Cellular internalization and detailed toxicity analysis of protein-immobilized iron oxide nanoparticles Purva Sanganeria,1 Shilpee Sachar,2* Sudeshna Chandra,3 Dhirendra Bahadur,3 Pritha Ray,4 Aparna Khanna1 1
Department of Biological Sciences, School of Science, NMIMS University, Vile Parle (West), Mumbai 400056, India Department of Chemical Sciences, School of Science, NMIMS University, Vile Parle (West), Mumbai 400056, India 3 Department of Metallurgical Engineering and Materials Science, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India 4 Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Navi, Mumbai 410210, India 2
Received 31 December 2013; revised 26 March 2014; accepted 5 April 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33178 Abstract: Iron oxide nanoparticles (IONPs) have been extensively used for biomedical applications like in the diagnosis and treatment of various diseases, as contrast agents in magnetic resonance imaging, and in targeted drug delivery. Despite several attempts, there is a dearth of information with respect to the cellular response and in-depth toxicity analysis of the nanoparticles. Considering the potential benefits of IONPs, there is a need to study the potential cellular damage associated with IONPs. The size and surface of the particles are some critical factors that should be analyzed when evaluating cytotoxicity. Therefore, in this study, we synthesized and characterized bare (7–9 nm) and protein-coated IONPs of diameter 50–70 nm, and evaluated their toxicity on membrane integrity, intracellular accumulation of reactive oxygen species, and mito-
chondrial activity in mouse fibroblast cell line by lactate dehydrogenase, 20 ,70 -dichlorofluorescein diacetate, and [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assays, respectively. Our extensive cytotoxicity analysis demonstrated that the size of the IONPs and their surface coating are the critical determinants of cellular response and potential mechanism toward cytotoxicity. The study of the interactions and assessment of potential toxicity of the nanoparticles with cells/tissues is a key determinant when considering their transC 2014 Wiley Periodicals, Inc. J lation in biomedical applications. V Biomed Mater Res Part B: Appl Biomater 00B: 000–000, 2014.
Key Words: nanomaterials/nanophase, protein adsorption, cell culture, surface characterization, FTIR
How to cite this article: Sanganeria P, Sachar S, Chandra S, Bahadur D, Ray P, Khanna A. 2014. Cellular internalization and detailed toxicity analysis of protein-immobilized iron oxide nanoparticles. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Magnetic nanoparticles are of interest in various fields of biomedicine because of their unique physical and chemical properties. In a clinical setting, the requirement for cellular tracking is extremely important to understand the biodistribution and homing of cells in the human body. Hence, an urgent need exists to develop noninvasive and sensitive imaging techniques, which will prove valuable for optimizing cell therapy. In particular, iron oxide nanoparticles (IONPs), a negative contrast agent in magnetic resonance imaging, have been utilized for various biomedical applications, such as cancer imaging, cellular labeling, and magnetofection.1,2 Because of their hydrophobic surface, instability in fluids, and inefficient prolonged retention with cells, their use in long-term cell tracking is limited. It has been reported
that surface modifications of magnetic nanoparticles can significantly improve their stability, biocompatibility, and shelf life.3 Various researchers have reported modifications of IONPs surface with diverse moieties,4 which can be categorized as: bifunctional surfactants like distearoyl-sn-glycero3-phospho ethanolamine-N-[carboxy (polyethylene glycol) 2000] and biotin5,6 inorganic coatings like silica and gold.7,8 Silica-coated IONPs are commercially available as 9 R and NanothermV R , which are currently used for FerumoxilV 10 hyperthermia therapy and have also proven to be among the promising nanomaterials for cell tracking. Gold-coated IONPs are commercially applied in drug delivery and immobilizing IgG for detection of hepatitis B antigen in blood sample; however, studies have proven that gold coating weakens the inherent magnetic properties of these nanoparticles and hence limits their use as an imaging modality.
Additional Supporting Information may be found in the online version of this article. Correspondence to: A. Khanna (e-mail: [email protected]) *Present address: Shilpee Sachar, Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai 400 098, India Contract grant sponsor: Department of Science and Technology, Government of India, under the “Nanomission” scheme; contract grant number: SR/NM/NS-1114/2011(G)
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Similarly, organic monomeric coatings like lauric acid, citric acid, gluconic acid, dimercaptosuccinic acid, oleic acid, and mercaptopropionic acid11–13 have shown cellular toxicity.14 Research has also been done on using organic polymers like dextran, polyethylene glycol, chitosan, and alginate as coating materials for IONPs.4,9,15 Dextran-coated IONPs were R , FeridexV R , and extensively used commercially as EndoremV 4,7 R for magnetic resonance imaging. ResovistV However, studies later revealed their toxic effects on the cells in terms of DNA damage and oxidative stress,8,14 which was found to be due to their poor retention on the surface of nanoparticles. Inspite of several attempts made in the past to overcome the limitations of using IONPs for biomedical applications, the need of a suitable coating with desired stability and biocompatibility still exists. The focus of the present study was to synthesize IONPs using optimal coatings like surfactants and proteins in order to achieve the desired properties. Along with the biocompatibility, the protein surface of IONPs will significantly increase the cellular uptake of these particles. In the present study, the synthesized IONPs [bare, cetyl trimethylammonium bromide (CTAB)-coated, and bovine serum albumin (BSA) and human serum albumin (HSA) coated nanoparticles] were compared with the commercially available polyvinylpyrrolidone-coated IONPs. Further, a dosedependent evaluation of cytotoxicity of the BSA- and HSAcoated nanoparticles was undertaken. This has utmost significance if the nanoparticles are intended to be translated from laboratory research to clinical applications.
thoroughly with ethanol and subjected to magnetic separation using a permanent magnet. Protein coating of IONPs CTAB-coated IONPs were stirred with different concentrations of proteins (BSA and HSA) in phosphate buffer (pH 7.0). The solutions were incubated for 24 h at 4 C. The colloidal stability of each solution was measured and was found to be maximum for the formulation containing proteins and IONPs in the ratio of 1000:62.5 mg. Particle characterization Crystalline structure by X-ray diffraction. The crystal structure of the synthesized IONPs and CTAB-coated IONPs was obtained by analyzing the X-ray diffraction (XRD) pattern of each sample recorded using XRD (PANalytical X’Pert Pro, with Cu Ka radiation, k 5 1.54 Å). Size analysis by transmission electron microscopy. The size and morphology of the synthesized nanoparticles was analyzed by transmission electron microscopy (TEM) using JEOL, Phillips CM 200. The sample specimen was prepared by dispersing the CTAB-coated IONPs in Milli-Q water followed by 5 min sonication. A drop of well-dispersed sonicated sample was drop cast on a carbon-coated copper grid, followed by drying the sample under suitable conditions. Functional group detection by Fourier transform infrared spectroscopy. The Fourier transform infrared (FTIR) spectrum was analyzed using JASCO, FT-IR 300E spectrometer, and the spectrum was studied from 4000 to 400 cm21.
MATERIALS AND METHODS
Synthesis of CTAB-coated IONPs Ferric chloride hexahydrate (FeCl36H2O) and ferrous chloride tetrahydrate (FeCl24H2O) were purchased from Sigma Aldrich (St. Louis, MO). CTAB and hydrazine hydrate (NH2NH2) were procured from Thomas Baker (India). BSA and HSA were purchased from Sigma. All chemicals were of analytical grade and were used as received. Surfactant-coated magnetic nanoparticles were synthesized by co-precipitation method as described earlier,16 where 0.2M FeCl36H2O and 0.15M FeCl24H2O were dissolved in 2:1 ratio in 80 mL of de-ionized water in a roundbottom flask, and the temperature was gradually increased to 80 C in nitrogen atmosphere with constant mechanical stirring at 1000 rpm. The temperature was initially maintained at 80 C for 30 min, and subsequently, the addition of hydrazine hydrate was done drop wise till a black precipitate formation was observed. The reaction mixture was again stirred at same conditions for 30 min, which was then followed by the addition of CTAB at various concentrations above and below critical micellar concentration (cmc 5 0.8 mM) viz. 0.4, 1.0, 1.5, 2.0, and 2.5 mM. The concentration of 1.5 mM was optimized for further studies. The reaction temperature was slowly raised to 90 C, and the reaction mixture was allowed to stir continuously for 60 min. The CTABcoated black precipitate was obtained by cooling the reaction mixture at room temperature, which was further rinsed
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Surface charge detection by f-zeta-potential measurements. Surface charge analysis of the CTAB-coated IONPs was performed in distilled water at various pH (pH 2–10) using Zeta Plus zeta potential analyzer (Brookhaven Instrument) at room temperature. Magnetization analysis by vibrating sample magnetometer. Magnetic properties of IONPs and CTABcoated IONPs were analyzed using a vibrating sample magnetometer (Lakeshore Model No. 7410, USA), which measures the magnetization of applied magnetic field (02T) at room temperature. Cell culture conditions Mouse embryonic fibroblast cell line, NIH 3T3, was obtained as a gift from IIT, Mumbai, and was used for cytotoxicity analysis. The doubling time for NIH 3T3 cell line used was observed to be 16–18 h for all the experiments in standard culture conditions. NIH 3T3 was chosen as controls for this study as they represent a standard and sensitive cell line. NIH 3T3 mimics the stromal cells found in embryonic tissue and hence represents a good model system to evaluate early toxicity. The cell line was maintained in high-glucose Dulbecco’s modified eagle medium (Cell Clone, Genetix) supplemented with 10% fetal bovine serum (Cell Clone, Genetix) and 1% penicillin, streptomycin, and amphotericin (Sigma)
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with media change at regular intervals. The cells were trypsinized using 0.25% trypsin-1 mM ethylenediaminetetraacetic acid, and a split ratio of 1:3 was followed at a subconfluency of 70%–80%. The cytotoxicity analysis was done using MTT reagent (Himedia, cell culture tested), dimethyl sulfoxide (DMSO) (MP Biomedicals, SDFCL), lactate dehydrogenase (LDH) assay (Roche, USA), and 20 ,70 -dichlorofluorescein diacetate (DCFDA) assay (Abcam). All chemicals, reagent, media, buffer, and plastic ware used were of cell culture grade (Cell Clone, Genetix). Cell exposure to IONPs and uptake studies For uptake studies, cells were seeded at a density of 5 3 105 cells/well in six-well plate and were maintained in incubator with 5% CO2 at 37 C. As the nanoparticle concentration of 60 mg/mL was found to be colloidally stable, it was used for further experiments. The uptake studies were done at 3, 6, 12, and 24 h intervals. Time-dependent cellular uptake studies were done for all synthesized nanoparticles (bare, CTAB-coated, and protein-coated IONPs) along with the commercial IONPs (Sigma). Prussian blue staining was performed for analyzing the internalization of nanoparticles. Cells were fixed using 4% paraformaldehyde solution for 40 min followed by Dulbecco’s phosphate buffered saline (DPBS) wash. The wells were then treated with Perl’s reagent (4% potassium ferrocyanide/12% HCl, 1:1 v/v) for a period of 30 min, with a further DPBS wash to remove the excess stain. The cells were then counterstained using 0.02% neutral red. The internalization of particles was analyzed using Carl Zeiss phase contrast microscope. Cellular viability analysis Mitochondrial impairment analysis by MTT assay. For MTT assay, 80%–90% confluent flasks were trypsinized using prewarmed 3–4 mL 0.25% trypsin–EDTA for a period of 5 min. Further cells were added to 96-well plate, with a cell count of 1 3 105 cells/well and kept overnight. After the addition of nanoparticles, 100 mL of MTT (0.5 mg/mL) was added to each well. The cytotoxicity analysis was done at different time points such as 24, 48, and 72 h. At each time point, 200 mL of DMSO was added to the treated cells. The readings were analyzed using 570-nm filter in an enzymelinked immunosorbent assay reader, and the percentage cytotoxicity was calculated using Eq. (1). The experiments were performed in triplicates. Control well using only media and fetal bovine serum were also included. % Cell Viability 5 Abs sample =Abs control 3100
(1)
Membrane leakage analysis by LDH assay. Membrane integrity after IONPs uptake was studied in terms of the extent of LDH production using LDH assay. After the IONPs uptake, the study was performed at an interval of 72 h in a 96-well plate in accordance with the kit guidelines using 490-nm filter in an enzyme-linked immunosorbent assay
reader. The studies were done for all the synthesized IONPs in comparison with commercial IONPs. Cytotoxicity was measured using the basal LDH released from untreated control cells. All the experiments were performed in triplicates. Reactive oxygen species formation by DCFDA assay. Production of reactive oxygen species (ROS) and hence promoting endogenous oxidative stress is reported in IONPs.17,18 Surface modifications of nanoparticles can minimize the ROS production and hence can reduce nanoparticleinduced cytotoxicity. In the present study, for the synthesized IONPs, the extent of ROS generation was studied by the DCFDA assay (a kit-based method). Intracellular ROS generation was determined by using the cell permeating reagent DCFDA, cell permeable compound, and a fluorescein dye that measures the hydroxyl, peroxyl, and other ROS activities within the cell. On penetrating the cell, it gets deacetylated by cellular esterases to a nonfluorescent compound, which is later oxidized by ROS into 20 ,70 -dichlorofluorescein and can be further analyzed by flow cytometry analysis. The cells were grown in a six-well plate at a density 5 3 104 cells/well. Culture media was replaced with 200 mL of IONPs at a concentration of 60 mg/mL for 3 h (optimized time for IONPs uptake), and ROS generation was evaluated at 72 h. After internalization, the cells were harvested using 0.5% trypsin for 5 min and were pelleted down at 1200 rpm for 7 min at 20 C. The debris was removed by 13 DPBS wash. After decanting phosphate buffered saline, 300–400 mL of DPBS was subsequently added. The cells were then incubated with 2 mM DCFDA dye (20 mM in DMSO) at 37 C for 30 min in dark. The ROS formation was analyzed using flow cytometry (FACS Calibur, BD Biosciences), and approximately 10,000 events/sample were analyzed after gating. RESULTS
Particle characterization using XRD, VSM, TEM, and high-resolution TEM Figure 1 shows the XRD pattern of the synthesized bare and CTAB-coated IONPs. The pattern clearly indicates the formation of magnetite Fe3O4 with the presence of characteristic peaks corresponding to planes (220), (311), (400), (422), (511), and (440). Figure 2(a,b) TEM and highresolution TEM shows that the synthesized CTAB-coated IONPs are spherical in shape. The size of IONPs (200 particles) was calculated using Image J software, and it was found to be uniform in the range of 7–9 nm (polydispersity index was low [see inset of Figure 2(a)]. The selected area electron diffraction pattern recorded for the synthesized IONPs was found to be similar to the reflections of inverse spinel structure of Fe3O4 [see inset of Figure 2(b)]. Figure 3 illustrates the hysteresis loop of synthesized IONPs. The CTAB-coated IONPs demonstrated superparamagnetic behavior with negligible remanence and coercivity in the hysteresis loop. The maximum magnetization of CTABcoated IONPs was found to be 45 emu/g at 20 kOe, which was slightly lower than that of the bare IONPs (54.7 emu/ g). It shows that the inherent magnetic property was retained even after surfactant coating. Figure 4(a) shows
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tein alone at a pH range 2–10. Isoelectric point (pI) of protein was recorded as 4.0. Protein bears a positive surface charge below pI and negative surface charge above pI. A zeta potential value of protein alone was found to be 20 mV at pH 2 and 229 mV at pH 10. The pI value of bare IONPs was calculated to be 6.8, and similarly its zeta potential values are 40 mV at pH 2 and 225 mV at pH 10.19 Figure 6 shows the protein coating on surfactant-functionalized IONPs, which was indicated from FTIR plots. Free unbound HSA shows a broad peak centered at 3450 cm21 owing to free carboxylic acid groups. HSA-coated IONPs exhibited a peak at 1655 cm21 (amide I), 1538 cm21 (amide II), and 1451 cm21 (amide III).
FIGURE 1. XRD patterns of (a) bare IONPs and (b) CTAB-coated IONPs.
the TEM micrographs of the protein-coated IONPs. The spherical-shaped protein-coated IONPs were analyzed using Image J software and were found to be in the size range of 50–70 nm [see inset of Figure 4(a)]. Figure 4(b) shows that the selected area electron diffraction pattern analyzed for the protein-coated IONPs reflects the crystalline nature of IONPs even after protein coating. The hydrodynamic diameter analyzed by the dynamic light scattering spectra demonstrated a single peak, showing the particle size to be 86 nm [inset Figure 4(b)]. The protein-coated IONPs were found to have colloidal stability for more than 6 months.
Surface charge and protein coating evaluation using zeta potential and FTIR Figure 5 shows the surface charge analysis of the proteincoated IONPs in comparison with the bare IONPs and pro-
Internalization of various IONPs by Prussian blue staining Figure 7 shows time-dependent cell uptake of synthesized nanoparticles: commercial, bare IONPs, CTAB-coated IONPs, and BSA- and HSA-coated IONPs. Prussian blue stained cells showed deep blue granules in the cytoplasm of cells treated with IONPs due to cytoplasmic accumulation of iron deposits. Further, no staining in the nucleus was seen. In vitro cytotoxicity analysis using MTT, LDH, and DCFDA assays Figure 8(a–c) shows the results of 24, 48, and 72 h MTT assay, indicating consistent cellular viability of >95% for protein-coated IONPs for a concentration of 60 mg/mL over the regular time intervals of 24, 48, and 72 h, whereas in case of CTAB-coated IONPs the viability decreased slightly from 92% for first 48 h to 90% in 72 h. Whereas, a clear decline in cell viability in case of commercial IONPs (54% for 24 h, 24% for 48 h, and 23% for 72 h) was observed, which shows that the synthesized IONPs with surfactant and protein functionalization are more biocompatible. Results are represented as 62 SD of the mean. Figure 9
FIGURE 2. (a) TEM micrograph of CTAB-IONPs. (b) high-resolution TEM of CTAB-IONPs. Inset 2(a): size distribution of CTAB-IONPs. Inset 2(b): electron diffraction pattern of CTAB-IONPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 3. M versus H plots of (a) bare IONPs and (b) CTAB-coated IONPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
shows the results of the 72 h LDH assay, which were found to be in accordance with the MTT results and showed maximum cellular viability for protein-coated IONPs (>80%) in comparison with commercial IONPs (60%) and bare IONPs (50%). Results are represented as 62 SD of the mean. Figure 10(a–f) shows the results of 72 h DCFDA assay and the amount of ROS produced in response to the uptake of commercial IONPs and synthesized IONPs with different surface modifications. Highest shift in the side scatter intensities was observed for commercial and bare IONPs that indicate maximum increase in internal density that can lead to morphological alterations. However, protein-coated IONPs showed a slight shift in side scatter intensities showing tolerable morphological alterations. DISCUSSION
In this work, we present the synthesis and characterization of surface-modified IONPs in terms of their crystallinity, its magnetic behavior, and colloidal stability. Further, detailed
cytotoxicity analysis by MTT, LDH, and DCFDA assays was done using NIH 3T3 (mouse embryonic fibroblast cell line). IONPs were synthesized using co-precipitation method. The XRD pattern of bare and CTAB-coated IONPs shows the presence of intense peaks, which confirmed the formation of highly crystalline nanoparticle. Also, magnetization loops of bare and CTAB-coated IONPs demonstrated slight decrease in magnetic property of CTAB-coated IONPs, but even after surface modification the magnetic behavior of the particle was found to be persistent. The presence of CTAB molecules over the surface of bare IONPs provides hydrophilicity without affecting the crystallinity and magnetization of the IONPs. The different characterization techniques presented in this work demonstrate average particle size of 7–9 nm of CTAB-coated IONPs and 50–70 nm for proteincoated IONPs with spherical morphologies, and hence an appropriate size for future biological applications. Uncoated nanoparticles have a tendency to aggregate. In order to overcome this, the surface of nanoparticles is coated with the proteins.11 Surface modifications affect the cell viability and increase the stability of the nanoparticles once internalized.20,21 The coating also helps in prolonged retention of the nanoparticles within the cells and thus increases its efficiency as contrast agent for long-term tracking.22,23 From the zeta plots, the zone between pH 4.0 and pH 7 was considered to be the interaction zone, as both the moieties were bearing opposite surface charges giving rise to surface adsorption and hence colloidally stable solution of protein-coated IONPs. The FTIR spectra of protein-coated IONPs revealed change of protein structure from a-helices to b-sheets due to immobilization.24 The important observation here is the presence of functional characteristics peak of high intensity at 1638 cm21 (amide I) in HSA-modified CTAB-coated IONPs25,26 which in turn indicates the fact that the functionality of immobilized HSA in comparison with free HSA is maintained. The Prussian blue staining revealed certain morphological alterations in the cells at 24 h interval of incubation in commercial as well as bare IONPs. However, the synthesized protein-coated IONPs did not show any such morphological
FIGURE 4. (a) TEM micrograph of protein-IONPs. (b) The selected area electron diffraction. Inset 4(a): protein corona around IONPs and size distribution of protein-coated IONPs. Inset 4(b): dynamic light scattering spectra. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 5. Zeta plots. Pictorial representation of surface charge and colloidal stability in solutions with various nanoparticle concentrations. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
alterations even after 24 h.27,28 The incubation period of 3 h was optimized for further cytotoxicity analysis. Because it has been earlier reported that IONPs have shown to generate cytotoxicity,29,30 detailed evaluation was
FIGURE 6. FTIR spectra of bare IONPs, HSA, and HSA-coated IONPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
done. The cell line used for cytotoxicity studies is NIH 3T3, which represents a mouse embryonic fibroblast cell line mimicking the stromal cells present throughout the connective tissue in the body31 and hence was used in the study. Various causes of cellular toxicity leading to cell death can
FIGURE 7. Prussian blue staining pictographs showing various IONPs internalization at 3, 6, 12, and 24 h, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 8. MTT plots showing toxicity analysis performed on mouse fibroblast cell line NIH 3T3 for 24, 48, and 72 h. Results are represented as 62 SD of the mean. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
be attributed to lysosomal destabilization, loss of mitochondrial membrane integrity,32–34 or membrane leakage. IONPs are known for the formation of ROS species in the acidic environment that results in the formation of free ions (Fe21 and Fe31). These free ions interact with H2O2 to generate hydroxyl radicals (Fenton reaction), which leads to oxidative stress indirectly causing cell death by damaging protein and DNA, which was evaluated by DCFDA assay.19
It is important to first evaluate and study the effects of commercial and synthesized IONPs in a time- and dosedependent manner by MTT and LDH assay. The results of MTT assay indicated consistent cellular viability of >95% for protein-coated IONPs for a concentration of 60 mg/mL after 72 h, whereas in case of CTAB-coated IONPs, the viability decreased slightly (2%) in a duration of 72 h. However, substantially low cellular viability of 23% was
FIGURE 9. LDH plots showing membrane leakage analysis performed on mouse fibroblast cell line NIH 3T3 at 72 h. Results are represented as 62 SD of the mean. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 10. DCFDA assay: (a–e) Scatter plots and peak histograms; (f) Histogram showing ROS generation of various IONPs for 72 h. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
observed in case of commercial IONPs. Cell viability evaluation at 24 and 48 h has demonstrated no cytotoxicity with protein-coated IONPs. LDH assay was performed to check whether LDH, a stable cytosolic enzyme in normal cells, can leak into the extracellular fluid only after the membrane damage and thus can be an effective measure to check the cytotoxicity.35 The results of the LDH assay were found to be in accordance with the MTT results and showed maximum cellular viability for protein-coated IONPs (>80%). The prominent decrease in cellular viability of commercial IONPs seen in LDH as well as MTT assay in comparison with proteincoated IONPs shows the significant role played by protein coating over IONPs. Various reports36,37 showed that uncoated particles induce greater toxicity than coated IONPs. A significant increase in LDH release was observed in 72 h for commercial and bare IONPs showing only 60%
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and 50% viability, respectively, whereas protein-coated IONPs consistently showed higher viability (>80%). Therefore, protein surface functionalization of nanoparticles helps in regulating IONPs interfacial properties by altering the hydrophobic nature of bare IONPs to hydrophilic by imparting ANH2 groups, which plays a vital role in interactions with biological systems.19 Literature reveals that flow cytometry has been widely applied in quantifying the cellular uptake of nanoparticles.38,39 However, less attention has been paid to the use flow cytometry in examining ROS generation in nanoparticle-induced cytotoxicity analysis. In the present study, along with the cellular uptake we have performed flow cytometry analysis in comparing the extent of ROS generation in bare and surface-modified IONPs. In the scatter plots, the side scatter depicts the enhanced intracellular granularity, and the forward scatter indicates the cell size.
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An increase in the side scatter is an indicator for the uptake of IONPs and intracellular density.40 Subsequent increase in the side scatter of the cells was observed in comparison with control plots, which signifies the increase in ROS production due to nanoparticle internalization. Highest shift in the side scatter intensities was observed for commercial and bare IONPs, which indicates maximum increase in internal density that can lead to morphological alterations, which can also be observed in the Prussian blue staining experiments (72 h) for the same systems. With surfactant and further protein coating, the ROS generation was not minimized; however, we observed a decrease in the mean fluorescent intensity in comparison with commercial and bare IONPs. Keeping in view the observations of flow cytometry analysis along with MTT and LDH assay analysis, we can conclude that internalization of protein-modified IONPs exhibits tolerable complexity alteration and reasonable intracellular ROS formation causing no harm to the proliferative capacity of the cells. SUMMARY
Colloidally stable surfactant and protein coated IONPs were synthesized and compared with bare and commercially available IONPs in terms of their cellular interactions and toxicity effects. Detailed in vitro cytotoxicity analysis in terms of mitochondrial impairment, membrane leakage, and ROS generation suggests the potential of HSA-coated IONPs as a suitable biocompatible coating for long-term biomedical applications such as cell/stem cell tracking. Studies are underway to understand whether HSA-coated nanoparticles affect viability, proliferation, and differentiation potential of stem cells isolated from human umbilical cord. Further, the potential of the synthesized biocompatible IONPs for stem cell labeling and tracking will be evaluated. ACKNOWLEDGMENTS
The authors acknowledge Ms. Snehal Gaikwad (ACTREC) for assisting in the flow cytometry analysis. REFERENCES 1. Nigam S, Barick KC, Bahadur D. Development of citrate stabilized Fe3O4 nanoparticles: Conjugation and release of doxorubicin for therapeutic applications. J Magn Magn Mater 2011;323:237–243. 2. Yang C, Wu J, Hou Y. Fe3O4 nanostructures: Synthesis, growth mechanism, properties and applications. Chem Commun 2011;47: 5130–5141. 3. Busch W, Bastian S, Trahorsch U, Iwe M, K€ uhnel D, Meißner T, Springer A, Gelinsky M, Richter V, Chrysanthy I. Internalisation of engineered nanoparticles into mammalian cells in vitro: Influence of cell type and particle properties. J Nanopart Res 2012;13:293– 310. 4. Mailander V, Landfester K. Interaction of nanoparticles with cells. Biomacromolecules 2009;10:2379–2400. 5. Xu C, Sun S. Monodisperse magnetic nanoparticles for biomedical imaging. Polym Int 2007;56:821–826. 6. Sperling RA, Parak WJ. Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles. Phil Trans R Soc A 2010;368:1333–1383. 7. Markides H, Rotherham M, El Haj AJ. Biocompatibility and toxicity of magnetic nanoparticles in regenerative medicine. J Nanomater 2012;2012:1–11.
8. Mahmoudi M, Hofmann H, Rutishauser BR, Fink AP. Assessing the in vitro and in vivo toxicity of superparamagnetic iron oxide nanoparticles. Chem Rev 2012;112:2323–2338. 9. Mahmoudi M, Sant S, Wang B, Sen T. Superparamagnetic iron oxide nanoparticles (SPIONS): Development, surface modification and applications in chemotherapy. Adv Drug Deliv Rev 2011;63: 24–46. 10. Laurent S, Mahmoudi M. Superparamagnetic iron oxide nanoparticles: Promises for diagnosis and treatment of cancer. Int J Mol Epidemiol Genet 2011;2:367–390. 11. Safi M, Berret JF. The role of the coating and aggregation state. Phys Procedia 2010;9:266–269. 12. Pisanic TR II, Blackwell JD, Shubayev VI, Finones RR, Jin S. Nanotoxicity of iron oxide nanoparticle internalization in growing neurons. Biomaterials 2007;28:2572–2581. 13. Pradhan P, Jyotsnendu G, Samanta G, Sarma HD, Mishra KM, Bellare J, Banerjee R, Bahadur D. Comparative evaluation of heating ability and biocompatibility of different ferrite based magnetic fluids for hyperthermia application. J Biomed Mater Res Part B: Appl Biomater 2006;81:12–22. 14. McCarthy JR, Weissleder R. Multifunctional magnetic nanoparticles for targeted imaging and therapy. Adv Drug Deliv Rev 2008; 60:1241–1251. 15. Wilhelm C, Billotey C, Roger J, Pons JN, Bacri JC, Gazeau F. Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating. Biomaterials 2003;24:1001–1011. 16. Ling D, Hyeon T. Iron oxide nanoparticles: Chemical design of biocompatible iron oxide nanoparticles for medical applications. Small 2012;9:1–17. 17. Karlsson HL, Gustafsson J, Cronholm P, Moller L. Size dependent toxicity of metal oxide particles—A comparison between nano and micrometer size. Toxicol Lett 2009;188:112–118. 18. Papanikolaou G, Pantopoulos K. Iron metabolism and toxicity. Toxicol Appl Pharmacol 2005;202(2):199–211. 19. Peng ZG, Hidajat K, Uddin MS. Adsorption of bovine serum albumin on nanosized magnetic particles. J Colloid Interface Sci 2004; 271:277–283. 20. Singh N, Jenkins GJS, Asadi R, Doak SH. Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION). Nano Rev 2010;1:1–15. 21. Hu L, Mao Z, Gao C. Colloidal particles for cellular uptake and delivery. J Mater Chem 2009;19:3108–3115. 22. Varna M, Ratajczak P, Ferreira I, Leboeuf C, Bousquet G, Janin A. In vivo distribution of inorganic nanoparticles in preclinical models. J Biomater Nanobiotechnol 2012;3:269–279. 23. Mahmoudi M, Laurent S, Shokrgozar MA, Hosseinkhani M. Toxicity evaluations of superparamagnetic iron oxide nanoparticles: Cell “vision” versus physicochemical properties of nanoparticles. ACS Nano 2011;5:7263–7276. 24. Mikhaylova M, Kim DK, Berry CC, Zagorodni A, Toprak M, Curtis ASG, Muhammed M. BSA immobilization on amine functionalized superparamagnetic iron oxide nanoparticles. Chem Mater 2004;16:2344–2354. 25. Hoskins C, Cuschieri A, Wang L. The cytotoxicity of polycationic iron oxide nanoparticles: Common endpoint assays and alternative approaches for improved understanding of cellular response mechanism. J Nanobiotechnol 2012;10:1–11. 26. Soenen SJH, Nuytten N, Meyer SFD, Smedt SCD, Cuyper MD. High intracellular iron oxide nanoparticle concentrations affect cellular cytoskeleton and focal adhesion kinase-mediated signaling. Small 2010;6:832–842. 27. Panariti A, Miserocchi G, Rivolta I. The effect of nanoparticle uptake on cellular behavior: Disruption or enabling functions? Nanotechnol Sci Appl 2012;5:87–100. 28. Canete M, Soriano J, Villanueva A, Roca AG, Veintemillas S, Serna CJ, Miranda R, Morales MDP. The endocytic penetration mechanism of iron oxide magnetic nanoparticles with positively charged cover: A morphological approach. Int J Mol Med 2010; 26:533–539. 29. Huang G, Chen H, Dong Y, Luo X, Yu H, Moore Z, Bey EA, Boothman DA, Gao J. Superparamagnetic iron oxide nanoparticles: Amplifying ROS stress to improve anticancer drug efficacy. Theranostics 2013;3(2):116–126.
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30. Bahring F, Schlenk F, Wotschadlo J, Buske N, Liebert T, Bergemann C, Heinze T, Hochhaus A, Fischer D, Clement JH. Suitability and viability assays for testing biological effects of coated superparamagnetic nanoparticles. IEEE Trans Magn 2013;49:383– 388. 31. Elsaesser A, Howard CV. Toxicology of nanoparticles. Adv Drug Deliv Rev 2012;64:129–137. 32. Zhu X, Tian S, Cai Z. Toxicity assessment of iron oxide nanoparticles in zebrafish (Danio rerio) early life stages. PLoS One 2012;7: 1–6. 33. Arora S, Rajwade JM, Panikar KM. Nanotoxicology and in vitro studies: The need of an hour. Toxicol Appl Pharmacol 2012;258: 151–165. 34. Yah CS, Simate GS, Iyuke SE. Nanoparticles toxicity and their routes of exposures. Pak J Pharm Sci 2012;25(2):477– 491. 35. Sohaebuddin SK, Thevenot PT, Baker D, Eaton JW, Tang L. Nanomaterial cytotoxicity is composition, size, and cell type dependent. Part Fibre Toxicol 2010;7:1–17.
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36. Naqvi S, Samin M, Abdin MZ, Ahmed FJ, Maitra AN, Prashant CK, Dinda AK. Concentration-dependent toxicity of iron oxide nanoparticles mediated by increased oxidative stress. Int J Nanomed 2010;5:983–989. 37. Mahmoudi M, Simchi A, Imani M, Shokrgozar MA, Milani AS, Haefli UO, Stroeve P. A new approach for the in vitro identification of the cytotoxicity of superparamagnetic iron oxide nanoparticles. Colloid Surf B Biointerfaces 2010;75:300–309. 38. Sanders KM, Degn LL, Mundy WR, Zucker RM, Dreher K, Zhao B, Roberts EJ, Boyes WK. In vitro phototoxicity and hazard identification of nano-scale titanium dioxide. Toxicol Appl Pharmacol 2012;258:2262236. 39. Kumar A, Pandey AK, Singh SS, Shanker R, Dhawan A. A flow cytometric method to assess nanoparticle uptake in bacteria. Cytometry 2011;79:7072712. 40. Toduka Y, Toyooka T, Ibuki Y. Flow cytometric evaluation of nanoparticles using side scattered light and reactive oxygen species mediated fluorescence-correlation with genotoxicity. Environ Sci Technol 2012;46:7629–7636.
PROTEIN-COATED IRON OXIDE NANOPARTICLES SHOW MINIMAL TOXICITY
Department of Biological Sciences, School of Science, NMIMS University, Vile Parle (West), Mumbai 400056, India Department of Chemical Sciences, School of Science, NMIMS University, Vile Parle (West), Mumbai 400056, India 3 Department of Metallurgical Engineering and Materials Science, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India 4 Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Navi, Mumbai 410210, India 2
Received 31 December 2013; revised 26 March 2014; accepted 5 April 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33178 Abstract: Iron oxide nanoparticles (IONPs) have been extensively used for biomedical applications like in the diagnosis and treatment of various diseases, as contrast agents in magnetic resonance imaging, and in targeted drug delivery. Despite several attempts, there is a dearth of information with respect to the cellular response and in-depth toxicity analysis of the nanoparticles. Considering the potential benefits of IONPs, there is a need to study the potential cellular damage associated with IONPs. The size and surface of the particles are some critical factors that should be analyzed when evaluating cytotoxicity. Therefore, in this study, we synthesized and characterized bare (7–9 nm) and protein-coated IONPs of diameter 50–70 nm, and evaluated their toxicity on membrane integrity, intracellular accumulation of reactive oxygen species, and mito-
chondrial activity in mouse fibroblast cell line by lactate dehydrogenase, 20 ,70 -dichlorofluorescein diacetate, and [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assays, respectively. Our extensive cytotoxicity analysis demonstrated that the size of the IONPs and their surface coating are the critical determinants of cellular response and potential mechanism toward cytotoxicity. The study of the interactions and assessment of potential toxicity of the nanoparticles with cells/tissues is a key determinant when considering their transC 2014 Wiley Periodicals, Inc. J lation in biomedical applications. V Biomed Mater Res Part B: Appl Biomater 00B: 000–000, 2014.
Key Words: nanomaterials/nanophase, protein adsorption, cell culture, surface characterization, FTIR
How to cite this article: Sanganeria P, Sachar S, Chandra S, Bahadur D, Ray P, Khanna A. 2014. Cellular internalization and detailed toxicity analysis of protein-immobilized iron oxide nanoparticles. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Magnetic nanoparticles are of interest in various fields of biomedicine because of their unique physical and chemical properties. In a clinical setting, the requirement for cellular tracking is extremely important to understand the biodistribution and homing of cells in the human body. Hence, an urgent need exists to develop noninvasive and sensitive imaging techniques, which will prove valuable for optimizing cell therapy. In particular, iron oxide nanoparticles (IONPs), a negative contrast agent in magnetic resonance imaging, have been utilized for various biomedical applications, such as cancer imaging, cellular labeling, and magnetofection.1,2 Because of their hydrophobic surface, instability in fluids, and inefficient prolonged retention with cells, their use in long-term cell tracking is limited. It has been reported
that surface modifications of magnetic nanoparticles can significantly improve their stability, biocompatibility, and shelf life.3 Various researchers have reported modifications of IONPs surface with diverse moieties,4 which can be categorized as: bifunctional surfactants like distearoyl-sn-glycero3-phospho ethanolamine-N-[carboxy (polyethylene glycol) 2000] and biotin5,6 inorganic coatings like silica and gold.7,8 Silica-coated IONPs are commercially available as 9 R and NanothermV R , which are currently used for FerumoxilV 10 hyperthermia therapy and have also proven to be among the promising nanomaterials for cell tracking. Gold-coated IONPs are commercially applied in drug delivery and immobilizing IgG for detection of hepatitis B antigen in blood sample; however, studies have proven that gold coating weakens the inherent magnetic properties of these nanoparticles and hence limits their use as an imaging modality.
Additional Supporting Information may be found in the online version of this article. Correspondence to: A. Khanna (e-mail: [email protected]) *Present address: Shilpee Sachar, Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai 400 098, India Contract grant sponsor: Department of Science and Technology, Government of India, under the “Nanomission” scheme; contract grant number: SR/NM/NS-1114/2011(G)
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Similarly, organic monomeric coatings like lauric acid, citric acid, gluconic acid, dimercaptosuccinic acid, oleic acid, and mercaptopropionic acid11–13 have shown cellular toxicity.14 Research has also been done on using organic polymers like dextran, polyethylene glycol, chitosan, and alginate as coating materials for IONPs.4,9,15 Dextran-coated IONPs were R , FeridexV R , and extensively used commercially as EndoremV 4,7 R for magnetic resonance imaging. ResovistV However, studies later revealed their toxic effects on the cells in terms of DNA damage and oxidative stress,8,14 which was found to be due to their poor retention on the surface of nanoparticles. Inspite of several attempts made in the past to overcome the limitations of using IONPs for biomedical applications, the need of a suitable coating with desired stability and biocompatibility still exists. The focus of the present study was to synthesize IONPs using optimal coatings like surfactants and proteins in order to achieve the desired properties. Along with the biocompatibility, the protein surface of IONPs will significantly increase the cellular uptake of these particles. In the present study, the synthesized IONPs [bare, cetyl trimethylammonium bromide (CTAB)-coated, and bovine serum albumin (BSA) and human serum albumin (HSA) coated nanoparticles] were compared with the commercially available polyvinylpyrrolidone-coated IONPs. Further, a dosedependent evaluation of cytotoxicity of the BSA- and HSAcoated nanoparticles was undertaken. This has utmost significance if the nanoparticles are intended to be translated from laboratory research to clinical applications.
thoroughly with ethanol and subjected to magnetic separation using a permanent magnet. Protein coating of IONPs CTAB-coated IONPs were stirred with different concentrations of proteins (BSA and HSA) in phosphate buffer (pH 7.0). The solutions were incubated for 24 h at 4 C. The colloidal stability of each solution was measured and was found to be maximum for the formulation containing proteins and IONPs in the ratio of 1000:62.5 mg. Particle characterization Crystalline structure by X-ray diffraction. The crystal structure of the synthesized IONPs and CTAB-coated IONPs was obtained by analyzing the X-ray diffraction (XRD) pattern of each sample recorded using XRD (PANalytical X’Pert Pro, with Cu Ka radiation, k 5 1.54 Å). Size analysis by transmission electron microscopy. The size and morphology of the synthesized nanoparticles was analyzed by transmission electron microscopy (TEM) using JEOL, Phillips CM 200. The sample specimen was prepared by dispersing the CTAB-coated IONPs in Milli-Q water followed by 5 min sonication. A drop of well-dispersed sonicated sample was drop cast on a carbon-coated copper grid, followed by drying the sample under suitable conditions. Functional group detection by Fourier transform infrared spectroscopy. The Fourier transform infrared (FTIR) spectrum was analyzed using JASCO, FT-IR 300E spectrometer, and the spectrum was studied from 4000 to 400 cm21.
MATERIALS AND METHODS
Synthesis of CTAB-coated IONPs Ferric chloride hexahydrate (FeCl36H2O) and ferrous chloride tetrahydrate (FeCl24H2O) were purchased from Sigma Aldrich (St. Louis, MO). CTAB and hydrazine hydrate (NH2NH2) were procured from Thomas Baker (India). BSA and HSA were purchased from Sigma. All chemicals were of analytical grade and were used as received. Surfactant-coated magnetic nanoparticles were synthesized by co-precipitation method as described earlier,16 where 0.2M FeCl36H2O and 0.15M FeCl24H2O were dissolved in 2:1 ratio in 80 mL of de-ionized water in a roundbottom flask, and the temperature was gradually increased to 80 C in nitrogen atmosphere with constant mechanical stirring at 1000 rpm. The temperature was initially maintained at 80 C for 30 min, and subsequently, the addition of hydrazine hydrate was done drop wise till a black precipitate formation was observed. The reaction mixture was again stirred at same conditions for 30 min, which was then followed by the addition of CTAB at various concentrations above and below critical micellar concentration (cmc 5 0.8 mM) viz. 0.4, 1.0, 1.5, 2.0, and 2.5 mM. The concentration of 1.5 mM was optimized for further studies. The reaction temperature was slowly raised to 90 C, and the reaction mixture was allowed to stir continuously for 60 min. The CTABcoated black precipitate was obtained by cooling the reaction mixture at room temperature, which was further rinsed
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Surface charge detection by f-zeta-potential measurements. Surface charge analysis of the CTAB-coated IONPs was performed in distilled water at various pH (pH 2–10) using Zeta Plus zeta potential analyzer (Brookhaven Instrument) at room temperature. Magnetization analysis by vibrating sample magnetometer. Magnetic properties of IONPs and CTABcoated IONPs were analyzed using a vibrating sample magnetometer (Lakeshore Model No. 7410, USA), which measures the magnetization of applied magnetic field (02T) at room temperature. Cell culture conditions Mouse embryonic fibroblast cell line, NIH 3T3, was obtained as a gift from IIT, Mumbai, and was used for cytotoxicity analysis. The doubling time for NIH 3T3 cell line used was observed to be 16–18 h for all the experiments in standard culture conditions. NIH 3T3 was chosen as controls for this study as they represent a standard and sensitive cell line. NIH 3T3 mimics the stromal cells found in embryonic tissue and hence represents a good model system to evaluate early toxicity. The cell line was maintained in high-glucose Dulbecco’s modified eagle medium (Cell Clone, Genetix) supplemented with 10% fetal bovine serum (Cell Clone, Genetix) and 1% penicillin, streptomycin, and amphotericin (Sigma)
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with media change at regular intervals. The cells were trypsinized using 0.25% trypsin-1 mM ethylenediaminetetraacetic acid, and a split ratio of 1:3 was followed at a subconfluency of 70%–80%. The cytotoxicity analysis was done using MTT reagent (Himedia, cell culture tested), dimethyl sulfoxide (DMSO) (MP Biomedicals, SDFCL), lactate dehydrogenase (LDH) assay (Roche, USA), and 20 ,70 -dichlorofluorescein diacetate (DCFDA) assay (Abcam). All chemicals, reagent, media, buffer, and plastic ware used were of cell culture grade (Cell Clone, Genetix). Cell exposure to IONPs and uptake studies For uptake studies, cells were seeded at a density of 5 3 105 cells/well in six-well plate and were maintained in incubator with 5% CO2 at 37 C. As the nanoparticle concentration of 60 mg/mL was found to be colloidally stable, it was used for further experiments. The uptake studies were done at 3, 6, 12, and 24 h intervals. Time-dependent cellular uptake studies were done for all synthesized nanoparticles (bare, CTAB-coated, and protein-coated IONPs) along with the commercial IONPs (Sigma). Prussian blue staining was performed for analyzing the internalization of nanoparticles. Cells were fixed using 4% paraformaldehyde solution for 40 min followed by Dulbecco’s phosphate buffered saline (DPBS) wash. The wells were then treated with Perl’s reagent (4% potassium ferrocyanide/12% HCl, 1:1 v/v) for a period of 30 min, with a further DPBS wash to remove the excess stain. The cells were then counterstained using 0.02% neutral red. The internalization of particles was analyzed using Carl Zeiss phase contrast microscope. Cellular viability analysis Mitochondrial impairment analysis by MTT assay. For MTT assay, 80%–90% confluent flasks were trypsinized using prewarmed 3–4 mL 0.25% trypsin–EDTA for a period of 5 min. Further cells were added to 96-well plate, with a cell count of 1 3 105 cells/well and kept overnight. After the addition of nanoparticles, 100 mL of MTT (0.5 mg/mL) was added to each well. The cytotoxicity analysis was done at different time points such as 24, 48, and 72 h. At each time point, 200 mL of DMSO was added to the treated cells. The readings were analyzed using 570-nm filter in an enzymelinked immunosorbent assay reader, and the percentage cytotoxicity was calculated using Eq. (1). The experiments were performed in triplicates. Control well using only media and fetal bovine serum were also included. % Cell Viability 5 Abs sample =Abs control 3100
(1)
Membrane leakage analysis by LDH assay. Membrane integrity after IONPs uptake was studied in terms of the extent of LDH production using LDH assay. After the IONPs uptake, the study was performed at an interval of 72 h in a 96-well plate in accordance with the kit guidelines using 490-nm filter in an enzyme-linked immunosorbent assay
reader. The studies were done for all the synthesized IONPs in comparison with commercial IONPs. Cytotoxicity was measured using the basal LDH released from untreated control cells. All the experiments were performed in triplicates. Reactive oxygen species formation by DCFDA assay. Production of reactive oxygen species (ROS) and hence promoting endogenous oxidative stress is reported in IONPs.17,18 Surface modifications of nanoparticles can minimize the ROS production and hence can reduce nanoparticleinduced cytotoxicity. In the present study, for the synthesized IONPs, the extent of ROS generation was studied by the DCFDA assay (a kit-based method). Intracellular ROS generation was determined by using the cell permeating reagent DCFDA, cell permeable compound, and a fluorescein dye that measures the hydroxyl, peroxyl, and other ROS activities within the cell. On penetrating the cell, it gets deacetylated by cellular esterases to a nonfluorescent compound, which is later oxidized by ROS into 20 ,70 -dichlorofluorescein and can be further analyzed by flow cytometry analysis. The cells were grown in a six-well plate at a density 5 3 104 cells/well. Culture media was replaced with 200 mL of IONPs at a concentration of 60 mg/mL for 3 h (optimized time for IONPs uptake), and ROS generation was evaluated at 72 h. After internalization, the cells were harvested using 0.5% trypsin for 5 min and were pelleted down at 1200 rpm for 7 min at 20 C. The debris was removed by 13 DPBS wash. After decanting phosphate buffered saline, 300–400 mL of DPBS was subsequently added. The cells were then incubated with 2 mM DCFDA dye (20 mM in DMSO) at 37 C for 30 min in dark. The ROS formation was analyzed using flow cytometry (FACS Calibur, BD Biosciences), and approximately 10,000 events/sample were analyzed after gating. RESULTS
Particle characterization using XRD, VSM, TEM, and high-resolution TEM Figure 1 shows the XRD pattern of the synthesized bare and CTAB-coated IONPs. The pattern clearly indicates the formation of magnetite Fe3O4 with the presence of characteristic peaks corresponding to planes (220), (311), (400), (422), (511), and (440). Figure 2(a,b) TEM and highresolution TEM shows that the synthesized CTAB-coated IONPs are spherical in shape. The size of IONPs (200 particles) was calculated using Image J software, and it was found to be uniform in the range of 7–9 nm (polydispersity index was low [see inset of Figure 2(a)]. The selected area electron diffraction pattern recorded for the synthesized IONPs was found to be similar to the reflections of inverse spinel structure of Fe3O4 [see inset of Figure 2(b)]. Figure 3 illustrates the hysteresis loop of synthesized IONPs. The CTAB-coated IONPs demonstrated superparamagnetic behavior with negligible remanence and coercivity in the hysteresis loop. The maximum magnetization of CTABcoated IONPs was found to be 45 emu/g at 20 kOe, which was slightly lower than that of the bare IONPs (54.7 emu/ g). It shows that the inherent magnetic property was retained even after surfactant coating. Figure 4(a) shows
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tein alone at a pH range 2–10. Isoelectric point (pI) of protein was recorded as 4.0. Protein bears a positive surface charge below pI and negative surface charge above pI. A zeta potential value of protein alone was found to be 20 mV at pH 2 and 229 mV at pH 10. The pI value of bare IONPs was calculated to be 6.8, and similarly its zeta potential values are 40 mV at pH 2 and 225 mV at pH 10.19 Figure 6 shows the protein coating on surfactant-functionalized IONPs, which was indicated from FTIR plots. Free unbound HSA shows a broad peak centered at 3450 cm21 owing to free carboxylic acid groups. HSA-coated IONPs exhibited a peak at 1655 cm21 (amide I), 1538 cm21 (amide II), and 1451 cm21 (amide III).
FIGURE 1. XRD patterns of (a) bare IONPs and (b) CTAB-coated IONPs.
the TEM micrographs of the protein-coated IONPs. The spherical-shaped protein-coated IONPs were analyzed using Image J software and were found to be in the size range of 50–70 nm [see inset of Figure 4(a)]. Figure 4(b) shows that the selected area electron diffraction pattern analyzed for the protein-coated IONPs reflects the crystalline nature of IONPs even after protein coating. The hydrodynamic diameter analyzed by the dynamic light scattering spectra demonstrated a single peak, showing the particle size to be 86 nm [inset Figure 4(b)]. The protein-coated IONPs were found to have colloidal stability for more than 6 months.
Surface charge and protein coating evaluation using zeta potential and FTIR Figure 5 shows the surface charge analysis of the proteincoated IONPs in comparison with the bare IONPs and pro-
Internalization of various IONPs by Prussian blue staining Figure 7 shows time-dependent cell uptake of synthesized nanoparticles: commercial, bare IONPs, CTAB-coated IONPs, and BSA- and HSA-coated IONPs. Prussian blue stained cells showed deep blue granules in the cytoplasm of cells treated with IONPs due to cytoplasmic accumulation of iron deposits. Further, no staining in the nucleus was seen. In vitro cytotoxicity analysis using MTT, LDH, and DCFDA assays Figure 8(a–c) shows the results of 24, 48, and 72 h MTT assay, indicating consistent cellular viability of >95% for protein-coated IONPs for a concentration of 60 mg/mL over the regular time intervals of 24, 48, and 72 h, whereas in case of CTAB-coated IONPs the viability decreased slightly from 92% for first 48 h to 90% in 72 h. Whereas, a clear decline in cell viability in case of commercial IONPs (54% for 24 h, 24% for 48 h, and 23% for 72 h) was observed, which shows that the synthesized IONPs with surfactant and protein functionalization are more biocompatible. Results are represented as 62 SD of the mean. Figure 9
FIGURE 2. (a) TEM micrograph of CTAB-IONPs. (b) high-resolution TEM of CTAB-IONPs. Inset 2(a): size distribution of CTAB-IONPs. Inset 2(b): electron diffraction pattern of CTAB-IONPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 3. M versus H plots of (a) bare IONPs and (b) CTAB-coated IONPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
shows the results of the 72 h LDH assay, which were found to be in accordance with the MTT results and showed maximum cellular viability for protein-coated IONPs (>80%) in comparison with commercial IONPs (60%) and bare IONPs (50%). Results are represented as 62 SD of the mean. Figure 10(a–f) shows the results of 72 h DCFDA assay and the amount of ROS produced in response to the uptake of commercial IONPs and synthesized IONPs with different surface modifications. Highest shift in the side scatter intensities was observed for commercial and bare IONPs that indicate maximum increase in internal density that can lead to morphological alterations. However, protein-coated IONPs showed a slight shift in side scatter intensities showing tolerable morphological alterations. DISCUSSION
In this work, we present the synthesis and characterization of surface-modified IONPs in terms of their crystallinity, its magnetic behavior, and colloidal stability. Further, detailed
cytotoxicity analysis by MTT, LDH, and DCFDA assays was done using NIH 3T3 (mouse embryonic fibroblast cell line). IONPs were synthesized using co-precipitation method. The XRD pattern of bare and CTAB-coated IONPs shows the presence of intense peaks, which confirmed the formation of highly crystalline nanoparticle. Also, magnetization loops of bare and CTAB-coated IONPs demonstrated slight decrease in magnetic property of CTAB-coated IONPs, but even after surface modification the magnetic behavior of the particle was found to be persistent. The presence of CTAB molecules over the surface of bare IONPs provides hydrophilicity without affecting the crystallinity and magnetization of the IONPs. The different characterization techniques presented in this work demonstrate average particle size of 7–9 nm of CTAB-coated IONPs and 50–70 nm for proteincoated IONPs with spherical morphologies, and hence an appropriate size for future biological applications. Uncoated nanoparticles have a tendency to aggregate. In order to overcome this, the surface of nanoparticles is coated with the proteins.11 Surface modifications affect the cell viability and increase the stability of the nanoparticles once internalized.20,21 The coating also helps in prolonged retention of the nanoparticles within the cells and thus increases its efficiency as contrast agent for long-term tracking.22,23 From the zeta plots, the zone between pH 4.0 and pH 7 was considered to be the interaction zone, as both the moieties were bearing opposite surface charges giving rise to surface adsorption and hence colloidally stable solution of protein-coated IONPs. The FTIR spectra of protein-coated IONPs revealed change of protein structure from a-helices to b-sheets due to immobilization.24 The important observation here is the presence of functional characteristics peak of high intensity at 1638 cm21 (amide I) in HSA-modified CTAB-coated IONPs25,26 which in turn indicates the fact that the functionality of immobilized HSA in comparison with free HSA is maintained. The Prussian blue staining revealed certain morphological alterations in the cells at 24 h interval of incubation in commercial as well as bare IONPs. However, the synthesized protein-coated IONPs did not show any such morphological
FIGURE 4. (a) TEM micrograph of protein-IONPs. (b) The selected area electron diffraction. Inset 4(a): protein corona around IONPs and size distribution of protein-coated IONPs. Inset 4(b): dynamic light scattering spectra. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 5. Zeta plots. Pictorial representation of surface charge and colloidal stability in solutions with various nanoparticle concentrations. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
alterations even after 24 h.27,28 The incubation period of 3 h was optimized for further cytotoxicity analysis. Because it has been earlier reported that IONPs have shown to generate cytotoxicity,29,30 detailed evaluation was
FIGURE 6. FTIR spectra of bare IONPs, HSA, and HSA-coated IONPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
done. The cell line used for cytotoxicity studies is NIH 3T3, which represents a mouse embryonic fibroblast cell line mimicking the stromal cells present throughout the connective tissue in the body31 and hence was used in the study. Various causes of cellular toxicity leading to cell death can
FIGURE 7. Prussian blue staining pictographs showing various IONPs internalization at 3, 6, 12, and 24 h, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 8. MTT plots showing toxicity analysis performed on mouse fibroblast cell line NIH 3T3 for 24, 48, and 72 h. Results are represented as 62 SD of the mean. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
be attributed to lysosomal destabilization, loss of mitochondrial membrane integrity,32–34 or membrane leakage. IONPs are known for the formation of ROS species in the acidic environment that results in the formation of free ions (Fe21 and Fe31). These free ions interact with H2O2 to generate hydroxyl radicals (Fenton reaction), which leads to oxidative stress indirectly causing cell death by damaging protein and DNA, which was evaluated by DCFDA assay.19
It is important to first evaluate and study the effects of commercial and synthesized IONPs in a time- and dosedependent manner by MTT and LDH assay. The results of MTT assay indicated consistent cellular viability of >95% for protein-coated IONPs for a concentration of 60 mg/mL after 72 h, whereas in case of CTAB-coated IONPs, the viability decreased slightly (2%) in a duration of 72 h. However, substantially low cellular viability of 23% was
FIGURE 9. LDH plots showing membrane leakage analysis performed on mouse fibroblast cell line NIH 3T3 at 72 h. Results are represented as 62 SD of the mean. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 10. DCFDA assay: (a–e) Scatter plots and peak histograms; (f) Histogram showing ROS generation of various IONPs for 72 h. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
observed in case of commercial IONPs. Cell viability evaluation at 24 and 48 h has demonstrated no cytotoxicity with protein-coated IONPs. LDH assay was performed to check whether LDH, a stable cytosolic enzyme in normal cells, can leak into the extracellular fluid only after the membrane damage and thus can be an effective measure to check the cytotoxicity.35 The results of the LDH assay were found to be in accordance with the MTT results and showed maximum cellular viability for protein-coated IONPs (>80%). The prominent decrease in cellular viability of commercial IONPs seen in LDH as well as MTT assay in comparison with proteincoated IONPs shows the significant role played by protein coating over IONPs. Various reports36,37 showed that uncoated particles induce greater toxicity than coated IONPs. A significant increase in LDH release was observed in 72 h for commercial and bare IONPs showing only 60%
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and 50% viability, respectively, whereas protein-coated IONPs consistently showed higher viability (>80%). Therefore, protein surface functionalization of nanoparticles helps in regulating IONPs interfacial properties by altering the hydrophobic nature of bare IONPs to hydrophilic by imparting ANH2 groups, which plays a vital role in interactions with biological systems.19 Literature reveals that flow cytometry has been widely applied in quantifying the cellular uptake of nanoparticles.38,39 However, less attention has been paid to the use flow cytometry in examining ROS generation in nanoparticle-induced cytotoxicity analysis. In the present study, along with the cellular uptake we have performed flow cytometry analysis in comparing the extent of ROS generation in bare and surface-modified IONPs. In the scatter plots, the side scatter depicts the enhanced intracellular granularity, and the forward scatter indicates the cell size.
PROTEIN-COATED IRON OXIDE NANOPARTICLES SHOW MINIMAL TOXICITY
ORIGINAL RESEARCH REPORT
An increase in the side scatter is an indicator for the uptake of IONPs and intracellular density.40 Subsequent increase in the side scatter of the cells was observed in comparison with control plots, which signifies the increase in ROS production due to nanoparticle internalization. Highest shift in the side scatter intensities was observed for commercial and bare IONPs, which indicates maximum increase in internal density that can lead to morphological alterations, which can also be observed in the Prussian blue staining experiments (72 h) for the same systems. With surfactant and further protein coating, the ROS generation was not minimized; however, we observed a decrease in the mean fluorescent intensity in comparison with commercial and bare IONPs. Keeping in view the observations of flow cytometry analysis along with MTT and LDH assay analysis, we can conclude that internalization of protein-modified IONPs exhibits tolerable complexity alteration and reasonable intracellular ROS formation causing no harm to the proliferative capacity of the cells. SUMMARY
Colloidally stable surfactant and protein coated IONPs were synthesized and compared with bare and commercially available IONPs in terms of their cellular interactions and toxicity effects. Detailed in vitro cytotoxicity analysis in terms of mitochondrial impairment, membrane leakage, and ROS generation suggests the potential of HSA-coated IONPs as a suitable biocompatible coating for long-term biomedical applications such as cell/stem cell tracking. Studies are underway to understand whether HSA-coated nanoparticles affect viability, proliferation, and differentiation potential of stem cells isolated from human umbilical cord. Further, the potential of the synthesized biocompatible IONPs for stem cell labeling and tracking will be evaluated. ACKNOWLEDGMENTS
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