Radio frequency plasma assisted surface modification of Fe3O4 nanoparticles using polyaniline/polypyrrole for bioimaging and magnetic hyperthermia applications

Surface modification of superparamagnetic Fe3O4 nanoparticles using polymers (polyaniline/polypyrrole) was done by radio frequency (r.f.) plasma polymerization technique and characterized by XRD, TEM, TG/DTA and VSM. Surface-passivated Fe3O4 nanoparticles with polymers were having spherical/rod-shaped structures with superparamagnetic properties. Broad visible photoluminescence emission bands were observed at 445 and 580 nm for polyaniline-coated Fe3O4 and at 488 nm for polypyrrole-coated Fe3O4. These samples exhibit good fluorescence emissions with L929 cellular assay and were non-toxic. Magnetic hyperthermia response of Fe3O4 and polymer (polyaniline/polypyrrole)-coated Fe3O4 was evaluated and all the samples exhibit hyperthermia activity in the range of 42–45 °C. Specific loss power (SLP) values of polyaniline and polypyrrole-coated Fe3O4 nanoparticles (5 and 10 mg/ml) exhibit a controlled heat generation with an increase in the magnetic field.


Introduction
Magnetic, optical, semiconducting and biocompatible properties of magnetite (Fe 3 O 4 ) are tunable, which make it as a promising candidate for magnetic resonance imaging, targeted drug delivery and cancer therapy [1][2][3][4][5][6][7]. Magnetite nanoparticles can be functionalised in a relatively easy way and this makes it an ideal candidate for various biological applications [1]. Controlling the particle size and subsequent surface modification to achieve a core-shell nanostructure makes it a synergetic multifunctional material for imaging and as a therapeutic agent in cancer therapy [8][9][10][11]. However, Fe 3 O 4 nanoparticles easily get agglomerate due to their inter-nanoparticle magnetic dipolar interactions and low surface potential and will adversely affect their useful magnetic properties. Agglomeration of Fe 3 O 4 nanoparticles can be prevented to a certain extent by coating a thin layer of nonmagnetic materials like graphite, polyethylene, polyvinyl alcohol, polyaniline and silica [2,12]. Previous studies reported the use of cross-linked polymers as a surface coating for Fe 3 O 4 nanoparticles, which resulted in the modification of its magnetic, optical and physical properties [13,14].
Superparamagnetic Fe 3 O 4 nanoparticles release thermal energy when subjected to an alternating magnetic field which is due to the Brownian and Neel relaxation [5,15]. This property of superparamagnetic iron oxide nanoparticles (SPIONs) can be used for hyperthermia generation and is effective for the treatment of tumours [7]. It is reported that cancerous cells are more sensitive to temperature in the range of 42-45°C than normal tissues [6]. However, studies show that an uncontrolled increase in temperature up to 60°C leads to irreversible cell injury and damage to the healthy tissue [16,17]. Therefore, a well-controlled heat generating system is required for initiating desired death of malignant cells by hyperthermia therapy. Heating efficiency of the superparamagnetic nanoparticles suspended in a medium depends on external parameters like frequency, strength of external alternating magnetic field and internal intrinsic properties (core size, shape, shell thickness, colloidal stability) of the material [18,19]. Fine tuning of these parameters will help to develop new combination of materials with improved heating efficiency suitable for magnetic hyperthermia therapy. Among the superparamagnetic materials, Fe 3 O 4 and γ-Fe 2 O 3 has the advantage of providing better heating rates with minimum dosage and can also retain smooth circulation in the bloodstream [18,19].
The hyperthermia response of the Fe 0 /PANI/polycaprolactone nanofibres synthesized by electrospinning is reported previously [20]. Eddy current loss from PANI and Neel's relaxation from magnetic Fe 0 nanoparticles together contribute to the power dissipation in composite fibres and provide better heating efficiency in this system [20]. Beeran et al. observed an enhancement in hyperthermia activity in HeLa cell population using ferrofluid-based superparamagnetic iron oxide substituted with Mn 2+ and stabilized using trisodium citrate surface coating [21]. Fe 3 O 4 nanoparticles showed a decrease in the magnetic moment and saturation magnetization on coating with a thermosensitive polymer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) [22]. However, an enhancement in the heating efficiency of PDMAEMA coated nanoparticles is observed, compared to bare Fe 3 O 4 nanoparticles [22]. Nemati et al. synthesized iron oxide nano-octopodes by thermal decomposition and studied its hyperthermia response [23]. A large improvement in heating efficiency (up to 70%) was obtained by changing the size and shape of the nanoparticles [23]. Studies indicate that higher heating rates for magnetic hyperthermia can be achieved by increasing the Brownian relaxation (since V H ≫ V M ) [18,19]. SPIONs coated with polymers can heat better due to increase in Brownian relaxation even at higher concentration and help to generate temperature elevation in a controlled manner.
Apart from the magnetic and hyperthermia properties, Fe 3 O 4 is also a semiconducting material in which intervalence and intersublattice charge transfer occur on optical excitation, which contributes to its light emission properties [24]. Similar to other semiconducting materials, presence of defects, oxygen vacancies or excitons are responsible for the optical properties of these Fe 3 O 4 [25]. One of the major challenges in the synthesis of magnetic-fluorescent nanomaterials is the quenching of photoluminescence (PL) emission by the magnetic core, contributed to the electron transfer between Fe 2+ and Fe 3+ ionic states in the tetrahedral and octahedral sites with the ligands [24][25][26][27]. This type of PL quenching can be controlled by coating the ferrite using polymers/silica layer [26,27]. Passivation of magnetite nanoparticles using conjugated polymers can also serve as a good photothermal agent for cancer therapy for both in vivo and in vitro applications [9,10]. Moreover, the conjugated polymer backbone can act as an array of light-harvesting units, having larger optical cross-sections compared to other organic dyes [8]. The modifications of magnetic, semiconducting and optical properties by encapsulation of magnetite with polymers result in efficient fluorescent probes having both imaging and targeting capabilities.
Various methods like chemical co-precipitation, sol-gel method, radio frequency (r.f.) plasma polymerization etc. can be adopted to provide a thin layer of polymer coating to superparamagnetic Fe 3 O 4 [1,13]. Among these methods, r.f. plasma polymerization is a single-step and cost-effective technique. Plasma treated surface modifications promote adhesion between polymer and Fe 3 O 4 nanoparticles, and contributes hydrophilicity to the composite [28]. The plasma process also ensures a sterile surface for the as synthesized material which makes the material more suitable for biological applications due to its biocompatibility [28,29]. The thickness of the coating (~few nm) over the material can be controlled by monitoring the monomer flow rates, pressure, current and time of deposition. Sethulakshmi et al. reported an enhancement in the saturation magnetization of polyaniline (PANI) coated ferromagnetic magnetite and γ-Fe 2 O 3 spherical nanoparticles having a size of 20-30 nm prepared by r.f. plasma polymerization [13].
Various reports indicate that thin polymer coating over bare Fe 3 O 4 nanoparticles adds multi-functionality, reduces agglomeration and provides better dispersibility in the aqueous media [13,22]. In this context, the present work describes the synthesis and surface modification of superparamagnetic Fe 3 O 4 nanoparticles with polyaniline and polypyrrole by r.f. plasma polymerization process. Magnetic and PL/fluorescence emission properties, magnetic hyperthermia response and cytotoxicity of the as synthesized samples are also investigated.

Preparation of iron oxide nanoparticles by coprecipitation method
Superparamagnetic Fe 3 O 4 nanoparticles were prepared by controlled co-precipitation method [30]. For this, 0.2 M FeCl 3 and 0.1 M FeSO 4 .7H 2 O (Merk) each in 120 ml prepared in distilled water were taken and stirred for 30 min. The pH was adjusted to 10 by adding drop-wise aqueous ammonia into the above solution at room temperature. The resulting solution was heated to a temperature of 80°C for about 90 min. This sample was then washed with distilled water several times for the removal of water soluble byproducts and kept it for gravity settling of nanoparticles. It was again washed with distilled water and centrifuged (3500 rpm). The residue obtained was black in colour and kept for drying at 80°C for 3 h in a hot air oven.
2.2 Surface modification of iron oxide nanoparticles using r.f. plasma polymerization method Fe 3 O 4 nanoparticles were coated with polymers by taking double-distilled monomers (aniline/pyrrole) with r.f. plasma polymerization process by using a home-made r.f. plasma polymerization setup [13,14]. It consists of a borosilicate glass tube of dimension (length 50 cm, diameter 5 cm) with provisions for evacuation as well as an inlet for monomer.
The r.f. plasma polymerization chamber was evacuated (0.028 millibar) using a rotary pump after placing Fe 3 O 4 powder inside the chamber in between the copper electrodes separated between a distance of 5 cm. In between the electrodes, a glow discharge of plasma was produced by providing an r.f. frequency of 7-13 MHz. The current for generating plasma was maintained at 74 mA and pressure inside the chamber was 0.032 millibar after monomer injection. Iron oxide powder placed inside the chamber exactly in between the electrodes was continuously stirred using a magnetic stirrer placed below the chamber, so that magnetic grains belonging to the experimental sample are renewed and exposed to plasma during polymerization.
Monomer sprayed in the region of plasma undergoes polymerization process and deposited on the surface of the iron oxide nanoparticles. The deposition process was carried out for about 7 min inside the chamber. After that, the sample was taken out from the reactor and washed with distilled water several times and kept for drying at 80°C. The sample thus obtained was powdered using a mortar and pestle, and used for further studies. Both PANI-coated Fe 3 O 4 and polypyrrole-coated Fe 3 O 4 samples were prepared with above-mentioned r.f. plasma polymerization setup.

Characterizations
X-ray diffraction of the samples was carried out using Bruker D8 Advance Twin-Twin equipment using Cu-K α (1.5404 Å) radiation. Powdered samples were dispersed and pressed on the glass plate before taking XRD of the sample.

Biocompatibility studies using MTT assay
For cell studies, samples were sterilized initially by keeping under UV light (30 W UV lamp of intensity 337.5 lumen) for 4 h. The U87 cells (Glioma cells) were seeded at a density of 10 4 cells ml −1 in a flat bottomed 96-well polystyrene-coated plate and was incubated at 37°C for 24 h in a 5% CO 2 incubator. Different concentrations (0.25, 0.5, 0.75 and 1 mg/ ml) of Fe 3 O 4 , PANI-and polypyrrole-coated Fe 3 O 4 in the medium was added to the plates in hexaplets. This system was kept for 24 hrs incubation, 20 μl (5 mg/ml) MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) solution was added to each well which is incubated for another 3 hrs. Formazan crystals were solubilised in dimethyl sulphoxide and absorbance was monitored at 595 nm in a microplate reader (BIO-RAD iMark TM Microplate Reader). Wells with complete media, nanoparticles and MTT reagent without cells were used as control. The untreated U87 cells and cells treated for 24 hrs with the above-mentioned concentration of nanoparticles were used for the MTT assay for cell viability determination. Percentage cell viability was calculated using the following formula [31]: 2.5 Uptake studies L929 cell lines were used for the cellular uptake studies of the nanoparticles, fluorescence images were taken using DAPI excitation. The cells were initially seeded on to culture plates, which were allowed to reach 80% confluency by incubating the plates in CO 2 incubator at 37°C. The cells were treated with 0.5 mg/ml concentration of respective samples (Fe 3 O 4 , PANI-coated Fe 3 O 4 and polypyrrolecoated Fe 3 O 4 ) since this concentration gives the best cell viability as understood from MTT assay. Wells were treated with the material for different time intervals ranging from 1, 2, 4 h. The aliquots were made in distilled water, since all the samples are dispersible in water. Bright field and fluorescent images (×40 magnification) of the samples were taken after the incubation of the materials with the cells using Olympus IX73 optical imaging system. A control experimental system was maintained for comparison with the treated materials.

Hyperthermia studies
The suitability of materials for hyperthermia applications was studied using Ambrell EASY HEAT induction system. The experimental setup consists of a solenoid coil (6 turns) with a diameter of 4 cm and length 2.6 cm in which ac (alternating current) frequency was set to 280 kHz. The heating efficiency of the sample at different concentrations (1 and 3 mg) and current (alternating magnetic field) were evaluated for 5 min (300 s) [21].  [31,32]. These observations demonstrate that Fe 3 O 4 phase of iron oxide is stable up to 250°C. From the TGA curve ( Supplementary Fig. S1), the polymer component percentage present in the sample is estimated, which is 4.28% (283-456°C) in PANI-coated Fe 3 O 4 and 3.48% (296-519°C) in polypyrrole-coated Fe 3 O 4 .
TEM images show the presence of spherical particles along with a few rod-shaped iron oxide nanostructures (Fig. 2a), some of these nanostructures are agglomerated. The average particle size of spherical-shaped Fe 3 O 4 nanoparticles is~11 nm (Fig. 2b) and that of PANI-coated Fe 3 O 4 is~14 nm (Fig. 4b). But spherical structures in the case polypyrrole-coated Fe 3 O 4 , the average particle size is 18 nm for (Fig. 5b). PL emission band observed at 513 nm is associated with radiative recombination of electrons from the crystal field site of Fe 3 O 4 to its octahedral site [25]. The broad peak at 632 nm can be attributed to the recombination of electrons  [33].
PL emission from the PANI and polypyrrole-coated Fe 3 O 4 samples is dominated by the polymer shell. Relatively weak emission peak observed at 445 nm (PANI-coated Fe 3 O 4 ) and peak at 398 nm (polypyrrole-coated Fe 3 O 4 ) arises due to the π−π* transitions occurring in the benzoid units of the polymer chain [34]. The strong PL emission band observed around 580 nm from the PANI-coated Fe 3 O 4 is attributed to the direct interband transitions occurs in the polymer chain [35]. Another broad emission band at 488 nm of polypyrrolecoated Fe 3 O 4 contributed to de-excitations from the polaron band [34]. Shift in emission bands observed in the PANI and polypyrrole-coated samples is probably due to the presence of polymer chain aggregation formed during r.f. plasma polymerization process. Cellular uptake studies of the Fe 3 O 4 , PANI and polypyrrole-coated Fe 3 O 4 nanoparticles are carried out in normal cell line, L929 (Fig. 7). Fluorescence emissions from the cellular assay at different time intervals are compared, maximum uptake of the samples are observed after 4 h of treating the L929 cell with the samples. One can see from the fluorescence images that there occurs a gradual increase in the fluorescence emission from the cells from the lowest to highest time interval. The brightfield images obtained also show that the cell morphology is not much varied and cells are found to be healthy even after 4 h of incubation. These observations show that Fe 3 O 4 , PANI and polypyrrole-coated Fe 3 O 4 nanoparticles are non-toxic and its fluorescence properties without the addition of any labelling agents or dyes can be exploited for bioimaging applications. Room temperature M-H curve (300 K) of Fe 3 O 4 , PANIcoated and polypyrrole-coated Fe 3 O 4 nanoparticles shows superparamagnetic behaviour (Fig. 9). The saturation magnetization of Fe 3 O 4 (M s ) sample is observed to be 51 emu/gm, which is less than the value reported for bulk magnetite (88 emu/gm). The reduced magnetization of Fe 3 O 4 may be attributed to the non-contribution of the surface to magnetic property, which arises due to the disordered alignment of surface spins [13,36]. The saturation magnetization (M s ) of PANI-coated and polypyrrole-coated  (Fig. 10) indicates that the current of 200 A is not enough to generate hyperthermia temperature of 42°C. Minimum hyperthermia activity is also observed for a current of 250 A for 1 mg concentration of all the 3 samples. But on increasing the alternating magnetic field (AMF) strength via adjusting the current strength to 300 A, the 1 mg of Fe 3 O 4 shows an enhancement in its temperature from 25 to 47°C. However, polypyrrole-coated iron oxide nanoparticles with 1 mg concentration give an increase in temperature from 25 to 42°C. Hyperthermia activity is exhibited by PANI-coated iron oxide (1 mg) at a current 400 A and its temperature is raised from 25 to 45°C.
Hyperthermia activity is absent in all the three samples with 3 mg up to a current of 200 A (Fig. 11). The rise in temperature is observed from 25 to 49°C for Fe 3 O 4 and 25 to 51°C for PANI-coated Fe 3 O 4 nanoparticles (3 mg) with an applied current of 250 A. However, in the case of 3 mg polypyrrole-coated Fe 3 O 4 , enhancement in temperature is observed from 25 to 46°C only with a current of 300 A. These studies shows that for achieving hyperthermia activity for a minimum quantity of about 1 mg Fe 3 O 4 and From the above results, one can conclude that Fe 3 O 4 magnetic nanoparticles show relatively more heating efficiency than polymer-coated magnetite nanoparticles with the same concentration and current strength. The rate of increase in temperature and saturation temperature is related to the applied current strength and the quantity of the experimental sample. However, at a current strength of 250 A (19.33 mT), 3 mg PANI-coated Fe 3 O 4 samples shows relatively more heating rate (0.293°C/minute) compared to Fe 3 O 4 (0.287°C/minute) and polypyyrole-coated Fe 3 O 4 (0.168°C/minute) with same concentration and current. Samples with higher concentration usually show more heating ability and saturation rate as compared to sample with lower concentration with the same current.
Heating efficiency of the samples under investigation is studied by determining specific loss power (SLP) of the particles by suspending a known quantity of samples in distilled water. The sample is subjected to an alternating magnetic field of specific strength and frequency. The change in temperature with respect to time is monitored continuously for a period of three minutes. SLP values of  Table 1. The values obtained in the present study are comparable with the previous reports [21,37]. It is observed that for the same current, SLP values of Fe 3 O 4 nanoparticles are decrease with increase in the concentration of the sample. While for PANI-and polypyrrole-coated Fe 3 O 4 show an increase in SLP value for higher concentration compared to a low concentration of the sample at constant field strength.
It is observed that SLP values have direct dependence on magnetic field strength and concentration of the experimental sample (Table 1). SLP values increase with respect to applied field strength for Fe 3 O 4 , PANI and polypyrrolecoated Fe 3 O 4 with 5 and 10 mg concentration of the sample. Heating efficiency of magnetic nanoparticles has a strong dependence on dipolar-dipolar interactions among the particles. The increase in the quantity of magnetic particles in a system leads to a decrease of interparticle distance,  [21,38,39]. The increase in dipolar-dipolar interactions among the particles enhances the Neel relaxation time consequently SLP values will decrease in agreement with our observation from Fe 3 O 4 with respect to higher concentration (10 mg) .
It is observed that polymer capping of Fe 3 O 4 also plays a major role in the heating efficiency of iron oxide nanoparticles. The hydrodynamic volume of the particle has direct dependence on the thickness of the polymer shell, a thin layer of surfactant coating has significant influence on the relaxation constant of the nanoparticles and also its heat generation capacity [15]. The polymer coating on Fe 3 O 4 resulted in controlled heat generation unlike that observed in the case of bare Fe 3 O 4 . An increase in SLP value for 10 mg concentration of PANI and polypyrrolecoated Fe 3 O 4 with respect to 5 mg concentration of the sample with same field strength and frequency is observed. This variation in the heating efficiency of polymer-coated Fe 3 O 4 NPs is attributed to the relaxation of individual NPs inside the polymer shell which results in an increase in heating rate than uncoated magnetic nanoparticles [23]. Also, the presence of polymer shell can make the matrix rigid and can arise friction between magnetic nanoparticles and shell. This can further generate heat and give higher SLP values for polymer-coated samples at higher concentrations. Hyperthermia studies show that heat dissipation value of the nanoparticles has a strong dependence on the particle concentration, size, shape anisotropy, strength of the magnetic field and the chemical nature of surface passivating polymers. All these parameters are related to the relaxation process of the superparamagnetic Fe 3 O 4 nanoparticles which influences the SLP values. TEM images (Figs. [2][3][4][5] of the samples used in the investigation also show the presence of spherical as well as rod-shaped particles. Rodshaped nanoparticles show better hyperthermia performance compared to spherical-shaped ones [40]. Relatively asymmetric morphological spherical and rod-shaped particles can kill cancer cells more effectively than perfect spherical nanoparticles when exposed to an ac magnetic field due to their mechanical oscillations [41]. From this study, it can be concluded that PANI and polypyrrole-coated Fe 3

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