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Polyvinyl alcohol functionalized cobalt ferrite nanoparticles for biomedical applications
Applied Surface Science 264 (2013) 598–604
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Polyvinyl alcohol functionalized cobalt ferrite nanoparticles for biomedical applications A.B. Salunkhe a , V.M. Khot a , N.D. Thorat a , M.R. Phadatare a , C.I. Sathish b , D.S. Dhawale b , S.H. Pawar a,∗ a b
Center for Interdisciplinary Research, D. Y. Patil University, Kolhapur 416006, Maharashtra, India National Institute for Materials Science, 1-1 Namiki, Tsukuba, 305-0044 Ibaraki, Japan
a r t i c l e
i n f o
Article history: Received 25 July 2012 Received in revised form 6 October 2012 Accepted 12 October 2012 Available online 23 October 2012 Keywords: CoFe2 O4 nanoparticles Polyvinyl alcohol coatings Cytotoxicity Magnetic diameter
a b s t r a c t In the present work, cobalt ferrite nanoparticles (CoFe2 O4 NPs) have been synthesized by combustion method. The surface of the CoFe2 O4 NPs was modified with biocompatible polyvinyl alcohol (PVA). To investigate effect and nature of coating on the surface of CoFe2 O4 NPs, the NPs were characterized X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The transmission electron microscopy (TEM) and dynamic light scattering (DLS) results demonstrate the monodispersed characteristics of CoFe2 O4 NPs after surface modification with PVA. The decrease in contact angle from 162◦ to 50◦ with PVA coating on NPs indicates the transition from hydrophobic nature to hydrophilic. The Magnetic properties measurement system (MPMS) results show that the NPs have ferromagnetic behavior with high magnetization of 75.04 and 71.02 emu/g of uncoated and coated CoFe2 O4 NPs respectively. These PVA coated NPs exhibit less toxicity over uncoated CoFe2 O4 NPs up to 1.8 mg mL−1 when tested with mouse fibroblast L929 cell line. © 2012 Elsevier B.V. All rights reserved.
1. Introduction MNPs because of their outstanding physical and chemical properties have received considerable attention in various fields from technology to biomedical applications [1]. The dimensions of these nanoparticles make them ideal candidates for nano engineering of surfaces and production of functional nanostructures. Such modification facilitates their use in biomedical applications for example as contrast agents for magnetic resonance imaging (MRI), for targeted drug delivery and as heating mediators in hyperthermia therapy application etc. [2]. Among various MNPs, spinel CoFe2 O4 NPs are extensively studied due to their ability to form an ideal magnetic system toward understanding and controlling magnetic properties at the atomic level through chemical manipulation. CoFe2 O4 has large anisotropy compared to other oxide ferrites [3,4]. Though they are proposed for biomedical application their use in medicine is restricted due to numerous problems such as toxicity due to the remarkable amount of cobalt release in aqueous solutions, aggregation in solution, and poor accessibility of the surface when surfactants are used. This problem can be overcome by surface coating of CoFe2 O4 with a compatible, nontoxic, and water-stable/dispersing material [5–9].
From this point of view, encapsulating MNPs by PVA is a promising and important approach. The rich and well documented chemistry of biocompatible PVA coatings may allow practical implementation of MNPs in pharmaceutical and biomedical applications [10,11]. PVA served as the protective polymer as it has the desired solution properties in water and contains many isolated hydroxyl functional groups, which can adsorb and complex with metal ions [12]. It is assumed that PVA chain adsorbed on the surface of magnetic core of CoFe2 O4 NPs and form a shell as shown in Fig. 1 [13]. In the present investigation, CoFe2 O4 NPs were first synthesized by combustion method and further coated with PVA. The effect of coating on structural, morphological and magnetic properties of CoFe2 O4 NPs was studied in detail. The attachment of PVA on the surface of CoFe2 O4 core and stability were investigated by XRD, FTIR, TEM and DLS respectively. The biocompatibility of coated and uncoated CoFe2 O4 NPs was tested for different concentrations (0.3–1.8 mg/mL) with mouse fibroblast L929 cell line by trypan blue assay.
2. Experimental 2.1. Materials
∗ Corresponding author. Tel.: +91 231 2601202/2601235; fax: +91 231 2601595. E-mail addresses: salunkhe [email protected], pawar s [email protected] (S.H. Pawar). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2012.10.073
Analytical grade cobalt nitrate [Co(NO3 )2 ·6H2 O] (99%), ferric nitrate [Fe(NO3 )3 ·9H2 O](99.9%), Glycine [CH2 NH2 COOH] (99%) and polyvinyl alcohol (-C2 H4 O)n (99%) were purchased from LOBA
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using a PSS/NICOMP 380 ZLS (Particle Sizing System, Santa Barbara, ˚ All CA, USA). The light source was He–Ne laser operated at 632.8 A. measurements were carried out at 25.0 ± 0.1 ◦ C using a circulating water bath. Cylindrical cells of 10 mm diameter were used in all of the light scattering experiments. The autocorrelation function was obtained using a 192-channel photon correlator. For reproducibility, at least three measurements were conducted for each reading. Magnetic properties of the NPs were measured by the MPMS XL. 2.5. Cytotoxicity study Fig. 1. Synthesis of CoFe2 O4 NPs and layering of PVA polymers on the surface of the NPs.
CHEM, India. All chemicals listed above are water soluble and used as received. 2.2. Synthesis of CoFe2 O4 NPs The CoFe2 O4 NPs (S1) were prepared by simple and inexpensive combustion method using corresponding metal nitrates and glycine. Metal nitrates were employed both as metal precursors and oxidizing agents while glycine serves as reducing agent and fuel for combustion. The stoichiometric amounts of metal nitrates and glycine were mixed in large beaker which results in slurry due to hygroscopicity of metal nitrates [14]. The obtained slurry was then introduced to hot plate preheated to 250 ◦ C. After evaporation of water content, the mixture starts frothing and ignited with flame within few seconds, giving voluminous and foamy CoFe2 O4 powder. The detailed synthesis and reaction mechanism of CoFe2 O4 nanoparticles was reported elsewhere [15]. 2.3. Surface coating of CoFe2 O4 nanoparticles The mixture of CoFe2 O4 nanoparticles and water was vigorously stirred for 10 min by dispersing the nanoparticles in double distilled water (DDW). PVA solution was prepared by dissolving 0.6 M PVA in 40 mL water. PVA solution was added to above mixture and stirred further for 30 min. The obtained mixture was kept overnight at room temperature, then supernatant was discarded and PVA coated CoFe2 O4 nanoparticles were collected. Surface modified CoFe2 O4 NPs (S2) are easily dispersible in DDW after 10 min sonication. 2.4. Characterizations Structure and phase of as synthesized and PVA coated CoFe2 O4 NPs was studied by XRD (Philips-3710) with Cu-K␣ radiation ˚ in the 2 range from 20 to 100◦ . The patterns were ( = 2.2897 A) evaluated by Panalytical X’pert high score software and compared with standard JCPDS (card no. 22-1086). Presence of the magnetic core and polymer coating of the MNPs was confirmed by FTIR spectroscopy (Perkin Elmer spectrometer model no. 783, USA). Elemental composition of the MNPs was determined by TGA with Trans analytical instruments (SDT 2960) operated in temperature from 35 ◦ C to 1000 ◦ C with heating rate of 10 ◦ C/min in flowing nitrogen ambiance. The particle size of coated and uncoated CoFe2 O4 NPs was measured with TEM (Philips CM 200 model, oper˚ Water contact angles of ating voltage 20–200 kV, resolution 2.4 A). coated and uncoated MNPs were measured using the sessile drop method by deposition of 4–6 L droplets of DI water on a horizontal surface and their observation in cross-section. Each drop was observed directly with an Olympus BX-41 microscope objective lens, whereas its image was digitally captured using a 1.4 megapixel computer-controlled digital CCD camera. The values reported are averages of more than 20 measurements performed in different areas of each sample surface. DLS measurements were carried out
2.5.1. Cell culture In vitro cell viability of coated and uncoated CoFe2 O4 MNPs was studied using mouse fibroblast L929 cell line purchased from National Center for Cell Science (NCCS), Pune, India. L929 cells were grown in minimal essential medium supplemented with 10% (v/v) FBS (fetal bovine serum), kanamycin (0.1 mg/mL), penicillin G (100 U/mL), and sodium bicarbonate (1.5 mg/mL) at 37 ◦ C in a 5% CO2 atmosphere. In brief, cells (1 × 105 cells/mL) were grown for 24 h. After 24 h, the old media was replaced by fresh media and different particle concentrations (0.3, 0.6, 1.2 and 1.8 mg/mL) of uncoated and coated CoFe2 O4 NPs. 2.5.2. Cell viability by trypan blue dye exclusion assay For this experiment, L929 cells were seeded at a density of 1 × 105 cells/mL in a culture plate. After 24 h of incubation, the old media was replaced with a media containing different concentrations of MNPs. And the cells were exposed for 48 h incubation time. Then, the dishes were washed thrice with PBS to remove the MNPs. Both the attached and unattached cells were harvested and combined after trypsinization (0.025% trypsin, 10 min). The cells were then stained with trypan blue dye and counted with a hemocytometer. The experiments were replicated three times and the data was graphically presented as mean. The total amount of cells, stained and unstained is counted. The calculated percentage of unstained cells will represent the percentage of viable cells. 3. Results and discussion 3.1. Effect of surface coating on structural properties 3.1.1. XRD analysis The formation of uncoated and PVA coated CoFe2 O4 NPs was confirmed from XRD pattern (Fig. 2(a)). CoFe2 O4 nanoparticles display several relatively strong reflection peak in the 2 region of 20–100◦ , which is quite similar to the other groups, confirming that prepared NPs are CoFe2 O4 with spinel cubic structure having fd3 m space group [16,17]. The obtained peaks are well matched with standard JCPDS card no. 22-1086. The determination of crystallite size (D) of CoFe2 O4 powder was based on X-ray diffraction line broadening and calculated by using Scherrer formula [18], D=
0.9 ˇ cos
(1)
where ˇ is the full-width at half maxima of the strongest intensity diffraction peak (3 1 1), is the wavelength of radiation and is the angle of strongest characteristic peak. From Fig. 2(a) it can be revealed that all resultant NPs are pure CoFe2 O4 with an inverse spinel structure. No change in phase was observed in case of particles coated with PVA while slight suppression of diffraction peaks can be clearly observed. There is no pronounced change in the lattice constant however crystallite size varies slightly after surface modification. The calculated crystallite size (D) for uncoated and coated CoFe2 O4 NPs are 38 nm and 35 nm respectively. The corresponding selected area electron diffraction (SAED) pattern of coated CoFe2 O4 is shown in Fig. 2(b). Figure shows spotty
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Fig. 2. (a) XRD patterns of uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs and (b) SAED pattern of PVA coated CoFe2 O4 NPs.
ring patterns for CoFe2 O4 phase consistent with XRD. According to the diffraction patterns in Fig. 2(b), the measured lattice constants and interplanar spacing (dh k l ) agree with those in the XRD results. The electron diffraction pattern consisting of rings indicates the good crystal structure of PVA coated CoFe2 O4 NPs. 3.1.2. Infrared analysis FTIR is an appropriate technique to analyze the attachment of polymer to surface of MNPs. Fig. 3 shows the FTIR spectra of uncoated and PVA coated CoFe2 O4 NPs. The series of characteristic IR bands are summarized in detail in Table 1 [19] and only the salient features are discussed below. In case of uncoated CoFe2 O4 NPs (S1), the band at 3214 cm−1 is assigned to stretching () vibrations due to adsorbed water on the surface of CoFe2 O4 NPs. The band observed at 578 cm−1 is corresponds to stretching vibrations of Fe O. In PVA coated CoFe2 O4 NPs (S2), the M O stretching band at 578 cm−1 , the alcoholic O H stretching band at 3260 cm−1 are observed. The additional bands at 2982 cm−1 corresponding to C H stretching vibrations, at 1385 cm−1 corresponding to C C stretching vibrations, at 1104 cm−1 attributable to M O C (M=Fe) bond and
at 996 cm−1 corresponding to CH2 rocking are observed in PVA coated CoFe2 O4 NPs. Therefore attachment of PVA onto CoFe2 O4 nanoparticles surface is confirmed and supported by contact angle measurements [20]. 3.1.3. Thermo-gravimetric analysis TGA provides additional quantitative evidence on structure of the NPs coating. In this experiment, MNPs are heated from 35 to 1000 ◦ C under flowing N2 and changes in mass loss of organic material are recorded. Although it is an extremely valuable technique for surface characterization of NPs, it is not commonly applied in literature for ferrite MNPs. The information one can retrieve from TGA measurements is mutual. First of all, TGA allows us to determine the bonding strength of the ligand to the NPs surface and its thermal stability. TGA (Fig. 4) for sample S1 shows only 2.5% weight loss in the temperature range of 25–250 ◦ C which may be due to evaporation of hydroxyl groups adsorbed on the surface of nanoparticles during synthesis. Such a very less weight loss suggests that, pure phase of CoFe2 O4 NPs can be achieved easily by combustion method. Whereas for sample S2, the weight loss process observed in two stage. In the first, ∼4% weight loss in the temperature range of 150–200 ◦ C is observed which is probably related to the outer layer of the polymer. However in the 200–400 ◦ C temperature range, ∼18.5% weight loss is noticed in the second stage attributed to the detachment of chemisorbed polymer layer from the surface which needs higher temperature for removal [21].
Table 1 Characteristic FTIR Vibrations for uncoated and PVA coated CoFe2 O4 NPs.
Fig. 3. FTIR spectra for uncoated (S1) and PVA coated CoFe2 O4 nanoparticles (S2).
Samples
IR region or bands (cm−1 ) Characteristics vibrations
CoFe2 O4
3214 578,571
(O H) (M O)
PVA coated CoFe2 O4
3260 2982 2850 1640 1385 1225 1104 996 578
(O H) a (CH2 ) s (CH2 ) ı (H O H) (C C) (OCH3 ) rocking (M O C (M Fe)) CH2 rocking (M O)
a , asymmetric stretching; s , symmetric stretching; ı, scissoring.
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Therefore, material can absorb water and its structure gets deteriorated with time. The contact angle for the hydrophilic surface is less than 90◦ and for hydrophobic surface is greater than 90◦ . Surface behavior of the coated and uncoated CoFe2 O4 NPs toward hydrophobicity or hydrophilicity was tested in terms of contact angle () of the water droplet with the solid surface, measured using the formula, = 2 tan−1
Fig. 4. Thermogravimetric curves for uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs.
3.2. Effect of surface coating on morphological properties 3.2.1. TEM analysis Fig. 5 shows the TEM images for samples S1 and S2. From Fig. 5, it can be observed that the uncoated NPs (S1) are in highly agglomerated form whereas PVA coated MNPs show well dispersed characteristic. The dipole–dipole interaction in magnetic NPs results in the agglomeration of these NPs. The use of polymer during the synthesis process can reduce this interaction through physical or chemical adsorption on the surface of particles. The size of PVA coated and uncoated NPs is 37 and 34 nm respectively. 3.2.2. Contact angle measurements For biomedical applications, MNPs should be hydrophilic in nature so that it forms stable dispersion in physiological saline medium like water. One of the fundamental methods of characterizing the hydrophobic or hydrophilic properties of a solid surface is to determine the contact angle. The contact angle is a measure of the wetting behavior of a particular liquid on the surface under investigation and directly relates to the interfacial energies of the systems. The contact angle on the solid surface gets changed merely by changing the chemistry of the outermost monolayer. Depending on affinity of material to be tested toward water, it classified into two groups such as hydrophobic and hydrophilic. The hydrophilicity of the material is due to presence of amphiphilic groups on the material surface.
2h
(2)
where, h is the height and is the width of water droplet touching the solid surface of sample. Fig. 6 shows a water droplet in equilibrium over a horizontal solid surface of samples. From Fig. 6 it is observed that, values of contact angles for uncoated and PVA coated MNPs are 162◦ and 50◦ respectively. Contact angle values of uncoated NPs clearly suggest their hydrophobic nature. Surface modification with PVA causes large decrease in contact angle to 50◦ . Such decrease in contact angle after surface modification occurred due to the presence of amphiphilic groups of PVA on the surface of CoFe2 O4 magnetic core. Generally, when water contact angle is less than 50◦ , MNPs could be easily dispersed in aqueous solution because of hydrophilic character of present endgroups [22,23]. These findings support the idea that surface hydrophilicity plays a pivotal role in creating water-dispersible nanoparticles useful for biomedical applications. 3.3. Effect of surface coating on magnetic properties The room temperature M–H curves of samples S1 and S2 are shown in Fig. 7. From Fig, it can be seen that the magnetization of sample S2 (71.02 emu/g) is smaller as compared to sample S1 (75.94 emu/g) at applied field of ±15 kOe at 300 K. However, the magnetization for both samples is close to that of bulk CoFe2 O4 NPs [24]. The reduction in magnetization for sample S2 may be attributed to the presence of non-magnetic polymer layer on to the surface of NPs which reduces the particle-particle interaction and lowers the exchange coupling energy which in turn reduces the magnetization. Both samples, S1 and S2 exhibit typical ferromagnetic nature with low coercivity (∼94 Oe and ∼102 Oe respectively). In case of ferrofluids, the particle size distribution follows a lognormal distribution function [25]. Using the value of magnetization, magnetic particle size and size distribution can be calculated from following equation,
Dm =
18kT Md
i 3εMd H0
1/2 1/3
Fig. 5. TEM images for uncoated (S1) and PVA coated CoFe2 O4 NPs (S2).
(3)
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Fig. 6. Images of the static water contact angle showing the effect of chemical functionalization and micro/nanostructure on the surface hydrophilicity of combustion synthesized uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs.
where Md is the domain magnetization of the bulk CoFe2 O4 (80 emu/g), Dm is the magnetic diameter, i is the initial magnetic susceptibility i ∼ (dM/dH)H → 0, and ε is the volume fraction of magnetic oxide. Here ε is defined as 1, and H0 is obtained from the experimental data at a high external field where M versus 1/H is linear with an intercept on the M-axis of 1/H0 . By using the experimental values for above parameters the diameter of uncoated and PVA coated CoFe2 O4 nanoparticles are calculated and found to be 6.78 nm (±1.2 nm) and 6.77 nm (±1.1 nm) respectively. The values calculated from the magnetization data are found to be lower than the values calculated by XRD and TEM, which may be attributed to a magnetically ineffective PVA layer on the particle surface [26]. 3.4. Effect of surface coating on hydrodynamic size of CoFe2 O4 nanoparticles DLS measurements have been taken to elucidate the binding of groups remained stably bound to the magnetic core CoFe2 O4 as well as to study effect of coating (magnetic core + organic coating) on the overall size of the nanoparticles when dispersed in the DDW. DLS cannot discriminate between inorganic and organic material and thus measures the overall particle size. Even in the absence of
external magnetic field, the magnetic dipole–dipole interactions between particles can cause their agglomeration. Therefore, the hydrodynamic size distributions are larger than those observed by TEM. The measurement of hydrodynamic size is important for biomedical applications of MNPs. For example, the MNPs larger than 30 nm are used for phagocytosis imaging in MRI [27,28]. On the other hand, the NPs (∼10 nm) are able to escape from the phagocytes; such MNPs conjugated with a target specific biomolecules are used to detect the target tissues through molecular interactions between NP–biomolecule conjugates and molecular markers expressed by target tissues [29,30]. It is also well known that the hydrodynamic size has significant influence on the relaxation mechanisms. [1] Fig. 8 illustrates the hydrodynamic size of the uncoated and PVA coated CoFe2 O4 in DW. The observed average particle size of the uncoated and PVA coated CoFe2 O4 NPs are ∼1676 nm and 126 nm. From figure, it can be concluded that uncoated nanoparticles are in agglomerated form with non-uniform size distribution when dispersed in DW whereas PVA coated nanoparticles exhibit nearly uniform and narrow size distribution over uncoated NPs. The PVA coating on NPs surface reduces the interparticle interactions and therefore prevents agglomeration between them. Therefore the narrow particle size distribution in case of particles coated with PVA is observed in DLS. 3.5. Cytotoxicity study
Fig. 7. Magnetization (M) loop for uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs at 298 K.
For biomedical purposes, especially in in vivo applications, toxicity is a critical factor to consider when evaluating their potential. NPs for hyperthermia are often purposely coated with biocompatible materials and monoclonal antibodies to target specific cells. As these nanoparticles are intentionally engineered to interact with cells, it is important to ensure that these enhancements are not causing any adverse effects. More significant is whether either naked or coated NPs will undergo biodegradation in the cellular environment and what cellular responses degraded NPs induce. For example, biodegraded NPs may accumulate within cells and lead to intracellular changes such as disruption of organelle integrity or gene alterations. While in vitro NPs applications afford less stringent toxicological characterization, in vivo use of NPs requires thorough understanding of the kinetics and toxicology of the particles. [31] In vitro cytocompatibility studies of coated and uncoated CoFe2 O4 NPs were tested using L929 cell line by trypan blue
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Fig. 8. DLS particle size distribution data (a) uncoated and (b) PVA coated CoFe2 O4 NPs.
assay with different particle concentrations. Uncoated (S1) and PVA coated CoFe2 O4 (S2) nanoparticles are tested with L929 cell lines incubated for 48 h with the concentration of 0.3, 0.6, 1.2 and1.8 mg mL−1 in the 5% CO2 atmosphere. Fig. 9 shows the effect of PVA coated and uncoated CoFe2 O4 NPs on L929 cell line. It is found that, PVA coated NPs (S2) do exhibit dose dependant cell viability. It has been noticed that, the percentage viability of L929 cells for control as well as for different magnetic particle concentration after 48 h incubation is different. Cell viability of PVA coated and uncoated CoFe2 O4 NPs is above 70% even at higher concentration of 1.8 mg mL−1 . As compared to coated NPs, uncoated NPs show higher toxic effect to L929 cells. This may be due to absence of biocompatible coating on the surface of CoFe2 O4 nanoparticles. Also it is well known that when MNPs expose to the healthy cells, presence of NPs on the cell surface affect the plasma membrane over a period of time and causes lysis of the cells hence causes cell death as compared to control. The PVA coated MNPs shows very high cell viability as compared to uncoated NPs for all tested concentrations. Therefore these PVA coated NPs with concentrations up to 0.6 mg mL−1 can be used for biomedical applications as it shows viability more than 90% after incubation for 48 h.
4. Conclusion In summary, CoFe2 O4 NPs were successfully synthesized by combustion method and surface modified with biocompatible PVA. Effect of PVA coating on the structural, morphological and magnetic properties is studied. XRD and FTIR studies show pure ferrite phase formation of with and without coated NPs. Thermogravimetric and FTIR analysis confirms the successful attachment of functional groups on the surface of NPs. Contact angle measurements show that hydrophobic surface of CoFe2 O4 nanoparticles transferred into hydrophilic due to attachment of amphiphilic groups provided by PVA. In vitro cytotoxicity evaluations based on trypan blue assay showed that PVA capping on the surface of MNPs effectively improved their stability and cytocompatibility. Acknowledgements Authors are grateful to Department of Science and Technology, Delhi and Board of Research in Nuclear Sciences, Mumbai for their financial assistance in the form of Research Projects. Authors would like to thank Dr. J. L. Gunjakar for providing TEM images. References
Fig. 9. Cell viability of uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs.
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Polyvinyl alcohol functionalized cobalt ferrite nanoparticles for biomedical applications A.B. Salunkhe a , V.M. Khot a , N.D. Thorat a , M.R. Phadatare a , C.I. Sathish b , D.S. Dhawale b , S.H. Pawar a,∗ a b
Center for Interdisciplinary Research, D. Y. Patil University, Kolhapur 416006, Maharashtra, India National Institute for Materials Science, 1-1 Namiki, Tsukuba, 305-0044 Ibaraki, Japan
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Article history: Received 25 July 2012 Received in revised form 6 October 2012 Accepted 12 October 2012 Available online 23 October 2012 Keywords: CoFe2 O4 nanoparticles Polyvinyl alcohol coatings Cytotoxicity Magnetic diameter
a b s t r a c t In the present work, cobalt ferrite nanoparticles (CoFe2 O4 NPs) have been synthesized by combustion method. The surface of the CoFe2 O4 NPs was modified with biocompatible polyvinyl alcohol (PVA). To investigate effect and nature of coating on the surface of CoFe2 O4 NPs, the NPs were characterized X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The transmission electron microscopy (TEM) and dynamic light scattering (DLS) results demonstrate the monodispersed characteristics of CoFe2 O4 NPs after surface modification with PVA. The decrease in contact angle from 162◦ to 50◦ with PVA coating on NPs indicates the transition from hydrophobic nature to hydrophilic. The Magnetic properties measurement system (MPMS) results show that the NPs have ferromagnetic behavior with high magnetization of 75.04 and 71.02 emu/g of uncoated and coated CoFe2 O4 NPs respectively. These PVA coated NPs exhibit less toxicity over uncoated CoFe2 O4 NPs up to 1.8 mg mL−1 when tested with mouse fibroblast L929 cell line. © 2012 Elsevier B.V. All rights reserved.
1. Introduction MNPs because of their outstanding physical and chemical properties have received considerable attention in various fields from technology to biomedical applications [1]. The dimensions of these nanoparticles make them ideal candidates for nano engineering of surfaces and production of functional nanostructures. Such modification facilitates their use in biomedical applications for example as contrast agents for magnetic resonance imaging (MRI), for targeted drug delivery and as heating mediators in hyperthermia therapy application etc. [2]. Among various MNPs, spinel CoFe2 O4 NPs are extensively studied due to their ability to form an ideal magnetic system toward understanding and controlling magnetic properties at the atomic level through chemical manipulation. CoFe2 O4 has large anisotropy compared to other oxide ferrites [3,4]. Though they are proposed for biomedical application their use in medicine is restricted due to numerous problems such as toxicity due to the remarkable amount of cobalt release in aqueous solutions, aggregation in solution, and poor accessibility of the surface when surfactants are used. This problem can be overcome by surface coating of CoFe2 O4 with a compatible, nontoxic, and water-stable/dispersing material [5–9].
From this point of view, encapsulating MNPs by PVA is a promising and important approach. The rich and well documented chemistry of biocompatible PVA coatings may allow practical implementation of MNPs in pharmaceutical and biomedical applications [10,11]. PVA served as the protective polymer as it has the desired solution properties in water and contains many isolated hydroxyl functional groups, which can adsorb and complex with metal ions [12]. It is assumed that PVA chain adsorbed on the surface of magnetic core of CoFe2 O4 NPs and form a shell as shown in Fig. 1 [13]. In the present investigation, CoFe2 O4 NPs were first synthesized by combustion method and further coated with PVA. The effect of coating on structural, morphological and magnetic properties of CoFe2 O4 NPs was studied in detail. The attachment of PVA on the surface of CoFe2 O4 core and stability were investigated by XRD, FTIR, TEM and DLS respectively. The biocompatibility of coated and uncoated CoFe2 O4 NPs was tested for different concentrations (0.3–1.8 mg/mL) with mouse fibroblast L929 cell line by trypan blue assay.
2. Experimental 2.1. Materials
∗ Corresponding author. Tel.: +91 231 2601202/2601235; fax: +91 231 2601595. E-mail addresses: salunkhe [email protected], pawar s [email protected] (S.H. Pawar). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2012.10.073
Analytical grade cobalt nitrate [Co(NO3 )2 ·6H2 O] (99%), ferric nitrate [Fe(NO3 )3 ·9H2 O](99.9%), Glycine [CH2 NH2 COOH] (99%) and polyvinyl alcohol (-C2 H4 O)n (99%) were purchased from LOBA
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using a PSS/NICOMP 380 ZLS (Particle Sizing System, Santa Barbara, ˚ All CA, USA). The light source was He–Ne laser operated at 632.8 A. measurements were carried out at 25.0 ± 0.1 ◦ C using a circulating water bath. Cylindrical cells of 10 mm diameter were used in all of the light scattering experiments. The autocorrelation function was obtained using a 192-channel photon correlator. For reproducibility, at least three measurements were conducted for each reading. Magnetic properties of the NPs were measured by the MPMS XL. 2.5. Cytotoxicity study Fig. 1. Synthesis of CoFe2 O4 NPs and layering of PVA polymers on the surface of the NPs.
CHEM, India. All chemicals listed above are water soluble and used as received. 2.2. Synthesis of CoFe2 O4 NPs The CoFe2 O4 NPs (S1) were prepared by simple and inexpensive combustion method using corresponding metal nitrates and glycine. Metal nitrates were employed both as metal precursors and oxidizing agents while glycine serves as reducing agent and fuel for combustion. The stoichiometric amounts of metal nitrates and glycine were mixed in large beaker which results in slurry due to hygroscopicity of metal nitrates [14]. The obtained slurry was then introduced to hot plate preheated to 250 ◦ C. After evaporation of water content, the mixture starts frothing and ignited with flame within few seconds, giving voluminous and foamy CoFe2 O4 powder. The detailed synthesis and reaction mechanism of CoFe2 O4 nanoparticles was reported elsewhere [15]. 2.3. Surface coating of CoFe2 O4 nanoparticles The mixture of CoFe2 O4 nanoparticles and water was vigorously stirred for 10 min by dispersing the nanoparticles in double distilled water (DDW). PVA solution was prepared by dissolving 0.6 M PVA in 40 mL water. PVA solution was added to above mixture and stirred further for 30 min. The obtained mixture was kept overnight at room temperature, then supernatant was discarded and PVA coated CoFe2 O4 nanoparticles were collected. Surface modified CoFe2 O4 NPs (S2) are easily dispersible in DDW after 10 min sonication. 2.4. Characterizations Structure and phase of as synthesized and PVA coated CoFe2 O4 NPs was studied by XRD (Philips-3710) with Cu-K␣ radiation ˚ in the 2 range from 20 to 100◦ . The patterns were ( = 2.2897 A) evaluated by Panalytical X’pert high score software and compared with standard JCPDS (card no. 22-1086). Presence of the magnetic core and polymer coating of the MNPs was confirmed by FTIR spectroscopy (Perkin Elmer spectrometer model no. 783, USA). Elemental composition of the MNPs was determined by TGA with Trans analytical instruments (SDT 2960) operated in temperature from 35 ◦ C to 1000 ◦ C with heating rate of 10 ◦ C/min in flowing nitrogen ambiance. The particle size of coated and uncoated CoFe2 O4 NPs was measured with TEM (Philips CM 200 model, oper˚ Water contact angles of ating voltage 20–200 kV, resolution 2.4 A). coated and uncoated MNPs were measured using the sessile drop method by deposition of 4–6 L droplets of DI water on a horizontal surface and their observation in cross-section. Each drop was observed directly with an Olympus BX-41 microscope objective lens, whereas its image was digitally captured using a 1.4 megapixel computer-controlled digital CCD camera. The values reported are averages of more than 20 measurements performed in different areas of each sample surface. DLS measurements were carried out
2.5.1. Cell culture In vitro cell viability of coated and uncoated CoFe2 O4 MNPs was studied using mouse fibroblast L929 cell line purchased from National Center for Cell Science (NCCS), Pune, India. L929 cells were grown in minimal essential medium supplemented with 10% (v/v) FBS (fetal bovine serum), kanamycin (0.1 mg/mL), penicillin G (100 U/mL), and sodium bicarbonate (1.5 mg/mL) at 37 ◦ C in a 5% CO2 atmosphere. In brief, cells (1 × 105 cells/mL) were grown for 24 h. After 24 h, the old media was replaced by fresh media and different particle concentrations (0.3, 0.6, 1.2 and 1.8 mg/mL) of uncoated and coated CoFe2 O4 NPs. 2.5.2. Cell viability by trypan blue dye exclusion assay For this experiment, L929 cells were seeded at a density of 1 × 105 cells/mL in a culture plate. After 24 h of incubation, the old media was replaced with a media containing different concentrations of MNPs. And the cells were exposed for 48 h incubation time. Then, the dishes were washed thrice with PBS to remove the MNPs. Both the attached and unattached cells were harvested and combined after trypsinization (0.025% trypsin, 10 min). The cells were then stained with trypan blue dye and counted with a hemocytometer. The experiments were replicated three times and the data was graphically presented as mean. The total amount of cells, stained and unstained is counted. The calculated percentage of unstained cells will represent the percentage of viable cells. 3. Results and discussion 3.1. Effect of surface coating on structural properties 3.1.1. XRD analysis The formation of uncoated and PVA coated CoFe2 O4 NPs was confirmed from XRD pattern (Fig. 2(a)). CoFe2 O4 nanoparticles display several relatively strong reflection peak in the 2 region of 20–100◦ , which is quite similar to the other groups, confirming that prepared NPs are CoFe2 O4 with spinel cubic structure having fd3 m space group [16,17]. The obtained peaks are well matched with standard JCPDS card no. 22-1086. The determination of crystallite size (D) of CoFe2 O4 powder was based on X-ray diffraction line broadening and calculated by using Scherrer formula [18], D=
0.9 ˇ cos
(1)
where ˇ is the full-width at half maxima of the strongest intensity diffraction peak (3 1 1), is the wavelength of radiation and is the angle of strongest characteristic peak. From Fig. 2(a) it can be revealed that all resultant NPs are pure CoFe2 O4 with an inverse spinel structure. No change in phase was observed in case of particles coated with PVA while slight suppression of diffraction peaks can be clearly observed. There is no pronounced change in the lattice constant however crystallite size varies slightly after surface modification. The calculated crystallite size (D) for uncoated and coated CoFe2 O4 NPs are 38 nm and 35 nm respectively. The corresponding selected area electron diffraction (SAED) pattern of coated CoFe2 O4 is shown in Fig. 2(b). Figure shows spotty
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Fig. 2. (a) XRD patterns of uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs and (b) SAED pattern of PVA coated CoFe2 O4 NPs.
ring patterns for CoFe2 O4 phase consistent with XRD. According to the diffraction patterns in Fig. 2(b), the measured lattice constants and interplanar spacing (dh k l ) agree with those in the XRD results. The electron diffraction pattern consisting of rings indicates the good crystal structure of PVA coated CoFe2 O4 NPs. 3.1.2. Infrared analysis FTIR is an appropriate technique to analyze the attachment of polymer to surface of MNPs. Fig. 3 shows the FTIR spectra of uncoated and PVA coated CoFe2 O4 NPs. The series of characteristic IR bands are summarized in detail in Table 1 [19] and only the salient features are discussed below. In case of uncoated CoFe2 O4 NPs (S1), the band at 3214 cm−1 is assigned to stretching () vibrations due to adsorbed water on the surface of CoFe2 O4 NPs. The band observed at 578 cm−1 is corresponds to stretching vibrations of Fe O. In PVA coated CoFe2 O4 NPs (S2), the M O stretching band at 578 cm−1 , the alcoholic O H stretching band at 3260 cm−1 are observed. The additional bands at 2982 cm−1 corresponding to C H stretching vibrations, at 1385 cm−1 corresponding to C C stretching vibrations, at 1104 cm−1 attributable to M O C (M=Fe) bond and
at 996 cm−1 corresponding to CH2 rocking are observed in PVA coated CoFe2 O4 NPs. Therefore attachment of PVA onto CoFe2 O4 nanoparticles surface is confirmed and supported by contact angle measurements [20]. 3.1.3. Thermo-gravimetric analysis TGA provides additional quantitative evidence on structure of the NPs coating. In this experiment, MNPs are heated from 35 to 1000 ◦ C under flowing N2 and changes in mass loss of organic material are recorded. Although it is an extremely valuable technique for surface characterization of NPs, it is not commonly applied in literature for ferrite MNPs. The information one can retrieve from TGA measurements is mutual. First of all, TGA allows us to determine the bonding strength of the ligand to the NPs surface and its thermal stability. TGA (Fig. 4) for sample S1 shows only 2.5% weight loss in the temperature range of 25–250 ◦ C which may be due to evaporation of hydroxyl groups adsorbed on the surface of nanoparticles during synthesis. Such a very less weight loss suggests that, pure phase of CoFe2 O4 NPs can be achieved easily by combustion method. Whereas for sample S2, the weight loss process observed in two stage. In the first, ∼4% weight loss in the temperature range of 150–200 ◦ C is observed which is probably related to the outer layer of the polymer. However in the 200–400 ◦ C temperature range, ∼18.5% weight loss is noticed in the second stage attributed to the detachment of chemisorbed polymer layer from the surface which needs higher temperature for removal [21].
Table 1 Characteristic FTIR Vibrations for uncoated and PVA coated CoFe2 O4 NPs.
Fig. 3. FTIR spectra for uncoated (S1) and PVA coated CoFe2 O4 nanoparticles (S2).
Samples
IR region or bands (cm−1 ) Characteristics vibrations
CoFe2 O4
3214 578,571
(O H) (M O)
PVA coated CoFe2 O4
3260 2982 2850 1640 1385 1225 1104 996 578
(O H) a (CH2 ) s (CH2 ) ı (H O H) (C C) (OCH3 ) rocking (M O C (M Fe)) CH2 rocking (M O)
a , asymmetric stretching; s , symmetric stretching; ı, scissoring.
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Therefore, material can absorb water and its structure gets deteriorated with time. The contact angle for the hydrophilic surface is less than 90◦ and for hydrophobic surface is greater than 90◦ . Surface behavior of the coated and uncoated CoFe2 O4 NPs toward hydrophobicity or hydrophilicity was tested in terms of contact angle () of the water droplet with the solid surface, measured using the formula, = 2 tan−1
Fig. 4. Thermogravimetric curves for uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs.
3.2. Effect of surface coating on morphological properties 3.2.1. TEM analysis Fig. 5 shows the TEM images for samples S1 and S2. From Fig. 5, it can be observed that the uncoated NPs (S1) are in highly agglomerated form whereas PVA coated MNPs show well dispersed characteristic. The dipole–dipole interaction in magnetic NPs results in the agglomeration of these NPs. The use of polymer during the synthesis process can reduce this interaction through physical or chemical adsorption on the surface of particles. The size of PVA coated and uncoated NPs is 37 and 34 nm respectively. 3.2.2. Contact angle measurements For biomedical applications, MNPs should be hydrophilic in nature so that it forms stable dispersion in physiological saline medium like water. One of the fundamental methods of characterizing the hydrophobic or hydrophilic properties of a solid surface is to determine the contact angle. The contact angle is a measure of the wetting behavior of a particular liquid on the surface under investigation and directly relates to the interfacial energies of the systems. The contact angle on the solid surface gets changed merely by changing the chemistry of the outermost monolayer. Depending on affinity of material to be tested toward water, it classified into two groups such as hydrophobic and hydrophilic. The hydrophilicity of the material is due to presence of amphiphilic groups on the material surface.
2h
(2)
where, h is the height and is the width of water droplet touching the solid surface of sample. Fig. 6 shows a water droplet in equilibrium over a horizontal solid surface of samples. From Fig. 6 it is observed that, values of contact angles for uncoated and PVA coated MNPs are 162◦ and 50◦ respectively. Contact angle values of uncoated NPs clearly suggest their hydrophobic nature. Surface modification with PVA causes large decrease in contact angle to 50◦ . Such decrease in contact angle after surface modification occurred due to the presence of amphiphilic groups of PVA on the surface of CoFe2 O4 magnetic core. Generally, when water contact angle is less than 50◦ , MNPs could be easily dispersed in aqueous solution because of hydrophilic character of present endgroups [22,23]. These findings support the idea that surface hydrophilicity plays a pivotal role in creating water-dispersible nanoparticles useful for biomedical applications. 3.3. Effect of surface coating on magnetic properties The room temperature M–H curves of samples S1 and S2 are shown in Fig. 7. From Fig, it can be seen that the magnetization of sample S2 (71.02 emu/g) is smaller as compared to sample S1 (75.94 emu/g) at applied field of ±15 kOe at 300 K. However, the magnetization for both samples is close to that of bulk CoFe2 O4 NPs [24]. The reduction in magnetization for sample S2 may be attributed to the presence of non-magnetic polymer layer on to the surface of NPs which reduces the particle-particle interaction and lowers the exchange coupling energy which in turn reduces the magnetization. Both samples, S1 and S2 exhibit typical ferromagnetic nature with low coercivity (∼94 Oe and ∼102 Oe respectively). In case of ferrofluids, the particle size distribution follows a lognormal distribution function [25]. Using the value of magnetization, magnetic particle size and size distribution can be calculated from following equation,
Dm =
18kT Md
i 3εMd H0
1/2 1/3
Fig. 5. TEM images for uncoated (S1) and PVA coated CoFe2 O4 NPs (S2).
(3)
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Fig. 6. Images of the static water contact angle showing the effect of chemical functionalization and micro/nanostructure on the surface hydrophilicity of combustion synthesized uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs.
where Md is the domain magnetization of the bulk CoFe2 O4 (80 emu/g), Dm is the magnetic diameter, i is the initial magnetic susceptibility i ∼ (dM/dH)H → 0, and ε is the volume fraction of magnetic oxide. Here ε is defined as 1, and H0 is obtained from the experimental data at a high external field where M versus 1/H is linear with an intercept on the M-axis of 1/H0 . By using the experimental values for above parameters the diameter of uncoated and PVA coated CoFe2 O4 nanoparticles are calculated and found to be 6.78 nm (±1.2 nm) and 6.77 nm (±1.1 nm) respectively. The values calculated from the magnetization data are found to be lower than the values calculated by XRD and TEM, which may be attributed to a magnetically ineffective PVA layer on the particle surface [26]. 3.4. Effect of surface coating on hydrodynamic size of CoFe2 O4 nanoparticles DLS measurements have been taken to elucidate the binding of groups remained stably bound to the magnetic core CoFe2 O4 as well as to study effect of coating (magnetic core + organic coating) on the overall size of the nanoparticles when dispersed in the DDW. DLS cannot discriminate between inorganic and organic material and thus measures the overall particle size. Even in the absence of
external magnetic field, the magnetic dipole–dipole interactions between particles can cause their agglomeration. Therefore, the hydrodynamic size distributions are larger than those observed by TEM. The measurement of hydrodynamic size is important for biomedical applications of MNPs. For example, the MNPs larger than 30 nm are used for phagocytosis imaging in MRI [27,28]. On the other hand, the NPs (∼10 nm) are able to escape from the phagocytes; such MNPs conjugated with a target specific biomolecules are used to detect the target tissues through molecular interactions between NP–biomolecule conjugates and molecular markers expressed by target tissues [29,30]. It is also well known that the hydrodynamic size has significant influence on the relaxation mechanisms. [1] Fig. 8 illustrates the hydrodynamic size of the uncoated and PVA coated CoFe2 O4 in DW. The observed average particle size of the uncoated and PVA coated CoFe2 O4 NPs are ∼1676 nm and 126 nm. From figure, it can be concluded that uncoated nanoparticles are in agglomerated form with non-uniform size distribution when dispersed in DW whereas PVA coated nanoparticles exhibit nearly uniform and narrow size distribution over uncoated NPs. The PVA coating on NPs surface reduces the interparticle interactions and therefore prevents agglomeration between them. Therefore the narrow particle size distribution in case of particles coated with PVA is observed in DLS. 3.5. Cytotoxicity study
Fig. 7. Magnetization (M) loop for uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs at 298 K.
For biomedical purposes, especially in in vivo applications, toxicity is a critical factor to consider when evaluating their potential. NPs for hyperthermia are often purposely coated with biocompatible materials and monoclonal antibodies to target specific cells. As these nanoparticles are intentionally engineered to interact with cells, it is important to ensure that these enhancements are not causing any adverse effects. More significant is whether either naked or coated NPs will undergo biodegradation in the cellular environment and what cellular responses degraded NPs induce. For example, biodegraded NPs may accumulate within cells and lead to intracellular changes such as disruption of organelle integrity or gene alterations. While in vitro NPs applications afford less stringent toxicological characterization, in vivo use of NPs requires thorough understanding of the kinetics and toxicology of the particles. [31] In vitro cytocompatibility studies of coated and uncoated CoFe2 O4 NPs were tested using L929 cell line by trypan blue
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Fig. 8. DLS particle size distribution data (a) uncoated and (b) PVA coated CoFe2 O4 NPs.
assay with different particle concentrations. Uncoated (S1) and PVA coated CoFe2 O4 (S2) nanoparticles are tested with L929 cell lines incubated for 48 h with the concentration of 0.3, 0.6, 1.2 and1.8 mg mL−1 in the 5% CO2 atmosphere. Fig. 9 shows the effect of PVA coated and uncoated CoFe2 O4 NPs on L929 cell line. It is found that, PVA coated NPs (S2) do exhibit dose dependant cell viability. It has been noticed that, the percentage viability of L929 cells for control as well as for different magnetic particle concentration after 48 h incubation is different. Cell viability of PVA coated and uncoated CoFe2 O4 NPs is above 70% even at higher concentration of 1.8 mg mL−1 . As compared to coated NPs, uncoated NPs show higher toxic effect to L929 cells. This may be due to absence of biocompatible coating on the surface of CoFe2 O4 nanoparticles. Also it is well known that when MNPs expose to the healthy cells, presence of NPs on the cell surface affect the plasma membrane over a period of time and causes lysis of the cells hence causes cell death as compared to control. The PVA coated MNPs shows very high cell viability as compared to uncoated NPs for all tested concentrations. Therefore these PVA coated NPs with concentrations up to 0.6 mg mL−1 can be used for biomedical applications as it shows viability more than 90% after incubation for 48 h.
4. Conclusion In summary, CoFe2 O4 NPs were successfully synthesized by combustion method and surface modified with biocompatible PVA. Effect of PVA coating on the structural, morphological and magnetic properties is studied. XRD and FTIR studies show pure ferrite phase formation of with and without coated NPs. Thermogravimetric and FTIR analysis confirms the successful attachment of functional groups on the surface of NPs. Contact angle measurements show that hydrophobic surface of CoFe2 O4 nanoparticles transferred into hydrophilic due to attachment of amphiphilic groups provided by PVA. In vitro cytotoxicity evaluations based on trypan blue assay showed that PVA capping on the surface of MNPs effectively improved their stability and cytocompatibility. Acknowledgements Authors are grateful to Department of Science and Technology, Delhi and Board of Research in Nuclear Sciences, Mumbai for their financial assistance in the form of Research Projects. Authors would like to thank Dr. J. L. Gunjakar for providing TEM images. References
Fig. 9. Cell viability of uncoated (S1) and PVA coated (S2) CoFe2 O4 NPs.
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