Design of pH-responsive antimicrobial nanocomposite as dual drug delivery system for tumor therapy

Applied Clay Science 141 (2017) 23–35

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Applied Clay Science journal homepage: www.elsevier.com/locate/clay

Design of pH-responsive antimicrobial nanocomposite as dual drug delivery system for tumor therapy Fatemeh Bazmi Zeynabad a,b, Roya Salehi c,⁎, Mehrdad Mahkam a,⁎⁎ a b c

Chemistry Department, Azarbaijan Shahid Madani University, Tabriz, Iran Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Reseach Center for Pharmaceutical Nanotechnology, School of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran

a r t i c l e

i n f o

Article history: Received 14 May 2016 Received in revised form 10 February 2017 Accepted 12 February 2017 Available online xxxx Keywords: Antibacterial nanocomposite Nano-clay Methotrexate Cell cycle Cationic polymer Stimuli responsive nanocomposite

a b s t r a c t A novel antibacterial clay/polymer nanocomposite with average particle size of 20–40 nm and two cationic compartments in polymer was synthesized via ion exchange. The structure of the nanocomposites was characterized by XRD, FT-IR, TG-DTA, and SEM. This multifunctional nanocomposite was used for dual drug delivery of anticancer drug methotrexate (MTX) and an antibacterial agent ciprofloxacin (CIP) with encapsulation efficiency of N 90% for both drugs. The in vitro antimicrobial activity of the clay/polymer nanocomposites was studied against Escherichia coli and Pseudomonas aeruginosa bacteria by a well diffusion method. The nanocomposite showed good or moderate antimicrobial activities. However, CIP loaded nanocomposites showed enhanced antimicrobial activity in comparison to free CIP. The potential antitumoral activity of this clay/polymer nanocomposite system was evaluated against MCF7 cell lines by MTT assay and cell cycle studies. The cytotoxicity studies demonstrated enhanced cytotoxicity of developed MTX loaded nanocomposite in comparison to free MTX. Cell cycle study showed that MTX-loaded nanocomposite caused S-phased arrest in MCF-7 cells compared to control nontreated cells (P b 0.001). Therefore, dual drug-loaded antibacterial nanocomposite has the potential to be used for cancer therapy. © 2017 Published by Elsevier B.V.

1. Introduction One of the most important health threats is multi-drug resistance of human cancer cells and pathogens (Housman et al., 2014; Fair and Tor, 2014; Tanwar et al., 2014). The pathogenic bacteria have evolved mechanisms of resistance to most commercially produced antibiotics (Tanwar et al., 2014; Alekshun and Levy, 2007). It is necessary to develop novel methods of antibacterial treatment which do not use traditional therapeutic systems. Latest progresses in nanotechnology have offered the basis for using metallic nanoparticles in the fight against MDR bacteria (Miller et al., 2015; Ray et al., 2012). Other researches have proved the antibacterial potential of some polymers like polyhexamethylene guanide (PHMB) (Chindera et al., 2016) or cationic polymers (Carmona-Ribeiro and de Melo Carrasco, 2013; Xue et al., 2015; Salehi et al., 2015a). Chemotherapy antibiotics are prescribed to patients due to susceptibility to infectious diseases (Salehi et al., 2015a).

⁎ Correspondence to: R. Salehi, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ⁎⁎ Correspondence to: M. Mahkam, Chemistry Department, Azarbaijan Shahid Madani University, Tabriz, Iran. E-mail addresses: [email protected] (R. Salehi), [email protected] (M. Mahkam).

http://dx.doi.org/10.1016/j.clay.2017.02.015 0169-1317/© 2017 Published by Elsevier B.V.

Cancer cells can enlarge resistance to chemotherapy drugs, but are less likely to develop resistance while chemotherapy drugs are applied in nanoformulation (Ma et al., 2012; Shen et al., 2013). Nanoparticle drug delivery has added more progress in the efficacy of cancer therapeutics and diminishes toxic side effects, therefore revealing a major guarantee in cancer therapy (Shen et al., 2013; Salehi et al., 2015c, 2014b, 2014a). Laponite is a 2:1 synthetic clay mineral, consisting of one magnesium octahedral layer between two silicon tetrahedral layers. It is a biocompatible non-toxic synthetic disk-shaped clay mineral with a thickness of approximately 1 nm and a diameter of 25 nm (Thomas et al., 2011) which has attracted attention in drug delivery and biomedical fields because of its high specific surface area (370 m2·g− 1) (Mustafa et al., 2015). High loading capacity of Laponite for anticancer drug DOX (98.3%) and pH-dependent sustain release profile indicated superior therapeutic efficiency of LAP/DOX systems as compared to free DOX (Wang et al., 2013). In aqueous dispersion, the Laponite RD particles have negative charges on the faces and a weakly positive charge on the edges. Laponite RD potential in pharmaceutical applications is due to cationic exchange capacity as well as adsorption properties (Thomas et al., 2011). Sodium ion in Lapnoite can be exchanged with organic cations and the degree of intercalation of organic cationic parts related to sodium ion concentration.

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Clay nanocomposites (NC) are used as scaffolds in tissue engineering (Haroun et al., 2009), wound healing (Sandri et al., 2014), and as drug delivery systems (Salcedo et al., 2012)and their positive effect on cancer cell death protection have been proven by several studies (Abbes et al., 2008; Maisanaba et al., 2014). Cervini-Silva et al. (2016) reported the effect of clay on cell growth of cancer cells, cell growth inhibition and/or cell growth increase for different cancer cell types (Cervini-Silva et al., 2016). Despite the beneficial effect of Laponite RD clay, its application in drug delivery is limited due to some inherent drawbacks such as instability and its tendency to flocculate and precipitate under physiological conditions. The ion exchange of inorganic cations with organic cations, especially with quaternary ammonium compounds, and synthesis of polymer-clay NCs not only would alleviate this disadvantage, but also introduce antibacterial property in polymer-clay NCs (Nigmatullin et al., 2008). Clay polymer have been used as drug delivery systems (Suresh et al., 2010; Jafarbeglou et al., 2016), antimicrobial agents (Nigmatullin et al., 2008), excipients and active agents in pharmaceutical applications (Khurana et al., 2015; Aguzzi et al., 2007). Hydrogels are known as three-dimensional polymeric networks with extraordinary capacities to absorb and keep water, with potential applications in biomedical fields. Due to the restricted molecular motion of polymer chains caused by the large amount of cross-links arranged randomly, the majority of developed hydrogels are soft, weak, and fragile, which seriously restrict their applications. Therefore, polymeric hydrogel NCs would ease this drawback. NCs hydrogels (first produced by Haraguchi et al.) (Haraguchi and Takehisa, 2002) are class of nanoclays that have attracted considerable attention in biomedical fields because of their superiority to traditional hydrogels. Incorporating Laponite particles into polymers and hydrogels improves mechanical strength, elasticity, hardness, swelling, and biological behaviors of the NC compared to the polymer alone (Ghadiri et al., 2013; Schexnailder et al., 2010; Yang et al., 2011). MTX is a known competitive inhibitor of dihydrofolate reductase enzyme (Spurlock et al., 2012). In lower doses MTX is used as synchronization agent, but in higher doses, exhibits cytotoxic effects on proliferating cells at S-phase by inhibiting DNA synthesis (Sen et al., 1990). Driven by this need, a nanotechnology-based approach was used to prepare a novel nanocarrier with potent antibacterial property and ability to be used as dual drug delivery system for cancer therapy. The article is divided into two parts. In the first part, two kinds of cationic vinyl monomers were synthesized and intercalated in Laponite particles by cationic exchange with sodium ions. The modified NCs were dispersed in water and used as seeds by in situ emulsion polymerization to synthesize Laponite/polymer NC with potent antibacterial property. The obtained NC was used as smart carrier for dual delivery of MTX as chemotherapy and ciprofloxacin (CIP) as antibacterial agents. 2. Materials & methods 2.1. Synthesis of 2-chloroethyl acrylate (CEA) A novel monomer of 2-chloroethyl acrylate was synthesized from chloroethanol and acryl chloride as follows: Firstly 0.06 mol chloroethanol and an equimolar of acryl chloride (0.06 mol) were reacted in 17 mL dichloromethane followed by dropwise addition of triethylamine (0.06 mol) which was used as HCl scavenger at room temperature for 12 h. the produced salt (three ethyl ammonium chloride) was filtered and organic phase was washed with deionized water several time, after separation of two phase (water and organic phase) the solvents removed by rotary evaporation. 2.2. Synthesis of cationic 3-methyl 1-[2-(acryloxy)-ethyl] imidazolium chloride ionic liquid monomer (AcImIL) A novel imidazolium based ionic liquid monomer was synthesized by mixing 0.041 mol CEA and 0.048 mol methyl imidazole in a 50 mL

two-necked round bottom flask under stirring and refluxed for 72 h at 45 °C under argon flow. At the end the product was extracted with acetonitrile and the solvent was removed by rotary evaporation The product was dried under vacuum, and stored at 4 °C. 2.3. Synthesis of 2-(methacryloyloxy) ethyl trimethyl ammonium chloride (MADQUAT) Cationic quaternary ammonium alkyl halide monomer (MADQUAT) was prepared as described in previous work (Salehi et al., 2015c). Firstly, 19.1 mmol of MADQUAT was added to 5 mL of dry THF and stirred for 5 min, then 22 mmol of CH3I was added dropwise to the solution. The reaction was continued for 12 h at room temperature during stirring. The final products were filtered, washed with 20 mL hexane and put in vacuum dry oven overnight to obtain the MADQUAT product (white solid powder, yield: 95%). 2.4. Synthesis of organo-modified Laponite RD (MADQ-AcImIL&LP) The organo modified Laponite-RD (MADQ-AcImIL&LP) was prepared by adding two kind of cationic ionic liquids, 1.2 g of AcImIL and 0.36 g MADQUAT in 1 g of Laponite-RD dispersion in 1000 mL of deionized water at 50 °C and ultrasonically dispersed for 5 min with sonication (400 W) by using probe-type ultrasonic generator. Then the resultant dispersion was vigorously stirred for 72 h. The precipitate was repeatedly filtered and washed with hot deionized water until all of chloride ion was removed (detected with 0.1 N AgNO3 solutions). It was dried at 50 °C for 24 h. 2.5. Synthesis of multifunctional stimuli-responsive nanocomposite with organo-modified Laponite RD 0.3 g organo modified Laponite-RD (MADQ-AcImIL&LP) was added into 20 mL dioxane after 1 h dispersion at 70–80 °C, 0.14 g Nisopropyl acryl amide (NIPAAm) and 0.1 g methacrylic acid (MAA) were added to the mixture. The obtained mixture was placed in a clean polymerization tube, and the reaction mixture was degassed for 10 min with argon. Then 0.016 g AIBN was added to the mixture as an initiator in argon gas condition. Then, the tubes were sealed under argon atmosphere and the mixture polymerized for 96 h at 70–72 °C. After completion of reaction, the product P(NIPAAm-MAA)&MADQAcImIL&LP was rinsed and completely milled in a mortar to get fine powder. 2.6. Instrumentation 2.6.1. Hydrogen nuclear magnetic resonance (1H NMR) spectroscopy 1 H NMR spectroscopy AcImIL was recorded in d6–DMSO solvent on a Bruker DRX-400 spectrometer with tetramethylsilane as internal reference. 2.6.2. Fourier transforms infrared (FTIR) spectroscopy The chemical structures of the Laponite-RD, AcImIL IL and P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposites were studied by FTIR spectroscopy (mix with KBr and press to disk) (Equinox 55 LS 101, Bruker, Germany). 2.6.3. X-ray diffraction (XRD) Powder X-ray diffraction patterns of the Laponite-RD and P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposite were recorded on a Bruker AXS model D8 Advance diffractometer using CuKα radiation (λ = 1.542 Å), with the Bragg angle ranging from 2 to 70 °C. 2.6.4. Scanning electron microscopy studies The surface morphology, average diameter, particle size and pore volume of organo modified Laponite (MADQ-AcImIL&LP) and

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P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposites were assessed by a field emission scanning electron microscope with energydispersive analysis using X-ray (FESEM-EDX), S4160 Hitachi, Japan. Particle size was calculated by measuring the diameters of at least 60 particles, using image analysis software (Image-Pro plus 4.5; Media Cybernetics, Silver Spring, USA). 2.6.5. TGA Thermogravimetric analysis (TGA) of Laponite-RD, organo modified Laponite (MADQ-AcImIL&LP) and P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposites was carried out with a Mettler-Toledo model 822 instrument. Disintegration patterns of TGA were achieved under a nitrogen atmosphere at a heating rate of 10 °C per minute from 50 to 850 °C.

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in vitro drug release study, cell culture and cytotoxicity test against MCF7 cell lines. MTX@CIP loaded P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposite were re-dispersed in 2 mL buffer solutions with pH values of 5.8 and 7.4 at 37 °C. The medium was stirred continuously (200 rpm) during the release study. During the appropriate time intervals, the whole solution (2 mL) was collected by centrifugation at 12,000 rpm for 10 min and the same amount of the fresh PBS solution was added to the precipitant. The amount of MTX and CIP in the release medium was measured by HPLC-UV method at the wavelength of 300 nm and 267 nm, respectively as previously described (Salehi et al., 2015c; Davaran et al., 2015; Rasouli et al., 2014b). All measurements were performed in triplicate. The releasing content was calculated by the following formula:

2.7. Preparation of inoculum The standard strain of P. aeruginosa (ATCC: 25922) and Escherichia coli (ATCC: 27853) were obtained in lyophilized form from Institute of pasture, Iran. These strains were activated by 48 h culturing at 37 °C in sterile nutrient agar (Liofilchem, Italy) followed by transfer of a single colony from grown plate into nutrient broth and incubated over night at 37 °C. Finally, the cells were gathered by centrifugation at 800 rpm for 10 min and redispersed in Ringer solution to get an optical density of around 0.08–0.1 at 540 nm which provide a bacterial dispersion with concentration around 1 to 2 × 108 CFU/mL for each test organism (Salehi et al., 2015a).

Drug encapsulation efficiency ð%; w=wÞ Mass of drug in nanogels  100 ¼ Mass of feed drug

Drug loading efficiency ð%; w=wÞ ¼

Drug released% ¼

Mass of drug in nanogels Mass of nanogels  100

Amount of drug in release medium Amount of drug in nano−formulation

2.8. Evaluation of the antimicrobial activity The antimicrobial activities of prepared P(NIPAAm-MAA)&MADQAcImIL&LP and CIP@ P(NIPAAm-MAA)&MADQ-AcImIL&LP nanoformulations were studied by Minimum Inhibitory Concentration (MIC) determination against standard strain of Pseudomonas aeruginosa and Escherichia coli using the serial dilution method. Briefly bacterial inoculum in Muller-Hinton Broth medium in the concentrations equal to 0.5 of McFarland standard, mixed with serially diluted P(NIPAAm-MAA)&MADQ-AcImIL&LP dispersion with different concentrations of 195–1 × 105 μg·mL−1 for Escherichia coli and Pseudomonas aeruginosa. CIP@ P(NIPAAm-MAA)&MADQ-AcImIL&LP dispersion with concentration in the range of 0.195–12.5 μg·mL− 1 and 0.006– 3.125 μg·mL− 1 for Pseudomonas aeruginosa and Escherichia coli, respectively. After 24 h incubation at 37 °C, streak cultures were made onto the Muller-Hinton agar plates from the content of the tubes. The first concentration with no mark of bacterial growth on plates regarded as MIC. All tests were done in three separate times. This measurement for MIC was done as recommended by the Clinical and Laboratory Standards Institute (CLSI) M27-A3 and CLSI M100-S22 (12-14). Tubes with the Muller Hilton agar (Merck) (without ginger extract) were used as control. 2.9. Drugs loading and release Predetermined amount of P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposite was dispersed in 5 mL of solution containing 10 mg CIP and sonicated for 3 min with using the probe-type ultrasonic generator at 300 W. Ciprofloxacin-loaded nanocomposite was collected by centrifugation at 12000 rpm for 10 min in order to remove the unloaded Ciprofloxacin. After that, the mixture was dispersed at 5 mL solution containing 10 mg MTX and the mixture was sonicated by probe-type ultrasonic generator at 300 W for 5 min and further stirred at room temperature for another 24 h. The unloaded MTX was removed by centrifugation at 12000 rpm for 10 min. MTX@CIP -loaded P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposite was collected by centrifugation at 12000 rpm for 10 min and freeze dried. The obtained MTX@CIP -loaded P(NIPAAmMAA)&MADQ-AcImIL&LP nanocomposite was used for latter tests of

2.10. Cell culture and in vitro cytotoxicity assay MCF7 breast cancer cell line were obtained from National Cell Bank of Iran and maintained in 75 cm2 culture flask, in Roswell Park Memorial Institute 1640 medium (RPMI 1640 medium; Gibco BRL Life Technologies) supplemented with 10% (v/v) fetal bovine serum and antibiotics (100 mg/mL penicillin–streptomycin). The cells were incubated at 37 °C in an atmosphere of 5% CO2 and 95% air with N 95% humidity; medium was changed every two days for cell feeding. MCF7 cell lines were used as the target cells to evaluate the cytotoxicity of nanocarriers by MTT assay. Also the antitumor activity of the free MTX and MTXloaded P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposites were evaluated by MTT method as previously described (Rasouli et al., 2014a). Briefly, when cell population reached to 70% confluence, they were dissociated with 0.25% trypsin in PBS (pH 7.4) and centrifuged at 800 g for 7 min at room temperature. MCF7 cells were seeded in 96well microplates (7 × 103 cells per well in 180 μL RPMI 1640), and incubated in a humidified atmosphere containing 5% CO2 at 37 °C for 24 h to let the cells attachment to the bottom of the wells. Then, free MTX and MTX-loaded P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposites with various concentrations (100, 50, 25, 10 and. 5 μg/mL) were prepared in fresh cell growth medium and added into the wells containing MCF7 cell lines. In order to investigate the cytotoxicity of nanocomposite, drug-free nanocarriers (P(NIPAAm-MAA)&MADQ-AcImIL&LP) with varying concentrations (200, 100 and 50 μg·mL−1) were added wells attached by MCF7 cell lines. After incubation for 72 h, a microplate was withdrawn for MTT assay. The MTT assay was done as follows: 20 μL of MTT solution (5 mg/2 mL) in PBS (pH 7.4) was added to all wells. The incubation was done for a further 4 h and then the solution was removed carefully from wells and then the cells treated with Sorenson buffer. MCF7 cells growth inhibition was calculated by spectrophotometric method by reading optical density of every well at a wavelength of 570 nm using a microplate reader (Multiskan MK3, Thermo Electron Corporation, USA), All of the tests were repeated three times and statistical analyses were performed using SPSS 15; P b 0.05 was considered significant.

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2.11. Cell cycle analysis About 1 × 105 MCF-7 cells were seeded into each well of 6 well plates. When cells reached 80% confluency, the treatments were performed on CIP@MTX-loaded P(NIPAAm-MAA)&MADQ-AcImIL&LP (named as ALD), P(NIPAAm-MAA)&MADQ-AcImIL&LP (named as AL) and MTX. After 72 the cells were subjected to cell cycle analysis: The medium was aspirated and the MCF-7 cells washed with appropriate volume of PBS, then the trypsin-EDTA solution was added and the cells harvested, after centrifugation for 5 min at 1200 rpm, supernatant removed and pellet redispersed with PBS. Cells numbers were estimated by counting with a hemocytometer. For each tube 1 × 106 cells were included. The pelleted cells were redispersed in PBS. For fixation of the cells, ethanol was added in a dropwise manner. The cells centrifuged and washed with cold PBS. After re- dispersion of pellet, 10 μL of 20 mg/mL RNase A was added and incubated at 37 °C for 45 min. 10 μL of Propidium Iodide solution in the dark was added and incubated in the dark at 4 °C for 30 min. Finally, the DNA content of the cells was analyzed (Alizadeh et al., 2015). 2.12. Statistical analysis Analysis of variance (ANOVA) and Student's t-test were used to determine the significant differences among groups. The difference was considered statistically significant at P values b 0.05. Our data were shown as mean ± standard deviation (SD). 3. Results and discussion The synthesis route of novel developed multifunctional P(NIPAAmMAA)&MADQ-AcImIL&LP nanocarrier is shown schematically in Fig. 1. At first, a new monomer of 2-chloroethyl acrylate was synthesized by the reaction of chloroethanol and acryl chloride (named as CEA). At the second step, a novel cationic imidazolium based ionic liquid monomer was synthesized by mixing CEA and methyl imidazole (named as AcImIL). This part is indicated as intermediate (A) in Fig. 1. At the

third stage, a cationic quaternary ammonium alkyl halide monomer (MADQUAT) was synthesized by the reaction between DMAEMA and methyl Iodide, indicated as intermediate B. Sodium ion in interlayer space of Laponite can be exchanged with cationic ionic liquids. Therefore, these two cationic monomers (A and B in Fig. 1) were effectively exchanged into the interlayer space of silicate layers of Laponite-RD after being exchanged with the sodium ion (MADQ-AcImIL) (Fig. 1) and lying flat on the surface of Laponite-RD as monolayer (intermediate C in Fig. 1). Finally, these monomers participated in free radical polymerization in the presence of NIPAAm and MAA, which led to polymer chain growth between galleries of silicate layers of Laponite-RD. 3.1. Characterization Fig. 2 shows 1H NMR spectra of Imidazolium-based ionic liquid in CDCl3, in which the characteristic peaks of two components of ImIL were observed. The peaks at δ (ppm) 9.11 (1H, N\\CH\\N), 7.8 (2H, NCHCH), 7.6(2H, NCHCH), and 3.6 (3H, N\\CH3) represented the protons of 1-Methylimidazole moiety of IL, and peaks at δ (ppm) 3.9 (2H, N\\CH2\\CH2\\O), 4.5 (2H, N\\CH2\\CH2\\O), 7.5 (1H, CH2_CH\\C_O), and 6.8 and 7.11 (2H, CH2_CH\\C_O) were attributed to the protons of 2-chloroethyl acrylate (CEA) moiety of IL. 3.1.1. Fourier transforms infrared spectroscopy Fig. 3a represents Laponite characteristic peaks at 1008 and 3451 cm−1 correspond to the stretching vibrations of Si\\O and O\\H, respectively. The peak at 1634 cm−1 was characteristic of the O\\H deformation band of physisorbed water of Laponite. In FTIR spectra of CEA (Fig. 3b), the strong peak at 1731 cm−1 was attributed to stretching vibration of kenton carbonyl group (C_O). The relatively weak peak at 1638 cm− 1 was recognized as stretching vibration of aliphatic C_C. Stretching vibration of C\\O band was revealed as strong peak at 1083 cm− 1. The strong peak at 800 cm−1 was characteristic peak of C\\Cl in CEA, and the C\\H stretching vibration of the aliphatic section was manifested through the strong peak at 2950 cm−1. Fig. 3c shows the spectrum of the AcImIL ionic liquid. The broad and strong peaks

Fig. 1. Synthesis root of P(NIPAAm-MAA)&MADQ-AcImIL&LP.

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Fig. 2. 1H NMR spectra of AcImIL.

appearing at 3100–3500 cm−1 show the presence of water in the ionic liquid system. Absorption peaks in the areas of 2985 and 3095– 3150 cm−1 are stretching vibrations of aliphatic and aromatic hydrogen bonds, respectively. The strong and sharp peaks at 1736 cm−1 reflect the esteric carbonyl group. The strong peaks at 1644 and 1574 cm−1 represent C_C bands of CEA moiety and imidazolium ring, respectively. The peak at 1571 cm−1 is also attributed to the stretching vibration of C_N in imidazolium heteroatoms ring. The peak at 1100 cm−1 is ascribed to the stretching vibration of C\\O. The absence of C\\Cl strong absorption peak and presence of C_N and C_C of imidazolium heteroatoms ring proved the successful synthesis of AcImIL. Qualitative evidence of the presence of either AcImIL or MADQUAT in the interlayer space of Laponite was provided by FTIR spectroscopy. The FTIR spectra shown in Fig. 3d display the characteristic signals of the cationic AcImIL ionic liquid and cationic monomer C_O at 1722 cm− 1 and C_N at 1580 cm− 1, respectively. It is worth noting that the peak at 1644 cm−1, characteristic of the C_C double bond stretching vibration of MADQUAT and AcImIL, overlaps the O\\H deformation band of physisorbed water of Laponite. The FTIR spectra of P(NIPAAmMAA)&MADQ-AcImIL&LP(Fig. 3e) revealed all peaks presented at Fig. 2d, and stretching vibration of NIPAAm amide group appeared at 1645 cm−1. 3.1.2. XRD Laponite-RD was modified with multifunctional P(NIPAAmMAA)&MADQ-AcImIL&LP NC. XRD pattern was used to demonstrate this modification. At 2θ = 7.04°, the basal spacing of Laponite-RD was 12.55 Å, but following the modification, this angle moved to 2θ = 6.08°, and the basal spacing of planes altered to 14.52 Å (Fig. 4). This is indicative for intercalation of MADQ-AcImIL, that is lying flat on the surface in the interlayer space. (Lagaly and Dekany, 2005; Katti et al., 2010).

Fig. 3. FTIR spectra of a) Laponite-RD, b) CEA, c) AcImIL, d) AcImIL and MADQUAT modified Laponite RD and e) P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposites.

3.1.3. Morphology The SEM micrographs of organo modified Laponite-RD (AEIMIL&LP) (Fig. 5A(a)), and P(NIPAAm-MAA)&MADQ-AcImIL&LP are displayed (Fig. 5A(b)) in Fig. 5A. The particles sizes were in the range of 30– 100 nm. EDX spectrums of AEIMIL&LP (Fig. 5B(a)) and P(NIPAAmMAA)&MADQ-AcImIL&LP (Fig. 5B(b)) indicated the presence of Al, Si, Mg, C, N, and O, all elements which were predicted to be present in

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Fig. 4. XRD pattern of Laponit RD (a) and P(NIPAAm-MAA)&MADQ-AcImIL&LP (b).

the structure of MADQ-AcImIL&LP and the final nanocomposite. In EDX spectra of MADQ-AcImIL&LP (Fig. 5B(a)), the inorganic part percentage was higher than the organic part (Table 1). The intensity of Al peaks at MADQ-AcImIL&LP was higher than other elements, but at final NC due

to the addition of polymeric comportment to NC, the organic part percentage was more than the inorganic part. And the intensity of Al peaks decreased, but the intensity of Si, C, N, and O increased (Figure 5B(b)) (Table 1).

Fig. 5. (A) SEM micrographs of a) organo modified Laponite-RD (AEIMIL&LP) and b) P(NIPAAm-MAA)&MADQ-AcImIL&LP, (B) EDX spectrums of a) AEIMIL&LP and b) P(NIPAAmMAA)&MADQ-AcImIL&LP.

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Table 1 Results of Minimum Inhibitory Concentration (MIC) determination (μg·mL−1) of P(NIPAAm-MAA)&MADQ-AcImIL&LP and Ciprofloxacin loaded P(NIPAAm-MAA)&MADQ-AcImIL&LP Nanocomposite. Escherichia coli Concentrations (μg·mL−1) P(NIPAAm-MAA)&MADQ-AcImIL&LP Nanocomposite Ciprofloxacin loaded P(NIPAAm-MAA)&MADQ-AcImIL&LP

5

−1

195–1 × 10 μg·mL 0.006–3.125 μg·mL−1

Pseudomonas aeruginosa Concentrations (μg·mL−1)

MIC⁎ −1

390.625 μg·mL 0.048 μg·mL−1

5

−1

195–1 × 10 μg·mL 0.195–12.5 μg·mL−1

MIC⁎ – 3.125 μg·mL−1

⁎ All MIC experiences were repeated for three times and mean of the results were considered as the final MIC.

Fig. 6. TGA thermogram of a) Laponite-RD, b) organo modified Laponite-RD (MADQUAT-AcImIL&LP) and c) P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposite.

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3.1.4. Thermal analysis Fig. 6 depicts the thermogravimetric analysis (TGA/DTG) profile for Laponite-RD (Fig. 6a), organo modified Laponite-RD (MADQUATAcImIL&LP) (Fig. 6b), and P(NIPAAm-MAA)&MADQ-AcImIL&LP NC (Fig. 6c). The onset degradation temperatures of organo modified Laponite-RD and NCs were about 330 and 230 °C, respectively. The weight loss of pure clay at temperature around 100 °C corresponds to the removal of water coordinated with Na+ in the interlayer space (Greesh, 2011). TGA thermograms of organo modified Laponite-RD (Fig. 6b) exhibit the three-stage degradation behavior. The little weight loss below 120 °C was due to physically adsorbed water (first step degradation) (Greesh, 2011; Bippus et al., 2009). The second stage between 150 °C and 600 °C is attributed to the combustion of intercalated cationic monomers (Bippus et al., 2009). The final stage degradation at temperature range of 600 °C to 800 °C is ascribed to the dehydroxylation of structural OH groups of Laponite. TGA thermograms of NC (Fig. 6c) showed in four steps in the temperature region of below 120, 120–250, 250–450, and 600–750 °C, that decomposition of intercalated organic parts (pending chains and polymer backbone) took place in the temperature range of 120 to 450 °C. A weight loss below 120 °C is due to evaporation of adsorbed water and organic solvents and above 500 °C is related to loss of structural hydroxyl group (Bajaj and Jasra, 2007). The difference between the weight loss of the unmodified and the modified clay confirmed the grafting of MPTM onto Laponite. From the TGA curves calculation and EDX results evaluation, organic parts percentage of organo modified Laponite RD were obtained around 22.9% and 26.25%, respectively. While the percentage of organic parts evaluation for final nanocomposite which extracted from TGA curves and EDX results, became approximately 49.75% and of 59.1%. The calculated results from two different methods were very close and confirmed each other. 3.2. Drug loading Smart multifunctional nanocarrier P(NIPAAm-MAA)&MADQAcImIL&LP of different functional groups with potent ability for simultaneous interaction by MTX plus CIP was developed to explore potential combination therapy applications. The drugs loading experiments

were performed at pH 7.4; carboxylic acid groups of grafted PMAA in the carrier were deprotonated (pKa = 6) (Salehi et al., 2015b), and QMADQUAT part of NPs was positively charged (Salehi et al., 2015c; Chen et al., 1990). In aqueous dispersion, in addition to the presence of exchangeable cations of Laponite RD and OH groups at the broken edges of the clay mineral layers, the Laponite RD has considerable negative charge on the surfaces and infirm positive charge on the boundaries, offering an easy route to interact with different kinds of drugs by ionic interaction and hydrogen bonding. At pH of 7.4, the net charge of MTX was negative (pKa of MTX is 3.8, 4.8 and 5.6) (Rasouli et al., 2014a, 2014b) due to the de-protonation of two carboxylic acid groups, while CIP, the second selected drug in this study (pKa value of 6.09 for carboxylic acid group and 8.74 for the nitrogen on the piperazinyl ring) (Davaran et al., 2015), had both negative and positive charges at pH 7.4 due to the de-protonation of the carboxylic acid and protonation of the nitrogen. The MTX and CIP were loaded to the P(NIPAAm-MAA)&MADQAcImIL&LP NC (Fig. 7a) by following reasons: a) electrostatic attraction between anionic and cationic parts of NC and drugs, b) hydrogen bonding, and c) drug intercalation into the clay structure. The drug loading results indicated that conjugation of MTX in modified NPs did not affect the loading capacity of the modified nanoclay for conjugation with the second drug CIP. Both drugs were loaded separately with encapsulation efficiency of N98%. Therefore, high loading efficiency values of the developed nano-clay pointed out that low amount of carrier is needed for efficient drug delivery. 3.3. In vitro drug release study In order to achieve a successful targeted anticancer drug delivery, a drug release needs to be restricted at physiological condition (normal tissues) while becoming dominant at tumor tissue. On the other hand, the sustained anticancer drug release from carrier is an essential requirement for cancer therapy (Singh et al., 2011). As earlier declared, extracellular pH in cancer tissues are lower than normal tissues. Therefore, pH-sensitive nanocarriers are in the heart of interests in the research based on cancer therapy. Fig. 7b shows the drug release profile

Fig. 6 (continued).

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31

the NPs. However, at pH 7.4, electrostatic interaction between MTX, CIP, and the multifunctional NPs led to incomplete drug release from P(NIPAAm-MAA)&MADQ-AcImIL&LP NPs at the end of the studied release profile. This could be a possible reason for reservoir effect of the present newly prepared formulation for simultaneous release of multiple drugs in longer time intervals. This property of P(NIPAAmMAA)&MADQ-AcImIL&LP NPs should be very useful to effectively treat tumors with acidic microenvironments and reduce the side effects of the drug on the normal tissues. 3.4. In-vitro antibacterial efficiencies of novel developed nanoparticles

Fig. 7. a) Scheme of simultaneous loading of methotroxate (MTX) and ciprofloxacine (CIP) on pH-responsive P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposite, b) Cumulative release of methotroxate (MTX) and ciprofloxacine (CIP) from MTX@CIP loaded pHresponsive P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposite at different pH (4, 5.5 and 7.4) at 37 °C.

of MTX plus CIP loaded smart P(NIPAAm-MAA)&MADQ-AcImIL&LP NC, incubated in a simulative normal body fluid (50 mM PBS, pH 7.4) and an acidic environment (50 mM PBS, pH 5.8) for 25 days at 37 °C. The release profile for P(NIPAAm-MAA)&MADQ-AcImIL&LP was studied at four stages: initial burst release at the first 48 h (stage I), decelerating release from 50 to 200 h (stage II), followed by fast release from 200 to 400 h (stage III), and finally constant release from 400 to 600 h (stage IV). At the initial stage (stage I), the release followed a diffusing pathway of the release (first 48 h); around 30% of MTX and CIP released no matter in buffer with natural or mild acidic pH. There was a high concentration gradient between the NPs surface and the release medium which acted as the driving force for drug diffusion at the first 48 h. Also, the part of drugs physically loaded on NPs was released in this stage. Around 15–20% of both drugs were released at time between 50 and 200 h (stage II) without significant pH dependent release profile. Possibly, the intercalated CIP and MTX were released and substituted with water at this stage. Between 200 and 400 h (stage III), the pHdependent release from modified nano-clay became dominant (20– 40% of both drugs were released in this stage). In was assumed that the part of MTX and CIP loaded by pH-dependent ionic interaction and hydrogen bonding in NPs started to release in stage III. MTX and CIP released amounts at pH value of 7.4 reached 55 and 70%, respectively, while at pH 5.8, MTX and CIP release reached 80 and 85%, respectively. By decreasing the pH value, an obvious increase in the drug release rate was observed. The constant release profile was obtained in the range of 400–600 h (stage IV) in which CIP and MTX release was completed and reached 100% at pH 5.8, while 65 and 75% of MTX and CIP were released at pH 7.4. The mutual interactions between the MTX plus CIP and the P(NIPAAm-MAA)&MADQ-AcImIL&LP NPs affected the differences in the release profiles. In fact, interactions between amine, hydroxyl, and carboxylic groups in the drugs and the NPs at pH 7.4 have been proven to entrap and greatly restrict the remaining drug release. Carrier/drugs interactions decrease the drug release rate by increasing drug/carrier adsorption capacity/entrapment efficiency (Pongjanyakul and Suksri, 2009). MTX and CIP show that the degree of ionization depends on pH conditions and presence of repulsive force among CIP, MTX, and NPs at pH 5.8 led to drugs releases from

The novel developed multifunctional P(NIPAAm-MAA)&MADQAcImIL&LP NCs have two cationic segments: cationic quaternary amine sections in QMADQUAT and imidazolium ionic liquid (MADQAcImIL) blocks intercalated in Laponit-RD which is recognized to have antibacterial property (Parolo et al., 2011; Vigliotta et al., 2012; Mohammed et al., 2010; He et al., 2011). In order to investigate the probable antibacterial ability of NC, the antibacterial test of the novel developed nanocomposite was performed against standard strains of Pseudomonas aeruginosa and Escherichia coli. The mentioned bacteria were treated with P(NIPAAm-MAA)&MADQ-AcImIL&LP and CIP@ P(NIPAAm-MAA)&MADQ-AcImIL&LP NCs with different concentrations (Table 2). Fig. 8 shows the results of antibacterial ability of the novel developed nanoparticles whereas MIC value of P(NIPAAm-MAA)&MADQAcImIL&LP was 390.625-μg·mL−1 for Escherichia coli, confirming the antibacterial activities of the novel developed nanoparticle sample. Moreover, P(NIPAAm-MAA)&MADQ-AcImIL&LP NC did not show any antibacterial ability when treated with Pseudomonas aeruginosa at concentration range of 195–1 × 105 μg·mL− 1. MIC values of CIP@ P(NIPAAm-MAA)&MADQ-AcImIL&LP NC were measured 3.125 and 0.048-μg·mL−1 for Pseudomonas aeruginosa and Escherichia coli, respectively. In the control sample, dense bacterial colonies were monitored with no vesicle treatment. Also, in samples treated with pristine ciprofloxacin, the bacteria colonies were detectable at highest concentration of 12.5 and 3.125 μg·mL−1 for Pseudomonas aeruginosa and Escherichia coli, respectively. As a result, antibacterial activity of CIP@ P(NIPAAmMAA)&MADQ-AcImIL&LP NC in comparison with CIP alone increased more and the MIC value of novel developed NC as antimicrobial agent varied based on the microorganism species. 3.5. In vitro cell assay Several studies have revealed the interaction between different cell types (cancer cells, stem cells, and primary cells) and clay minerals surfaces without modification (Nones et al., 2015), and positive effects of clays minerals on cancer cell death protection have been approved by different studies (Abbes et al., 2008; Maisanaba et al., 2014). For example, toxicity of anticancer drug 6-mercaptopurine to human neuroblastoma cells was reduced by administration of drug loaded bentonite clay minerals (Kevadiya et al., 2013). Other studies have revealed that aflatoxin B1 loaded clays minerals do not affect the viability of neural crest stem cells (Nones et al., 2015) and colon-cancer cell lines (Abbes Table 2 EDX quantification element normalized of the MADQUAT-AcImIL&LP and P(NIPAAmMAA)&MADQ-AcImIL&LP. Element

C N O Mg Al Si

MADQUAT-AcImIL&LP

P(NIPAAm-MAA)&MADQ-AcImIL&LP

A%

W%

A%

W%

37.34 4.98 9.25 0.90 45.11 2.43 100.0

22.72 3.53 7.50 1.11 61.68 3.45 100.0

29.19 9.24 32.52 8.41 4.93 15.72 100.0

39.13 10.62 32.73 5.57 2.94 9.01 100.0

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Fig. 8. Streak cultures on the surfaces of Muller-Hinton agar plates, P(NIPAAmMAA)&MADQ-AcImIL&LP nanocomposite treated with a) Pseudomonas aeruginosa, b) Escherichia coli. The first concentration with no sign of bacterial growth on plates considered as Minimum Inhibitory Concentration (MIC).

et al., 2008). No significant apoptosis has been observed in human fibroblast, embryonic kidney cells, (Liu et al., 2011) and murine fibroblasts (Corrales et al., 2012) exposed to polymer modified clay minerals. Unmodified and organically modified clay minerals do not induce any DNA damage in human colon cancer (Sharma et al., 2010) and human colon carcinoma cells (Maisanaba et al., 2014). Although studies indicate the biocompatibility of clay minerals, but other in vitro studies demonstrate clay minerals cytotoxic effects on neural crest stem cell (Nones et al., 2015), human lung fibroblasts (Geh et al., 2006), macrophage cell lines (Elmore, 2002), human endothelial cells and human B lymphoblast cells (Zhang et al., 2011, 2010) in vitro by cell membrane damage, cell lysis (Williams and Environmental, 2005) and DNA and chromosome damage (Zhang et al., 2011, 2010). In an interesting study conducted by Cervin-Silva et al., high specificity of interaction among clay minerals and cell surface was observed by exposing bentonite clays to two kinds of cancer cell lines, U251 (central nervous system) and SKLU-1 (lung adenocarcinoma). These interactions led to growth inhibition of U251 and growth augmentation of SKLU-1 cells due to inhibiting the growth of high grade glioma and swelling, gathering of solutes, and their hydration and transformation (Cervini-Silva et al., 2016), cytotoxicity of the novel developed NC was assessed via MTT assay by exposing MCF7 cells to various concentrations of P(NIPAAmMAA)&MADQ-AcImIL&LP NC. After incubation of 50, 100, and 200 μg·mL−1 of P(NIPAAm-MAA)&MADQ-AcImIL&LP NCs (AL), an absorbance value of 99.5% of the control, even in the presence of high NC concentration (200 μg·mL−1), was observed, which is in in agreement with several studies indicating the nontoxicity and in some case the proliferative effect of clays on cancer cell lines (Abbes et al., 2008; Maisanaba et al., 2014; Sharma et al., 2010). It seems that P(NIPAAmMAA)&MADQ-AcImIL&LP NCs (AL) was nontoxic to the MCF7 cells (Fig. 9a). Anti-proliferation efficacy of the MTX@ P(NIPAAmMAA)&MADQ-AcImIL&LP in comparison with free MTX at MTX concentration ranges of 5 to 100 μg/mL was investigated after 72 h exposure to MCF7 breast cancer cells, and the cell viabilities were quantified by MTT assay (Fig. 9b). The cell viability of MCF7 cell lines exposed to MTX loaded NC showed dose-dependent manner; lower cancer cell viability was achieved by decreasing drug concentration (Salehi et al., 2015a). While, for free drugs, the results were reverse, and lower MCF7 cells viability was achieved by increasing drug concentration. This finding may be due to the fact that by increasing the drug content in nanoformulation, the amount of clay NC increases too, leading to cell proliferation as explained in literature. Significant growth inhibition of MCF7 cells was observed when the cells were treated with the dispersion of MTX@

Fig. 9. Cell growth inhibition rates by different concentration of a) AL and b) MTX and ALD on MCF7 cell lines after 72 h incubation. AL: P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposite, MTX: Methotrexate and ALD: methotrexate loaded P(NIPAAmMAA)&MADQ-AcImIL&LP nanocomposite.

P(NIPAAm-MAA)&MADQ-AcImIL&LP NCs with MTX concentration below 25-μg·mL-1. Only 36 and 42% of the MCF7 cells remained viable at a MTX dose of 5 and 10-μg·mL−1 after incubation with MTX-loaded NC. Hence, MTX antitumor efficacy was maintained while encapsulated in nanoparticles where higher efficacy was obtained at lower dose. Consequently, the applied dose for the developed MTX loaded NCs can be reduced to have similar clinical response when higher amounts of MTX are utilized. 3.6. Cell cycle results In the present study, the effects of free MTX and MTX loaded P(NIPAAm-MAA)&MADQ-AcImIL&LP NCs (ALD) and P(NIPAAmMAA)&MADQ-AcImIL&LP NCs (AL) on cell cycle were investigated. The effect of two concentrations of NC (AL) (50 and 100 μg·mL−1) without MTX on MCF-7 cells cycle was examined (Fig. 10A). The results showed that the AL polymer has no significant effect on cell cycle. Additionally, the study showed that MTX administration caused S-phased arrest in MCF-7 cells as compared to the control non-treated cell cycle (Fig. 10B). Despite the non-toxic property of AL NC, the MTX loaded NC caused significant (P b 0.001) accumulation of cells in S-phase in MTX loaded NCs (Fig. 10C–D). These findings emphasize the enhancing anti-cancer effects of drugs by polymer composites. 4. Conclusion In the present inquiry, a novel quaternary ammonium containing clay/polymer nanocomposite was synthesized, and an increase in the d-spacing of XRD spectra conformed the intercalation of copolymer part into the Laponite RD. TGA spectra of NC showed a sharp weight loss at about 200–270 °C due to decomposition of intercalated copolymer. This novel developed NC showed suitable inhibition properties over E. coli growth, and the antibacterial activity was boosted by CIP loaded clay/polymer NC in comparison with those from the antibiotic solutions. The antibacterial property of the synthesized materials make them an interesting material for scientific research of antibacterial

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Fig. 10. (A) The cell cycle analysis graphs in a) control, b) 50 μg·mL−1 AL, c) 100 μg·mL−1 AL, (B) The cell cycle analysis graphs a) 10 μg·mL−1 MTX, b) 50 μg·mL−1 MTX, c) 100 μg·mL−1 MTX, (C) The cell cycle analysis graphs a) 10 μg·mL−1 ALD, b) 50 μg·mL−1 ALD and c) 100 μg·mL−1 ALD obtained by flow Cytometry, (D) The frequencies of the cells in cell cycle phases. ALD composites as combinations of MTX and AL polymer showed higher S-phase arrest as compared to cells treated with MTX alone. * P b 0.05, ***P b 0.01 AL: P(NIPAAm-MAA)&MADQAcImIL&LP nanocomposite, MTX: Methotrexate and ALD: methotrexate loaded P(NIPAAm-MAA)&MADQ-AcImIL&LP nanocomposite.

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composites, providing a promising field of application in several areas related to human and animal health, especially drug delivery system in cancer therapy. The dual CIP/MTX formulated P(NIPAAmMAA)&MADQ-AcImIL&LP NC provided the opportunity to deliver both MTX and CIP drugs in combination and pH dependent, sustained release manner, and substantiating dose-dependent cytotoxic activity in MCF-7 cell lines.

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