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BIOPHA-3465; No. of Pages 9 Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx

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Original article

Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy Rouhollah Khodadust a,*, Gozde Unsoy a, Ufuk Gunduz a,b,** a b

Middle East Technical University, Department of Biotechnology, 06800 Ankara, Turkey Middle East Technical University, Department of Biology, 06800 Ankara, Turkey

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 September 2014 Accepted 16 October 2014

The objective of this study was to develop and evaluate the anticancer activity and the safety of a combinational drug delivery system using polyamidoamine (PAMAM) dendrimer-coated iron oxide nanoparticles for doxorubicin and poly I:C delivery in vitro. Dendrimer-coated magnetic nanoparticles (DcMNPs) are suitable for drug delivery system as nanocarriers with their following properties, such as surface functional groups, symmetry perfection, internal cavities, nano-size and magnetization. These nanoparticles could be targeted to the tumor site under a magnetic field since they have a magnetic core. DcMNPs were found as a convenient vehicle for targeted doxorubicin delivery in cancer therapy. Poly (I:C) binding on doxorubicin loaded DcMNPs (DcMNPs-Dox) was reported for the first time in the literature. It was also demonstrated that loading of doxorubicin into the cavities of DcMNPs increases the binding efficiency of poly (I:C) to the surface functional groups of dendrimer up to 10 times. When we compare the in vitro cytotoxic properties of doxorubicin, poly (I:C) and poly (I:C) bound doxorubicin loaded DcMNPs (PIC-DcMNPs-Dox), it was observed that PIC-DcMNPs-Dox show the highest cytotoxic effect by passing the cell resistance mechanisms on doxorubicin resistant MCF7 (MCF7/Dox) cells. Results demonstrated that applying PIC-DcMNPs-Dox would improve the efficacy by increasing the biocompatibility of system in blood stream and the toxicity inside tumor cells. These results provide invaluable information and new insight for the design and optimization of a novel combinational drug delivery system for targeted cancer therapy. ß 2014 Published by Elsevier Masson SAS.

Keywords: Cancer Drug targeting Dendrimers Nanoparticles Efflux pumps Toxicity

1. Introduction Most common pharmacological approach to cancer therapy is based on conventional chemotherapy protocols, which generally exhibit high cytotoxicity and poor specificity [1]. In the recent years, iron oxide magnetic nanoparticles (MNPs) are used in clinical applications of targeted drug delivery system. MNPs can be targeted to the tumor site by the help of an implanted permanent magnet or an externally applied magnetic field, which results, with the accumulation of the drug [1,2]. MNPs could be coated with a suitable polymer shell, such as PEG, chitosan, dextran, polyethyleneimine and PAMAM dendrimer

* Corresponding author. Department of Biotechnology, Middle East Technical University, Department of Biological Sciences, Middle East Technical University, Ankara 06800, Turkey, Tel.: +90 312 210 51 84/83; fax: +90 312 210 79 76. ** Corresponding author. Department of Biological Sciences, Middle East Technical University, Ankara 06800, Turkey. E-mail addresses: [email protected] (R. Khodadust), [email protected] (U. Gunduz).

for an efficient drug loading and carrying [3,4]. By this way, polymer coated MNPs can circulate in the blood stream for a long time without being cleared by macrophages [5]. Besides, chemotherapeutic drugs, targeting ligands, having high selectivity for specific cancer cell receptors, are also loaded to make these MNPs more functional [6]. Well-known polyamidoamine (PAMAM) dendrimers possess various useful characteristics, which make them suitable carriers for drug and gene delivery applications [7,8]. PAMAM dendrimers are synthesized by producing concentric shells of branched cells (generations) around a central initial iron oxide core. The PAMAM dendrimers have primary amine groups at each branch end and tertiary amine groups at each branching point [9]. A critical property of PAMAM dendrimers is the increased density of surface groups with higher generations of coating. At some generation level, the surface groups will reach the so-called ‘‘de Gennes dense packing’’, which limits and seals the interior of the nanoparticles from the bulk solution. This limitation depends on the strength of molecular interactions between adjacent surface groups, which may be affected by pH, polarity, and temperature of

http://dx.doi.org/10.1016/j.biopha.2014.10.009 0753-3322/ß 2014 Published by Elsevier Masson SAS.

Please cite this article in press as: Khodadust R, et al. Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.009

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the bulk solution. This feature can be utilized to tailor the encapsulation and release properties of dendrimers in drug delivery applications [10]. In lower generations of PAMAM dendrimers, the polarity is higher while the toxicity is low. It was also demonstrated that only large cationic PAMAM dendrimers induce aggregation of human platelets in plasma in vitro. The aggregation caused by large cationic dendrimers was proportional to the number of surface amines [11]. Generally, PAMAM dendrimers possess empty internal cavities and can encapsulate hydrophobic drug molecules [12]. Furthermore, the terminal amine groups of PAMAM dendrimers which are the main reason for the toxicity in blood steam can be modified with different functional groups and can be linked with various biomolecules, such as drugs, vitamins (folic acid, biotin, etc.), antibodies, growth factors and imaging agents [13]. Studies in the literature suggest that the cytotoxicity of PAMAM dendrimers can be reduced by the complexation and functionalization of the dendrimer surface with nucleic acids, such as ssRNA, dsRNA, DNA and inorganic polyphosphates [14,15]. Doxorubicin is an effective anticancer chemotherapeutic reagent. However, the wide range application of this drug is prevented due to the serious side effects. When applied in conventional method, the main problem with this drug is the cardiotoxicity and heart damage. The second important problem is the gain of resistance to doxorubicin, which limits its application [16]. In order to overcome the resistance mechanism, doxorubicin was loaded into the cavities of synthesized DcMNPs [17– 19]. Loading of drug into the cavities of PAMAM dendrimers enhances its aqueous solubility, bioavailability, and controls its release profile [18,20]. At the same time, loading of drug into nanoparticles will help the doxorubicin to bypass Pgp-mediated efflux proteins [19,21,22]. Thus, doxorubicin resistance would be prevented. In this study, doxorubicin was loaded onto the newly synthesized fourth generation dendrimeric magnetic nanoparticles (G4DcMNPs) and the surface amine groups were modified with polyinosinic:polycytidylic acid (poly I:C). Poly I:C is a synthetic analogue of double stranded RNA which directly triggers apoptosis in many types of human malignant cells, including breast cancer, melanoma and hepatoma cells by activating endosomal (TLR3) or cytosolic (MDA-5, RIG-1) receptors [23,24]. Poly (I:C) cannot be taken up into the cells by itself. Naturally occurring polyphosphates, such as extracellular DNA, RNA, and inorganic polyphosphates like poly (I:C) have been shown to pave the way for the blood coagulation. However, DcMNPs inhibit nucleic acids to induce thrombosis [14]. DcMNPs act as antithrombotic agents and prevent the coagulation and blood clotting by binding to these nucleic acids [14,15]. On the other hand, it was demonstrated by Parker et al. that, although nucleic acids like poly (I:C) decrease the cytotoxic effect of nanoparticles, they do not quench the cell penetration efficiency of PAMAM dendrimers [25]. Immobilization of poly (I:C) onto the nanoparticles will be an important tool for the fabrication of active targeted delivery systems since they can contribute to the targeting of DcMNPs at the tumor site through the application of external magnetic field and prevent serious side effects on healthy tissues [26].

2. Materials and methods 2.1. Materials Ferric chloride hexahydrate (FeCl36H2O), ferrous chloride tetrahydrate (FeCl24H2O), ammonia solution (NH3) (32%), 3aminopropyl trimethoxysilane (APTS) [NH2(CH2)3-Si-(OCH3)3],

methylacrylate, methanol, ethanol, ethylenediamine, phosphate buffer saline (PBS), ethylenediaminetetraacetic acid, trisaminomethane, EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride), 1-methylimidazole, polyinosinic–polycytidylic acid (poly (I:C)), sodium carbonate and acetic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA) then were used in the synthesis of PAMAM-coated MNPs. The 2,3-bis(2-methoxy-4nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide (XTT) reagent was purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). Doxorubicin was kindly provided by Gu¨lhane Military Academy (School of Medicine, Ankara, Turkey). Human serum was donated by the authors of this manuscript for physiological stability studies. MCF7 cell line was obtained from SAP Institute (Ankara, Turkey). Doxorubicin resistant MCF7 cell line was developed by Kars et al. in 2008 [27]. 2.2. Synthesis of DcMNPs Iron oxide (Fe3O4) core was synthesized with co-precipitation method. The surface of iron oxide was modified with 3aminopropyltrimethoxysilane (APTS) and coating was carried out with PAMAM dendrimers through Michael reaction. The synthesis, aminosilane modification and PAMAM dendrimer coating of iron oxide nanoparticles were performed as described by Khodadust et al. in 2013 [28,29]. The stepwise growth of dendrimers was repeated until the G4DcMNPs were achieved using the methylacrylate and ethylenediamine steps. The obtained DcMNPs were then washed with methanol and with distilled water by magnetic decantation. 2.3. Doxorubicin loading on G4DcMNPs Doxorubicin at different drug concentrations was loaded on G4DcMNPs, in PBS (pH 7.2) at room temperature. The mixture of buffer, drug, and G4DcMNPs were rotated at 10 rpm with fivesecond vibration intervals for 24 h while being protected from light. After the incubation period, doxorubicin loaded G4DcMNPs were separated by magnetic decantation and the doxorubicin entrapment efficiency was quantified by measuring the absorbance values at 481 nm by a Shimadzu UV spectrophotometer (Columbia, USA). Drug entrapment (%, w/w) was calculated from the following equation; [30] Drug entrapmentð%; w=wÞ ¼

ðMass of the total drug  Mass of free drug Þ  100 Mass of total drug

2.4. Poly (I:C) binding on G4DcMNPs Poly (I:C) was dissolved in nuclease free water to a final concentration of 10 mg/mL as stock. In order to obtain efficient binding between 50 -phosphate group of poly (I:C) and NH2 groups at the surface of G4DcMNPs, poly (I:C) was first activated. In this procedure, first poly (I:C) was heated to 55 8C and then cooled to room temperature to prepare double stranded poly (I:C) according to manufacturer’s instructions (Sigma–Aldrich). Then, the activation procedure was continued in the presence of EDC and 1methylimidazole [26,31]. Then newly synthesized G4DcMNPs were dissolved in 0.1 M 1methyl-imidazole (MeIm) buffer (pH 6 and pH 6.5) to a final concentration of 10 mg/mL. The EDC solution of 0.013 M was prepared using 0.1 M MeIm buffer (pH 6 and pH 6.5) and used fresh. Poly (I:C) was diluted as 200 mg/mL in MeIm buffer (pH 6 and pH 6.5) and 4 mg of Poly (I:C) was put into microcentrifuge tubes. Freshly prepared EDC solution was added immediately and

Please cite this article in press as: Khodadust R, et al. Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.009

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incubated for 15 min at room temperature. Then, DcMNPs/MeIm solutions were added to the reaction with final volume as 100 mL and rotated for 2 h at room temperature. The applied PIC:G4DcMNPs ratios were as 1:10, 1:15, 1:20, 1:30, and 1:35. Poly (I:C) loaded DcMNPs were washed with distilled water by using magnetic separation in order to remove EDC from the solution. Agarose gel electrophoresis was applied to analyze the binding efficiency and the stability of the poly (I:C) functionalized dendrimer-coated magnetic nanoparticles (PIC-G4DcMNPs). 2.5. Poly (I:C) binding on G4DcMNPs-Dox and binding optimization It was previously demonstrated that the loading was higher at lower generations of dendrimer and G4DcMNPs calculated as the optimal generation for doxorubicin loading. The surface functional groups at the surface of G4DcMNPs were flexible and the distance between these groups was adequate for doxorubicin to enter into the cavities inside the derdrimer [18]. In order to obtain the most effective delivery systems, here poly (I:C) modified, doxorubicin loaded G4DcMNPs (PIC-G4DcMNPs-Dox) were prepared for targeted cancer therapy. Doxorubicin was loaded on G4DcMNPs at 400 mg/mL and 500 mg/mL concentrations. The doxorubicin loaded G4DcMNPs were washed with 0.1 M MeIm (pH 6) to remove the PBS buffer and make the doxorubicin loaded G4DcMNPs ready for poly (I:C) binding. Then, poly (I:C) binding was performed on G4DcMNPs-Dox. The G4DcMNPs-Dox was sensitive to acidic pH. Therefore, to determine the most efficient pH for binding of poly (I:C), binding was studied at pH 6 and pH 6.5 which provide slightly acidic conditions for the protonation of amine groups represented by PAMAM dendrimer. This would lead not only efficient poly (I:C) binding but also less doxorubicin release during the poly (I:C) binding. 2.6. Stability and release of doxorubicin from G4DcMNPs Stability of G4DcMNPs-Dox and PIC-G4DcMNPs-Dox were studied in PBS buffer (pH 7.2) up to 8 weeks in human serum

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and up to 4 days at 37 8C. The release of doxorubicin from the nanoparticles in PBS buffer was determined by measuring the absorbance at 481 nm by a UV spectrophotometer. Doxorubicin release in human serum was measured by Varian Cary Eclipse fluorescence spectrophotometer (CA, USA) (excitation at 480 nm, emission maximum at 560–590 nm). 2.7. Cellular internalization of G4DcMNPs and G4DcMNPs-Dox The internalization of dendrimer-coated iron oxide nanoparticles were previously confirmed by light microscopy [18]. In this study, the internalization of free doxorubicin and G4DcMNPs-Dox were analyzed by confocal microscopy, in order to see the effect of delivery system on internalization efficacy. The free doxorubicin and G4DcMNPs-Dox were incubated with breast cancer MCF7 cell lines in 6-well plates. After 24 h incubation, the medium was removed from the plates and the plates were washed with PBS for several times so that the nanoparticles, which were not internalized, were removed from the environment. 2.8. Cytotoxicity analysis Cytotoxicity analysis of free poly (I:C), free doxorubicin, PICG4DcMNPs, G4DcMNPs-Dox and finally PIC-G4DcMNPs-Dox was performed on 1 mM doxorubicin resistant MCF7 (MCF7/Dox) breast cancer cells via XTT proliferation assays. 3. Results 3.1. G4DcMNPs synthesis Synthesized bare MNPs, aminosilane modified MNPs, and DcMNPs were characterized by TEM, VSM, FT–IR, XPS, TGA, and DLS analyses. Results demonstrated that the DcMNPs can be used as a suitable drug nanocarrier for cancer therapy [28,29]. In addition, comparison of TEM results of DcMNPs and PIC-DcMNPs demonstrated that poly (I:C) modification improves the dispersivity of the

Fig. 1. DLS results of DcMNPs (a) and PIC-DcMNPs (b). TEM images of DcMNPs (c) and PIC-DcMNPs (d). The scale bar in the TEM images show 200 nm.

Please cite this article in press as: Khodadust R, et al. Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.009

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complex. DLS results demonstrated that after poly (I:C) modification the size of DcMNPs increased about 20 nm (Fig. 1). 3.2. Doxorubicin loading on G4DcMNPs Our previous study about the loading, release and stability of doxorubicin on different generations (G2-G7) of DcMNPs was demonstrated that G4DcMNPs seems to be the most suitable generation to overcome the doxorubicin resistance mechanism in tumor cells [18]. In this study, drug entrapment efficiencies of G4DcMNPs were calculated with different doxorubicin concentration at room temperature (Eq (1)). The optimum drug entrapment was obtained with 500 mg/mL doxorubicin concentration (Table 1). In order to confirm the loading of doxorubicin on G4DcMNPs, FT–IR analysis was performed. The strong stretching absorption band between 408 and 673 cm1 corresponds to Fe–O bond of Fe3O4 core [32]. The stretching vibrations of Si–O–Fe at 950 cm1 shifted to 1050 cm1 on G4DcMNPs. The vibrations of –CO–NH2 bonds were observed at 1450, 1490, 1530, and 1620 cm1 [18]. The FT–IR spectrum of doxorubicin shows multiple peaks at 2932 (C– H), 1730 (C–O), 1618 and 1577 (N–H), 1414 (C–C) and 1071 (C–O) cm1. These peaks are also present in the FT–IR spectrum of doxorubicin loaded G4DcMNPs (Fig. 2). According to these results, doxorubicin was succesfully loaded onto the G4DcMNPs. 3.3. Poly (I:C) binding on G4DcMNPs Poly (I:C) was bound onto the surface of synthesized dendrimer-coated magnetic nanoparticles. Agarose gel electrophoresis was used to analyze poly (I:C) binding efficiencies of G4DcMNPs. The results demonstrated that the highest amount of poly (I:C) bound onto the surface of G4DcMNPs, was achieved with the ratios of 1:30 and 1:45 poly (I:C):G4DcMNPs for pH 6 and pH 6.5, respectively (Fig. 3). 3.4. Poly (I:C) binding on G4DcMNPs-Dox In order to obtain the most efficient system for cancer therapy, poly (I:C) bound doxorubicin loaded G4DcMNPs (PIC-G4DcMNPsDox) was improved. First of all, doxorubicin was loaded into the cavities of G4DcMNPs. Because, when poly (I:C) was bound to the surface of G4DcMNPs first, doxorubicin could not be loaded into the cavities of these surface modified nanoparticles since poly (I:C) covers the dendrimeric surface of G4DcMNPs. So, doxorubicin entrance into the cavities becomes impossible. On the other hand, when doxorubicin was loaded first, poly (I:C) was successfully bound to the surface of dendrimers. Amount of loaded doxorubicin and pH of the solution for poly (I:C) binding are the important factors to obtain the poly (I:C) modified doxorubicin loaded G4DcMNPs. The schematic representation of poly (I:C) modification of doxorubicin loaded G4DcMNPs was shown in Fig. 4. Positions of functional groups on the surface of DcMNPs are very important for binding of anticancer reagents, such as poly (I:C). The distance between the surfaces functional groups increase with the increased amount of loaded doxorubicin in the cavities of G4DcMNPs. This would facilitate an improvement on the binding

Fig. 2. FT–IR analysis of doxorubicin, G4DcMNPs, and doxorubicin loaded G4DcMNPs.

efficiency of poly (I:C). In order to observe the effect of loaded doxorubicin amount on poly (I:C) binding, drug was loaded at 400 mg/mL and 500 mg/mL concentrations. Then, poly (I:C) binding procedure was performed on G4DcMNPs-Dox. It was observed that the total amount of released doxorubicin during washing steps and poly (I:C) binding procedure was only 8  2% of total amount of loaded doxorubicin. Agarose gel electrophoresis was applied to analyze the binding efficiency of poly (I:C) on 400 and 500 mg/mL doxorubicin loaded G4DcMNPs and the ratio of poly (I:C):G4DcMNPs was found as 1:20 and 1:10, respectively in order to efficiently bind poly (I:C) on G4DcMNPs-Dox (Fig. 5). The binding of poly (I:C) was more efficient on G4DcMNPs-Dox500 as compared with G4DcMNPs-Dox400 because of the increased distance between the functional groups. Results demonstrated that when the doxorubicin was loaded into the cavities of G4DcMNPs, the surface functional groups became more prone to combine with poly (I:C). The doxorubicin loaded G4DcMNPs is sensitive to acidic pH. Higher drug release is obtained at the lower pH conditions. Therefore, to determine the optimum binding pH of poly (I:C) on G4DcMNPs-Dox500, loading studies were performed at slightly acidic conditions, such as pH 6 and pH 6.5. This provides not only an efficient poly (I:C) binding but also prevents significant doxorubicin release during the poly (I:C) binding process. Agarose gel electrophoresis was applied to analyze the pH effect on binding efficiency of poly (I:C) on G4DcMNPs-Dox500. The results showed that the most efficient binding occurs at pH 6 (Fig. 5b). When the pH of MeIm buffer was changed from 6 to 6.5, it resulted with a slight decrease in the binding efficiency. 3.5. Stability and Release of Doxorubicin from G4DcMNPs-Dox and PIC-G4DcMNPs-Dox Doxorubicin release studies were performed in acetate buffer at pH 5.2 and 4.5, which mimics the pH of endosomal conditions. The release profiles of doxorubicin from G4DcMNPs-Dox500 and PICG4DcMNPs-Dox500 at pH 5.2 and pH 4.5 are given in Fig. 6. The release studies were continued up to 24 h. Release of doxorubicin from G4DcMNPs-Dox500 and PIC-G4DcMNPs- Dox500 were analyzed using UV spectrophotometer. There was no significant difference in doxorubicin release from G4DcMNPs-Dox500 and

Table 1 Doxorubicin entrapment efficiency of G4DcMNPs calculated from Eq (1).

Drug entrapment (w/w) (%) Entrapped amount of drug (mg/mL)

90 mg/mL

120 mg/mL

180 mg/mL

240 mg/mL

300 mg/mL

400 mg/mL

500 mg/mL

600 mg/mL

98 88

97 116

97 175

97 233

97 291

96 384

96 480

79 474

Please cite this article in press as: Khodadust R, et al. Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.009

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Fig. 3. Agarose gel electrophoresis for poly (I:C) loading optimization of G4DcMNPs at pH 6 and pH 6.5. First eight wells demonstrate the loading of poly (I:C) at pH 6. Wells from 9 to 16 demonstrate poly (I:C) loading at pH 6.5. Wells between 17 and 20 show the different dilutions of control poly (I:C). 1 in the ratio represents 4 mg of poly (I:C).

PIC-G4DcMNPs-Dox500 (Fig. 6a). In general, doxorubicin release was higher at pH 4.5 compared with pH 5.2. Results demonstrated that, the stability of doxorubicin in G4DcMNPs-Dox and PIC-G4DcMNPs-Dox were similar in PBS buffer (data not shown). However, considering human serum as releasing condition, the stability of doxorubicin in PIC-G4DcMNPsDox was higher with respect to G4DcMNPs-Dox (Fig. 6b). 3.6. Cellular internalization of G4DcMNPs-Dox It was observed by fluorescent microscopy that the G4DcMNPsDox were highly taken up by 1 mM doxorubicin resistant MCF7 (MCF7/Dox) cells after 4 h while less of the free doxorubicin was taken up by these cells (Fig. 7). 3.7. Cytotoxicity analysis In order to quantify the cytotoxic effects of PIC-G4DcMNPs-Dox on doxorubicin resistant MCF7 cells, XTT cell viability assays were performed and inhibitory concentration 50 (IC50) values were calculated. The IC50 value of doxorubicin and poly (I:C) for 1 mM doxorubicin resistant MCF7 cells by applying PIC-G4DcMNPs-Dox were calculated as 12 mg/mL doxorubicin and 7 mg/mL poly (I:C). The IC50 value for 1 mM doxorubicin resistant MCF7 cells by applying free doxorubicin and G4DcMNPs-Dox were also calculated as 100 mg/mL and 21 mg/mL (Table 2). In addition, the cytotoxicity analysis of free poly (I:C) and PIC-DcMNPs were calculated as 450 mg/mL and 28 mg/mL. Comparison of these results with G4DcMNPs-Dox (IC50 Dox = 21 mg/mL) and PICG4DcMNPs-Dox (IC50 Dox = 12 mg/mL and IC50 poly (I:C) = 7 mg/ mL) demonstrated that the loading of doxorubicin can be a suitable way to bypass the resistance mechanisms (Table 2).

Combination of doxorubicin with poly (I:C) on G4DcMNPs would be more effective than doxorubicin loaded DcMNPs. Poly (I:C) bound G4DcMNPs-Dox treatment provides improvements for drug resistance and more effective than G4DcMNPs-Dox on resistant MCF7 cells. Application of PIC-G4DcMNPs-Dox on MCF7/Dox cells decreases required amount of doxorubicin and ensures that resistant cells get 10 times more sensitive to drug. Poly (I:C) binding onto the doxorubicin loaded DcMNPs also modifies the toxic amine groups at the surface of dendrimeric nanoparticles into the non-toxic phosphate and hydroxyl groups. The in vitro cytotoxicity studies demonstrated that poly (I:C) binding on the surface of G4DcMNPs-Dox decreased the required amount of drug to kill 50% of cancer cells (IC50 values). Therefore, required amount of DcMNPs to deliver sufficient amount of anticancer drugs was also decreased, that provides a decrease in the cytotoxicity of delivery system in the blood stream (Table 2). Obtained results demonstrated that, combination of poly (I:C) and doxorubicin on DcMNPs (PIC-DcMNPs-Dox) will be superior to both PIC-DcMNPs and DcMNPs-Dox. 4. Discussion Detailed characterization of the PAMAM dendrimer-coated nanoparticles by XRD, XPS, FT–IR, TEM, DLS, z potential, VSM, and TGA-FT–IR analyses demonstrated that the synthesized nanoparticles could be used in magnetic targeted drug delivery systems [28,29]. Dendrimer coating of MNPs provides cavities for the loading of different anticancer drugs and reduces the agglomeration [33]. It also provides surface functional groups to bind signalling molecules or anticancer agents [34,35]. TEM results demonstrated that poly (I:C) binding improves the dispersivity of DcMNPs and prevents their agglomeration. DcMNPs can be

Fig. 4. Schematic representation of poly (I:C) modification of Dox-loaded G4DcMNPs.

Please cite this article in press as: Khodadust R, et al. Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.009

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Fig. 5. a: agarose gel electrophoresis for the loading optimization of poly (I:C) on 500 mg/mL and 400 mg/mL doxorubicin loaded G4DcMNPs (G4DcMNPs-Dox500 and G4DcMNPs-Dox400) at pH 6. Wells 1–8 demonstrate the loading of poly (I:C) on 500 mg/mL Dox-loaded G4DcMNPs. Wells 9–16 demonstrate the loading of poly (I:C) on 400 mg/mL Dox-loaded G4DcMNPs. Wells 17–20 demonstrate the control poly (I:C) and different dilutions. 1 in the ratio represents 4 mg of Poly (I:C); b: agarose gel electrophoresis for binding optimization of poly (I:C) on G4DcMNPs-Dox500 at pH 6 and pH 6.5. Wells 1–8 demonstrate the loading of poly (I:C) on G4DcMNPs-Dox500 at pH 6. Wells 9–16 demonstrate the loading of poly (I:C) on G4DcMNPs-Dox500 at pH 6.5. Wells 17–20 demonstrate the control poly (I:C) at different dilutions. 1 in the ratio represents 4 mg of poly (I:C).

Fig. 6. a: release of doxorubicin from G4DcMNPs-Dox and PIC-G4DcMNPs-Dox at pH 5.2 and pH 4.5; b; stabilities of 500 mg/mL doxorubicin from G4DcMNPs and G4DcMNPsPIC in human serum up to 10 h at 37 8C.

Please cite this article in press as: Khodadust R, et al. Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.009

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Fig. 7. Bright field microscopy image (a) and fluorescent microscopy image (b) of MCF7/Dox cells treated with 30 mM (18 mg/mL) doxorubicin as loaded on G4DcMNPs. Bright field microscopy image (c) and fluorescent microscopy image of MCF7/Dox cells treated with 30 mM free doxorubicin (d).

targeted to the specific cancer cells by its magnetic core in the presence of magnetic field [1,2]. In this study, fourth generation of dendrimer (G4DcMNPs) was chosen to be applied as combinational delivery system. It was because of the fact that at higher generations of DcMNPs (G5, G6, G7) due to the increased terminal groups, compact packing limitation was reached, which is called ‘‘de Gennes’s dense packing’’. This situation will limit the drug loading into the cavities of NPs [28,29,36]. Considering low generations of DcMNPs (G2 and G3), the distance between surface branches is such that the doxorubicin will be released and escaped from endosome near to the membrane of the cells. Therefore, doxorubicin will be caught and pumped out by resistance mechanism. It is known that Pglycoprotein (multidrug resistance protein-1) acts as a pump to extrude the doxorubicin out of the cell [27,37]. Therefore, doxorubicin was loaded into the cavities of newly synthesized G4DcMNPs in PBS buffer. According to the chemical rule ‘‘likes dissolves like’’, doxorubicin was efficiently dissolved and loaded on to DcMNPs in PBS buffer [18,38]. Different environmental conditions, such as polarity, pH, and temperature can influence the loading efficiencies of drugs onto the dendrimers [36]. Doxorubicin loading on G4DcMNPs was performed at different concentrations at room temprature. The maximum entrapment

efficiency was observed up to 500 mg/mL drug concentration. PAMAM dendrimers exhibit extended conformations upon lowering the pH due to electrostatic repulsion between surface primary amines. This conformational extension results in forcing of the dendritic branches apart and releasing of the loaded drug [8,39]. The repulsions of the primary and tertiary amine groups of the G4DcMNPs at late endosomal pH 4.5 were optimal for drug release. G4DcMNP, releasing the drug in low pH (late endosome) away from the cell membrane efflux pump and near to nucleus, seems to be the most suitable generation for efficient doxorubicin delivery to treat drug resistant tumor cells. This also means that at slightly acidic pH, there will not be significant release of doxorubicin from G4DcMNPs which makes this generation suitable for surface modification even after drug loading. Combinational drug delivery systems for the treatment of cancer are becoming more popular because they generate synergistic anticancer effects, reduce drug-related toxicity and suppress multidrug resistance through different mechanisms of action [41,42]. In order to obtain combinational and more effective delivery system, poly (I:C) binding on G4DcMNPs and G4DcMNPsDox at pH 6 and pH 6.5 values were investigated. The poly (I:C) binding on G4DcMNPs-Dox was 3 times more efficient than G4DcMNPs. Poly (I:C) binding was 1.5 times more efficient at pH

Table 2 IC50 values of free doxorubicin, poly (I:C), G4DcMNP-Dox, PIC-G4DcMNP, and PIC-G4DcMNP-Dox.

Doxorubicin amount Poly (I:C) amount G4DcMNP amount

IC50 of free dox (mg/mL)

IC50 of free PIC (mg/mL)

IC50 of G4DcMNP-Dox (mg/mL)

IC50 of PIC-G4DcMNP (mg/mL)

IC50 of PIC-G4DcMNP-Dox (mg/mL)

170 – –

– 450 –

21 – 270

– 28 400

12 7 145

Please cite this article in press as: Khodadust R, et al. Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.009

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6 than pH 6.5 because the EDC activation of poly (I:C) was more efficient at pH 6 [31]. The surface amine functional groups of DcMNPs were more prone for poly (I:C) binding at slightly acidic pH than basic and neutral pH. This is because the pH induces conformational change of PAMAM dendrimers from a ‘‘dense core’’ (high pH) to a ‘‘dense shell’’ (low pH) [40]. Poly (I:C) binding on G4DcMNPs were investigated at pH 6 and pH 6.5 and binding was more efficient at pH 6. This would be because of the fact that, the lower the pH, the denser the shell of the DcMNPs [40]. In addition, for an efficient binding with amine and carboxyl groups, the activation of nucleic acids needs to be performed in the presence of EDC at acidic pH [31]. G4DcMNPs, which have more surface amine functional groups, were more prone to binding of poly (I:C) than G2DcMNPs, G3DcMNPs which have very few functional groups at the surface (data not shown). The surface functional groups of PAMAM dendrimers will tend to bind poly (I:C) at slightly acidic pH than in neutral and basic pH. Since the most efficient activation of the phosphate groups of nucleic acids with EDC occurs at acidic environment [31]. The binding of poly (I:C) was more efficient at highest amount (500 mg/ mL) of doxorubicin loaded G4DcMNPs. Fourth generation of PAMAM dendrimers have lower rigidity and smaller number of surface functional groups [43], which makes them unsuitable for efficient surface modification. However, in the case that the cavities of the G4DcMNPs were fully loaded with doxorubicin, the DcMNPs-Dox became more rigid and the surface functional groups became more feasible for binding of poly (I:C) as in the case of higher generations (G7DcMNPs). On the other hand, doxorubicin release from G4DcMNPs-Dox and PIC-G4DcMNPs-Dox was not significantly different. In general, doxorubicin release was higher at acidic conditions. Therefore, only small amount (8  2%) of doxorubicin was lost during washing with MeIm buffer and binding of poly (I:C) at pH 6. The release of doxorubicin from PIC-G4DcMNPs-Dox starts at acidic pH and maximum release was achieved in late endosomal pH 4.5. Because at neutral pH, only the primary amine groups were protonated which makes the gyration radius suitable for the entrance of drugs while at pH 4.5, the gyration radius is so high which lets most of the drugs to escape and release from the cavities of nanoparticles [40]. Combining of poly (I:C) with G4DcMNPs-Dox will decrease the toxic effects of the nanoparticles in blood stream by modifying the toxic amine groups to water soluble and non-toxic phosphate and hydroxyl groups without affecting the penetration of nanoparticles. The surface amine (NH2–) functional groups are the main reason of PAMAM dendrimer toxicity [25,44,45]. By covering them with poly (I:C), they are converted to phosphate and hydroxyl groups, which are not toxic. The surface hydroxyl and phosphate functionalization provides low toxicity, high solubility and biocompatibility [25,46]. Poly (I:C) binding on the surface of DcMNPs through phosphoramide bound would increase the stability of doxorubicin loaded nanoparticles by preventing the drug release in blood stream. The phosphoramide bond is a very stable in blood stream [47]. However, after entering the tumor cells, the poly (I:C) will be released from the surface by phosphoramidase enzyme which is highly expressed in tumor cells and will increase the cytotoxicity of the system inside the tumor cells. Literature studies demonstrated that small amounts of the phophoramidase enzymes are present in many normal tissues; however, large amounts are found in the grey matter of the central nervous system and in malignant epithelial tumors [44,48]. By entering the cell through endosomes, poly (I:C) binds to the TLR3 receptors and activate apoptotic pathways. It was known that naturally occurring polyphosphates, such as extracellular DNA, RNA, and inorganic polyphosphates like Poly

(I:C) have been shown to pave the way for the blood coagulation when in the blood stream [14,15]. Combining of these nucleic acids on cationic dendrimers like PAMAM can prevent their coagulation properties without influencing the penetration properties of nanoparticles [14,15,25]. Therefore, loading of poly (I:C) on G4DCMPNs-Dox will prevent the coagulation and blood clotting properties of poly (I:C) making the system safer in blood stream and more toxic inside the tumor cells. Consequently, PIC-DcMNPsDox complex can be used as a novel and suitable combinational therapy targeting system for the sensitization of doxorubicin resistant cells. In vivo characterization of this system will help to overcome the drug resistance mechanism of breast cancer, which is one of the main obstacles in breast cancer therapy. It may open new insights on combinational-targeted cancer therapy. 5. Conclusions This is the first report in the literature for the successful poly (I:C) binding on DcMNPs-Dox. Our findings revealed that loading of doxorubicin on G4DcMNPs facilitates poly (I:C) binding. Loading of doxorubicin into the cavities of G4DcMNPs increases the binding efficiency of poly (I:C) to the surface functional groups up to 10 times. Application of PIC-G4DcMNPs-Dox seems to be superior to G4DcMNPs-Dox and PIC-G4DcMNPs in terms of its cytotoxic effect on doxorubicin resistant MCF7 cells. When we compare in vitro cytotoxic effects of free doxorubicin, free poly (I:C), DcMNPsDox, PIC-DcMNPs and PIC-G4DcMNPs-Dox, it is observed that PICG4DcMNPs-Dox has the highest efficacy on doxorubicin resistant MCF7 cells. Binding of poly (I:C) to the surface amine groups of DcMNPs-Dox decreases the cytotoxicity of dendrimers in the blood stream and increases its biocompatibility. This prevents the coagulation effect of poly (I:C) and increases the solubility of drug delivery system in the blood stream. TEM results demonstrated that poly (I:C) binding on DcMNPs increases their dispersivity. Phosphoramidase enzyme which can break the linkage between P groups of poly (I:C) and N groups of DcMNPs are highly active in tumor cells [14,15,25]. Therefore, poly (I:C) bound doxorubicin loaded DcMNPs as a novel combinational delivery system will not only sensitize doxorubicin resistant cells but also make the system specific to tumor cells and cause apoptosis of the drug resistant MCF7 cells at lower levels of doxorubicin. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This study was supported by TUBITAK (TBAG-109T949 and TBAG-2215) and Middle East Technical University (BAP-07-022010-06). References [1] Colombo M, et al. Biological applications of magnetic nanoparticles. Chem Soc Rev 2012;41:4306–34. [2] O’Mahony AM, Godinho BMDC, Cryan JF, O’Driscoll CM. Non-viral nanosystems for gene and small interfering RNA delivery to the central nervous system: formulating the solution. J Pharm Sci 2013;102:3469–84. [3] Unsoy G, Yalcin S, Khodadust R, Gunduz G, Gunduz U. Synthesis optimization and characterization of chitosan-coated iron oxide nanoparticles produced for biomedical applications. J Nanoparticle Res 2012;14.11:1–13. [4] Zou A, Chen Y, Huo M, Wang J, Zhang Y, Zhou J, et al. In vivo studies of octreotide-modified N-octyl-O, N-carboxymethyl chitosan micelles loaded with doxorubicin for tumor-targeted delivery. J Pharm Sci 2013;102(1): 126–35.

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Please cite this article in press as: Khodadust R, et al. Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.009