Synthesis, Characterization and Imaging of Fluorescine Isothiocyanate Conjugated Magnetite Nanoparticles in MCF 7 Breast Cancer Cell Lines

In this work we describe fabrication, characterization and possible application of FITC (fl uorescine isothiocyanate) conjugated magnetite nanoparticles (MNPs) for biomedical applications such as imaging of cancer cells. The MNPs possessed octahedral-like geometry with almost completely dispersed distribution with high saturation magnetization. The fi nal FMNPs (fl uorescine isothiocyanate magnetite nanoparticles) absorption band showed 30 nm red shift towards longer wavelength compared to bare MNP and the laser-induced fl uorescence was observed at 518 nm. The MTT (3-(4,5-Dimethylthiazol-2yl)-2,5-Diphenyltetrazolium Bromide) results showed that FITC conjugation diminishes the toxicity MNPs mainly due to the reduction of surface charge. Fluorescence microscopy confi rmed the uptake and distribution of FMNPs in MCF 7 (Michigan Cancer Foundation-7) breast cancer cells, which suggests that it can be utilized for applications such as a magnetic fl uorescent probe for bioassay. Research Article


Introduction
It is well known that biomarkers (or biological markers) are measurable sign of an organism evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention.
Some of potential applications of biomarkers include oncology, screening, differential diagnosis, prediction of response to treatment, and monitoring of progression of disease [1]. Equally important is the critical and effective role that nanotechnology, particularly the nanobiomaterials that can play in achieving the above goals. Indeed, the design and development of a reliable and green chemistry process for synthesis of nanomaterials is an important aspect of ongoing nanotechnology research namely biomedical engineering. Cancer nanotechnology has already shown its capability in revolutionizing the current techniques in cancer early diagnosis, imaging, treatment and prevention [2].
Magnetite, Fe 3 O 4 , is a common magnetic iron oxide that has a cubic inverse spinel structure with oxygen forming an fcc closed packing and Fe cations occupying interstitial tetrahedral sites and octahedral sites. The electrons can hop between Fe 2+ and Fe 3+ ions in the octahedral sites at room temperature, rendering magnetite an important class of halfmetallic materials. With proper surface coating, these magnetic nanoparticles can be chemically stable, well dispersed with uniform size distribution. When the size of these nanoparticles becomes so small that their dimension can be considered as a single domain, they lack a hysteresis loop and a possess high fi eld irreversibility, high saturation fi eld and extra anisotropy contributions called superparamagnetic iron oxide nanoparticle (SPION) [3,4]. Over the past decades SPIONs with size and morphology dependent physical and chemical properties including biocompatibility, biodegradability, long blood retention time, chemical composition, magnetic behaviour, surface structure, adsorption properties, solubility, low toxicity, and good magnetic response have attracted worldwide research attention [5][6][7]. These unique materials have been utilized successfully for number of applications including contrast-enhanced imaging [8,9]  imaging, photoacoustic imaging and ultrasound imaging [12][13][14][15][16]. In clinical detection and therapy, all modalities play a   great role with signifi cant contributions, however, each also   has its advantages and disadvantages defi ned by different   parameters such as sensitivity, spatial and temporal resolution, invasiveness (e.g., radiation ionization, dose of radioactive material). Despite the limiting factor of optical technique in deep lying cancer screening within the body's organ, it has key advantages including wavelength sensitivity of tissue intrinsic chromophores, low energy radiation and ability to monitor multiple independent optical biomarkers. Generally, optical imaging is performed in the range of ultravioletvisible-infrared radiation where it can be in the form of radiative as heat and radiative as fl uorescence response. When nanoparticles or probing ligands are suitably conjugated by optical or fl uorescent agents, they become smart in such a way that they can be used to identify or target specifi c biological organs [17]. The fl uorescence spectroscopy [18,19] and imaging [20,21] have also been widely used in studying cancer diagnosis.  [23,24]. In our recent report, it was shown that the emission of FITC intensity is pH-dependent which can be considered as an advantage since tumors or infl amed tissues are relatively acidic, about 6.5, due to hypoxic conditions [25] thus, the fl uorescence intensity can be used as an easy monitoring probe. Also, it was recently reported by our group that SPION-based plasmonic nanodendrimers can have the potential capability to be utilized for thermal and bioimaging applications [26,27]. In this research, the results of synthesized SPIONs functionalized by fl uorescine isothiocynate (FITC) used as biomarker for fl uorescence imaging of L929 and MCF 7 cancer cell lines are described.

Synthesis of Magnetite nanoparticles MNPs
1.28 M ferric chloride hexahydrate, 0.64 M ferrous hetrahydrate and 0.4 M hydrochloric acid were prepared as a source of iron by dissolving the respective chemicals in 18.2 M milli-Q water and deoxygenated by bubbling N 2 gas for 1 h prior to the use under vigorous stirring. In the same way, an optimized value of 1.5 M NaOH were prepared as the alkali sources [7,28,29]. Aqueous dispersion of magnetic nanoparticles was prepared by alkalinizing an aqueous mixture of ferric and ferrous salts with NaOH at room temperature. 25 mL of iron source was added drop-wise into 250 mL of alkali source under vigorous magnetic stirring at 1500 rpm for 30 min at ambient temperature. The molar ratio of Fe 2+ : Fe 3+ = 1:2 and complete precipitation of Fe 3 O 4 between 7.5-14 pH was maintained under a non-oxidizing environment, since this would critically affect the physical and chemical properties of the nanosized magnetic particles. The precipitated powder was isolated by applying an external magnetic fi eld, and the supernatant was removed from the precipitate by decantation. The powder was washed and the solution was decanted twice after centrifugation at 5000 rpm for 15 min. Then 0.01M HCl was added to neutralize the anionic charge on the particle surface.

FITC conjugation of APTS-SPONs
This was obtained between isothiocyanate functional group in FITC structure which has high tendency to react with amine groups on Fe 3 O 4 nanoparticles as shown in below: Before the measurements, the samples were dried at 15 °C in a vacuum for 6 h. Fluorescence microscopy (Zeiss Axioshop-Germany) was used to study the cells fl uorescence).

Results and discussion
As shown in fi gure 2A, the mean Fe 3 O 4 particle size examined by TEM imaging are almost dispersed and fi gure 2Bshows the APTS functionalized MNPs. It is notable that, the particles have an octahedral-like geometry ( Figure 2B). The histogram size distribution covers sizes between (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) nm with about 25% count on <12> nm diameter. The infl uence of the chemical potential on the shape evolution of crystals has been elucidated by Jin et al. [30]. In the case of crystal growth, it would be benefi cial to have a higher chemical potential, which is mainly determined by the NaOH concentration.
Octahedral    [19]. It is reported in the literature that Ms of iron-oxide nanoparticles increases with increase in particle size [5,7,32]. In addition, it may also be due to different chemical compositions on the surface like oxidation of Fe 3 O 4 to Fe 2 O 3 , surface effect such as nonlinearity of spins of magnetically inactive layer with the magnetic fi eld. Also, the discrepancy could be explained by the variation of synthesizing methods which can produce particles of different size [33]. Pinning is one of the main sources of the H c . Grain size dependence of H c and permeability (GSDCP) theory [34] predicts: where A denotes the exchange constant, K is a magneto crystalline anisotropy constant, P 1 and P 2 are dimensionless factors. Therefore, reducing the grain size, D g , creates more pining sites and increases H c . For ultrafi ne particles, the modifi ed form of theory predicts: The difference between equations (2) and (3)    value of 10 μg/mL, the cell viability was about 63% and 70% and as the concentration further increased to 500 μg/mL, the viability was reduced to 23% and 60% for MNPs and FMNPs respectively. It is worth note that up to 100 μg/mL, the viability remained almost constant for FMNPs. Thus, the conjugation of MNPs with FITC signifi cantly reduced the toxicity mainly due to the reduction of surface charge. Therefore, with FITC on the surface, the cytotoxicity effect due to direct contact of cell membrane with MNP surface can be reduced. Brownian motion using Lorentzian distribution as normally is considered in soft matter physics. Therefore, one may assume that (r′)=d3r′ND(r′) where (r′) is the probability of fi nding a NP at a distance, r′ in a volume d3r′ and ND (r′) is a density distribution of NPs, a function which decays according to the spatially damped radial function [36]. Thus, two possible sources could be sought, fi rstly the diffusion or mobility due to stronger Brownian motion within the dish which takes place radially and secondly the extra eddy micro currents acting as non-uniform source of distribution when the NPs were added in the experiment. In either case, the fact remains that the images prove their conceptual applications as molecular probes which can provide a better detection and hence understanding of cellular mechanisms.