Biochimica et Biophysica Acta 1830 (2013) 3005–3010

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Biochimica et Biophysica Acta

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Iron nanoparticles from animal blood for cellular imaging and targeteddelivery for cancer treatment

M. Chamundeeswari a,⁎, T.P. Sastry b,⁎, B.S. Lakhsmi c, V. Senthil d, Enzo Agostinelli e

a St. Joseph's College of Engg, Sholinganallur, Chennai-600 119, Indiab Bio-Products Lab, Central Leather Research Institute, Adyar, Chennai-600 020, Indiac Centre for Biotechnology, Anna University, Chennai-600 025, Indiad Gemini Scans, Chennai-600 029, Indiae Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, SAPIENZA University of Rome and CNR, Institute Biology and Molecular Pathology, Piazzale Aldo Moro 5,00185 Rome, Italy

⁎ Corresponding authors. Tel.: +91 9994752386.E-mail addresses: chamundeeswari@gmail.com (M.

sastrytp@hotmail.com (T.P. Sastry).

0304-4165/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbagen.2012.12.031

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 17 August 2012Received in revised form 27 December 2012Accepted 29 December 2012Available online 10 January 2013

Keywords:Breast cancer cellChitosanFolic acidMRI scanIron nanoparticle

Background: Iron nanoparticles (INPs) are usually prepared from inorganic sources, but we have prepared itfrom goat blood using incineration method. These INPs are then coated with chitosan (C) and coupled withfolic acid (F) to form bionanocomposite for folate receptors.Methods: The bionanocomposite was characterized for its physicochemical properties and cancer celltargeting studies using Fourier transform infrared spectroscopy, transmission electron microscopy, Zetapotential analysis, scanning electron microscopy–energy dispersive X-ray spectroscopy and magneticresonance imaging analyses.Results: The results have shown that the particle size of the INP-CF was found to be 80–300 nm and con-firmed the presence of chitosan and folic acid in the bionanocomposite. Cancer and normal mouse embryoniccell line study confirmed the internalization of INP-CF and this phenomenon was also supported by physico-chemical studies.

Conclusion: Thus, nanobiocomposite prepared using natural sources as a raw material will be beneficialcompared to commercially available synthetic sources and can be used as receptor targeting agent for cancertreatment. This nanobiocomposite when coupled with substances such as monoclonal antibodies might actas a theranostic nanoagent for cancer therapy in the years to come.General significance: The prepared novel nanobiocomposite containing INPs isolated from natural source maybe used as multifunctional agent due its paramagnetic property apart from its drug delivery effect.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Nowadays, nanoparticles are being extensively used as carriers fordrugs, proteins, vaccines and genes due to their smaller size when com-pared to themicrometer-sizedmammalian cells [1]. Cancer nanotechnol-ogy is an interdisciplinary field which provides a wide range of potentialapplication such as molecular imaging, molecular diagnosis and targetedtherapy [2–5]. Among the commercially available nanoparticles,magneticnanoparticle plays a versatile role in the field of oncology for both diagno-sis and treatment of cancer. Apart from this, it acts as a theranostic agentto solve the complexity of oncology related disorders [6]. Themain focus-es of these applications are to destroy selectively disseminated tumorcellswhile sparing the normal tissues [7]. The knowledge of cellular inter-action of nanoparticles with normal and cancer cells in vitro helps inthe development of improved diagnostics and better treatment methods

Chamundeeswari),

rights reserved.

for imaging and targeted drug delivery [8]. Several methods have beenadapted for preparing magnetic nanoparticles in various forms suchas liposomes, solid lipid nanoparticles, dendrimers, nanocomposites,nanobrushes, nanotubes, micelles, nanogels and nanorods for biomedicalapplications [9,10]. Recently, a thermo-sensitive magnetic polymer basedhybrid nanogel has been reportedwhich finds its wide role in a variety ofmagnetic based applications [11].

Breast cancer, the most frequently diagnosed cancer affectingwomen as well as the second leading cause of cancer death requiresearly detection [12,13]. A wide variety of breast cancer cell lines suchas BT-20, MDA-MB-231, MDA-MB-435, MDA-MB-468, MCF-7, SkBr 3,T47D and ZR75.1 [14] have been used as an experimental tool fortargeted delivery and imaging. Normal mouse embryonic fibroblastcells such as NIH 3T3 which have low expression of folate receptorscan be used as a negative control [15,16]. Magnetic Resonance Imaging(MRI) is one of the non-invasive techniques used to visually track mag-netic nanoparticles in vivo and in vitro [17]. Magnetic nanoparticlesdue to their non-invasive character help to improve the contrast ofMRI by providing changes in longitudinal (T1-recovery) and transverse

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relaxation (T2 decay). Among these two relaxations the T2-weightedimages (T2*W) are preferably useful for diagnosing the internalinjuries and cancer lesions [18–20]. Folic acid, an essential watersoluble vitamin, is used as a target ligand for folate receptor which isover-expressed in human cancer cells such as ovarian, breast and pros-tate cancers while only minimally distributed in normal tissues [16,21].Chitosan, a natural polycationic biopolymer is biodegradable, biocom-patible and non-toxic polymeric material which has been particularlyselected as a potential carrier for drugs [22,23]. Though magneticnanoparticles are coated with biopolymers such as starch and collagen,chitosan coated magnetic nanoparticle finds its vital role in biomedicalfield and cell separation techniques [24]. Many studies were conductedto test the toxicity of Gadolinium (Gd)-based MRI contrast agent andresults have shown that Gd could not be completely cleared from thebody even after 2 weeks, thus increasing its toxic effect in the organssuch as liver, kidney etc [25–27].

Considering the above aspects, targeted nanocarriers have beendeveloped for cancer imaging, diagnosis and therapy. In this study,we have developed a folate conjugated chitosan coated magneticbionanocomposite which can be detected by MRI. This magneticbionanocompositemaybe an economical alternative to the commerciallyavailable toxicMRI contrast agents and provide high resolution images todetect the cancer cells (MCF-7— human breast adenocarcinoma).

2. Materials and methods

2.1. Materials and reagents

Chitosan and folic acid were purchased from Sigma-Aldrich St.Louis, MO, USA; goat blood was collected from nearby municipalslaughter house; MCF-7 and NIH 3T3 cell lines were purchased fromthe National Centre for Cell Science Pune; all other chemicals usedwere of analytical grade.

2.2. Preparation of INPs from goat blood

1 liter of goat blood was collected and mechanically stirred using aglass rod for 15 min continuously to isolate the fibrin. The defibrinatedblood was centrifuged at 10,000 rpm (7155 g) for 20 min; the super-natant (serum portion) was discarded and the RBC collected at bottomof the tube was removed, washed withwater for 10 times and stored at4 °C. The RBC was incinerated in a silica crucible using a muffle furnaceat 800 °C for 2 h. After cooling the residue (INPs) were collected andstored in a glass container. The preparation and characterization ofINPs from blood were reported in our earlier publication and theexperiment was designed with the approval of Institutional EthicalCommittee [28].

2.3. Preparation of folate conjugated INP bionanocomposite

50 mg of INPs was dissolved in 500 μl of 6 N HCl and this INPsolution was treated with 200 μl of 0.5% chitosan solution, vortexedand incubated for 1 h at 25 °C with frequent vortexing for every15 min. The varying concentration of folic acid 1–5 mg was preparedand checked for maximum loading with INP-C using UV–vis spectros-copy. Finally the INP-C was then treated with 400 μl (4 mg) of folicacid solution which shows maximum loading and incubated at25 °C for 2 h with frequent vortexing. The folate conjugated INP-C(INP-CF) was then precipitated by raising the pH to 7 using 1 MNaOH solution. The precipitate was then separated by centrifugingat 10,000 rpm (7155 g) for 10 min, washed with 500 μl of distilledwater and dried at 37 °C (INP-CF). The sterilization of INP-CF wasdone by exposing to UV radiation for 2 h.

2.4. Characterization

Fourier transform infrared (FT-IR) analysis of the samples wasrecorded on a Nicolet 360 FT-IR spectroscope using KBr pellet. Thesize and morphology of the bionanocomposite were investigatedusing Tecnai 10, Philips Transmission electron microscope at an accel-erating voltage of 80 kV. The zeta potential of the uncoated and coatednanoparticles was analyzed using laser light scattering technique byMalvern Zetasizer (v2; SL.No. MAL1066495), UK. The MCF-7 cellswere cultured to carry out in vitro experiments such as cellular uptakeof Fe through folate receptor mediated endocytosis, superficial mor-phological observations using phase contrast microscopy, the cytoplas-mic distribution using SEM–EDX and MRI studies.

2.5. Cell culturing

The MCF-7 and NIH 3T3 cells were grown in folate-free DulbeccoModified Eagles Medium (FFDMEM) containing 10% fetal calfserum, 100 units/mL penicillin, 100 μg/mL streptomycin and 4 mML-glutamine, incubated at 37 °C in a 5% CO2/95% air humidifiedatmosphere.

2.6. In vitro MRI imaging, SEM–EDX and phase contrast microscopystudies

The MCF-7 cells were grown for 24 h in FFDMEM, followed by theaddition of INP, INP-C, and INP-CF solutions containing varyingconcentrations of Fe 50 μl (25 μg), 100 μl (50 μg), 150 μl (75 μg),200 μl (100 μg) and 250 μl (125 μg). After 24 h incubation, (untreatedand treated cells with: INP, INP-C, INP-CF were incubated at 37 °C andone set of INP-CF was incubated at 4 °C). A negative control wasconducted with NIH 3T3 cells using INP-CF solution containing varyingconcentrations of Fe, which was then incubated at 37 °C for 24 h. Thecells were viewed under phase contrast microscope and lifted usingSaline:Trypsin:Versene solution; centrifuged at 4000 rpm (2862 g)for 5 min and was washed thrice with 200 μL of phosphate bufferedsaline. Then each pellet was suspended in 500 μL of 1% agarose solutionfollowed by the measurement of spin–spin relaxation time T2*W MRimages for each sample using 1.5 T Avanto high field magnetic reso-nance image analyzer Siemens Erlanger, Germany. The imaging param-eters were given in our earlier publication [28]. One set of pellets wasused for SEM–EDX analysis.

3. Results

3.1. FT-IR analysis

The FT-IR spectra of INP, INP-C and INP-CF are presented inFig. 1A–C. The FT-IR spectrum of INP (Fig. 1A) exhibits a characteristicpeak at 564 cm−1 which confirms the presence of iron in oxide formmaghemite [γ-Fe2O3]; INP-C (Fig. 1B) the characteristic peaks ofchitosan that appear at 3038 to 3200 cm−1 represent\OH stretchingabsorption band, peak at 1645 cm−1 represents N\H stretchingvibration of amide I band, peak at 1003 cm−1 represents free primaryamino group at C2 position of chitosan molecule, bands at 1404 and1431 cm−1 represent C\O stretching of chitosan; INP-CF (Fig. 1C)characteristic absorption bands of folic acid were observed. Peaks at1606 cm−1 representing the benzene ring absorption, 1674 cm−1

representing ester bond and 1484 cm−1 showing the hetero ringand conjugated double bond confirm the presence of folic acid.

3.2. TEM analysis

TEM study (Fig. 2A and B) reveals the size and shape of theprepared INP-CF before and after coating. The INP-CF exhibits spher-ical shaped nanoparticles before coating with chitosan. After coating

Table 1Zeta potential of INP, INP-C and INP-CF.

S. No Sample Zeta potential (mv)

1 INP +5.3±0.42 INP-C +7.7±0.93 INP-CF +6.1±0.5

Fig. 1. A–C. FT-IR spectrum of INP, INP-C and INP-CF representing the presence ofcharacteristic functional groups and shift in peaks due to interaction between the func-tional groups of the molecules in chitosan.

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most of the nanoparticles seem to agglomerate forming nanoclustersprobably due to the magnetism and surface tension of the droplet onthe carbon coated TEM grid as it dries during preparation for imaging[28].

3.3. Zeta potential analysis

The charge on the surface of the coated and uncoated nanoparticleswas analyzed using Malvern Zetasizer. The zeta potential values ofINP, INP-C and INP-CF are given in Table 1. The zeta potential valuesof INPs are found to be increased after coating with chitosan and aslight decrease was observed after conjugating them with folic acid.

3.4. MRI analysis

In ourMRI in vitro detection experiments (Fig. 3A–F) the untreatedMCF-7 cells (A), nanobiocomposite treated cells: INP (B), INP-C (C)and INP-CF incubated at 4 °C (D) show no drop in signal anddarkening of pixels due to the absence of Fe (untreated MCF-7 cells)and very less uptake of Fe which is not detectable incase of INP,INP-C and INP-CF incubated at 4 °C. MCF-7 cells treated with INP-CFsolution incubated at 37 °C (F) containing varying concentration ofFe (25 μg to 125 μg/ml) have shown an increased drop in signal anddarkening of pixels with respect to concentration of cellular uptake

Fig. 2. A–B. TEM micrograph showing the spherical shaped INP-CF particles beforecoating (A) and after coating (B) formation of nanoclusters due to agglomeration.

of iron through the folate receptor mediated endocytosis. A negativecontrol experiment (E) conducted using NIH 3T3 cells treated withINP-CF solution containing varying concentrations of Fe showed ab-sence of drop in signal and darkening of pixels due to very less uptakeof Fe.

3.5. SEM–EDX analysis

SEM–EDX reveals the cellular uptake of Fe in INP, INP-C, INP-CF in-cubated at 4 °C and INP-CF incubated at 37 °C (Fig. 4). The untreatedcells show the absence of Fe but in the case of treated cells the uptakeof Fe was found to be: INP — 0.21 μg, INP-C — 12.87 μg, INP-CF incu-bated at 4 °C — 7.14 μg and INP-CF incubated at 37 °C — 46.25 μgwhich seem to be a high uptake when compared to INP, INP-C andINP-CF incubated at 4 °C and it may be due to the entry of thebionanocomposite through folate receptor mediated endocytosis. Incase of NIH 3T3 cells used as a negative control, treated with INP-CFand incubated at 37 °C showed a less uptake of Fe — 4.99 μg. This isdue to low level expression of folate receptor on the surface of theNIH 3T3 cells.

3.6. Phase contrast microscopy studies

MCF-7 and Hela cells express human folate receptor, which canundergo a cellular uptake of Methotrexate (MTX) conjugated to

Fig. 3. A–F. MRI T2*W images of MCF — 7 and NIH 3T3 cells. Untreated MCF 7 cells(A); bionanocomposite treated MCF 7 cells: INP (B), INP-C (C), INP-CF incubated at4 °C (D), INP-CF treated with NIH 3T3 cells, incubated at 37 °C (E) and INP-CF treatedwith MCF 7 cells, incubated at 37 °C (F) respectively containing varying concentrationof Fe (1–25 μg, 2–50 μg, 3–75 μg, 4–100 μg and 5–125 μg/ml). A–E show no drop in sig-nal and darkening of pixel due to the absence of uptake of Fe by the cells and F showshigh drop in signal and darkening of pixel due to the uptake of Fe through folate receptormediated endocytosis.

Fig. 4. A–F. SEM–EDX images of MCF-7 and NIH 3T3 cells. Untreated cells (A) show theabsence of Fe; MCF 7 and NIH 3T3 treated cells (B–F) show the cellular uptake of Fe:INP (B), INP-C (C), INP-CF incubated at 4 °C (D) and NIH 3T3 cells (E) show lesser up-take of Fe. The INP-CF treated MCF 7 cells, incubated at 37 °C (F) show the maximumuptake of Fe when compared with that of controls respectively.

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magnetic nanoparticles for providing apoptosis in diseased cells [29].In our study, an entry through folate receptor was observed throughphase contrast microscopy images of cells with high expression of fo-late receptors (MCF-7) and with normal cells which have low

Fig. 5. A–D. Phase contrast microscopy images of untreated (A) and INP-CF treated MCF 7 (Band the thick arrow ( ) indicates INP-CF cellular uptake. Inset in B shows the magnifieduntreated (C) and INP-CF treated NIH 3T3 (D) control cells show live adherent cells withou

expression of folate receptors (NIH 3T3). Untreated MCF-7 cells(Fig. 5A) show live adherent cells, whereas INP-CF treated MCF-7cell lines (Fig. 5B) show live adherent cells interacting withnanobiocomposite. The NIH 3T3 cells are used as a negative controlfor the experiment which lacks the over expression of folate receptorswhen compared with folate receptor over expressing cells [16]. Theuntreated NIH 3T3 (Fig. 5C) and INP-CF treated NIH 3T3 (Fig. 5D)cells show live adherent cells without any interaction on the surfaceof the cells. This shows that the nanobiocomposite may be enteredthrough folate surface receptor expressed on their surfaces and thisphenomenon was also supported by SEM–EDX and MRI analyses.

4. Discussion

The molecular interactions of the prepared INPs after coating withchitosan coupled folic acid were studied from the FT-IR spectral dataobtained. The iron oxide exhibits strong bands in the low frequencyregion 1000–500 cm−1 due to the iron oxide skeleton. This patternis consistent with the maghemite [γ-Fe2O3] spectrum (broad bandat 520–610 cm−1) [30]. In our study, the Fe\O stretching vibrationband confirms that the prepared INPs were maghemite. The coatingof the chitosan on INPs was confirmed by the presence of the NHstretching, since the natural polymer chitosan provides a free aminoterminal for the coupling of the carboxylic acid terminal present inthe folic acid. The folic acid coupling as a ligand with chitosan wasfurther confirmed with a band which shows a characteristic absorp-tion for benzene ring and conjugated double bonds. This IR data re-veals the coating of folic acid conjugated chitosan onto the INPs. Onthe basis of the results obtained for the coated INP-CF, it proves thatthe prepared particles were nanosized with spherical shape. Butmost of the nanoparticles are agglomerated forming nanoclustersprobably due to the magnetism and surface tension of the dropleton the carbon coated TEM grid as it dries during preparation for imag-ing [19]. The surface charge of nanocomposite is important because it

) cells. The thin arrow (→) indicates INP-CF interacting to the surface of folate receptorimage of a particular area of INP-CF treated MCF-7 cells and incubated at 37 °C. Thet any interaction on the surface of the cells.

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correlates with the stability of composite suspension and its interac-tion with cellular membrane. The zeta potential of INPs was foundto be positive and increased with the coating of chitosan (INP-C).This may be due to the NH3

+ ions on the surface of chitosan. However,INP-CF has exhibited lesser zeta potential, because of the carboxylgroups in the folic acid [31,32].

A high performance MR imaging can be produced by a target spe-cific MRI contrast agent with unique superparamagnetism. In thenanobiocomposite, the folic acid was used as a target for folate recep-tor and γ-Fe2O3 with unique paramagnetism helped in achieving anenhanced T2*W image of treated MCF-7 cells. Appearance of darkerpixels and a substantial MRI drop in signal in general have generateda darkening contrast in T2*W images as reported in literature [19,33]and a similar effect was reported in MRI study with respect to in-crease in concentration of Fe. The cellular uptake of INP-CF (incubatedat 37 °C) was high when compared to that of INP, INP-C and INP-CFincubated at 4 °C. A drop in signal with appearance of darker pixelsis only seen in INP-CF incubated at 37 °C which may be due to theincreased uptake of bionanocomposite through folate receptor medi-ated endocytosis. In case of MCF-7 cells incubated with INP, INP-C andINP-CF at 4 °C it shows the absence of signal and darkening of pixelsdue to lesser amount of Fe uptake which is not detectable throughMRI scanning methods. The SEM–EDX provides the evidence for thecellular uptake of INP-CF solution, which reveals that the folic acidis a potential carrier for folate receptor expressing cancer cells. Thisuptake is attributed to the targeting effects of folate receptor [17].The EDX analysis confirms that the major uptake of Fe was found infolic acid containing preparation rather than folic acid without. Thisconfirms that the cellular uptake of the bionanocomposite may bedue to receptor mediated rather than non-specific pinocytosis. Thephase contrast microscopy observation shows slight morphologicalchange on the surface of the cell lines when they were treated withINP-CF containing 125 μg Fe for 24 h (incubated at 37 °C), whichreveals the evidence for the internalization of INP-CF particles. TheINP, INP-C and INP-CF incubated at 4 °C were conducted as a controlexperiment to prove that the entry of INP-CF (incubated at 37 °C) wasmainly through folate receptor. The MCF 7 cells treated with INP-CFand incubated at 4 °C were mainly used as a control since the uptakeof INP-CF into cells through folate receptor is arrested at low temper-ature. This is because the entry of the particles through receptor is anenergy mediated process which cannot occur during the incubation atlow temperature. The targeted transportation of INP-CF into the cellsvia folate receptor using energy source will be produced duringincubation at 37 °C which was proved using MCF 7 cells. A negativecontrol experiment was conducted using NIH 3T3 cells as a supportiveexperiment to confirm that the entry of particles is through folatereceptor. The results obtained have proved that the presence of Fewithin cells after incubation with INP-CF at 37 °C has occurred inMCF 7 cells and is mainly due to over expression of folate receptorwhich is comparatively higher than that in NIH 3T3 cells [16]. The con-trol experiment results suggested that the uptake of nanobiocompositewas mainly through the folate receptor rather than non-specificpinocytosis [34].

In conclusion, the INP-CF bionanocomposite prepared in thisstudy was able to internalize into the cancer cells (MCF-7) and thisphenomenon was confirmed by the MRI and SEM–EDX analysis.This novel composite containing INPs isolated from natural sourcemay be used as a MRI contrast agent and as receptor targeting agentfor cancer treatment. This nanobiocomposite when coupled with sub-stances such as monoclonal antibodies and chemotherapeutic agentsmay act as a theranostic nanoagent for cancer therapy in the years tocome. Thus prepared nanobiocomposite using natural substances asraw material may act as multifunctional agent by incorporating thenecessary therapeutics to achieve desired functions by adapting sim-ple technique when compared with the other commercially availablepreparations.

Acknowledgement

The authors would like to thank Mr. Sridharan and KhemlalAdikarai, Technical assistants of St. Joseph's College of Engineering,Chennai for helping in the collection of goat blood.

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