http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, Early Online: 1–16 ! 2015 Informa UK Ltd. DOI: 10.3109/1061186X.2015.1016437

ORIGINAL ARTICLE

Enhancement of cytotoxicity of artemisinin toward cancer cells by transferrin-mediated carbon nanotubes nanoparticles Huijuan Zhang1*, Yandan Ji1*, Qianqian Chen1, Xiaojing Jiao1, Lin Hou1, Xiali Zhu1,2, and Zhenzhong Zhang1

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School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China and 2School of Pharmaceutical Sciences, Henan University of Traditional Chinese Medicine, Zhengzhou, China Abstract

Keywords

Artemisinin (ART) is a kind of drug with an endoperoxide bridge which tends to react with Fe2+ to generate radicals for killing cancer cells. However, simultaneous delivery of hydrophobic ART and Fe2+ ions into cancer cells remains a major challenge. In this study, a multi-functional tumor-targeting drug delivery system employing hyaluronic acid-derivatized multi-walled carbon nanotubes (HA-MWCNTs) as drug carriers, transferrin (Tf) as targeting ligand and ART as a model drug for cancer treatment was constructed. This delivery system (HA-MWCNTs/ Tf@ART) not only retained optical property of MWCNTs and cytotoxicity of ART but also demonstrated synergistic anti-tumor effect using ART and Tf. Compared with free ART, remarkably enhanced anti-tumor efficacy of this drug vehicle was realized both in cultured MCF-7 cells in vitro and in a tumor-bearing murine model in vivo, due to increased intracellular accumulation of ART and co-delivery of Tf and ART analogs. HA-MWCNTs/Tf@ART with laser irradiation demonstrated the highest inhibition effect compared to the other groups. This result may provide a new way of using promising natural drugs for cancer therapy.

Chinese herb artemisinin, co-delivery of Tf and ART, reactive oxygen species, tumor-targeting

Introduction Artemisinin (ART), a sesquiterpene lactone extracted from the Chinese herb Artemisia annua L., and its derivatives have been widely used as anti-malarial drugs for many years [1]. The potential application of ART analogs in cancer treatment has attracted international attention, and these compounds have been investigated in preclinical and clinical studies [2]. For 420 years, the activity of ART against cancer cells has been recognized [3,4]. The ART molecule contains an endoperoxide bridge (COOC) which can interact with a Fe(II) to form free radicals, resulting in macromolecular damage and cell death [5,6]. In mammals, iron in the blood is transported by transferrin (Tf). Tf is a 698-residue protein, and binds two Fe(III) in its active sites. Cells in need of iron express transferrin receptors (TfR) on their surface [7]. Binding of holo-Tf (i.e. iron-loaded Tf) to the receptor triggers endocytosis and traffics holo-Tf intracellularly in endosomes. When pH inside an endosome drops, iron is released and moves into the cytoplasm after it gets reduced to the Fe(II) state. Apo-Tf (iron-free Tf) is then recycled to the cell surface and release. Each serum Tf molecule undergoes 100–200 cycles of ion binding, intracellular transport and release *These authors contributed equally to this work. Address for correspondence: Prof. Zhenzhong Zhang, School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Kexue Road, Zhengzhou 450001, China. Tel: +86-371-6778-1910. Fax: +86-371-6778-1908. E-mail: [email protected]

History Received 28 October 2014 Revised 6 January 2015 Accepted 26 January 2015 Published online 10 March 2015

during its life time [8]. The level of TfR expression varies depending on the cell types. Non-dividing cells have relatively low levels of TfR expression, whereas rapidly proliferating cells can express up to 100 000 TfR per cell [9,10]. Most cancer cells express a high concentration of TfR on cell surface and have a high amount of Fe(III) ion uptake into the cells. For example, in the case of human breast cancer cells, 5–15 times more TfR are expressed on their cell surface compared to normal breast cells, and breast cancer cells take up more iron than normal breast cells [11]. As described above, cancer cells need Tf-mediated iron uptake to maintain their uncontrolled growth, and TfR is highly expressed on cancer cell surface. So Tf can be used as an effective drug targeting molecule [12–14]. Targeted drug delivery is an effective strategy to increase drug concentration at the targeted tumor site by variety of functionalized drug carriers, which thus improves tumor therapeutic efficacy and reduces side effects [15]. Nanoparticle-assisted drug delivery systems could improve water solubility, circulation time, pharmacokinetic properties, intracellular uptake profile and preserve the metabolic stability of drugs in cancer cells, while avoiding toxicity in normal cells via both passive and active targeting strategies [16]. Targeted drug delivery system using functionalized multi-walled carbon nanotubes (MWCNTs) is a promising strategy that augments the selectivity and dosage of drug to the tumor cells, enhancing therapeutic index [17,18]. MWCNTs have shown great potential in cancer treatments because of their controlled and targeted drug delivery

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Figure 1. Scheme for preparation of HA-MWCNTs/Tf@ART.

characteristics, which can simultaneously lead to higher therapeutic efficiency and minimize side effects [19]. Besides, MWCNTs have excellent photothermal conversion property in near-infrared (NIR) region, where biological systems have low absorption and high transparency [20], thus offering a possibility for cancer photothermal therapy [21]. They have been used for a wealth of applications [22], such as DNA biosensors, protein biosensors and ion channel blockers [23]. Moreover, MWCNTs can effectively shuttle various biomolecules, such as drugs, peptide, proteins, DNA and RNA, into cells by endocytosis [24]. Nevertheless, the surface of MWCNTs is highly hydrophobic and insoluble in either aqueous solution or common organic reagents. As a result, solubility under physiological conditions is a key prerequisite to make MWCNTs biocompatible [25]. Previous studies have shown that covalent functionalization could be used to modify MWCNTs to improve their dispersion in water and physiological environments [26]. Hyaluronic acid (HA) is a naturally occurring linear polysaccharide with negative charge, which exists widely in extracellular matrix and is primarily cleared by the lymphatic system [27]. HA has a series of excellent properties, such as biocompatible, biodegradable and non-immunogenic characteristics [28,29]. The expression of receptor for HA (CD44) was reported to have a close relation with the occasion of tumors [30]. Many tumor cells showed up-regulated expression of CD44, resulting in HA having a high affinity and good selectivity for tumors [31,32]. HA-based tumor-targeted drug delivery system has been an emerging and fast-growing field. It has been demonstrated that HA-modified nanoparticles showed rapid uptake into tumor cells through receptormediated endocytosis [33]. In this study, HA was linked on MWCNTs in order to obtain a water-soluble and tumortargeting drug carrier, HA-MWCNTs. In this study, we constructed a multifunctional tumortargeting drug delivery system employing HA-MWCNTs, with high aqueous solubility, neutral pH and tumor-targeting

activity, as drug carriers, Tf as targeting ligand and ART as a model hydrophobic chemotherapy drug for treatment of cancer, as shown in Figure 1. Owing to the absorption properties of the MWCNTs in NIR region, this system (HA-MWCNTs/Tf@ART) could simultaneously deliver both the heat and drug to the tumorigenic region to facilitate the combined chemotherapy and photothermal treatment. Meanwhile, the special action mechanism of ART and the specific expression of Tf receptor make this delivery system increasing pharmacological activity of the drug greatly at the target site, while reducing the toxicity of ART in non-target organs effectively. And the action mechanism was shown in Figure 2. In our study, the cytotoxicity and receptor-mediated tumor-targeting characteristics of HA-MWCNTs/Tf@ART was evaluated in vitro and in vivo. Finally, the systemic toxicity was assessed by histological examination.

Experimental Materials ART (purity 499.0 wt%) was purchased from Create-Life Biotech Limited Company (Zhengzhou, China). MWCNTs were purchased from Chengdu Organic Chemicals Co. Ltd, Chinese Academy of Sciences Corporation Ltd (Chengdu, China). Tf, HA (purity 498%, molecular weight 12 000– 14 000), ethylenediamine, formamide, N-hydroxysuccinimide (NHS), N-(3-dimethylamino propyl-N0 -ethylcarbodiimide) hydrochloride (EDC  HCl), fluorescein isothiocyanate (FITC), sulforhodamine B (SRB) and dimethyl sulfoxide (DMSO), triethylamine were obtained from Sigma-Aldrich (St Louis, MO). Penicillin, streptomycin and fetal bovine serum (FBS) were bought from Life Technologies (Carlsbad, CA). Oxidation of multi-walled carbon nanotubes After being purified with 12 M HNO3, MWCNTs (200 mg) were added to 80 ml of mixture acid (HNO3:H2SO4 ¼ 1:3),

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Figure 2. Targeting of HA-MWCNTs/Tf@ART to membrane CD44 and CD71 receptors of cancer cells and the special action mechanism of ART and transferrin under exposure of NIR.

then added 20 ml of H2O2 and stirred for 48 h at room temperature. The resulting solid products (COOH-MWCNTs) were then collected on a membrane filter (Millipore, Darmstadt, Germany, pore size 0.22 mm), followed by thorough rinsing with Milli-Q water and dried in vacuum at 60  C for 24 h. Amine-functionalized HA HA (molecular weight ¼ 46 000, 200 mg) was added to 30 ml of formamide, heating at 50  C until dissolved (2–3 h), cooled down at room temperature. Then EDC  HCl (518 mg) and NHS (310 mg) were added to this solution, activating for 30 min. After that 1.0 ml of ethylenediamine was added to the activated HA solution drop by drop in the ice bath and reacted at room temperature for 2 h. The reaction solution was cooled with excess pre-cooled acetone (3–4 times the amount of reaction solution) and then the resulting products would crystallize. The crystallization were then collected on a membrane filter (Millipore, pore size 0.22 mm), followed by thorough rinsing with acetone. And finally the precipitation were re-dissolved with water and dialyzed by a dialysis bag (molecular weight cutoff ¼ 3 500) for 12 h to remove acetone, EDC and NHS. At last, the final product (NH2-HA) was lyophilized to give the yield of 78.5% (157 mg). Synthesis of HA-MWCNTs COOH-MWCNTs (45 mg) and NH2-HA (90 mg) were added to 60 ml of formamide, sonicated until dissolved, then added EDC  HCl (173 mg) and NHS (103 mg) to this solution and stirred for 15 min at room temperature. After that 180 ml of triethylamine was added to the activated solution drop by drop in the ice bath and reacted at room temperature for 24 h. Cooled the reaction solution with excess pre-cooled acetone (3–4 times the amount of reaction solution) and centrifuged for 15 min at 10 000 revolutions/min. Finally the precipitation were re-dissolved with water and dialyzed by a dialysis bag

(molecular weight cutoff ¼ 12 000) for 48 h, then the synthesis products (HA-MWCNTs) were freeze-dried in vacuum for 24 h with the yielding efficiency 88.9%. Preparation of HA-MWCNTs/Tf The large specific surface area of carbon nanotube and the six-membered ring of its sidewalls with rich electron could interact with Tf by hydrogen bonding, hydrophobic interactions, Van der Waals force, electrostatic attraction and – conjugated effect. Combining Tf with HA-MWCNTs could cause autofluorescence quenching of Tf (ex ¼ 280 nm, em ¼ 332 nm), so we could obtain the best combining ratio while the maximum fluorescence quenching of Tf occurred. Three solution samples of Tf (10.0 mg/ml) were prepared and then HA-MWCNTs with different concentration of 0, 10.0 and 20.0 mg/ml were added, respectively. With the concentration increased, the fluorescence spectrum was recorded to confirm the best combining ratio. ART adsorption on HA-MWCNTs/Tf HA-MWCNTs/Tf (10 mg) was added to water (10 ml) and sonicated at room temperature for 2 h to get an excellent aqueous dispersion. ART (30 mg) was added to the dispersion and magnetically stirred for 48 h. Then, ultrasonicated using an ultrasonic cell disruption system (400 W, 20 times), and the nanosuspension was dialyzed by a dialysis bag (molecular weight cutoff ¼ 3500) for 12 h to remove free ART. The resulting HA-MWCNTs/Tf@ART nanosuspension was stored at 4  C until use. Characterization Dynamic light scattering (DLS) (Zetasizer Nano ZS-90, Malvern Instruments Ltd, Worcestershire, UK), transmission electron microscope (TEM) (Tecnai G2 20, FEI Co., Hillsboro, OR), atomic force microscope (AFM) (9500J3,

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Shimadzu Co. Ltd, Tokyo, Japan) and scanning electron microscope (SEM) (Quonxe-2000, FEI Co., Eindhoven, Netherlands) were used for characterizing particle size, zeta potential and morphological of HA-MWCNTs/Tf@ART, respectively. The optical properties of MWCNTs, HAMWCNTs and HA-MWCNTs/Tf were characterized using an ultraviolet–visible (UV–VIS) spectrometer (Lambda 35, PerkinElmer Inc., Waltham, MA). Fourier transform infrared spectroscopy (FT-IR) spectra was recorded on a Nicolet iS10 spectrometer (Thermo Fisher Scientific, Madison, WI). The relative amount of HA grafted to MWCNTs was tested using a thermal gravimetric analysis (TGA, PerkinElmer Inc.) with the experimental conditions of scanning from 25 to 800  C under nitrogen at a heating rate of 20  C/min and the quantity of MWCNTs was determined at 808 nm by UV–VIS spectrometer. The interaction occurred between HAMWCNTs and Tf was characterized by the fluorescence spectrum (Shimadzu Co. Ltd). Determination of ART loading and release HA-MWCNTs/Tf@ART nanosuspension was diluted with 10 times methanol and sonicated to ensure that the loading ART was dissolved completely, and then centrifuged for 30 min at 10 000 revolutions/min to separate HA-MWCNTs/Tf and ART to determine the amount of ART attached on HA-MWCNTs/Tf. After that, ART solution was hydrolyzed with a fivefold 0.2% sodium hydroxide for 30 min at 50 ± 1  C and then the drug-loading efficiency can be measured at 291 nm by UV–VIS spectrometer. The quantity of MWCNTs was determined at 808 nm by UV–VIS spectrometer. For release study, HA-MWCNTs/Tf@ART and ART samples were placed into dialysis bags (molecular weight cutoff ¼ 3500), which were dialyzed in 50 ml 20% ethanol aqueous buffer solution (ethanol:PBS, v/v ¼ 1:4), respectively. The release assay was performed at 37.0 ± 0.5  C with a stirring rate of 100 revolutions/min. About 0.2 ml dialyzate was drawn at various time points, being replaced by the same volume of aqueous buffer solution. The concentration of ART released from HA-MWCNTs/Tf was quantified by UV–VIS spectrometer under the above conditions. The cumulative percentage of drug release was calculated and the best fitting release kinetic model was evaluated. Cellular uptake and internalization To evaluate the intracellular uptake capacities of HAMWCNTs/Tf@ART in MCF-7 cell lines, fluorescence microscopy and flow cytometry analysis were used. FITC, a fluorescence probe, was incorporated into HA-MWCNTs@ ART as well as HA-MWCNTs/Tf@ART by mixing of these samples with FITC according to the following method. FITC in DMSO (1 mg/ml, 80 ml) was added to HAMWCNTs/Tf@ART nanosuspension (4.0 ml) and ultrasonicated with ultrasonic cell disruption system to obtain HAMWCNTs/Tf@ART-FITC. Excess FITC was removed by Sephadex G-25 column (Sigma Aldrich Co. LLC). MCF-7 cells (3  105 cells per well) were seeded on glass cover slips in six-well plates. When cells reached 70% confluence, they were treated with HA-MWCNTs/Tf@ART-FITC and HAMWCNTs@ART-FITC (HA-MWCNTs concentration: 20 mg/ml)

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for 0.5, 1 and 2 h, respectively. At the designated time points, cells were washed with phosphate-buffer saline (PBS) for three times followed by fixing with 70% ethanol for 30 min. The cells were observed under a fluorescence microscope (Eclipse 80i, Nikon Corporation, Tokyo, Japan). Cell culture and inhibition measurements MCF-7 human breast cancer cells line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in normal Roswell Park Memorial Institute (RPMI)-1640 culture medium with 10% FBS and 1% penicillin/streptomycin in 5% CO2 and 95% air at 37  C in a humidified incubator. For cell inhibition measurements, MCF-7 cells (5  103 cells per well) were plated in 96-well plates and then incubated for 24 h. After incubating MCF-7 cells with various concentrations of free ART, HA-MWCNTs@ART and HAMWCNTs/Tf@ART (ART dose: 30 mg/ml; HA-MWCNTs dose: 15 mg/ml) for 24, 48 and 72 h, respectively, standard SRB assay was carried out to determine cell viabilities. Moreover, in order to investigate cytotoxicity of the vehicle itself, cells were also treated with different concentrations of HA-MWCNTs for 72 h and then cell viabilities were determined. Meanwhile, for hyperthermia experiments, MCF-7 cells (5  103 cells per well) were seeded in 96-well plates for 24 h. Then, the medium was taken off and was treated with medium containing various concentrations of HA-MWCNTs/Tf and HA-MWCNTs/Tf@ART (ART dose: 30 mg/ml; HAMWCNTs dose: 15 mg/ml), respectively. After that, cells were irradiated with an 808 nm NIR laser at a powder density of 2.5 W/cm2 for 0.5 and 1.0 min. Then, cells were incubated at 37  C for a further 24 h. SRB assay was used to measure growth inhibition. The groups without laser irradiation acted as control groups. Intracellular reactive oxygen species and DNA fragmentation detection Reactive oxygen species (ROS) generation inside cells was detected using DCFH-DA Reactive Oxygen Species Assay Kit (Sigma, St. Louis, MO). MCF-7 cells were seeded in confocal dishes at a density of 5  104 cells/dish. Following incubation with ART, HA-MWCNTs@ART and HAMWCNTs/Tf@ART (ART dose: 60 mg/ml; HA-MWCNTs dose: 30 mg/ml) for 24 h, DCFH-DA was loaded into the cells. After 30-min incubation, cells were washed twice with PBS and then fluorescence images of treated cells were acquired using a fluorescence microscope (Zeiss LSM 510, Thuringia, Germany). Furthermore, in order to quantitatively determine the amount of ROS generated in cancer cells, cells with different treatment were washed with PBS then trypsinized, collected by centrifugation and suspended in PBS buffer for flow cytometry analysis. MCF-7 cells were incubated with ART, HA-MWCNTs@ ART and HA-MWCNTs/Tf@ART (ART dose: 60 mg/ml; HAMWCNTs dose: 30 mg/ml) for 24 h. Then, cells were collected and re-suspended in pre-warmed low-melting-point agarose (LMA) before 100 ml of the cell LMA suspension was placed on a slide, which had been pre-coated with normal-melting-

DOI: 10.3109/1061186X.2015.1016437

point agarose and lysed in cold lysis solution (Beijing CoWin Bioscience Co., Ltd, Beijing, China) for 2 h. After lysis, the samples were electrolyted for 25 min. Neutralization buffer was added to the samples at room temperature for 45 min, and stained with 2% ethidium bromide in the dark. Finally, the samples were observed by the fluorescence microscope with excitation wavelength of 633 nm.

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Cell cycle and apoptosis determined by flow cytometry MCF-7 cells were seeded in six-well plates (5  105 cells per well). Cells were incubated in the presence of ART, HAMWCNTs@ART and HA-MWCNTs/Tf@ART for 2 h (ART dose: 30 mg/ml; HA-MWCNTs dose: 15 mg/ml), respectively. The hyperthermia groups of HA-MWCNTs@ART and HAMWCNTs/Tf@ART were treated with 808 nm laser for 1.0 min. Then, culture medium containing drugs was removed and fresh culture medium was added to each well followed by incubation at 37  C for 24 h. Then, cells were collected for cell-cycle analysis. What’s more, cells with the same treatment were collected and suspended in 400 ml binding buffer to adjust cell density to 1  106 cell/ml, then further added 5 ml of recombinant human anti-Annexin V–FITC and 5 ml of propidium iodide (PI), staining for 15 min at room temperature in dark for cell apoptosis test. Subsequently, the samples

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were analyzed by a Partec PAS flow cytometer (Partec GmbH, Munster, Germany). Xenograft tumor mouse model All animal experiments were performed under a protocol approved by Henan Laboratory Animal Center, China. S180 tumor-bearing model was generated by subcutaneous injection of 2  106 cells into the right shoulder of female BALB/c mice (18–20 g, Henan Laboratory Animal Center). The mice were used when the tumor volume reached 100 mm3 (7 days after tumor inoculation). In vivo anti-tumor efficacy Based on the results of the experiments in vitro, anti-tumor efficacy was investigated in tumor-bearing mice. The mice (n ¼ 6) were treated with saline, free ART, HA-MWCNTs@ ART and HA-MWCNTs/Tf@ART by tail vein injection every 2 days for 10 days, at the dose of 37.5 mg ART/kg. Throughout the study, mice were weighed and tumors were measured with calipers every 2 days. Tumor volume (V) was calculated according to the formula: V ¼ [length  (width)2]/2. At the end of the experiment, the animals were sacrificed and the tumor tissues were soaked in 10% formalin solution, embedded with paraffin for hematoxylin and eosin

Figure 3. Characterization of HA-MWCNTs/Tf. (A) FT-IR spectrum of (a) HA-MWCNTs/Tf, (b) COOH-MWCNTs and (c) HA-MWCNTs; (B) UV spectrum of (a) HA-MWCNTs, (b) HA-MWCNTs/Tf and (c) Tf; (C) the fluorescence spectroscopy with increased mass concentration ratio of HA-MWCNTs of (a) 0, (b) 10.0 and (c) 20.0 mg/ml.

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(H&E) staining. Morphological changes were observed under microscope (Eclipse 80i, Nikon Corporation). For the hyperthermia groups, excepting for the above experimental describing, a laser of 808 nm was performed toward tumor site of tumor-bearing mice for 2.0 min after administration.

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Biodistribution studies To quantitatively assess the biodistribution characteristics of HA-MWCNTs/Tf@ART, tumor-bearing mice were treated by tail vein injection with ART solution, HA-MWCNTs@ART or HA-MWCNTs/Tf@ART at a matched dose of 100 mg ART/kg. At 0.08, 0.17, 0.5, 1, 2, 4 and 8 h after injection, six animals per time point were killed and tissues (heart, liver, spleen, lung, kidney, brain and tumor) were excised, weighed and frozen at 20  C. Then, tissues were homogenized in saline (w/v, 1:3), adding 3.5 ml ethyl ether to 200 ml tissue homogenate to extract ART. After centrifugation at 1  104 rpm for 10 min, organic layer was transferred to a new glass tube and evaporated to dryness under nitrogen. Finally, the residue was dissolved in 100 ml mobile phase and a 20 ml aliquot of each sample was injected for highperformance liquid chromatography (HPLC, Waters e2695, Milford, MA) analysis with the following conditions: a SymmetryÕ C18 column (150  4.6 mm, 5.0 mm); mobilephase acetonitrile/water 52:48; column temperature 25  C; detection wavelength 207 nm and flow rate 1.0 ml/min.

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Results and discussions Synthesis and characterization of HA-MWCNTs/Tf To improve the solubility of MWCNTs in water, HAderivatized MWCNTs was performed via an amido linkage between amine-functionalized HA (NH2-HA) and carboxylation-functionalized MWCNTs (COOH-MWCNTs). FT-IR results showed the C¼O stretching vibration absorption at 1735 cm1 after MWCNTs was oxidized, corresponding to pristine MWCNTs. HA grafted to MWCNTs was confirmed by the strong C–N stretching vibration absorption at 1423.31 cm1, C–H stretching vibration absorption at 2914.95 cm1, N–H stretching vibration absorption at 3430.84 cm1 and bending vibration band at 1631.75 cm1 (Figure 3A). The relative amount of HA grafted onto the surface of COOH-MWCNTs was tested by UV–VIS spectrometer and TGA. Because MWCNTs had a strong absorption at 808 nm and a good linear relationship, while HA had no absorption at 808 nm, so the quantity of MWCNTs in HAMWCNTs can be measured and then the relative amount of HA connected to MWCNTs can be calculated. The ratio of HA was 31.97% and this chemical modification could significantly improve the dispersibility of MWCNTs in water. The large specific surface area of MWCNTs could interact with Tf by hydrogen bonding, hydrophobic interactions, Van

Figure 4. Characterization of HA-MWCNTs/Tf. (A) TEM images of (a) COOH-MWCNTs, (b) HA-MWCNTs and (c) HA-MWCNTs/Tf; (B) TGA curves of (a) COOH-MWCNTs, (b) HA-MWCNTs and (c) NH2-HA; (C) zeta potential of HA-MWCNTs/Tf.

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der Waals force, electrostatic attraction and – conjugated effect. Combining Tf with HA-MWCNTs could cause autofluorescence quenching of Tf, so we could obtain the best combining ratio while the maximum fluorescence quenching occurred. Figure 3(C) showed a hill plot of fluorescence quenching due to the interaction of HAMWCNTs and Tf. As we can see, the best combining ratio was 1:1 (mass concentration ratio of HA-MWCNTs to Tf). Tf combining with HA-MWCNTs was verified by N–H stretching vibration absorption at 3293.09 cm1, C–N stretching vibration absorption at 1449.14 cm1, acylamide I band (1647.66 cm1), acylamide II band (1541.40 cm1) and acylamide III band (1355.58 cm1) (Figure 3A). Meanwhile, the characteristic absorption peaks were shifted from 245 (HA-MWCNTs) to 260 nm (HA-MWCNTs/Tf) (Figure 3B), suggesting there was a large conjugated system forming. And the zeta potential of the complex was 10.23 ± 0.42 mV (Figure 4C). In addition, the morphology of MWCNTs modified with HA with or without Tf was characterized by TEM, as shown in Figure 4(A). The COOH-MWCNTs (a) presented a smooth surface and stretched long chain, while HAMWCNTs (b) demonstrated a rough surface with dark spots due to the existence of HA. Combining Tf with HAMWCNTs (c) showed that Tf was adsorbed on the sidewall so that it could improve water dispersibility and biological compatibility of the carbon nanotubes [34]. At the same time, this modification could decrease the adsorption of blood protein to reduce the toxicity of MWCNTs in vivo [35].

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Preparation and characterization of HA-MWCNTs/Tf@ART We developed a HA-MWCNTs/Tf drug delivery system, and ART was adsorbed on MWCNTs through a simple physical adsorption by hydrophobic interactions, hydrogen bonding and Van der Waals force. Prime MWCNTs is highly hydrophobic and insoluble in either aqueous solution or common organic reagents. As a result, solubility under physiological conditions is a key prerequisite to make MWCNTs biocompatible. Covalent functionalizations (e.g. HA) could be used to modify MWCNTs to improve their dispersion in water and physiological environments. As Figure 5(C) showed, HA-MWCNTs/Tf@ART was stable in water (b) and cell culture medium (c) over multiple weeks without significant aggregation. AFM images (Figure 5A) and SEM images (Figure 5B) indicated that HA-MWCNTs/Tf@ART had an uniform size and tubular structure. The length of MWCNTs were 180 nm. Nanoparticles 55 nm have been reported to be cleared by kidney quickly [36], while larger nanoparticles have been reported to preferentially home into tumors through leaky tumor neovasculature as a result of the enhanced permeability and retention (EPR) effect. The size of HAMWCNTs/Tf@ART opened up the possibility of targeting tumors without being cleared rapidly by the kidney. To determine the saturation level of ART loading onto HA-MWCNTs/Tf, different feed ratios of ART/HAMWCNTs/Tf were performed, indicating that ART loading efficiency increased with increasing amount of ART. An ART

Figure 5. Characterization of HA-MWCNTs/Tf@ART. (A) AFM images of HA-MWCNTs/Tf@ART; (B) SEM images of HA-MWCNTs/Tf@ART; (C) photos of (a) pristine MWCNTs in water, (b) HA-MWCNTs/Tf@ART in water and (c) HA-MWCNTs/Tf@ART in cell culture media; (D) UV spectrum of (a) ART after alkaline hydrolysis and (b) pristine ART dissolved in methanol; (E) release profiles of ART from HA-MWCNTs/Tf@ART and ART solution; and (F) linear relationship of the cumulative release of ART Q (Q570%) versus square root of the time in the Higuchi model.

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loading of 200% (ART/HA-MWCNTs/Tf weight ratio of 3/1) was chosen for the following experiments. The ART was hydrolyzed with a fivefold amount of 0.2% sodium hydroxide for 30 min at 50 ± 1  C, then, the amount of ART loading on MWCNTs can be measured at 291 nm by UV–VIS spectrometer (Figure 5D). The quantity of MWCNTs was determined at 808 nm by UV–VIS spectrometer. So that the loading efficiency could be calculated (214.6 wt%). This result indicated HA-MWCNTs/Tf was a promising material for drug delivery. To investigate the release kinetics of HA-MWCNTs/ Tf@ART, we incubated this drug delivery system in 20% ethanol aqueous buffer solution (ethanol:PBS, v/v ¼ 1:4). As seen in Figure 5(E), release of ART in control group was very fast, while ART release from HA-MWCNTs/Tf was slow and sustained, suggesting that interaction between ART and HA-MWCNTs/Tf plays a critical role in the drug release. In order to analyze the mechanism of sustained-release process, kinetic models are usually used to describe the slow release kinetics feature. Higuchi model is an ideal model. The mathematical expression is Q ¼ Kt1/2, wherein Q is a cumulative release percentage at time t. Figure 5(F) showed Higuchi model fitting curve of ART released from HA-MWCNTs/Tf@ART. In Figure 5(F) Q and square root of t showed a good linear relationship with formula of Q ¼ 13.03t1/2+10.691 when the cumulative release percentage was 570%, suggesting the release kinetics followed Fick diffusion mechanism.

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Enhanced cell uptake of ART by MCF-7 cells To explore the uptake of this drug delivery system by MCF7 cells (Figure 6), we labeled the drug carriers with FITC, and tracked their internalization through co-localization of FITC signal (green fluorescence). The results showed that HA-MWCNTs/Tf@ART could effectively enhance uptake of ART by MCF-7 cells and uptake of HA-MWCNTs/ Tf@ART-FITC, HA-MWCNTs@ART-FITC and FITC by

Figure 7. Cytotoxicity of drug vehicle (HA-MWCNTs) on MCF-7 cells at 72 h. Data are presented as mean ± standard deviation (n ¼ 6).

Figure 6. Fluorescence microscopic images of MCF-7 cells. (A) FITC alone; (B) HA-MWCNTs@ART-FITC and (C) HA-MWCNTs/Tf@ART-FITC at 0.5, 1 and 2 h.

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Figure 9. Cell inhibition of HA-MWCNTs/Tf and HA-MWCNTs/ Tf@ART with 808-nm laser irradiation for 0 (control group), 0.5 and 1.0 min. Data are presented as mean ± standard deviation (n ¼ 6).

Inhibition efficiency on MCF-7 cells

Figure 8. Cytotoxicity of ART, HA-MWCNTs@ART and HAMWCNTs/Tf@ART on MCF-7cells at 24, 48 and 72 h. Data are presented as mean ± standard deviation (n ¼ 6).

MCF-7 cells were time-dependent manners (Figure 6). The uptake of HA-MWCNTs/Tf@ART-FITC was faster than that of HA-MWCNTs@ART-FITC by MCF-7 cells, and more HA-MWCNTs/Tf@ART-FITC was found in MCF-7 cells at 2 h than HA-MWCNTs@ART-FITC. This distinction in uptake was because receptors for HA (CD44) and Tf (CD71) were both highly expressed on the surface of MCF-7 cells [37,38], resulting in HA-MWCNTs/Tf@ART rapid uptake into tumor cells through receptor-mediated endocytosis.

Cytotoxicity of drug vehicle is one of the important evaluation factors for the application of biomaterials in vivo. The cytotoxicity study of HA-MWCNTs on MCF-7 cells was carried out at different concentrations. As shown in Figure 7, cell viability remained 490% even at 50 mg/ml after 72 h incubation. Because concentrations of HA-MWCNTs used in following experiments were 550 mg/ml, we may consider that HA-MWCNTs had no obvious toxicity to MCF-7 cells. This result suggested that the toxicity of MWCNTs had been reduced after modified by HA. The cytotoxicity study of ART, HA-MWCNTs@ART and HA-MWCNTs/Tf@ART was carried out on MCF-7 cells using SRB method. As shown in Figure 8, these formulations showed a time-dependent and dose-dependent increase in cytotoxicity. HA-MWCNTs@ART and HA-MWCNTs/ Tf@ART had higher inhibition efficiency on MCF-7 cells than ART (p50.05) at 24 h (Figure 8). This is probably due to HA-targeted delivery system can be transferred into cells faster. And when time periods were prolonged to 48 and 72 h, the cells also presented a higher inhibition rate by treatment with HA-MWCNTs@ART than ART, This is probably due to that ART releasing from HA-MWCNTs was slow and

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Furthermore, the chemo-photothermal synergetic therapy effect was accessed under irradiation of NIR laser. The result showed that HA-MWCNTs/Tf@ART with irradiation treatment displayed significantly higher cytotoxicity compared to those without irradiation treatment, suggesting a synergistic effect occurred. Intracellular ROS and DNA fragmentation detection The ART molecule contains an endoperoxide bridge (COOC) which can interact with Fe(II) to generate free radicals, leading to apoptosis of cancer cells. So the level of intracellular ROS was an important indicator for cancer treatment. ROS productions were observed in MCF-7 cells

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sustained, and as a result, the anti-tumor effect was significantly better than ART group in the other two stages. Furthermore, there was significant difference between HAMWCNTs@ART and HA-MWCNTs/Tf@ART at all time points. And, it was also found that HA-MWCNTs/Tf@ART had higher inhibition efficiency on MCF-7 cells than ART and HA-MWCNTs@ART (p50.05) at all time points, suggesting that the special action mechanism of ART and Tf making this drug delivery system enhancing the pharmacological activity greatly at the targeting cells. HA-MWCNTs/Tf and HA-MWCNTs/Tf@ART presented an obvious dose-dependent cytotoxicity under irradiation by NIR laser as seen in Figure 9, suggesting that HA-MWCNTs/ Tf were potential agent for photothermal therapy of cancer.

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Figure 10. ROS detection. (A) Images of intracellular ROS with fluorescence microscope: (a) control cells; (b) ART; (c) HA-MWCNTs@ART and (d) HA-MWCNTs/Tf@ART; (B) determination of intracellular ROS by flow cytometry; (C) the statistics of (B) determined by flow cytometry; and (D) images of single cell gel electrophoresis for DNA damage: (a) control cells; (b) ART; (c) HA-MWCNTs@ART and (d) HA-MWCNTs/Tf@ART.

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DOI: 10.3109/1061186X.2015.1016437

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Figure 11. Cell-cycle distribution at 24 h: (A) control cells; (B) ART; (C) HA-MWCNTs@ART; (D) HA-MWCNTs/Tf@ART; (E) HAMWCNTs@ART with 808-nm laser irradiation for 1.0 min and (F) HA-MWCNTs/Tf@ART with 808-nm laser irradiation for 1.0 min.

incubated with ART, HA-MWCNTs@ART and HAMWCNTs/Tf@ART using DCFH-DA fluorescent probe (Figure 10A). As shown in Figure 10(A), green fluorescence of DCFH was observed in cancer cells incubated with ART, HA-MWCNTs@ART or HA-MWCNTs/Tf@ART at the same presence of ART (60 mg/ml) and HA-MWCNTs (30 mg/ml) for 24 h, whereas control cells showed negligible DCFH fluorescence. Greener fluorescence of DCFH was observed in HA-MWCNTs/Tf@ART group, in comparison to those treated with ART or HA-MWCNTs@ART at the same HAMWCNTs and ART concentrations. Moreover, we also used flow cytometry to determine the amount of ROS generated in cancer cells. Similar result reproduced with 20.1% (ART), 40.4% (HA-MWCNTs@ART) and 88.0% (HA-MWCNTs/ Tf@ART), respectively (Figure 10B), indicating that HA-MWCNTs/Tf@ART greatly improved ROS generation efficacy of ART due to the presence of Tf. As revealed by electrophoretic analysis (Figure 10D), no DNA fragmentation was present in the total cells of control group (a), while MCF-7 cells treated with ART (b), HA-MWCNTs@ART (c) and HA-MWCNTs/Tf@ART (d) exhibited DNA fragmentation. In this study, images were analyzed by CASP software. The tail DNA content represents the degree of DNA damage on MCF-7 cells. Compared with ART group (18.6 ± 2.4%), tail DNA content in the HA-MWCNTs@ART group was 41.3 ± 5.1%, due to increased intracellular accumulation of ART; however, tail DNA content in HA-MWCNTs/Tf@ART group was 76.5 ± 6.3%. There is a significant difference in tail DNA contents between HA-MWCNTs@ART and HA-MWCNTs/ Tf@ART groups (p50.01), indicating that the existence of Tf caused much more cells damage. Also, these DNA

Table 1. Average percentage of cell-cycle distribution in each phase. Quantitative statistics (%, mean ± SD) Groups Control ART HA-MWCNTs@ART HA-MWCNTs/Tf@ART HA-MWCNTs@ART-808 nm HA-MWCNTs/Tf@ART-808 nm

G1

G2/M

S

68.4 ± 1.2 62.5 ± 1.9 50.4 ± 2.1 53.4 ± 2.8 40.5 ± 1.3 35.1 ± 0.8

9.8 ± 0.6 8.7 ± 0.3 17.2 ± 0.9 9.1 ± 0.5 19.8 ± 1.5 15.1 ± 1.4

21.8 ± 1.6 28.8 ± 1.1 32.4 ± 1.7 37.5 ± 0.9 39.7 ± 1.8 49.8 ± 2.1

Data are presented as mean ± standard deviation (n ¼ 3).

fragmentations confirmed the excellent anti-tumor effect of HA-MWCNTs/Tf@ART. This result was consistent with the cytotoxicity and intracellular ROS assay, and also revealed the synergistic enhancement effects of ART and Tf. Cell cycle and apoptosis determined by flow cytometry Treatment of HA-MWCNTs/Tf@ART with irradiation showed the strongest antitumor efficacy on MCF-7 cells in vitro because of the combination of chemotherapy and photothermal therapy. The mechanism of cell death with the tests of cell cycle and apoptosis by flow cytometry was further investigated. As shown in Figure 11 and Table 1, there were 21.8 ± 1.6 and 28.8 ± 1.1% of cells in S-phase in the control group and ART group, respectively. By contrast, HA-MWCNTs@ART and HA-MWCNTs/Tf@ART groups showed a more arrest of cell cycle at S-phase of 32.4 ± 1.7 and 37.5 ± 0.9%, respectively. Furthermore, after 808 nm laser irradiation for 1.0 min, the ratios of S-phase

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Figure 12. Cell apoptosis at 24 h: (A) control cells; (B) ART; (C) HA-MWCNTs@ART; (D) HA-MWCNTs/Tf@ART; (E) HA-MWCNTs@ART with 808-nm laser irradiation for 1.0 min and (F) HA-MWCNTs/Tf@ART with 808-nm laser irradiation for 1.0 min.

were significantly increased to 39.7 ± 1.8 and 49.8 ± 2.1%. This result may be the reason of chemo-photothermal synergetic therapy effect. The cell apoptosis experiment was used to test and verify the death of MCF-7 cancer cells. As seen in Figure 12, necrotic cells, early stage apoptotic cells and late stage apoptotic cells were observed after the drugs treatment. Compared with the ART group (10.7%), there were more early stage apoptotic cells in HA-MWCNTs@ART (17.3%) and HA-MWCNTs/Tf@ART groups (25.6%). Upon laser irradiation, the amount of late apoptotic cells and necrotic cells both increased. The ratio of late apoptotic cells and necrotic cells increased from 6.6% and 0.8% to 26.0% and 11.5%, respectively, indicating that the chemo-photothermal synergistic effect triggered cancer cells to die through late stage apoptosis and cell necrosis. The cell cycle represents a series of tightly integrated events that allow cells to grow and proliferate. Cancer represents the dysregulation of cell cycle such that cells that overexpress cyclins or do not express the CDKIs continue to undergo unregulated cell growth. The cell cycle also serves to protect the cell from DNA damage [39]. Thus, cell-cycle arrest, in fact, represents a survival mechanism that provides tumor cell the opportunity to repair its own damaged DNA. Thus, abrogation of cell-cycle checkpoints, before DNA repair is complete, can activate the apoptotic cascade, leading to cell death. One theme emerging in drug discovery is to

develop agents that target the cell-cycle checkpoints which are responsible for the control of the cell-cycle phase progression [40]. The results showed that photothermal therapy by NIR-excited MWCNTs triggered cancer cells to die through the S-phase arrest, the late stage apoptosis and cell necrosis associated with the effect of anticancer drugs. As we know, the S-phase is the most sensitive phase to hyperthermia [41], so when cancer cells are arrested in S-phase, hyperthermia would cause a large number of cancer cells apoptosis. Maybe this is one of the mechanisms of chemo-photothermal synergistic effect. In vivo anti-tumor efficacy The tumor-bearing mice were divided into six groups and treated with the protocols as summarized in method section ‘‘In vivo anti-tumor efficacy’’. The tumor sizes after various treatments were measured every 2 days after injection. The changes of relative tumor volume as a function of time were plotted in (Figure 13A). After 10 days of treatment, control group, ART, HA-MWCNTs@ART and HA-MWCNTs/ Tf@ART groups showed a relative tumor volume (V/V0) of 4.81 ± 0.62, 3.12 ± 0.33, 2.69 ± 0.26 and 1.71 ± 0.16, respectively, suggesting that HA-MWCNTs/Tf@ART was more effective than the other two therapeutic groups (p50.05) (Figure 14). There were two reasons leading to such a result. On one hand, Tf could mediate more ART to be transferred

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Figure 13. In vivo anti-tumor treatments (A) tumor growth of mice in different treatment groups within 12 days; (B) changes of body weight of mice in different groups during treatment; (C) H&E stained tumor tissues harvested from the mice with different treatments: (a) control cells; (b) ART; (c) HA-MWCNTs@ART; (d) HA-MWCNTs/Tf@ART; (e) HA-MWCNTs@ART with 808-nm laser irradiation for 2.0 min and (f) HA-MWCNTs/ Tf@ART with 808-nm laser irradiation for 2.0 min. Data were presented as mean ± standard deviation (n ¼ 6).

into tumors, inducing more drug accumulation at tumor site. On the other hand, iron in the blood could be transported to tumor site by Tf so that there were more Fe(II) in the tumor interacting with ART to form free radicals. Nevertheless, compared with HA-MWCNTs@ART and HA-MWCNTs/ Tf@ART groups, the tumor volume of mice treated with HA-MWCNTs@ART-808 nm and HA-MWCNTs/Tf@ART808 nm laser irradiation were both reduced with (V/V0) of 1.14 ± 0.13 and 0.67 ± 0.09, respectively. This result was consistent with the result in ‘‘Inhibition efficiency on MCF-7 cells’’ section, indicating a powerful chemo-photothermal synergistic therapy effect. Furthermore, tumor cells of saline control group (Figure 13C) (a) were in good condition of rapid proliferation and cell-to-cell closely spaced with a big cell size. But, it could be clearly observed in (b) ART and (c) HAMWCNTs@ART that tumor cells began to shrink and a clear gap was emerged between the cells, indicating the state of cell had begun to change and the number of cells had also been reduced. Obvious necrosis karyolysis phenomenon had been observed in (d) HA-MWCNTs/Tf@ART. Moreover, in (e) HA-MWCNTs@ART-808 nm and (f) HA-MWCNTs/ Tf@ART-808 nm, symptoms included obvious necrosis,

karyotheca dissolving and nucleolus disappearing all appeared and the HA-MWCNTs/Tf@ART-808 nm group was the most typical, demonstrating that combination with laser irradiation, HA-MWCNTs/Tf@ART had a strong antitumor effect in vivo. There may be some reasons as following: (i) endoperoxide bridge of ART was instability and it could easily generate free radicals by homolytic cleavage reaction under the condition of heating, then induce apoptosis of tumor cells; (ii) hyperthermia changes capillary blood flow perfusion to improve tumor perfusion, resulting in the change of distribution of drugs in tissues and improve plasma concentration of ART in tumor; (iii) hyperthermia can damage the cell membrane stability, leading to membrane permeability increased which is conducive to the delivery of chemical drugs to tumor cells; (iv) hyperthermia can inhibit activity of DNA polymerase and ligase in cancer cells, resulting in synthesis disorders of DNA and RNA; (v) hypoxic cells in solid tumors of animals are 10–20% for the relative proportion while the ratio in human solid tumors is much higher than this. Hypoxic cells are poor tolerance of heat and vulnerable to damage so the effect of hyperthermia increased with the ratio of hypoxic cells increased; (vi) at the early stage of heat stimulation, there

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Figure 14. Tumor after dissection: (A) control cells; (B) ART; (C) HA-MWCNTs@ART; (D) HA-MWCNTs/Tf@ART; (E) HAMWCNTs@ART with 808-nm laser irradiation for 2.0 min and (F) HA-MWCNTs/ Tf@ART with 808-nm laser irradiation for 2.0 min.

Table 2. AUC0 ! 8 h of tissue in mice after i.v. ART, HA-MWCNTs@ ART and HA-MWCNTs/Tf@ART (unit: mg g1h). AUC Sample

ART

Heart Liver Spleen Lung Kidney Brain Tumor

77.42 ± 8.31 48.14 ± 4.44 43.69 ± 3.89 94.16 ± 6.96 80.00 ± 4.68 13.71 ± 1.15 27.56 ± 1.78

HA-MWCNTs@ART HA-MWCNTs/Tf@ART 126.63 ± 5.56 275.26 ± 6.49 317.58 ± 9.76 116.26 ± 8.94 148.20 ± 6.18 29.95 ± 7.94 107.06 ± 7.31

109.68 ± 7.28 318.13 ± 12.43 265.82 ± 14.61 89.39 ± 6.24 180.01 ± 10.49 44.63 ± 8.05 226.33 ± 9.24

Data are presented as mean ± standard deviation (n ¼ 6).

are changes in tissues such as vasodilation, blood flow accelerating and the metabolism of tissue cells enhanced while then the blood flow slows down leading to stasis and accumulation of acidic metabolites which can cause a decrease in pH value within the tissue. The tumor is easily damaged due to the increased sensitivity of the cells in an acidic environment to heat which enhances the effect of hyperthermia. Allowing for high toxicity usually leads to weight loss, body weight of the mice for all groups were measured during the treatments and no weight loss was observed (Figure 13B), implying that the toxicity of formulations were not obvious. Figure 15. Biodistribution in S180 tumor-bearing mice injected with ART, HA-MWCNTs@ART and HA-MWCNTs/Tf@ART at 15 min and 2 h after injection (i.v.). Data are presented as mean ± standard deviation (n ¼ 6).

Biodistribution To understand the tumor treatment efficacy of various ART formulations (ART, HA-MWCNTs@ART and HAMWCNTs/Tf@ART), we investigated biodistribution of ART in tumor and various main organs. It showed significant differences of the three formulations (Figure 15). Differences

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in biodistributions of ART were obvious after injection; HA-MWCNTs@ART and HA-MWCNTs/Tf@ART with high ART concentrations in the RES organs (liver/spleen) were observed (Figure 15). Importantly, the concentration of ART in tumor was significantly higher of HA-MWCNTs/Tf@ART group than HA-MWCNTs@ART and ART groups (p50.05). The area under the curve (AUC) in tumor of HA-MWCNTs/ Tf@ART group (226.33 ± 9.24 mg g1 h) was higher ART group (27.56 ± 1.78 mg g1 h) and HA-MWCNTs@ART group (107.06 ± 7.31 mg g1 h) by 8.21- and 2.11-fold, respectively (Table 2). The ability of higher drug delivery efficiency to tumor by HA-MWCNTs/Tf was striking and directly responsible for the high tumor suppression efficacy of HA-MWCNTs/Tf@ART.

Conclusions A multi-functional tumor-targeting drug delivery system employing HA-derivatized MWCNTs as drug carrier, Tf as targeting ligand was successfully constructed. The nanoscale HA-MWCNTs/Tf showed neglectable toxicity, and could serve not only as a powerful ‘‘heater’’ under 808-nm laser irradiation but also as an effective drug targeting delivery carrier. A special drug ART used to treat malaria was loaded onto HA-MWCNTs/Tf with high-loading efficacy to form a drug delivery system for cancer treatment. HA-MWCNTs/ Tf@ART showed excellent anti-tumor efficacy in vitro and in vivo, indicating that there was a great potential of HA-MWCNTs/Tf@ART for cancer therapy.

Declaration of interest The authors declare no conflicts of interest in relation to this work. This work was supported by grants from the National Natural Science Foundation of China (No. 81273451 and 81101684).

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Enhancement of cytotoxicity of artemisinin toward cancer cells by transferrin-mediated carbon nanotubes nanoparticles.

Artemisinin (ART) is a kind of drug with an endoperoxide bridge which tends to react with Fe(2+) to generate radicals for killing cancer cells. Howeve...
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