Development and characterization of a novel conductive polyaniline-g-polystyrene/Fe3O4 nanocomposite for the treatment of cancer

Abstract The goal of this study is to synthesize, characterize and investigate some physicochemical properties of conductive polyaniline-g-polystyrene/Fe3O4 (Fe3O4/PSt-g-PANi) nanocomposites. For this purpose, initially, Fe3O4 nanoparticles were synthesized by a co-precipitation method. Then, the desired nanocomposite was synthesized in two steps. First, the atom transfer radical polymerization (ATRP) of styrene was performed using an ATRP initiator attached to the surface of Fe3O4 nanoparticles, followed by functionalization of the Fe3O4-PSt with amine groups (–NH2). Second, surface oxidative graft copolymerization of aniline was accomplished using the –NH2 moieties on the Fe3O4/PSt-NH2 as the anchoring sites. The prepared materials were characterized by various instruments, including TEM, SEM, TGA, EDX, FT-IR, XRD and conductivity measurements. The results indicated that the synthesized conductive polymer/Fe3O4 nanocomposites had higher electrical conductivity and thermal resistance than those of the corresponding homopolymers.


Introduction
In recent decades, inorganic/organic composites have attracted the attention of scientists in both scientific and industrial fields. These composites possess unique properties (such as thermal stability) and have various functional groups within a single material. These composite/hybrid materials present novel and wonderful properties (e.g. mechanical, chemical, electrical, rheological, magnetic, optical, catalytic and so on). They have different applications in areas such as drug delivery systems, diagnostics, coatings and catalysis [1][2][3][4][5][6][7][8][9][10].
Encapsulation of nanoparticles, such as metal oxides into conducting polymers, leads to nanohybrid organic/inorganic composites that demonstrate high thermal resistance, catalytic, electrochemical, optical, mechanical, electrical properties [24][25][26][27]. Also, metal oxide nanoparticles in conducting polymeric matrices allow for the development of products in many applications in sensors, microelectronics, cancer therapy, etc [28][29][30][31]. The unique properties of such compositions are essentially dependent on their size and shape. Diverse conducting polymer/inorganic particle composites have been synthesized by different methods and their properties and applications studied [32][33][34][35][36][37]. For example, Sadek et al. have reported In 2 O 3 -PANI composites as sensors for H 2 , NO 2 and CO gases [33,38]. Xu et al. published the synthesis of a Pt-PANI composite and its application in detecting glucose [34]. De et al. studied TiO 2 -PANI nanocomposites that can be used as electronic gadgets like capacitors [35]. V 2 O 5 -PANI composites have remarkable promise for their use as a cathode material for lithium secondary batteries [36]. One of the distinctive applications of polyaniline is its use in photothermal therapy of cancer. Xia et al. [39], Hong et al. [40] and Zhou et al. [20] have reported polyaniline as drug carriers for the photothermal therapy of cancer.
With the expansion of magnetic materials, significant research attempts have been created to synthesize conjugated polymers with magnetic materials to generate new magnetic and electrically conducting organic/inorganic materials, that can be used in electromagnetic interposition shielding, data storage and biomedical applications [41][42][43][44][45][46][47][48][49]. Different methods have been improved to make conducting magnetic nanocomposites, such as through the precipitation of iron salt in a polymer matrix [50,51], the in situ synthesis of the polymer by oxidative [52] or electrochemical [53] processes in the presence of well-dispersed magnetic nanoparticles and polymerization of the monomer [54][55][56].
A new method for the synthesis of organic/inorganic materials is controlled/living radical polymerization (CRP). CRP is a strong method for the synthesis of well-defined organic polymers because of a controlled molecular weight, narrow molecular weight distribution, combination and performance [57]. One of the methods of CRP is atom transfer polymerization (ATRP). ATRP has been commonly used for the preparation of polymers with a well-defined structure [58] by surface-initiated polymerization of various monomers, such as styrene, methacrylates, acrylates and dienes [59][60][61][62][63].
In the present study, we report a novel route for the synthesis of a brush polymer from the surface of Fe 3 O 4 as core/ shell nanoparticles. Therefore, we used the combination of two methods of ATRP of styrene and in situ chemical oxidation of aniline from the surfaces of the magnetic nanoparticles (Scheme 1). Characterization of this well-defined brush polymer has been investigated by performing FT-IR, thermogravimetric analysis, energy-dispersive X-ray (EDX), transmission electron microscopy (TEM), and X-ray diffraction (XRD). Considering the properties of the conductive polymer/Fe 3 O 4 nanocomposite, it has potential applications in the treatment of cancer.

ATRP of styrene with Fe 3 O 4 /Br-MPA as the initiator
In the ATRP polymerization, functional nanoparticles with Br-MPA (25 mg) were mixed with a styrene monomer (2.5 ml). After purgation of the mixture with nitrogen for 10 min, it is transferred to a bottle with CuCl (4.9 mg) and 2,2-bipyridine (19 mg). The reaction temperature was raised to 120 C. After finishing the reaction time (20 h), the mixture was diluted with tetrahydrofuran (THF). The final products were precipitated with methanol. For further characterization, it was dissolved in toluene to form a clear yellowish solution [65] (Scheme 3).

Preparation of poly(p-amino styrene)-coated Fe 3 O 4 (Fe 3 O 4 /PS-NH 2 )
First, the nitroxide group is produced on polystyrene backbone using strong nitric acid and sulphuric acid. In a roundbottomed flask containing strong nitric acid(15 ml), which was placed in the ice-water bath, the polystyrene coated iron oxide nanoparticles (Fe 3 O 4 /PS) (0.75 gr) was suspended in it. Then, 23 ml of strong sulphuric acid was slowly added to it and the temperature was enhanced to 30 C and continued for 10 h. After the desired time, the product was filtered and washed with sufficiently of water. It was transferred to a Soxhlet's extraction apparatus for reflux-extraction in 96% ethanol for 10 h. The product was dried under vacuum at 50 C over 48 h. The product obtained in this step was Fe 3 O 4 / PS-NO 2 (Scheme 3).
Next, the nitroxide group was reduced to an amino group. In a round-bottomed flask containing 20 ml of hydrochloric acid (6M) and 23

Preparation of Fe 3 O 4 /polystyrene-g-polyaniline, core/ shell nanoparticles
For surface oxidative graft copolymerization of aniline is the same as in the Abbasian et al [65]. Briefly, the Fe 3 O 4 /PSt-NH 2 (0.1 g), aniline monomers (0.01 mol) were added into the aqueous solution of hydrochloric acid (1.0 M). The oxidant solution (2.6 mol of (NH 4 ) 2 S 2 O 8 in 20 ml of 1.0 M HCl) was added into the reaction mixture at 0 C. The oxidative graft copolymerization was performed for 8 h. The PSt-NH 2 was used as the anchoring sites for polyaniline. The final product was dispersed into 1-methyl-2-pyrrolidone and centrifuged to remove the PANI homopolymer (Scheme 4).

Instrumentation
FT-IR spectra were recorded using a Shimadzu FT-IR-8101M; the spectrum was recorded at room temperature. The thermal properties of the Fe 3 O 4 /PSt-g-PANI nanocomposite measurement were determined by TGA-PL (England). XRD spectra were obtained using a Siemens D 5000 (Berlin, Germany), Xray generator (CuK a radiation with k ¼ 1.5406 Å) with a 2h scan range of 2 to 80 at room temperature. The size and morphologies of the products were examined by scanning electron microscopy (SEM; Cam Scan MV 2300). The samples for SEM imaging were coated with a thin layer of gold film to avoid charging. Transmission electron microscopy (TEM) images were performed on a Philips EM208 microscope (Phillips, Eindhoven, Netherlands) with a 100 kV accelerating voltage. The conductivity of the samples was measured at room temperature by a standard four-probe apparatus (Azar Electric Co., Urmia, Iran). Electrochemical experiments were conducted using Auto-Lab PGSTA T302N.

Preparation and surface modification of the magnetic nanoparticles
The Br-MPA attached to the surface of the iron oxide nanoparticles were used for ATRP polymerization. In addition, Br-MPA helps to stabilize nanoparticles in an organic solvent.
The XRD spectrum of Fe 3 O 4 is displayed in Figure 1(a). The XRD spectrum indicated that the nanoparticles do not have any impurities. The average size of the Fe 3 O 4 nanoparticles was calculated as $11 nm using the Debye Sherrer's formula and the peak from 311 reflection [66].
The equation uses the reference peak width at angle h, where k is the X-ray wavelength (1.54060 Å), b is the width of the XRD peak at half height and k is a shape factor, about 0.89 for magnetite.
The atom transfer radical polymerization (ATRP) initiator, Br-MPA, was attached on the Fe 3 O 4 nanoparticles through the esterification reaction. The evidence showed that Br-MPA was chemically bonded to Fe 3 O 4 nanoparticles. The FT-IR spectrum of Fe 3 O 4 (a), functional carboxylic acid (Br-MPA) (b), and Br-MPA -modified Fe 3 O 4 (c) are shown in Figure 2. The FT-IR spectrum of the Fe 3 O 4 nanoparticles was showed in Figure 2(a). The peak at 3417 cm À1 is related to the stretching vibrations of ÀOH or water molecules adsorbed onto the surface of the Fe 3 O 4 nanoparticles, and the peak at 565 cm À1 is attributed to the stretching vibration mode of Fe-O. In Figure2(b), due to the structure of functional carboxylic acid (Br-MPA), there are characteristic adsorptions bands in the FT-IR spectrum. The peak at 2944-2843 cm À1 region is attributed to stretching vibration of C-H, the band around 1700 cm À1 is due to the stretching vibration of C¼O, and the peak at 3008 cm À1 is ascribed to the stretching vibrations of the OH group.
The FT-IR spectrum of surface modification of the magnetic nanoparticles by Br-MPA was displayed in Figure 2(c). The bands at 1625 cm À1 is related to the C¼O stretch in the carboxylic acid. It showed that the Br-MPA was attached to the Fe 3 O 4 nanoparticles. Asymmetric and symmetric C¼O vibrations of carboxylate groups was displayed the double peaks at 1571-1535 and 1443 cm À1 . Unreacted hydroxyl groups (-OH) on the nanoparticle surface appeared in 3326 cm À1 . The strong band at 2925 cm À1 (asymmetric) and 2850 cm À1 (symmetric) is related to the -CH 2 stretching vibrations. Figure 3 and Table 1 showed the energy-dispersive X-ray (EDX) analysis spectrum of Fe 3 O 4 -Br-MPA. The presence of C, O, Fe and Br signals presented that the nanoparticle surfaces were modified by 2-bromo2-methyl propionic acid (Br-MPA).

ATRP polymerization of styrene with modified Fe 3 O 4 nanoparticles
One of the preferences of ATRP [67,68] is that an alkene monomer like styrene can be polymerized with molecular weight and PDI control, either in bulk or in solution [69]. FT-IR, SEM and XRD can prove that the "grafting from" reaction proceeds successfully. Figure 4(a) shows the FT-IR spectrum of Fe 3 O 4 /PSt. According to the FT-IR spectrum the characteristic absorption bands for polystyrene are as follows: the C-H aromatic stretching vibration at 3026 cm À1 , the C-H aliphatic stretching    Figure 5(b)). Therefore, the Fe 3 O 4 nanoparticle was coated with polystyrene and becomes core/shell nanocomposite. These materials with chemically bonds between organic and inorganic have improved properties. Figure 1(b) shows the X-ray diffraction pattern after growth of polystyrene on the Fe 3 O 4 nanoparticles. The appearance of peaks at 2h ¼ 10-24 is related to polystyrene. This shows that the polystyrene has an amorphous structure. The characteristic peak at 2h ¼ 35.5 is attributed to the presence of Fe 3 O 4 nanoparticles.

Preparation of poly(p-amino styrene) grafted onto Fe 3 O 4
FT-IR spectrum confirmed the structural characteristics of the obtained polymer-coated Fe 3 O 4 . In the spectrum line of Fe 3 O 4 -PS-NO 2 , the absorption bands at 1515 cm À1 and 1346 cm À1 were attributed to the -NO 2 group (Figure 4(b)), which, suggested that the nitration reaction occurred and -NO 2 groups were attached to a benzene ring. After a reduction reaction and the production of amino groups, the bands at 3363 cm À1 and 1608 cm À1 became stronger because of the stretching vibration of N-H as shown in the FT-IR spectra of Fe 3 O 4 /PSt-NH 2 (Figure 4c). Furthermore, we found that the characteristic bands of -NO 2 at 1515 and 1346 cm À1 apparently became weaker, suggesting that -NO 2 groups were reduced to -NH 2 .

Characterization of Fe 3 O 4 /polystyrene-g-polyaniline nanocomposites
The Fe 3 O 4 /polystyrene-g-polyaniline nanocomposite was characterized by FT-IR. Figure 6 shows the FT-IR spectrum of Fe 3 O 4 /polystyrene-g-polyaniline. The formation of polyaniline was shown by an absorption band at 3379 cm À1 which can be attributed to the N-H stretching mode; 1587, 1500 cm À1 is related to the C¼N stretching mode for quinoid and C¼C stretching mode for benzenoid units; and 1303, 1168 cm À1 can be attributed to the C-N stretching mode for the benzenoid unit. 1118 and 825 cm À1 can be attributed to the C-H in the benzene plane. Figure1(c) displays the X-ray diffraction pattern for the Fe 3 O 4 /PSt-g-PANI nanocomposite. This is characterized by the   appearance of peaks extending over the 2h range of 5 to 30 . This shows that the polymer has an amorphous structure and is a completely coated nanoparticle. SEM was used to study the morphology of Fe 3 O 4 /PSt-g-PANI as shown in Figure 7. Much larger particles were obtained when the polymer chains grew from the Fe 3 O 4 /PSt surface ( Figure 5(b)). The compacted and large amounts of polyaniline chains "clothe" the Fe 3 O 4 /PSt nanocomposite to become bulk materials. Figure 8 shows the thermal behavior and thermogravimetric analysis (TGA) of the Fe 3 O 4 , Fe 3 O 4 /PSt and Fe 3 O 4 /PSt-g-PANI nanoparticles. As shown in Figure 8(a), the Fe 3 O 4 nanoparticles exhibit high stability in the range of 50-800 C. These nanoparticles have lost about 6% of their total weight. Probably the evaporation of absorbed water on the surface of nanoparticles had led to weight loss. Figure 8(b) is related to PSt-grafted Fe 3 O 4 that shows a sharp weight loss (61%) at 300-350 C. This weight loss is attributed to evaporation of solvent trapped between the polymer chains and the burning-up of the grafted polystyrene. Figure 8(c) shows the thermal behavior of Fe 3 O 4 /PSt-g-PANI nanocomposite, i.e. 41% weight loss at 50-800 C. The initial weight loss is about 3.6% at 127 C that can be assigned to the decomposition of solvent molecules trapped between the polymer chains. The rapid weight decrease is in the second region at $340 C, which is attributed to the decomposition of homo-PANI. Moderate rate of weight loss in Fe 3 O 4 /PS-g-PANI could be related to the presence of the polyaniline chain on the backbone of polystyrene. Figure 9(a) shows the TEM image of the Fe 3 O 4 nanoparticles. The TEM image determines the size distribution and     Figure 9(b) shows the Fe 3 O 4 /PSt-g-PANI nanocomposite. This image illustrated that aggregation and the dispersion were decreased and improved, respectively. The Fe 3 O 4 /PSt-g-PANI nanocomposite exhibited very good dispersion in the solvent.

Conductivity measurements
The electrical conductivity of polyaniline and its composites makes it suitable for electrical applications. Conductivity measurements were carried out by a four-point probe method. The blends of PANI nanocomposites were compacted into pellets for measurement. All measurements were done in air at room temperature and converted to conductivity by the following equation: In this equation, q is the resistivity (X cm), d is the conductivity (s/cm), V is the potential difference (mV), I is the applied constant current (mA), and x is the thickness (cm). The conductivity of the nanocomposite was high compared to that of PANI. The improved conductivity of the composite is possibly due to the charge transfer between Fe 3 O 4 nanoparticles and PANI. The conductivity values of the pure PANI and its nanocomposites were 1.32 and 1.25 Scm À1 , respectively ( Table 2).
As shown in Figure 10, the effect of the potential scanning rate (V) on the peak currents for the chemically synthesized homo and nanocomposite of PANi were investigated under cyclic voltammetric (CV) conditions in the range of 10 to 90 mVs À1 , in DMSO between À0.20 and þ1.00 V. All potentials are given versus the reference (Ag/AgCl) electrode. The polymer film was prepared on glassy carbon (GC) microelectrode by casting the polymeric solution of samples in THF by ultrasonic treatment for 10 min.
Typical cyclic voltammograms (CVs) of the homoPANI film are shown in Figure 10(a). As seen, the CVs of the homoPANI indicated two anodic peaks at around 0.31 and 0.52 V versus the reference electrode. Moreover, as can be realized, the anodic peaks shifted in the direction of a positive potential with increasing scan rate, which indicated that the electrochemical doping/dedoping of the casted polymer film was chemically reversible. In Figure 10(b), the CVs of the nanocomposite of the PANI film exhibited two typical redox couples with anodic peaks at about 0.26 and 0.47 V against the Ag/AgCl electrode. A similar voltammogram of pure polyaniline for the nanocomposites of PANi showed that the anodic peaks currents linearly increased with increasing scan rate. Also, Figure 10(b) shows that despite the polystyrene chains, the PANI nanocomposite is electrically conductive.

Conclusion
A Fe 3 O 4 /PSt-g-PANi nanocomposite was successfully synthesized with a core-shell structure and a new path, combining surface-initiated atom transfer radical polymerization of styrene with pattern-guided oxidative polymerization of aniline. Immobilization of an ATRP initiator on the Fe 3 O 4 surface allowed for the subsequent surface-initiated ATRP to produce polystyrene grafted polymers. Then, functionalization of the polystyrene grafted to the amino group was completed. The resulting macroinitiator (Fe 3 O 4 /PSt-NH 2 ) was used for in-situ chemical oxidating of aniline. FT-IR and TGA data verify that the surface modification of Fe 3 O 4 by a chemical grafting approach was indeed successful. FT-IR, XRD, TGA, TEM, and SEM confirmed the success of the synthesis and polymerization processes. The synthesized Fe 3 O 4 /polystyrene-g-polyaniline showed better electrical conductivity and better thermal stability relative to homopolymers. Moreover, morphological studies by SEM and TEM presented direct and clear evidence for grafting of polystyrene and polyaniline to the Fe 3 O 4 surface nanoparticles. The method presented for the preparation of the Fe 3 O 4 /polymer nanocomposite is attractive because the chemical grafting of the polymers to Fe 3 O 4 has benefits over a composite prepared by the simple mixing of components.  Due to improved physicochemical properties relative to the homopolymers, the synthesized nanocomposite could be beneficial for numerous applications from supercapacitors or other electronic devices to medicine. This nanocomposite showed high electrical conductivity of about 1.25 Scm À1 .

Disclosure statement
No potential conflict of interest was reported by the authors.