Phase contrast (left) and fluorescence (right) images of MGH-U1 cells exposed to free-DOX (top) and SHMNC-DOX (bottom) for 4 h. (B) Effects of the endocytosis inhibitors filipin III (Fil), amiloride (Amil), and chlorpromazine (Cpz) on the uptake of SHMNCs by MGH-U1 cells. Phase contrast (top) and fluorescence (bottom) images of cellular uptake of SHMNCs. (C) Ratios of the relative fluorescence intensity of SHMNCs taken up by MGH-U1 cells treated with inhibitors to those without inhibitors. Cells were selected randomly for imaging analysis. Values are the means  S.D. (n ¼ 100), *: p ¼ 0.001, relative to the SHMNCs-treated group.

SHMNC-DOX could be concentrated and guided easily to the tumor site and display excellent monitoring. In addition, SHMNC-DOX was shown to permeate the tumor tissue with a wide distribution by passive local enhanced permeability and retention (EPR) (Fig. 10B). Prussian blue staining of tumor tissue slices confirmed that the deposition of SHMNC-DOX at the tumor site was higher than that of Resovist with MT (Fig. 10C). The results indicated that the higher magnetization of drug carriers could improve the efficiency of MT therapy and enhance the MRI sensitivity to precede the better chemotherapy and monitoring. In the in vivo fluorescence imaging study, we used LMNC to replace Resovist to prove the magnetization effects of magnetic drug carriers for MT therapy. After 18 h of the injection with 5 mg/ kg of LMNC-Cy5 and SHMNC-Cy5 and MT application, the tumor

site displayed obvious accumulation of carriers and the accumulation of SHMNC-Cy5 was w3.3-fold higher than that of LMNC-Cy5 (Fig. 11A). After the test, the mice were sacrificed and the fluorescence images of different organs showed noticeable changes in tumor and liver after injection of SHMNC-Cy5 and MT application, suggesting that nanocarriers would accumulate in liver and MT could enhance the local concentration at the tumor site (Fig. 11B). 4. Discussion In this work, we developed SHMNCs for DOX to enhance the efficiency of MT therapy for MGH-U1 bladder cancer cells. These nanocarriers used water-soluble conducted polymer as shell which had positive charge providing more carboxyl groups to conjugate

Fig. 6. TEM images of MGH-U1 cells co-incubated with (A) SHMNCs and (B) SHMNC-DOX for 4 h. (C) Diffraction pattern of the region indicated by the red ring in (A). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. (A) Viability of MGH-U1 cells after incubation with SHMNCs (-) and SHMNCs and exposure to a 900-Gauss magnetic field ( ). (B) MGH-U1 cells exposed to SHMNC-Cy5 for 24 h. Phase contrast (left) and fluorescence images of live cells (middle) and of Cy5 dye (right).

drugs. Besides, the positive charges also helped to reduce the aggregation of SHMNCs and to increase their stability in DI water [41,42]. The size, morphology and intracellular distribution of SHMNCs were checked by TEM. The hydrodynamic size of SHMNCs (87.2 nm) was greater than that measured by TEM (12.3 nm), mainly due to the process involved in preparation of samples (Fig. 1A,B). The hydrodynamic size was measured in the hydrated state, whereas TEM images depicted the size at the dried state of sample. Therefore, in the hydrated state, the nanocarriers had a higher hydrodynamic volume due to the hydrophilic polymer coatings of the nanocarriers and solvent effects [43]. Although, a slight aggregation of nanocarriers in suspension state would not be excluded, the range still maintained nano-scale size to apply in MT therapy. Even the nano-scale size could provide high surface area to conjugate more antitumor drug, the magnetization of nanocarriers must also be higher to enhance the efficiency for MT therapy due to magnetic gradients decreased rapidly as the distance between the target site and the external magnetic field increased. Also, leucopenia could be induced in patients exposed for longer than 24 h to field strengths greater than 1-T [44]. In order to solve the serious problem, we designed SHMNCs with a core of SHMNPs and a shell of SPAnH to affect the efficient MT with lower applied field strength. The magnetization of SHMNCs (89.7 emu/g) was greater than those of Resovist (73.7 emu/g) (Fig. 1C) and other reported magnetic drug carriers [33e35]. Moreover, the magnetization also affects the detection sensitivity of MRI. As expected, the higher sensitivity of SHMNCs than that of Resovist was obtained by R2 image of MRI and its in vitro characteristic measurements (i.e. higher detectability

with fewer concentrations of particles) (Fig. 2), allowing the assessment of localized concentration of DOX and its distribution to be detected and quantified accurately in vivo [45]. For more effective therapy, DOX must be immobilized on the SHMNCs as efficiently as possible so that it could provide an effective therapeutic dose without requiring so many SHMNCs to be injected that the iron in the particles induces liver damage [46,47]. However, the maximum amount of DOX immobilized per mg of SHMNCs depended on the availability of carboxyl functional groups on the surface of SHMNCs, and some residues might be inaccessible as a result of steric hindrance (Fig. 3). The quantification analysis showed that per mg of SHMNCs could immobilize 270.9 mg DOX (Fig. 4A). This high payload (27.1 wt%) of DOX was believed to be the result of the high surface area of the SHMNCs, which was about 2e4-fold higher than for other drug delivery systems [4,5,11]. In addition, the degradation degree of DOX increased as incubation time at 37  C increased, resulting in a failure of its ability to crosslink DNA. However, this defect could be improved by covalently conjugating DOX on the surface of SHMNCs due to the degree-of-freedom of DOX was limited to drastically prevent distortion of the molecule, thereby enhancing w40 h of its halflives (Fig. 4). This increased stability could maintain DOX’s activity to interact with topoisomerase II to form DNA-cleavable complexes producing cytocidal activity, and prove crucial to extending the period during which it could exert its therapeutic effect in vivo (Fig. 8). Free-DOX incubated with MGH-U1 cells for 4 h was transported rapidly into the cells by passive diffusion. However, free-DOX near the inner surface of the cell membrane was pumped out

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Fig. 8. (A) DNA interstrand crosslinking in MGH-U1 cells after treatment with free-DOX ( ) and SHMNC-DOX ( ) for 16 h. Values are the means  S.D. (n ¼ 3). (B) Relative DNA expression in MGH-U1 cells before and after treatment. Values are the means  S.D. (n ¼ 4), *: p ¼ 0.01, relative to without DOX treatment. (C) Viability of MGH-U1 cells after incubation with free-DOX ( ), SHMNC-DOX ( ), and SHMNC-DOX and exposure to a 900-Gauss magnetic field ( ). (D) Viability of MGH-U1 cells after incubation with LMNC-DOX (-) and SHMNC-DOX ( ) and exposure to a 300-Gauss magnetic field for 24 h.

immediately by P-gp pumps, reducing the intracellular concentration and limiting its efficiency of antitumor cells. Thereby, we developed the SHMNCs as carriers of DOX to change the pathway from P-gp pumping to endocytosis for the dodge of P-gp effects,

resulting in enhancing the intracellular concentration by 23% relative to free-DOX (Fig. 5A). Previous studies on cellular uptake of nanoparticles showed that some factors would affect the pathway such as particle size, shape, molecular weight and surface charge

Fig. 9. Fluorescence micrographs of MGH-U1 cells co-cultivated with (A) SHMNCs, (B) 6 mM free-DOX, (C) an effective dose of SHMNC-DOX equivalent to 6 mM free-DOX, and an effective dose of (D) SHMNC-DOX or (E) LMNC-DOX equivalent to 6 mM free-DOX and subjected to a 300-Gauss magnetic field applied in the region indicated by the red ring for 12 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 10. (A) Quantification of Fe accumulated in the tumors by ICP-OES after intravenous injection of Resovist and SHMNC-DOX. Values are means  SD (n ¼ 3). (B) In vivo imaging of Resovist (top) and SHMNC-DOX (bottom) distribution in hypodermic tumors with MT (left, T2-weighted images; right, combined R2 maps and T2-weighted images). (C) Staining with Prussian Blue (100x) shows the uptake of Fe in tumor tissue after the treatment of Resovist (top) and SHMNC-DOX (bottom).

[29]. Because the long-time of exposure would cause serious cytotoxicity to cells, the pathways of cellular uptake were examined by using three membrane entry inhibitors to pretreat the MGH-U1 cells for 1 h before co-culturing with SHMNC-Cy5. Thus, the low

uptake of SHMNC-Cy5 was caused by pretreatment of the cells with Fil, suggesting that the major uptake pathway for SHMNCs was caveolae-mediated endocytosis (Fig. 5B,C). The result possibly because the average hydrodynamic size of SHMNCs was w87.2 nm,

Fig. 11. (A) In vivo fluorescence images of MGH-U1 tumor bearing mice without treatment (i.e. control, left) and after intravenous injection of LMNC-Cy5 (middle), SHMNC-Cy5 (right) with 18 h of MT application. (B) The representative fluorescence images of different organs which were gotten from control mouse (top) and SHMNC-Cy5 treatment mouse (bottom).

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and most (70%) of SHMNCs was between 50 and 100 nm. Because Fil bound specifically tocholesterol(thus crosslinking cholesterol in the plasma membrane), it was sequestered to caveolae [48,49], which allowed particles with sizes between 50 nm and 100 nm to enter cells preferentially. Some seriously aggregated SHMNCs, with diameters may higher than 200 nm, were confined to clathrinmediated endocytotic pathways [50]. The results showed the internalization of nanoparticles via the endocytotic pathways was size-dependent. Therefore, the major endocytotic pathway of SHMNC-DOX may change to clathrin-mediated endocytosis due to its average hydrodynamic size was increased to w255.0 nm. Thus, conjugation of DOX to SHMNCs appeared to enhance the apoptotic effects of the drug in MGH-U1 cells (Fig. 6), presumably because drug delivery via SHMNCs bypassed the effects of P-gp pumps and enhanced the drug’s thermal stability, thus enhancing intracellular drug concentration. The DNA damaging potential and in vitro cytotoxicity of SHMNC-DOX was tested in MGH-U1 cell. The SHMNCs were shown to have no cytotoxic effects on the cells (Fig. 7). DOX damaged DNA by producing free radicals intracellularly and by direct interstrand intercalation. The ability of DOX to intercalate into DNA resulted from the covalent binding with the 2-amino group of a G-base in the minor groove of DNA on one strand using HCHO, and to the Gbase on the opposing strand using hydrogen bonds. The SHMNCDOX caused serious DNA crosslinking than free-DOX, resulting in lower DNA expression and inducing apoptosis [43]. The cytotoxicity for MGH-U1 cells was significantly enhanced by the combination of SHMNC-DOX and MT application (Fig. 8). However, the efficiency of MT therapy depended on the magnetization of carriers and the strength of applied magnetic field. The higher the applied magnetic field, the lower the concentration of SHMNC-DOX required for 50% inhibition of MGH-U1 cellular growth (Fig. 8C,D). At the constant applied magnetic field (300 Gauss), the MT therapy effects were more obvious for the drug carried by nanocarriers with the higher magnetization (Fig. 9D,E). This is also confirmed in in-vivo test that the SHMNCs displayed more excellent MT ability than LMNCs (Fig. 10). The fluorescence images of the nanocarriers distributed in different organs showed no obvious accumulation in heart (Fig. 11), suggestion that this system could reduce the cardiotoxicity caused by DOX. These results confirmed that the magnetization of carriers was one of the key factors for MT therapy and that even lowerstrength magnetic fields could be used to guide significant quantities of SHMNCs conjugated DOX to a specific area, enhancing its local concentration and reducing the injection doses to avoid the damages of other organs. 5. Conclusion In summary, the chemotherapeutic agent DOX was immobilized successfully on SHMNCs by covalent bonding between the eNH2 of DOX and the eCOOH of SHMNCs to enhance the thermal stability of DOX and MT efficiency. The half-life of DOX at 37  C was greatly improved (from 65 h to 105 h). The SHMNCs conjugated DOX was effectively at concentrations sufficient to induce cytotoxicity in cancer cells and overcame the resistance to the drug caused by P-gp pumps, resulting in increasing the intracellular concentration of DOX by 23% relative to free-DOX. Furthermore, the concentration of SHMNCs in vitro and in vivo could be detected easily by MRI R2 mapping. The high R2 relaxivity (434.7 mM
1s
1) should allow the assessment of the distribution and concentration of DOX in various tissues and organs. Most significantly, SHMNC-DOX could be easily targeted and concentrated to specific sites by applying a lower magnetic field strength to increase the level of DNA crosslinking and reduce the IC50 of DOX toward MGH-U1 cells. This advanced nanocarrier with high payload of drug, high magnetization,

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superparamagnetic property and high MRI relaxivity could not only be a most effective MT carrier of chemotherapeutic agent but be an excellent contrast agent of MRI. Magnetic delivery of SHMNC-DOX was a promising MT therapy that could provide more effective tumor treatment using lower therapeutic doses with potentially fewer side-effects.

Acknowledgments This work was supported by grants from National Science Council, Industrial Technology Research Institute of Republic of China, and Chang Gung Memorial Hospital for financial aid (NSC 100-2221-E-182-005, NSC 99-2221-E-182-068, NSC 98-2221-E182-045-MY3, NSC 94-2216-E-182-001, AF51RQ3100, and CMRPG391781). The authors thank Chang Gung Memorial Hospital Microscopy Core Laboratory and Molecular Imaging Center for the assistance of TEM, Confocal and 7T- MRI images.

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