Magnetic iron nanoparticles for in vivo targeted delivery and as biocompatible contrast agents

Abstract

Iron nanoparticles (NPs) of size less than 20 nm were synthesized using an in-house developed cryomill. These NPs exhibit values of saturation magnetisation (∼180 emu g⁻¹) close to that of pure iron. The particles were found to be nontoxic at concentrations required for MRI imaging as indicated by MTT assay. In vivo studies demonstrated the suitability of using these particles as contrast agents for MRI. The iron NPs were bio-capped with TRITC–dextran and injected into mice to study the transport behavior of the NPs under the influence of an external magnetic field. The iron NPs showed enhanced aggregation and contrast when a bar magnet was placed on the mice as observed by whole body fluorescence imaging.

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Introduction

Success in medical treatment significantly depends on rapid diagnosis and detection of abnormalities of the physiological condition. As a result, enormous importance is placed on developing non-invasive diagnostic procedures.^1,2 Among these, imaging techniques are often preferred because they provide a highly sensitive, rapid, reliable and site specific detection of diseases.^1–4 There are several imaging techniques such as positron emission tomography (PET), single photon emission computed tomography (SPECT), optical imaging using fluorescence or luminescence and magnetic resonance imaging (MRI)^1–4 PET and SPECT that have high sensitivity. However, many of these suffer from a relatively poor spatial resolution and the use of radioactive agents.^5–7 MRI can be attractive with the physicians as they do not require use of ionizing radiation.^5–8 In order to improve the signal quality, improvement in the contrast agents and instrument design are required. Improvements in the instrument design, however, often increase in the cost of diagnosis. Hence efforts are being made to develop robust magnetic and fluorescence contrast agents for MRI and fluorescence imaging (FLI).^9–11

The difference in signal from various body tissues enable MRI to differentiate organs and to discriminate benign and malignant tissue. However, the ability of MRI to detect the smallest tumors is impeded by its lack of sensitivity which can be increased by the use of improved contrast agents. Magnetic contrast agents are active substances generally loaded with magnetic metal ions to improves the differences in signals between tissues.^5–11 The most significant improvements can be realized when contrast agents having selective strategies can be devised: (i) using a specific administration route according to the targeted organ, (ii) size or ligand dependent delivery that takes into account the specific characteristics of the targeted organ using biochemical receptors. Based on these strategies, magnetic nanoparticles conjugated with targeting antibody have been synthesised to target a specific tissue.^11–15 Several magnetic nanoparticles of iron, iron oxide and gadolinium based materials have been developed in the recent past.^13–15 Gadolinium based contrast agents such as gadolinium meglumine, or pentacetate and their derivatives are frequently used for MRI imaging. It is also used for detecting the lesions and differential diagnosis between various disease. But it has very low magnetic saturation level resulting in reduced contrast and difficulty in differential diagnostics and hence not an efficient targeting contrast agent. Further, excess use may have effect on health.^13–15 Elemental iron has high saturation magnetisation and hence ideal as a contrast agent. However, the intrinsic nature of pure iron is to oxidize rapidly and hence present difficulties in synthesizing the nanoparticles of iron. As a result most of the research work have only reported iron oxide or iron core with iron oxide shell.^13–17 There are recent reports describing methods of synthesis of hybrids of graphene, silica and quantum dots that represents a new approach to improve the imaging technique.^18–20 However, a serious efforts towards developing an iron based contrast agent with reduced oxides may lead to significant improvement. This is due to the fact that iron oxide has ∼1/3 magnetic saturation value compared to pure iron. Since the magnetic contrast decreases with increasing fraction of iron oxide, lowering iron oxide content should lead to better image contrast. Apart from magnetic saturation, toxicity and hydrophobicity of nanoparticles represent major concerns in the application of iron oxide. This is often overcome by functionalization of the particles. Several organic functional materials that can be used with iron oxide has been developed. But addition of these non-magnetic materials further reduces magnetic control and saturation. In the current work, we prepared non-toxic magnetic nanoparticles of iron (iron NPs) for application as a contrast agent in optical imaging and MRI. The detailed processing of these nanoparticles with high purity is reported in our previous work.²¹ Extending the cryomilling processing route in this work, we have prepared iron nanoparticles of average size of 8 nm. These nanoparticles have been investigated for targeted delivery application.

Experimental details

Materials and methods

In the current study, we have used an in-house developed cryomill. The details of the mill is given elsewhere.^21–23 The starting iron shots had 99.95% purity and milling was carried out at a temperature of 154 K under high purity argon atmosphere. The milled power samples were preserved at sub-zero temperature during the course of the investigation to avoid coarsening and oxidation. The synthesized powders were subjected to X-ray diffraction studies (XRD, X'Pert PRO), scanning electron microscopy (SEM, FEI Sirion, USA), and FEG EPMA (JEOL JXA-8530F) for composition analysis. Transmission electron microscopy (TEM, FEI, FEG Technai F30, Netherlands) attached with energy dispersive X-ray spectroscopy (EDS) was used for local composition analysis and high resolution imaging. The magnetic behavior of dry particles was investigated using Vibrating Sample Magnetometer (Lake Shore 7400 Vibrating Sample Magnetometer).

Evaluation of biocompatibility of iron NPs. Cytotoxicity of iron NPs was determined by MTT assay on HeLa cell line and RAW 264.7 macrophage cell line. Mention one line about the two cell lines. The cells were seeded and incubated for 12 h in 96 well microtitre plates in DMEM supplemented with 10% fetal calf serum (FCS). The cells were treated with iron NPs with concentrations varying from 10 to 1000 μg ml⁻¹. After 24 h treatment with NPs, cells were washed with PBS and 20 μL of MTT reagent (5 mg ml⁻¹) was added to each well. The cells were incubated for 4 h at 37 °C and finally 200 μL of DMSO (Sigma) was added to each well and absorbance of treated and untreated samples were recorded at 570 nm using Elisa plate reader (Spectra max 340PC, Molecular devices, USA).

The study of cytotoxicity was carried out using LIVE/DEAD Viability kit (Life Technologies, India Ltd.) by quantifying the fluorescence intensity of the treated and untreated cells by fluorescence spectroscopy. The kit is composed of two dyes which stain live cells using calcein AM (green) and dead cells using ethidium homodimer-1 (red). HeLa cells were seeded in 96 well plate and incubation with the iron NPs sample at a concentration of 10 to 1000 μg ml⁻¹. After 24 h, the treated samples were washed with PBS twice and reagents added according to manufacturer's protocol. The fluorescence intensity of calcein AM (Ex: 494 nm, Em: 517 nm) and ethidium homodimer-1 (Ex: 528 nm, Em: 617 nm) was measured using fluorescence spectrophotometer (F-7000, Hitachi). The samples for microscopy were also treated similarly with 10 to 1000 μg ml⁻¹ of iron NPs for 24 h and images acquired using fluorescence microscope (Leica MM AF, place). The quantification of live and dead cells in the fluorescence images was carried out by calculating the ratio of live to dead cells using image analysis software (ImageJ).

Preparation of biocapped iron NPs. 1 mg ml⁻¹ concentration of iron NPs was incubated overnight with TRITC–dextran (1 mg ml⁻¹). After incubation for 12 h, samples were centrifuged at 2000 rpm to separate the particles and washed with MilliQ water three times to remove any unadsorbed dye. The treated NPs were again suspended in MilliQ water and used for further studies. The particles were scanned under a confocal microscope to confirm the adsorption of the dye on to the iron NPs.

Hemolysis by nanoparticles. To 3 ml of blood obtained from healthy volunteers, 50 μL of 5% disodium–EDTA was added to prevent coagulation. It was centrifuged at 3000 rpm for 10 min to separate serum from RBCs. The isolated RBCs were further washed five times with sterilized 0.9% sodium chloride solution and diluted 10 times. Appropriate volume of dextran coated iron NP nanoparticles stock solution was added to 300 μL (150 million) of diluted RBC solution in order to prepare concentrations ranging from 10 μg ml⁻¹ to 2000 μg ml⁻¹. Plain water and 0.9% NaCl solution were used as the positive and negative controls respectively. Percentage hemolysis from dextran coated iron NP was compared with uncoated iron NP and iron oxide nanoparticles. All the samples were prepared in triplicates. The samples were centrifuged at 10 000 rpm for 10 min and the absorbance of the supernatants were recorded at 415 nm using Nanodrop spectrophotometer. The percentage hemolysis was calculated by the following formula:

% hemolysis = (test sample − negative control)/(positive control − negative control)

Magnetic field response of iron NPs in vivo. Preliminary study of magnetic field assisted targeted delivery was studied in 4 to 5 week old female Balb/c mice which were bred at the Central Animal Facility (CAF), Indian Institute of Science, Bangalore, India. All procedures were followed as per CAF guidelines and in vivo studies were approved by the Institute Ethical Committee, IISc. Single mice was used per sample to study the efficiency of magnetic field enhanced targeted delivery assisted by an external magnetic field. Mice were treated with 5 mg kg⁻¹ body weight of free TRITC–dextran and equivalent amount of TRITC–dextran conjugated with iron NPs. These were injected through the tail vein. Mice injected with equal volume of saline was taken as control. Bar magnet (1 × 1 cm, 2 tesla, V. R. Technologies, Bangalore, India) was placed on the mice abdomen. Time dependent distribution of iron NPs was studied by whole body imaging technique. Mice were anesthetised using isoflurane mixed with oxygen and placed on in vivo imaging (Xenogen IVIS 200, Perkin Elmer, USA). Mice were exposed to fluorescent light for 20 s and images were acquired at emission wavelength range of TRITC which is 520 nm. The distribution of TRITC–dextran conjugated iron NPs was compared in the presence and absence of external magnetic field.

Results and discussion

Following our recent report of the methodology of large scale production of nanoparticles, milling in the present experiments were conducted using Fe powders of 99.99% purity in a specially designed mill under pure argon.²¹ The mill is capable of operating at temperatures close to liquid nitrogen (LN₂) temperature (∼150 K). The inset of Fig. 1a shows the X-ray diffraction patterns of the Fe powder milled at 150 K. The positions of the peaks are marked on the top of the figure. All the peaks can be indexed using reflections of bcc Fe (JCPDS-06-0696). No additional peaks of oxides or contaminant can be detected. The reflection of the most intense peak (110) has shifted to a higher angle of 2θ (from 44.62 degree for as received powder to a 44.7 degree for 6 h milled sample), suggesting a lattice compression after the milling. The lattice parameter calculated using all the peaks are found to be 2.79 Å, which is 2.3% lower compared to the standard lattice parameter of iron (2.856 Å). From the broadening of the observed peaks, we have estimated coherent diffracting domain size using the technique of Hall and Williamson.²² In the case of single domain free particles, these represent the particle size of our milled powders. This is plotted in Fig. 1a as a function of milling time. The analysis yields size of the milled powder to be 8 ± 2 nm after five hours of cryomilling. The SEM images shown in Fig. 1b reveal evolution of the particles during different durations of cryomilling till 5 hours. Comparing the results with crystallite size measurement from XRD analysis, it is apparent that the measured average size of crystallite is almost identical for both the cases. A detailed TEM study of 5 h cryomilled sample is shown in Fig. 1c–e. Fig. 1d shows a high resolution image of a 8 nm iron particle with lattice fringes that corresponds to alpha iron. During the milling process, the powder experiences repeated plastic deformation by the impact from the balls. The minimum grain size attainable during mechanical milling is controlled by a balance between the process of fracturing and cold welding. At low temperature, the process of fracturing dominates as ductile to brittle transition for Fe lies below room temperature (299 K).²³ Fig. 1e reveals a five fold twinned particle that confirms the hindrance of dislocation motions at these twin boundaries. Hence the iron fractures in a brittle manner. The SAD pattern also confirms the particle to be single phase bcc iron. A detailed large area chemical composition analysis using electron probe micro analyzer (EPMA) (JEOL JXA-8530F equipped with field emission gun) were performed using both energy dispersive and wave length dispersive spectroscopy (Fig. 1b). The large area scans of different particle agglomerates at different locations do not show any other element other than iron indicating no contamination from the milling container. No signal or peak corresponding to oxygen could also be found during the EPMA analysis. Composition analysis was also carried out using energy dispersive spectroscope (EDS) attached with a field emission transmission electron microscopy (FEI make, TECNAI F30) on particle agglomerates dispersed in a TEM grid. A representative spectrum is shown in Fig. 1f that confirms the absence of oxygen. A representative STEM image of the particle agglomerates and its color composition map (150 × 150 nm) for iron and oxygen are shown in Fig. 1g. The composition map of iron shows single color contrast while signals from oxygen are absent in the region.

Fig. 1 (a) Coherently diffracted domain size (nm) vs. milling hours for cryo temperature milling of Fe obtained from powder diffraction data. The inset shows the XRD plot of all the samples marking plans corresponding to bcc iron. No impurity peak could be observed (b) photographic images of the milled powders (images obtained with a digital camera) at different milling time. SEM images of the powder at each stage (marked by the arrow) are also presented to illustrate the particle morphology and size. (c) A bright field transmission electron microscope image of 5 h cryomilled sample. Inset shows particle size distribution obtained by DLS technique. (d) and (e) HRTEM image of the particles showing Fe lattice with inset showing selected area electron diffraction patterns. (f) EDS spectra from agglomerated NPs showing iron as well as Cu that comes from the grid. The absence of peaks of oxide and other impurities can be noted. (g) Composition maps using STEM showing regions with iron and oxygen in different colors.

Ball milling gives rise to reduction of grain size and results in nanocrystalline aggregate in the forms of micron size particles. As mentioned earlier, the reduction of particle size is dictated by the competition between the process of fracturing verses sintering or welding. Room temperature milling of micron size powder results in local heating which results in agglomeration or welding of the nanoparticles. In case of cryomilling, the fracturing process get enhanced while the processes of welding or sintering are reduced significantly at low temperature. This results in the formation of well separated nanocrystal or nanoparticles. The enhanced fracturing is rapid as evidenced by the reduction of mm size shots to submicron size in 30 min.

A plot for obtaining saturation magnetization from the nanoparticles is shown in Fig. 2a. The inset shows increase in saturation magnetization as the size decreases and reaches a saturation value of ∼210 emu g⁻¹. This is close to that observed for bulk pure iron (i.e. 220 emu g⁻¹) and the observed reduction is due to atomic layer surface coatings. The coercivity of the particle increases as size reduces. However, below a size of 30 nm, the trend reverses leading to a decrease in saturation magnetisation. The susceptibility of the nanoparticles increases as the size reduces. The iron NPs are dissolved in water and kept for 3 days and the HRTEM of the particle shows a thin (one or two atomic layer) amorphous layer (shown in Fig. 2b). The magnetic measurements of these particles indicate a decreased magnetic saturation of 180 emu g⁻¹ due to the oxygen enrichment on the surface. On the other hand these layers protects the nanoparticle. The major concern in introducing metallic nanoparticles in the body is its retention and the issue of toxicity to tissues. The characteristics of an ideal diagnostic agent is its ability to generate high signal strength, stability in the body fluids, biocompatibility and elimination from the body without any significant toxicity. Since the iron NPs are introduced into the body, its subsequent fate is of great importance. Iron is an important dietary micronutrient required for normal metabolism and erythropoiesis. The dietary requirement of iron is almost 1–2 mg day⁻¹, which is absorbed mainly from the intestine. Iron is required to form haemoglobin which is responsible for transporting oxygen in the body and also as component of the mitochondrial enzymes. In the body, iron complexes with transferrin and is stored mainly in liver as ferritin or homosiderin. Iron is also stored in immune macrophage cells and erythroid cells as transferrin complex.²⁴ Iron transport and metabolism is tightly regulated by the body. However, iron overload in the body can lead to a condition called as haemochromatosis which can result in pathologies such as cirrhosis, bone disorders and endocrine related disorders. Hence we need to study the toxicity of the iron NPs towards mammalian cells. In order to verify the stability of the colloid, the iron NPs are dissolved in water and kept for 3 days following which structural and magnetic analysis were performed. The HRTEM of the particle shows a thin (two to three atoms) amorphous layer (shown in Fig. 2b). The magnetic measurements of these particles reveal a saturation magnetization of 180 emu g⁻¹ that supports the presence of amorphous surface layer. These layers most likely protect the nanoparticles from further oxidation. The iron NPs coated with TRITC (tetramethylrhodamine)–dextran are characterized using TEM as shown in Fig. 2d–e. The dark field image of the agglomerates of coated NPs reveals development of stable NPs. The SAD patterns (rings) from the particles confirm the presence of bcc iron while STEM line profile confirms absence of iron oxide in coated particles (Fig. 2d). The HRTEM image of individual NPs shown in Fig. 2e reveal a thin amorphous coating that further protects the NPs. These coating also improves the stability of the nanoparticles.

Fig. 2 (a) M–H curves for 1–5 h cryomilled samples. The top inset shows the variation of saturation magnetization as a function of size while the bottom inset shows the variation of coercivity as a function of size for iron nanoparticles. (i) 30 min, (ii) 60 min, (iii) 3 h, (iv) 6 h. (b) HRTEM image of the iron NPs that were treated by keeping in water solution (c) M–H curve of as received sample before and after treating with water. (d) Dark field TEM image after the coating with insets showing SAD pattern of iron and composition line scan of the particle agglomerates indicating absence of oxygen. (e) HRTEM of iron NPs after coating.

To determine the viability of the cells after exposure to the nanoparticles, MTT assay was performed using macrophage cell line RAW 264.7 and HeLa cell line in triplicate over 24 h of treatment (Fig. 3). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is based on the reduction of the tetrazolium dye to insoluble formazan crystals by dehydrogenase enzymes present in viable cells. The insoluble formazan crystals formed are then dissolved in DMSO to form a purple coloured solution. The intensity of the purple solution formed is directly proportion to the number of viable cells. The absorbance of the solution is measured at 570 nm corresponding to the absorption maxima of the reduced dye. The absorbance of the iron NP treated sample is compared with PBS treated cells which is used as control (Fig. 3A and B).

Fig. 3 Cytotoxicity of the iron NP nanoparticles evaluated by MTT assay on (A) RAW 264.7 macrophage cell line and (B) HeLa cell line, showing significant toxicity above 50 μg ml⁻¹. (C) Toxicity study by measurement of fluorescent intensity of calcein AM and ethidium dye to evaluate percentage of live cells and dead cells after treatment with iron NP for 24 h. PC (positive control): cells treated with 0.1% saponin solution 10 min prior to staining. NC (negative control): cells to which no sample is added. (D) Hemocompatibility study of dextran coated iron NPs at concentration ranging from 10 μg ml⁻¹ to 2000 μg ml⁻¹, and its corresponding image.

The cytotoxicity of the iron NPs was also evaluated using HeLa cells by a live/dead assay. The dye calcein AM permeates into the cytoplasm of cells and becomes fluorescent by action of esterase enzyme resulting in green fluorescence. Ethidium dye however cannot diffuse into live cells and stains only the nuclear components of the dead cell and fluoresces in the red region of the spectrum. HeLa cells were treated for 24 h with iron NPs and its cytotoxicity evaluated by both spectroscopic techniques as well as by fluorescence microscopy. The negative control (NC) were cells which were untreated and positive control (PC) were cells which were treated with 0.1% saponin for 10 min just before adding the staining dyes. We found even at concentrations less than 50 μg ml⁻¹ the cells showed apoptosis (Fig. 3C) which was also seen in the fluorescence images. At concentrations higher than 50 μg ml⁻¹ significant toxicity was observed. At 50 μg ml⁻¹ viability decreased to 66% and apoptosis increased by almost 5 times the negative control. At 100 μg ml⁻¹ the cells exhibit almost 80% toxicity to iron NP particles, which remains constant till 1 mg ml⁻¹. We observed a concentration dependent hemolysis trend with dextran coated iron NP nanoparticles. The percentage hemolysis was less than 0.5% at concentrations less than 200 μg ml⁻¹. The hemolysis percentage increased sharply after 1000 μg ml⁻¹ (2%) to 9% at 2000 μg ml⁻¹. The hemolysis capacity of dextran coated iron NP was compared with uncoated iron NP and iron oxide nanoparticles (Fig. 3D). The dextran coated iron NPs exhibited lower toxicity at all concentrations compared to the iron NP or iron oxide nanoparticles. For a concentration less than 500 μg ml⁻¹ there was no significant difference between iron NP or iron oxide nanoparticles but above 500 μg ml⁻¹ the iron NPs were less toxic compared to iron oxide nanoparticles. The data is supported by literature which suggests significant blood incompatibility ascribed to iron oxide nanoparticles.^25,26 The presence of coating appears to be essential in all iron based contrast agents to reduce toxicity. However, our observation that the synthesized iron NP particles exhibit lower hemolysis compared to iron oxide nanoparticles is significant.

The DIC images of HeLa cells (Fig. 4A–D) show aggregation of iron NP nanoparticles, which may be due presence of serum in the media. The morphology of the cells do not show any significant changes with increasing concentration of sample. The positive control used for comparison of toxicity was 0.1% saponin, which shows complete cell damage (Fig. 4F). The percentage of dead cells was 20% after treatment with 10 μg ml⁻¹ as compared to <5% in negative control (Fig. 4G). At higher concentrations, 500 to 1000 μg ml⁻¹ the cell death was seen to be almost 50% (Fig. 4I and J). The viability data shows the expected negative trend at concentrations greater than 50 μg ml⁻¹. Hence, we observe concentration dependent toxicity of the iron NPs after 24 h of incubation. Since, we could not find any toxicity data for iron NPs, we compared our results to the closest material available which is iron oxide.

Fig. 4 Fluorescence microscopy images of HeLa cells after treatment with iron NP nanoparticles. (A)–(D): DIC images of cells treated with iron NP at a concentration of 10, 100, 500 and 1000 μg ml⁻¹ respectively. (E) Fluorescence image of cells with negative control, (F) positive control: 0.1% saponin for 10 minutes before staining. (G)–(J) cells treated with 10, 100, 500 and 1000 μg ml⁻¹ of iron NP particles, (inset) image based analysis of percentage of live cells to dead cells. We find that above 100 μg ml⁻¹ there is considerable toxicity observed in HeLa cells.

The cytotoxicity of iron oxide nanoparticles have been evaluated on different mammalian cells such as fibroblasts, liver cells, lung epithelial cells, breast cancer cells, etc. The iron oxide nanoparticles exhibited 60 to 80% toxicity in L929 fibroblast cells in a concentration range of 1 to 20 mM.²⁷ Octapod iron oxide nanoparticles synthesised by Zhao et al., exhibited negligible toxicity on HepG2 cells even at a concentration of 100 μg ml⁻¹.²⁸ No toxicity was reported by using ferromagnetic iron oxide on breast cancer cells (MDA-MB-231) even at a concentration of 200 μg ml⁻¹ by MTT assay.²⁹ Many reports have suggested using polymer coating to reduce the cytotoxicity while retaining the magnetic properties of the iron oxide nanoparticles.^30,31 However the use of only MTT assay in evaluating the cytotoxicity has been questioned in the recent literature, and it is imperative to complement it with other toxicological studies.³²

Our results indicate that the particles can be safely administered for MRI imaging purposes at concentrations less than 50 μg ml⁻¹ without any significant toxicity. The result from the MTT studies indicate that the RAW 264.7 macrophage cells are able to withstand higher concentration of iron NPs as compared to epithelial cells (HeLa). The percentage viability at 100 μg ml⁻¹ concentration was 90% for RAW cells while it decreased to almost 70% for HeLa cells. This may be because the macrophage cells can sequester and store iron. However, in the live dead fluorescence assay, toxicity was observed even at 50 μg ml⁻¹. However, in the image based analysis of the cells, we did not observe significant difference between the 10 μg ml⁻¹ and 100 μg ml⁻¹ treated samples. Hence, we used iron NPs at concentrations less than 50 μg ml⁻¹ for further biomedical applications.

Magnetic field assisted in vivo targeted delivery

Intravenous route of administration of iron NPs causes rapid systemic distribution to all the vascularised organs resulting in limited therapeutic efficacy at the target site and toxicity to other organs. To overcome these limitations, research on designing stimuli based responsive carriers based on stimuli such as pH, enzyme, temperature and magnetic field for controlled delivery are being explored.^33,34 In conventional therapy, delivery of therapeutic molecules to the target site is the major challenge as it can overcome the limitation of selectivity, imaging contrast and longer retention in the target site. Magnetic nanoparticles are promising candidate for targeted delivery as they respond to an external magnetic field which can be placed near the target site and does not depend upon biochemical signals from the tissues. In addition to ease of preparation, enhanced the cellular uptake make these magnetic nanoparticles an attractive choice for targeted delivery.³⁵

We have observed that in mice injected with free TRITC–dextran, the dye distribution and excretion occurred rapidly (<1 h) from the systemic circulation (Fig. 5B). Hence, we prepared and studied the controlled distribution of TRITC–iron NPs administered via tail vein injection. Images were taken 1 h post treatment in the presence and absence of an external magnetic field. Mice treated in the absence of magnetic field showed uncontrolled distribution of TRITC–iron NP (Fig. 5C). We placed a bar magnet (2 tesla) on the mice abdomen to ensure a constant magnetic field (Fig. 5D) and injected TRITC–iron NPs via the tail vein injection and imaged after 1 h. We observed better accumulation of TRITC–iron NPs in the abdominal site of the mouse in the presence of magnetic field (Fig. 5E). This proves that the TRITC–iron NPs were able to respond to external magnetic field and could be used for targeting any specific organ by changing the location of the magnetic field. The accumulation of iron-NPs in the abdominal area will result in greater imaging contrast between tissues during MRI. The experimental results prove that novel iron NPs efficiently respond to the external magnetic field and can successfully be utilized for in vivo delivery with enhanced therapeutic efficacy at the targeted site.

Fig. 5 Magnetic field assisted targeted delivery of TRITC–dextran conjugated iron NP nanoparticles in mice. CLSM images of iron NP (bright field) and after TRITC–Dex coating (scale bar: 5 μm). Mice injected with different samples (A) saline, (B) free TRITC–dextran, (D) the photographic image of bar magnetic is placed near abdomen for inducing the external magnetic field, (C) and (E) mice injected with TRITC–iron NP nanoparticles in the absence and presence of magnetic field respectively.

Conclusion

Current paper reports synthesis of nanoparticles of iron in large quantity using low temperature milling. These particles yield high saturation magnetization. The colloid of these nanoparticles can be safely administered as magnetic contrast agent for imaging purposes at concentrations less than 50 μg ml⁻¹ without any significant toxicity. The MTT studies indicate that the RAW 264.7 macrophage cells are able to withstand higher concentration of iron NPs as compared to epithelial cells (HeLa). The percentage viability at 100 μg ml⁻¹ concentration was 90% for RAW cells while it decreased to almost 70% for HeLa cells. Magnetic field assisted in vivo study shows efficient contrast during imaging due to the presence of nanoparticles. It is also shown that these particles can be used for targeted delivery to any specific part of the body by using external magnets. A detailed study for optimisation of MR contrast agent is needed in future.

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Footnote

† Equal contribution.

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