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Synthesis and characterization of recyclable clusters of magnetic nanoparticles as doxorubicin carriers for cancer therapy
Accepted Manuscript Title: Synthesis and characterization of recyclable clusters of magnetic nanoparticles as doxorubicin carriers for cancer therapy Author: Juan Wu Yujiao Wang Wei Jiang Shanshan Xu Renbing Tian PII: DOI: Reference:
S0169-4332(14)02184-9 http://dx.doi.org/doi:10.1016/j.apsusc.2014.09.184 APSUSC 28840
To appear in:
APSUSC
Received date: Revised date: Accepted date:
8-7-2014 27-9-2014 28-9-2014
Please cite this article as: J. Wu, Y. Wang, W. Jiang, S. Xu, R. Tian, Synthesis and characterization of recyclable clusters of magnetic nanoparticles as doxorubicin carriers for cancer therapy, Applied Surface Science (2014), http://dx.doi.org/10.1016/j.apsusc.2014.09.184 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and characterization of recyclable clusters of magnetic nanoparticles as doxorubicin carriers for cancer therapy
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Juan Wu, Yujiao Wang, Wei Jiang∗, Shanshan Xu, Renbing Tian National Special Superfine Powder Engineering Research Center, Nanjing University of Science
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and Technology, Nanjing 210094, China
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Abstract
This study focuses on the synthesis and characterization of recyclable clusters of magnetic
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nanoparticles (CMNPs) as doxorubicin carriers for cancer therapy. Fe3O4 nanoparticles were used as magnetically responsive carriers, the modified polyethylene glycol dicarboxylic acid
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(APS-PEG-TFEE) acted as a steady bridge between Fe3O4 and drug. The prepared CMNPs exhibited a size within 20 nm, good stability and super-paramagnetic responsibility (Ms 62.02
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emu/g); doxorubicin (DOX) can be successfully loaded to CMNPs at a loading rate of 76.19% by electrostatic interaction. Moreover, the release studies in vitro showed that the drug-loaded
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carriers (CMNPs-DOX) had excellent pH-sensitivity, 76.16% of DOX was released within 72 h at pH 4.0, and the secondary drug loading rate was nearly 52%. WST-1 assays in model breast cancer cells (MCF-7) demonstrated that CMNPs-DOX exhibited high anti-tumor activity, while the CMNPs were practically non-toxic. Thus, our results revealed that CMNPs would be a competitive candidate for drug delivery carriers and CMNPs-DOX could be used in targeted cancer therapy in the near future. Keywords: Fe3O4; magnetic targeted; pH-sensitive; doxorubicin; cancer therapy
∗
Corresponding author. Tel.: +86-25-84315042; Fax.: +86-25-84315042
E-mail address: [email protected] (W. Jiang) 1
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1. Introduction The application of iron oxide nanoparticles (Fe3O4) in biomedical researches such as
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magnetic drug delivery [1], tumor-specific cell targeting [2], gene therapy [3], bioseparation [4] and magnetic resonance imaging [5] has drawn considerable attention due to the ultra-fine size,
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biocompatibility and superparamagnetic behavior [6]. These properties are fully exploited when
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they are used as drug delivery carriers. The drug-loaded Fe3O4 can be specifically enriched in tumor area due to synergistic mechanisms such as (Ⅰ) the application of an external magnetic field
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[7], (Ⅱ) the size range (10-100 nm) is small enough to evade enhanced permeability and retention (EPR) of the body as well as penetrate the very small capillaries within the body tissues [8], (Ⅲ)
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internalization of nanoparticles within cancer cell compartments [9]. As a consequence, the chemotherapeutic drugs can be targeted to the lesion site in the body, and also the magnetic
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carriers not only decrease the systemic side effects but also promote the efficacy of chemotherapy. For effective tumor therapy, prolonged availability of the drug at the target site is the primary
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requisite of any drug carriers. In this regard, drug retention and drug release properties are indispensable features which should be taken into consideration. One of the promising drug carriers is the pH-sensitive drug carrier [10,12-14], and the drug release rate was responsive to the environmental pH value. As the pH value of most solid tumors (pH<6.0) was lower than the surrounding normal tissues (pH 7.4) [11], the anti-cancer drug which was connected by pH-sensitive bond could be triggered to release, so that to improve the therapeutic effect. In recent years, hydrazone bond [12-14] has emerged as a pH-sensitive bond to connect drugs.
While the experiments of loading drugs by hydrazone bond always involve multiple steps and cost a large amount of organic reagent which would cause pollution. Yang et al. [12] designed and 2
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synthesized a triblock copolymer for drug delivery, before connecting DOX, anhydrous dimethylformamide (DMF) and anhydrous hydrazine were used to handle triblock copolymer at
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40℃. After dialyzing and freeze-drying, DOX was conjugated onto the copolymers in the presence of DMF. Fang et al. [15] synthesized a biodegradable poly(beta-amino ester) (PBAE)
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copolymer to load DOX. In the process of drug connection, dimethylsulfoxide and ether were used
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as solvent, in order to remove any unreacted reactants and solvent, aqueous solution of PBAE-DOX was required to pass through column. As compared to hydrazone bonds, electrostatic
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interaction [16] which has desirable pH-responsivity usually exhibits easier operation in experimental procedure, and lower consumption and pollution. In addition, there are limited
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reports on using electrostatic interaction to connect DOX in magnetic targeted drug delivery carriers.
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Silanization is helpful to form bonds between inorganic and organic components for the modification of functional groups on the surface of Fe3O4 [19,23]. To date, various silane coupling
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agents such as 3-aminopropyltriethoxysilane [20] and tetraethoxysilane [21] have been used. At the same time, poly(ethylene glycol) (PEG) is used widely to modify Fe3O4 for long blood
circulation time [17,18]. For this, we synthesized and characterized of polyethylene glycol dicarboxylic
acid
modified
by
3-aminopropyltriethoxysilane
and
2,2,2-trifluoroethanol
(APS-PEG-TFEE). As a bridge linking, APS-PEG-TFEE could covalently link to the Fe3O4 through -Si-O- [24] and load DOX via electrostatic interaction, so as to form CMNPs that can deliver DOX and are recyclable (Fig. 1). Furthermore, the CMNPs-DOX could effectively release DOX at pH<6.0 and inhibit the growth of model breast cancer cells (MCF-7). Thus, our approach clearly indicates the CMNPs-DOX might be a promising candidate for targeted tumor therapy. 3
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2. Materials and methods 2.1. Materials All chemicals were analytical grade and used without further purification. Ferric chloride
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(FeCl3 • 6H2O), ferrous sulfate (FeSO4 • 7H2O), (3-aminopropy)triethoxysilane (APS) and
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N-hydroxyl succinamide (NHS) were supplied by Sinopharm Chemical Reagent Co., Ltd.,
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Shanghai, China. Polyethylene glycol (PEG) dicarboxylic acid (Mn 600) was purchased from Sigma-Aldrich. 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) and
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2,2,2-trifluoroethanol (TFEA) were purchased from Aladdin. DOX was purchased from Yuanye Biotechnology Co. Ltd., Shanghai, China. Dulbecco’s Modified Eagle medium (DMEM) was from
Thermo
Fisher
Biochemical
Products
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purchased
Co.,
Ltd.,
Beijing,
China.
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) and MCF-7
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were supplied by Beyotime Biotechnology Co., Ltd., Shanghai, China. Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd., Zhejiang, China. All other
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chemicals and solvents used in this study were of high analytical grade and commercially available.
2.2. Characterizations 1
H NMR spectrum was recorded on AV-III nuclear magnetic resonance spectrometer (NMR,
Bruker Co. Ltd., Germany). The size and morphology of the samples were investigated by transmission electron microscopy (TEM, Model Tecnai 12, Philips Co. Ltd., Holland). The thermal stability of the dry samples were measured by thermogravimetric analysis (TGA, Model TA2100, TA Instruments, USA) under N2 at a heating rate of 10℃/min over the range of 50~650 ℃. The crystalline phases were recorded using X-ray diffraction (XRD, Bruker Co. Ltd., Germany) 4
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with Cu Kα radiation. Surface charge was evaluated by the zeta potential analyzer (BI-90Plus, Brookhaven Co. Ltd., USA). Magnetic properties of the particles were detected at room
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temperature using vibrating sample magnetometer (VSM, Model 7410, Lake Shore Co. Ltd., USA). Fourier-transform infrared spectroscopy (FT-IR) spectra were recorded on Vector 22
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UV-visible spectrophotometry (UV-vis, Agilent Co. Ltd., USA).
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spectrometer (Bruker Co. Ltd., Germany). The drug loading and release rate were detected by
2.3. Experimental
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2.3.1. Synthesis of modified polyethylene glycol dicarboxylic acid (APS-PEG-TFEE) The synthetic process for APS-PEG-TFEE was modified from previous report [22]. To PEG
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dicarboxylic acid (116.6 mmol) was dropwise added 12 mL of thionyl chloride and refluxed for 4 h to obtain a colorless liquid. After that, 12 mL TFEA was dropwise added to the liquid and the
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obtained mixture was stirred for 30 min, and refluxed for additional 12 h to obtain a pale-yellow liquid which was dissolved in 20 mL of toluene. Finally, 3 mL APS in batches (3 batches, 1 batch
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per 30 min) was added after 500μL of triethylamine was added for reaction of 10 min, and the
mixture was stirred for 12 h to get APS-PEG-TFEE. The solvent and excess reactants in each step
during the whole reaction process were removed by rotary evaporation under vacuum. 2.3.2.
Preparation of Fe3O4
According to the improved chemical co-precipitation method [23], 1.1127 g FeSO4•7H2O
and 1.9182 g FeCl3•6H2O were dissolved in deionized water. The mixture was stirred under N2 at 80℃ for 1 h. Then, 40 mL of aqueous ammonia (25 wt.%) was injected into the mixture rapidly, continued for another 1 h and then cooled to room temperature. The precipitated particles were separated by magnet and washed several times with water. Finally, Fe3O4 nanoparticles were dried 5
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under vacuum at 50°C. 2.3.3.
Surface modification of Fe3O4 with APS-PEG-TFEE
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Initially, 100 mg dry Fe3O4 was dispersed into 50 mL of toluene, then 2 mL APS-PEG-TFEE was added after ultrasound of 30 min. The mixture was purged with N2 and sonicated at 50°C for
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4 h. The resulting colloidal suspension was washed twice in toluene and re-suspended in 50 mL of
2.3.4.
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toluene. Carboxylation of Fe3O4@APS-PEG-TFEE to form the CMNPs
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In order to effectively make the surface of Fe3O4@APS-PEG-TFEE carboxylated, ethylenediamine was used to substitute for the -CF3 of APS-PEG-TFEE, so that there was a large
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number of amino on the surface of Fe3O4. Briefly, 800μL of ethylenediamine was added into 50 mL of suspension, the mixture was purged with N2 and sonicated at 50°C for 2 h. Then, the
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product was magnetic decanted, washed with toluene and dichloromethane, and dried in fume hood to obtain amino-terminal Fe3O4. After that, 2 g PEG dicarboxylic acid was dissolved in 5 mL
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of deionized water which contained 200 mg EDC and 200 mg NHS. The activation reaction went on for 4 h. Finally, 50 mg amino-terminal Fe3O4 dispersed in 5 mL of deionized water was added
and the reaction continued for 24 h. The final product was collected by magnet, washed with deionized water and re-dispersed in it (10 mL) to form suspension of CMNPs. 2.3.5.
Drug loading (CMNPs-DOX)
2.1 mg DOX which was dissolved in deionized water (2 mL) was added to the suspension of CMNPs, and then the reaction went on for 24 h under dark. The product was collected by magnet and washed twice with deionized water. The final product was freeze-dried and storied in the dark at 5℃. 6
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2.4. In vitro release of DOX Briefly, 0.01 M of phosphate buffer solution (PBS) (pH 4.0, 5.0, 7.4) was prepared to imitate
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conditions like tumors and normal tissues. After that, 5 mg CMNPs-DOX was dispersed in 2 mL PBS and then transferred to a dialysis bag which was immersed in 4 mL of the same medium and
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kept in the oscillator at 37℃. At selected time intervals, PBS (2 mL) outside the dialysis bag was
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removed for UV-vis analysis at 482 nm and replaced by the fresh with the same volume. To determine the amount of DOX released, calibration curves were run with DOX in corresponding
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PBS at pH 4.0, 5.0 and 7.4, respectively. The release experiments were conducted in triplicate. The results presented are the average data with standard deviations. The drug loading rate (Lr) was
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calculated by the formula: Lr=(m1/m0)×100%, where m1 is the quality of DOX loaded onto CMNPs, m0 is the quality of DOX added before the reaction; the release rate (Rr) of DOX was
CMNPs-DOX.
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2.5. WST-1 assays
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d
calculated by the formula: Rr=(m2/m1)×100%, where m2 is the quality of DOX released from
MCF-7 cells were seeded in 96-well plates (5 × 103 cells/well) in 200 μ L DMEM
supplemented with 10% FBS for 24 h at 37℃ in a humidified 5% CO2-containing atmosphere.
Then, the media was aspirated and replaced by 200μL of fresh cell culture medium. 20μL of free DOX, CMNPs, or CMNPs-DOX at various concentrations in fresh cell culture medium was added and the cells were incubated for 24 h. After that, 20μL WST-1 solution (5 mg/mL) was added. The cells were incubated for 2 h, and the absorbance at a wavelength of 450 nm of each well was measured using a multifunctional microplate reader (TECAN Infinite 200 Pro, Austria). The relative cell viability (%) was determined by comparing the absorbance at 450 nm with control 7
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wells containing only cell culture medium. The bright-field images of MCF-7 cells were recorded on microscope (Olympus, IX81, Japan).
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3. Results and discussion 3.1. Characterization of sample
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The structure of the modified polyethylene glycol dicarboxylic acid (APS-PEG-TFEE) was
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identified by 1H NMR analysis. The chemical shifts for protons from 1 to 11 of APS-PEG-TFEE are shown in Fig. 2 and its inset table. The molecular structure of APS-PEG-TFEE was confirmed
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by the presence of all characteristic peaks and peak integration. To further verify the successful synthesis of APS-PEG-TFEE, FT-IR spectra were also collected at each step of the synthesis (Fig.
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3). The broad band above 3000 cm-1 corresponds to -OH of PEG dicarboxylic acid (Fig. 3a) and disappears in Fig. 3b, and then reappears in Fig. 3c, which result from the stretching vibration of
d
-NH in the amide linkage between APS and PEG dicarboxylic acid. In Fig. 3c, the peaks at 1779
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and 1675 cm-1 are attributed to the trifluoroacetate ester and the amide carbonyl respectively,
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confirming the formation of half-amide/ester structure. As shown in Fig. 4, the dispersion of Fe3O4 was improved effectively after coating (Fig. 4b),
it was because that APS-PEG-TFEE and PEG glycol dicarboxylic acid lowered the high surface energy of Fe3O4 nanoparticles. In addition, the average particle size of CMNPs in TEM image was about 20 nm, which was small enough both to evade reticuloe endothelin system (RES) of the body as well as penetrate the very small capillaries within the body tissues and therefore may offer the most effective distribution in certain tissues [8]. In order to confirm the existence of cladding layer and obtain the amount of APS-PEG-TFEE and PEG dicarboxylic acid so as to determine the amount of drug used in the further experiments, 8
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thermogravimetric analysis (TGA) had been used. In Fig. 5b, upon heating, amino-terminal Fe3O4 shows a weight loss of 2.10% at temperatures ranging from 50 to 100℃, mainly due to the loss of
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physically adsorbed water on the products. When being heated to 650℃, probably because of the decomposition of APS-PEG-TFEE, leading to a weight loss of 16.17%. In comparison, the weight
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loss of CMNPs is about 25.65% (Fig. 5c), the increased amount (9.48%) could attribute to the
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decomposition of PEG glycol dicarboxylic acid. While for the bare Fe3O4 (Fig. 5a), there is almost no weight loss. These results show that APS-PEG-TFEE and PEG dicarboxylic acid have coated
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to the surface of Fe3O4 successfully, and CMNPs can keep its structural and functional integrity at temperatures below 100℃, which is very important to the application in vivo.
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Fig. 6 shows that the diffraction peaks with 2θ values of 30.09, 35.44, 43.07, 54.35, 56.96, 62.55, 70.91 and 74.00ºare correspond to the (220), (311), (400), (422), (511), (440), (620) and
te
d
(533) reflection planes of magnetite Fe3O4 respectively (JCPDS no.19-629) [23]. And during the whole process of coating, there is almost no phase change of Fe3O4, this is very important to the
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application for magnetic targeted drug delivery. As can be seen from Fig. 7, the strong absorption band at about 582 cm-1 is due to Fe-O
stretching vibration for Fe3O4. The signals appear at 3430 cm-1 might be attributed to the
stretching vibration of -OH. Absorbance peaks at 1100 and 1395 cm-1 correspond to C-O-C symmetric and asymmetric stretching, respectively, and the broad band at 3130 cm-1 results from the -NH stretch band in the amide linkage between the APS and the PEG dicarboxylic acid, all of these confirm that APS-PEG-TFEE has coated onto Fe3O4. The absorbance peaks at 1641 and 1161 cm-1 (Fig. 7b) representδNH andνCN, which is a strong evidence of the primary amine group. Significant peak broadening at 1623 cm-1 is attributable to the amide linkage formed between the 9
Page 9 of 28
PEG dicarboxylic acid and primary amine. And the appearance of peak at 2929 cm-1 for -CH2 stretching vibration also confirms the existence of PEG dicarboxylic acid.
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As expected, in Fig.8, the zeta potential evolution of Fe3O4 supplemented with different modifications showed marked differences. For amino-terminal Fe3O4, the value changed from the
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as prepared value (-33.30 mV) to a positive value of 7.94 mV. In the case of CMNPs, the value
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decayed from positive to negative (-40.79 mV). These revealed that the surface of Fe3O4@APS-PEG-TFEE was successfully carboxylated, and a large number of carboxyl groups
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presented on the surface of CMNPs. After drug loading, the value changed to a less positive of
onto CMNPs via electrostatic interaction.
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0.87 mV, this may be caused by the residually excess DOX, indicating the DOX had been loaded
In Fig. 9, the saturation magnetization (Ms) value of CMNPs-DOX is 49.07 emu/g and
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d
without obvious pronounced hysteresis loops, indicating that CMNPs-DOX is superparamagnetic at room temperature. Meanwhile, we can also see that the Ms of Fe3O4 (74.79 emu/g) is reducing
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successively along with the coating (62.02 emu/g) and drug loading (49.07 emu/g). These also confirm that the polymers have coated onto Fe3O4 and DOX has effectively connected to the
drug-loading platform for both polymers and DOX are nonmagnetic. When a magnet was placed
outside the tube (Fig. 9b), CMNPs-DOX was concentrated at the magnet site, whereas the bulk solution became colorless. Therefore, it is expected that CMNPs-DOX can be easily controlled by an external magnetic field, and then the drug can be easily delivered to the target area. 3.2. pH dependent drug release As can be seen in Fig. 10, the release rate of DOX from loaded CMNPs was found to increase with decreasing pH, such that 28.70, 64.27 and 76.16% of DOX were released within 72 10
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h when stored in PBS of pH 7.4, 5.0 and 4.0, respectively. Thus, the pH-sensitive drug release could occur once the CMNPs-DOX was internalized into the tumor cells. At the end of the release
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experiment, CMNPs-DOX incubated in PBS at pH 4.0 was reclaimed and washed with water, then re-dispersed in water for secondary loading of DOX under the same conditions. The secondary Lr
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was 51.43% (initial Lr was 76.19%), indicating that CMNPs have good recycling performance.
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3.3. Cytotoxicity analysis
The cytotoxicity of CMNPs, free DOX and CMNPs-DOX (taken at the same drug
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concentrations) were evaluated by WST-1 assay against MCF-7 cancer cells. Fig. 11 shows the related cytotoxicity profiles, compared to DOX solution, no significant cytotoxicity can be seen
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for CMNPs, indicating that they can be regarded as safe drug delivery carriers and are promising for controlled drug delivery. On the contrary, CMNPs-DOX exhibited higher toxicity against
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MCF-7 cells, because the magnetism of CMNPs would help the internalization of CMNPs-DOX
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within cancer cell compartments, and also the Fe3+ had slightly bigger toxicity over cell then Fe2+
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due to the tendency of oxygen capture from the cell, this result agreed with what was reported by previous studies and showed that the presence of Fe3O4 led to increasing cytotoxic effects [25,26].
Also, WST-1 assay revealed that CMNPs-DOX showed the IC50 of 100-120 μg/mL compared to
DOX which showed the IC50 of 120-140 μg/mL. Correspondingly, the statuses of MCF-7 cells which were treated by CMNPs-DOX with different drug concentration are shown in Fig. 12. All the results show that CMNPs can be potentially useful for drug delivery and CMNPs-DOX can be applied in magnetic targeted cancer therapy. 4. Conclusion The clusters of magnetic nanoparticles (CMNPs) were successfully prepared for dual targeted 11
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(magnetic targeted and pH-sensitive) delivery of doxorubicin. All the results indicated that CMNPs had suitable in size within 20 nm, good stability and super-paramagnetic responsibility as
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well as good performance of recycling and reuse, the secondary drug loading rate was nearly 52%. The DOX that loaded onto CMNPs could be effectively released from the carriers under acidic
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conditions, 76.16% of DOX was released within 72 h at pH 4.0, and the CMNPs-DOX had
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satisfactory anti-tumor activity on MCF-7 cells. To this end, we have enough reasons to believe the application of CMNPs in the field of targeted drug delivery for tumor therapeutics.
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Acknowledgments
The authors are grateful for National Science Foundation of China (Project No. 50602024,
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No.50972060), the Scientific and Technical Supporting Programs of Jiangsu province (BE2012758), the Scientific Research Fund from NJUST Research Funding (No.2010ZDJH06),
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Dubois, M.L. Saboungi, L. Chourpa, Magnetic nanocarriers of doxorubicin coated with poly(ethyleneglycol) and folic acid: relation between coating structure, surface properties, colloidal stability, and cancer cell targeting, Langmuir 28 (2012) 1496-1505. [25] J. Varshosaz, H. Sadeghi-aliabadi, S. Ghasemi, B. Behdadfar, Use of magnetic folate-dextran-retinoic acid micelles for dual targeting of doxorubicin in breast cancer, Biomed Res Int 2013 (2013) 1-16. [26] H.S. Yoo, T. G. Park, Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicin-PEG-folate conjugate, J Control Release 100 (2004) 247-256.
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Figures caption:
for the preparation of CMNPs-DOX.
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Fig. 1. The description of experimental scheme, (a) the rationale for drug delivery and (b) method
Fig. 2. 1H NMR spectra of APS-PEG-TFEE dissolved in CDCl3 (500 MHz, 298 K).
and (c) APS-PEG-TFEE.
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Fig. 3. FT-IR spectra of (a) PEG dicarboxylic acid, (b) acyl chlorination of PEG dicarboxylic acid
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Fig. 4. TEM images of (a) Fe3O4 and (b) CMNPs.
Fig. 5. TG analysis results of (a) Fe3O4, (b) amino-terminal Fe3O4, and (c) CMNPs. Fig. 6. XRD patterns of (a) Fe3O4, (b) amino-terminal Fe3O4, and (c) CMNPs. Fig. 7. FT-IR spectra of (a) Fe3O4@APS-PEG-TFEE, (b) amino-terminal Fe3O4, and (c) CMNPs
in KBr pellet.
Fig. 8. Zeta potential data of Fe3O4, amino-terminal Fe3O4, CMNPs and CMNPs-DOX dispersed in deionized water at the concentration of 0.01 mg/mL. The measurements were conducted at least in triplicate for each sample. Fig. 9. Images of (a) VSM measurement results of (a1) Fe3O4, (a2) CMNPs, (a3) CMNPs-DOX, 14
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(b) the suspended aqueous solution of CMNPs-DOX (b1) before and (b2) after a magnet is placed outside the test tube for 30 min.
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Fig. 10. Release kinetics of DOX from CMNPs-DOX. Fig. 11. Cytotoxicity evaluation of DOX solution, suspensions of CMNPs and of CMNPs-DOX by
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WST-1 assay on MCF-7 cells after 24 h treatment. Data were expressed as means±SD.
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Fig. 12. Bright-field images of MCF-7 cells which were treated by CMNPs-DOX with drug concentration of (a) 0 μg/mL, (b) 10 μg/mL, (c) 20 μg/mL, (d) 40 μg/mL, (e) 80 μg/mL and (f)
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Highlights
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1 Silane-terminated PEG dicarboxylic acid was a steady bridge between Fe3O4 and drug; 2 The drug carriers had magnetic targeting and pH-response properties; 3 The drug carriers can be recovered for re-loading of drug in vitro studies; 4 Drug was loaded onto carriers by electrostatic interaction which was pollution-free; 5 Drug loading rate was 76.19% and drug loaded carriers had excellent pH-sensitivity; 6 Drug-loaded carriers exhibited high anti-tumor activity, but the carriers were not.
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S0169-4332(14)02184-9 http://dx.doi.org/doi:10.1016/j.apsusc.2014.09.184 APSUSC 28840
To appear in:
APSUSC
Received date: Revised date: Accepted date:
8-7-2014 27-9-2014 28-9-2014
Please cite this article as: J. Wu, Y. Wang, W. Jiang, S. Xu, R. Tian, Synthesis and characterization of recyclable clusters of magnetic nanoparticles as doxorubicin carriers for cancer therapy, Applied Surface Science (2014), http://dx.doi.org/10.1016/j.apsusc.2014.09.184 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and characterization of recyclable clusters of magnetic nanoparticles as doxorubicin carriers for cancer therapy
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Juan Wu, Yujiao Wang, Wei Jiang∗, Shanshan Xu, Renbing Tian National Special Superfine Powder Engineering Research Center, Nanjing University of Science
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and Technology, Nanjing 210094, China
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Abstract
This study focuses on the synthesis and characterization of recyclable clusters of magnetic
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nanoparticles (CMNPs) as doxorubicin carriers for cancer therapy. Fe3O4 nanoparticles were used as magnetically responsive carriers, the modified polyethylene glycol dicarboxylic acid
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(APS-PEG-TFEE) acted as a steady bridge between Fe3O4 and drug. The prepared CMNPs exhibited a size within 20 nm, good stability and super-paramagnetic responsibility (Ms 62.02
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emu/g); doxorubicin (DOX) can be successfully loaded to CMNPs at a loading rate of 76.19% by electrostatic interaction. Moreover, the release studies in vitro showed that the drug-loaded
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carriers (CMNPs-DOX) had excellent pH-sensitivity, 76.16% of DOX was released within 72 h at pH 4.0, and the secondary drug loading rate was nearly 52%. WST-1 assays in model breast cancer cells (MCF-7) demonstrated that CMNPs-DOX exhibited high anti-tumor activity, while the CMNPs were practically non-toxic. Thus, our results revealed that CMNPs would be a competitive candidate for drug delivery carriers and CMNPs-DOX could be used in targeted cancer therapy in the near future. Keywords: Fe3O4; magnetic targeted; pH-sensitive; doxorubicin; cancer therapy
∗
Corresponding author. Tel.: +86-25-84315042; Fax.: +86-25-84315042
E-mail address: [email protected] (W. Jiang) 1
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1. Introduction The application of iron oxide nanoparticles (Fe3O4) in biomedical researches such as
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magnetic drug delivery [1], tumor-specific cell targeting [2], gene therapy [3], bioseparation [4] and magnetic resonance imaging [5] has drawn considerable attention due to the ultra-fine size,
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biocompatibility and superparamagnetic behavior [6]. These properties are fully exploited when
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they are used as drug delivery carriers. The drug-loaded Fe3O4 can be specifically enriched in tumor area due to synergistic mechanisms such as (Ⅰ) the application of an external magnetic field
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[7], (Ⅱ) the size range (10-100 nm) is small enough to evade enhanced permeability and retention (EPR) of the body as well as penetrate the very small capillaries within the body tissues [8], (Ⅲ)
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internalization of nanoparticles within cancer cell compartments [9]. As a consequence, the chemotherapeutic drugs can be targeted to the lesion site in the body, and also the magnetic
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carriers not only decrease the systemic side effects but also promote the efficacy of chemotherapy. For effective tumor therapy, prolonged availability of the drug at the target site is the primary
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requisite of any drug carriers. In this regard, drug retention and drug release properties are indispensable features which should be taken into consideration. One of the promising drug carriers is the pH-sensitive drug carrier [10,12-14], and the drug release rate was responsive to the environmental pH value. As the pH value of most solid tumors (pH<6.0) was lower than the surrounding normal tissues (pH 7.4) [11], the anti-cancer drug which was connected by pH-sensitive bond could be triggered to release, so that to improve the therapeutic effect. In recent years, hydrazone bond [12-14] has emerged as a pH-sensitive bond to connect drugs.
While the experiments of loading drugs by hydrazone bond always involve multiple steps and cost a large amount of organic reagent which would cause pollution. Yang et al. [12] designed and 2
Page 2 of 28
synthesized a triblock copolymer for drug delivery, before connecting DOX, anhydrous dimethylformamide (DMF) and anhydrous hydrazine were used to handle triblock copolymer at
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40℃. After dialyzing and freeze-drying, DOX was conjugated onto the copolymers in the presence of DMF. Fang et al. [15] synthesized a biodegradable poly(beta-amino ester) (PBAE)
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copolymer to load DOX. In the process of drug connection, dimethylsulfoxide and ether were used
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as solvent, in order to remove any unreacted reactants and solvent, aqueous solution of PBAE-DOX was required to pass through column. As compared to hydrazone bonds, electrostatic
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interaction [16] which has desirable pH-responsivity usually exhibits easier operation in experimental procedure, and lower consumption and pollution. In addition, there are limited
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reports on using electrostatic interaction to connect DOX in magnetic targeted drug delivery carriers.
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Silanization is helpful to form bonds between inorganic and organic components for the modification of functional groups on the surface of Fe3O4 [19,23]. To date, various silane coupling
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agents such as 3-aminopropyltriethoxysilane [20] and tetraethoxysilane [21] have been used. At the same time, poly(ethylene glycol) (PEG) is used widely to modify Fe3O4 for long blood
circulation time [17,18]. For this, we synthesized and characterized of polyethylene glycol dicarboxylic
acid
modified
by
3-aminopropyltriethoxysilane
and
2,2,2-trifluoroethanol
(APS-PEG-TFEE). As a bridge linking, APS-PEG-TFEE could covalently link to the Fe3O4 through -Si-O- [24] and load DOX via electrostatic interaction, so as to form CMNPs that can deliver DOX and are recyclable (Fig. 1). Furthermore, the CMNPs-DOX could effectively release DOX at pH<6.0 and inhibit the growth of model breast cancer cells (MCF-7). Thus, our approach clearly indicates the CMNPs-DOX might be a promising candidate for targeted tumor therapy. 3
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2. Materials and methods 2.1. Materials All chemicals were analytical grade and used without further purification. Ferric chloride
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(FeCl3 • 6H2O), ferrous sulfate (FeSO4 • 7H2O), (3-aminopropy)triethoxysilane (APS) and
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N-hydroxyl succinamide (NHS) were supplied by Sinopharm Chemical Reagent Co., Ltd.,
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Shanghai, China. Polyethylene glycol (PEG) dicarboxylic acid (Mn 600) was purchased from Sigma-Aldrich. 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) and
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2,2,2-trifluoroethanol (TFEA) were purchased from Aladdin. DOX was purchased from Yuanye Biotechnology Co. Ltd., Shanghai, China. Dulbecco’s Modified Eagle medium (DMEM) was from
Thermo
Fisher
Biochemical
Products
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purchased
Co.,
Ltd.,
Beijing,
China.
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) and MCF-7
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were supplied by Beyotime Biotechnology Co., Ltd., Shanghai, China. Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd., Zhejiang, China. All other
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chemicals and solvents used in this study were of high analytical grade and commercially available.
2.2. Characterizations 1
H NMR spectrum was recorded on AV-III nuclear magnetic resonance spectrometer (NMR,
Bruker Co. Ltd., Germany). The size and morphology of the samples were investigated by transmission electron microscopy (TEM, Model Tecnai 12, Philips Co. Ltd., Holland). The thermal stability of the dry samples were measured by thermogravimetric analysis (TGA, Model TA2100, TA Instruments, USA) under N2 at a heating rate of 10℃/min over the range of 50~650 ℃. The crystalline phases were recorded using X-ray diffraction (XRD, Bruker Co. Ltd., Germany) 4
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with Cu Kα radiation. Surface charge was evaluated by the zeta potential analyzer (BI-90Plus, Brookhaven Co. Ltd., USA). Magnetic properties of the particles were detected at room
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temperature using vibrating sample magnetometer (VSM, Model 7410, Lake Shore Co. Ltd., USA). Fourier-transform infrared spectroscopy (FT-IR) spectra were recorded on Vector 22
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UV-visible spectrophotometry (UV-vis, Agilent Co. Ltd., USA).
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spectrometer (Bruker Co. Ltd., Germany). The drug loading and release rate were detected by
2.3. Experimental
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2.3.1. Synthesis of modified polyethylene glycol dicarboxylic acid (APS-PEG-TFEE) The synthetic process for APS-PEG-TFEE was modified from previous report [22]. To PEG
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dicarboxylic acid (116.6 mmol) was dropwise added 12 mL of thionyl chloride and refluxed for 4 h to obtain a colorless liquid. After that, 12 mL TFEA was dropwise added to the liquid and the
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obtained mixture was stirred for 30 min, and refluxed for additional 12 h to obtain a pale-yellow liquid which was dissolved in 20 mL of toluene. Finally, 3 mL APS in batches (3 batches, 1 batch
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per 30 min) was added after 500μL of triethylamine was added for reaction of 10 min, and the
mixture was stirred for 12 h to get APS-PEG-TFEE. The solvent and excess reactants in each step
during the whole reaction process were removed by rotary evaporation under vacuum. 2.3.2.
Preparation of Fe3O4
According to the improved chemical co-precipitation method [23], 1.1127 g FeSO4•7H2O
and 1.9182 g FeCl3•6H2O were dissolved in deionized water. The mixture was stirred under N2 at 80℃ for 1 h. Then, 40 mL of aqueous ammonia (25 wt.%) was injected into the mixture rapidly, continued for another 1 h and then cooled to room temperature. The precipitated particles were separated by magnet and washed several times with water. Finally, Fe3O4 nanoparticles were dried 5
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under vacuum at 50°C. 2.3.3.
Surface modification of Fe3O4 with APS-PEG-TFEE
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Initially, 100 mg dry Fe3O4 was dispersed into 50 mL of toluene, then 2 mL APS-PEG-TFEE was added after ultrasound of 30 min. The mixture was purged with N2 and sonicated at 50°C for
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4 h. The resulting colloidal suspension was washed twice in toluene and re-suspended in 50 mL of
2.3.4.
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toluene. Carboxylation of Fe3O4@APS-PEG-TFEE to form the CMNPs
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In order to effectively make the surface of Fe3O4@APS-PEG-TFEE carboxylated, ethylenediamine was used to substitute for the -CF3 of APS-PEG-TFEE, so that there was a large
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number of amino on the surface of Fe3O4. Briefly, 800μL of ethylenediamine was added into 50 mL of suspension, the mixture was purged with N2 and sonicated at 50°C for 2 h. Then, the
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product was magnetic decanted, washed with toluene and dichloromethane, and dried in fume hood to obtain amino-terminal Fe3O4. After that, 2 g PEG dicarboxylic acid was dissolved in 5 mL
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of deionized water which contained 200 mg EDC and 200 mg NHS. The activation reaction went on for 4 h. Finally, 50 mg amino-terminal Fe3O4 dispersed in 5 mL of deionized water was added
and the reaction continued for 24 h. The final product was collected by magnet, washed with deionized water and re-dispersed in it (10 mL) to form suspension of CMNPs. 2.3.5.
Drug loading (CMNPs-DOX)
2.1 mg DOX which was dissolved in deionized water (2 mL) was added to the suspension of CMNPs, and then the reaction went on for 24 h under dark. The product was collected by magnet and washed twice with deionized water. The final product was freeze-dried and storied in the dark at 5℃. 6
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2.4. In vitro release of DOX Briefly, 0.01 M of phosphate buffer solution (PBS) (pH 4.0, 5.0, 7.4) was prepared to imitate
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conditions like tumors and normal tissues. After that, 5 mg CMNPs-DOX was dispersed in 2 mL PBS and then transferred to a dialysis bag which was immersed in 4 mL of the same medium and
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kept in the oscillator at 37℃. At selected time intervals, PBS (2 mL) outside the dialysis bag was
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removed for UV-vis analysis at 482 nm and replaced by the fresh with the same volume. To determine the amount of DOX released, calibration curves were run with DOX in corresponding
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PBS at pH 4.0, 5.0 and 7.4, respectively. The release experiments were conducted in triplicate. The results presented are the average data with standard deviations. The drug loading rate (Lr) was
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calculated by the formula: Lr=(m1/m0)×100%, where m1 is the quality of DOX loaded onto CMNPs, m0 is the quality of DOX added before the reaction; the release rate (Rr) of DOX was
CMNPs-DOX.
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2.5. WST-1 assays
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calculated by the formula: Rr=(m2/m1)×100%, where m2 is the quality of DOX released from
MCF-7 cells were seeded in 96-well plates (5 × 103 cells/well) in 200 μ L DMEM
supplemented with 10% FBS for 24 h at 37℃ in a humidified 5% CO2-containing atmosphere.
Then, the media was aspirated and replaced by 200μL of fresh cell culture medium. 20μL of free DOX, CMNPs, or CMNPs-DOX at various concentrations in fresh cell culture medium was added and the cells were incubated for 24 h. After that, 20μL WST-1 solution (5 mg/mL) was added. The cells were incubated for 2 h, and the absorbance at a wavelength of 450 nm of each well was measured using a multifunctional microplate reader (TECAN Infinite 200 Pro, Austria). The relative cell viability (%) was determined by comparing the absorbance at 450 nm with control 7
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wells containing only cell culture medium. The bright-field images of MCF-7 cells were recorded on microscope (Olympus, IX81, Japan).
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3. Results and discussion 3.1. Characterization of sample
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The structure of the modified polyethylene glycol dicarboxylic acid (APS-PEG-TFEE) was
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identified by 1H NMR analysis. The chemical shifts for protons from 1 to 11 of APS-PEG-TFEE are shown in Fig. 2 and its inset table. The molecular structure of APS-PEG-TFEE was confirmed
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by the presence of all characteristic peaks and peak integration. To further verify the successful synthesis of APS-PEG-TFEE, FT-IR spectra were also collected at each step of the synthesis (Fig.
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3). The broad band above 3000 cm-1 corresponds to -OH of PEG dicarboxylic acid (Fig. 3a) and disappears in Fig. 3b, and then reappears in Fig. 3c, which result from the stretching vibration of
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-NH in the amide linkage between APS and PEG dicarboxylic acid. In Fig. 3c, the peaks at 1779
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and 1675 cm-1 are attributed to the trifluoroacetate ester and the amide carbonyl respectively,
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confirming the formation of half-amide/ester structure. As shown in Fig. 4, the dispersion of Fe3O4 was improved effectively after coating (Fig. 4b),
it was because that APS-PEG-TFEE and PEG glycol dicarboxylic acid lowered the high surface energy of Fe3O4 nanoparticles. In addition, the average particle size of CMNPs in TEM image was about 20 nm, which was small enough both to evade reticuloe endothelin system (RES) of the body as well as penetrate the very small capillaries within the body tissues and therefore may offer the most effective distribution in certain tissues [8]. In order to confirm the existence of cladding layer and obtain the amount of APS-PEG-TFEE and PEG dicarboxylic acid so as to determine the amount of drug used in the further experiments, 8
Page 8 of 28
thermogravimetric analysis (TGA) had been used. In Fig. 5b, upon heating, amino-terminal Fe3O4 shows a weight loss of 2.10% at temperatures ranging from 50 to 100℃, mainly due to the loss of
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physically adsorbed water on the products. When being heated to 650℃, probably because of the decomposition of APS-PEG-TFEE, leading to a weight loss of 16.17%. In comparison, the weight
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loss of CMNPs is about 25.65% (Fig. 5c), the increased amount (9.48%) could attribute to the
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decomposition of PEG glycol dicarboxylic acid. While for the bare Fe3O4 (Fig. 5a), there is almost no weight loss. These results show that APS-PEG-TFEE and PEG dicarboxylic acid have coated
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to the surface of Fe3O4 successfully, and CMNPs can keep its structural and functional integrity at temperatures below 100℃, which is very important to the application in vivo.
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Fig. 6 shows that the diffraction peaks with 2θ values of 30.09, 35.44, 43.07, 54.35, 56.96, 62.55, 70.91 and 74.00ºare correspond to the (220), (311), (400), (422), (511), (440), (620) and
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(533) reflection planes of magnetite Fe3O4 respectively (JCPDS no.19-629) [23]. And during the whole process of coating, there is almost no phase change of Fe3O4, this is very important to the
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application for magnetic targeted drug delivery. As can be seen from Fig. 7, the strong absorption band at about 582 cm-1 is due to Fe-O
stretching vibration for Fe3O4. The signals appear at 3430 cm-1 might be attributed to the
stretching vibration of -OH. Absorbance peaks at 1100 and 1395 cm-1 correspond to C-O-C symmetric and asymmetric stretching, respectively, and the broad band at 3130 cm-1 results from the -NH stretch band in the amide linkage between the APS and the PEG dicarboxylic acid, all of these confirm that APS-PEG-TFEE has coated onto Fe3O4. The absorbance peaks at 1641 and 1161 cm-1 (Fig. 7b) representδNH andνCN, which is a strong evidence of the primary amine group. Significant peak broadening at 1623 cm-1 is attributable to the amide linkage formed between the 9
Page 9 of 28
PEG dicarboxylic acid and primary amine. And the appearance of peak at 2929 cm-1 for -CH2 stretching vibration also confirms the existence of PEG dicarboxylic acid.
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As expected, in Fig.8, the zeta potential evolution of Fe3O4 supplemented with different modifications showed marked differences. For amino-terminal Fe3O4, the value changed from the
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as prepared value (-33.30 mV) to a positive value of 7.94 mV. In the case of CMNPs, the value
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decayed from positive to negative (-40.79 mV). These revealed that the surface of Fe3O4@APS-PEG-TFEE was successfully carboxylated, and a large number of carboxyl groups
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presented on the surface of CMNPs. After drug loading, the value changed to a less positive of
onto CMNPs via electrostatic interaction.
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0.87 mV, this may be caused by the residually excess DOX, indicating the DOX had been loaded
In Fig. 9, the saturation magnetization (Ms) value of CMNPs-DOX is 49.07 emu/g and
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without obvious pronounced hysteresis loops, indicating that CMNPs-DOX is superparamagnetic at room temperature. Meanwhile, we can also see that the Ms of Fe3O4 (74.79 emu/g) is reducing
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successively along with the coating (62.02 emu/g) and drug loading (49.07 emu/g). These also confirm that the polymers have coated onto Fe3O4 and DOX has effectively connected to the
drug-loading platform for both polymers and DOX are nonmagnetic. When a magnet was placed
outside the tube (Fig. 9b), CMNPs-DOX was concentrated at the magnet site, whereas the bulk solution became colorless. Therefore, it is expected that CMNPs-DOX can be easily controlled by an external magnetic field, and then the drug can be easily delivered to the target area. 3.2. pH dependent drug release As can be seen in Fig. 10, the release rate of DOX from loaded CMNPs was found to increase with decreasing pH, such that 28.70, 64.27 and 76.16% of DOX were released within 72 10
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h when stored in PBS of pH 7.4, 5.0 and 4.0, respectively. Thus, the pH-sensitive drug release could occur once the CMNPs-DOX was internalized into the tumor cells. At the end of the release
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experiment, CMNPs-DOX incubated in PBS at pH 4.0 was reclaimed and washed with water, then re-dispersed in water for secondary loading of DOX under the same conditions. The secondary Lr
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was 51.43% (initial Lr was 76.19%), indicating that CMNPs have good recycling performance.
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3.3. Cytotoxicity analysis
The cytotoxicity of CMNPs, free DOX and CMNPs-DOX (taken at the same drug
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concentrations) were evaluated by WST-1 assay against MCF-7 cancer cells. Fig. 11 shows the related cytotoxicity profiles, compared to DOX solution, no significant cytotoxicity can be seen
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for CMNPs, indicating that they can be regarded as safe drug delivery carriers and are promising for controlled drug delivery. On the contrary, CMNPs-DOX exhibited higher toxicity against
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MCF-7 cells, because the magnetism of CMNPs would help the internalization of CMNPs-DOX
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within cancer cell compartments, and also the Fe3+ had slightly bigger toxicity over cell then Fe2+
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due to the tendency of oxygen capture from the cell, this result agreed with what was reported by previous studies and showed that the presence of Fe3O4 led to increasing cytotoxic effects [25,26].
Also, WST-1 assay revealed that CMNPs-DOX showed the IC50 of 100-120 μg/mL compared to
DOX which showed the IC50 of 120-140 μg/mL. Correspondingly, the statuses of MCF-7 cells which were treated by CMNPs-DOX with different drug concentration are shown in Fig. 12. All the results show that CMNPs can be potentially useful for drug delivery and CMNPs-DOX can be applied in magnetic targeted cancer therapy. 4. Conclusion The clusters of magnetic nanoparticles (CMNPs) were successfully prepared for dual targeted 11
Page 11 of 28
(magnetic targeted and pH-sensitive) delivery of doxorubicin. All the results indicated that CMNPs had suitable in size within 20 nm, good stability and super-paramagnetic responsibility as
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well as good performance of recycling and reuse, the secondary drug loading rate was nearly 52%. The DOX that loaded onto CMNPs could be effectively released from the carriers under acidic
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conditions, 76.16% of DOX was released within 72 h at pH 4.0, and the CMNPs-DOX had
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satisfactory anti-tumor activity on MCF-7 cells. To this end, we have enough reasons to believe the application of CMNPs in the field of targeted drug delivery for tumor therapeutics.
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Acknowledgments
The authors are grateful for National Science Foundation of China (Project No. 50602024,
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No.50972060), the Scientific and Technical Supporting Programs of Jiangsu province (BE2012758), the Scientific Research Fund from NJUST Research Funding (No.2010ZDJH06),
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and the Fundamental Research Funds for the Central Universities (No.30920130112003).
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Figures caption:
for the preparation of CMNPs-DOX.
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Fig. 1. The description of experimental scheme, (a) the rationale for drug delivery and (b) method
Fig. 2. 1H NMR spectra of APS-PEG-TFEE dissolved in CDCl3 (500 MHz, 298 K).
and (c) APS-PEG-TFEE.
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Fig. 3. FT-IR spectra of (a) PEG dicarboxylic acid, (b) acyl chlorination of PEG dicarboxylic acid
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Fig. 4. TEM images of (a) Fe3O4 and (b) CMNPs.
Fig. 5. TG analysis results of (a) Fe3O4, (b) amino-terminal Fe3O4, and (c) CMNPs. Fig. 6. XRD patterns of (a) Fe3O4, (b) amino-terminal Fe3O4, and (c) CMNPs. Fig. 7. FT-IR spectra of (a) Fe3O4@APS-PEG-TFEE, (b) amino-terminal Fe3O4, and (c) CMNPs
in KBr pellet.
Fig. 8. Zeta potential data of Fe3O4, amino-terminal Fe3O4, CMNPs and CMNPs-DOX dispersed in deionized water at the concentration of 0.01 mg/mL. The measurements were conducted at least in triplicate for each sample. Fig. 9. Images of (a) VSM measurement results of (a1) Fe3O4, (a2) CMNPs, (a3) CMNPs-DOX, 14
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(b) the suspended aqueous solution of CMNPs-DOX (b1) before and (b2) after a magnet is placed outside the test tube for 30 min.
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Fig. 10. Release kinetics of DOX from CMNPs-DOX. Fig. 11. Cytotoxicity evaluation of DOX solution, suspensions of CMNPs and of CMNPs-DOX by
cr
WST-1 assay on MCF-7 cells after 24 h treatment. Data were expressed as means±SD.
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Fig. 12. Bright-field images of MCF-7 cells which were treated by CMNPs-DOX with drug concentration of (a) 0 μg/mL, (b) 10 μg/mL, (c) 20 μg/mL, (d) 40 μg/mL, (e) 80 μg/mL and (f)
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Highlights
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1 Silane-terminated PEG dicarboxylic acid was a steady bridge between Fe3O4 and drug; 2 The drug carriers had magnetic targeting and pH-response properties; 3 The drug carriers can be recovered for re-loading of drug in vitro studies; 4 Drug was loaded onto carriers by electrostatic interaction which was pollution-free; 5 Drug loading rate was 76.19% and drug loaded carriers had excellent pH-sensitivity; 6 Drug-loaded carriers exhibited high anti-tumor activity, but the carriers were not.
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