Current Nanoscience, 2011, 7, 000-000
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Biocompatibility Studies of Functionalized CoFe2O4 Magnetic Nanoparticles Pranav Kumar Prabhakar1, Swetha Vijayaraghavan2, John Philip2, and Mukesh Doble1* 1
Department of Biotechnology, Indian Institute of Technology Madras, Chennai-600 036, India, 2SMART Section, NDED, MMG, Indira Gandhi Centre for Atomic Research, Kalpakkam- 603 102, India Abstract: CoFe2O4 nanoparticles of different sizes were synthesised by controlling the digestion time using precipitation method and were characterised by X-ray diffraction, transmission electron microscopy, dynamic light scattering, and vibrating sample magnetometer. The average crystalline size increases from 13.9 to 19 nm as the digestion time is increased from 1.3 to 120 minutes. The CoFe2O4 nanoparticles were coated with two biological polymers, namely polyvinyl alcohol (PVA) and polyethylene glycol (PEG) at various ratios to enhance their biocompatibility. Coated nanoparticles were analysed for their cytotoxicity by MTT assay against 3T3-L1 adipocytes. Coated nanoparticles were found to be less cytotoxic when compared to uncoated one. The cell viability decreased as the concentration of the polymer (either PVA or PEG) coating increased. Cell viability decreases as the concentration of nanoparticle increases. At 5 μg/ml the cell viability with PEG coated nanoparticles (1:4) was 92.5%, with PVA coated nanoparticles (1:4) was 82.7% and with uncoated nanoparticles it was 46.4%. As the ratio of biopolymers (PVA and PEG) to nanoparticle increases, the viability of the cell increases. The difference between the effect of these two polymers increases as the concentration of the nanoparticle decreases. The antiinflammatory properties of these nanoparticles were determined by RTPCR by measuring the two pro-inflammatory cytokines (namely tumor necrosis factor and IL6). TNF- and IL6 were upregulated by 3.57- & 2.86 folds their base level with uncoated nanoparticles. Whereas it was upregulated by 1.54- & 1.68-folds with PEG coated and 1.9- & 2.18-folds with PVA coated nanoparticles. Thus the coated nanoparticles can be used for further biological experiments including magnetic resonance imaging, and in targeted drug delivery systems for various diseases.
Keyword: Nanoparticles, biocompatibility, inflammatory response, polyvinyl alcohol, polyethylene glycol. 1. INTRODUCTION Magnetic nanoparticles (MNP) are a major class of nanoscale materials with the potential to revolutionize current clinical diagnostic and therapeutic techniques. Due to their unique physical properties and ability to function at the cellular and molecular level of biological interactions, MNPs are being actively investigated as the next generation of magnetic resonance imaging (MRI) contrast [1] and as a vehicle for targeted drug delivery [2]. The recent interest in nanotechnology has significantly expanded the breadth and depth of MNP research because of their wide range of applications in the detection, diagnosis, and treatment of illnesses, including cancer [3], cardiovascular disease [4], and neurological disease [5], MNPs may soon play a significant role in meeting the healthcare for tomorrow. Different forms of MNP with various chemical compositions have been proposed and evaluated for biomedical applications to exploit nanoscale magnetic phenomena, such as enhanced magnetic moments and superparamagnetism. Like other nanomaterial-based systems, advances in nanotechnology now allow for precise engineering of the critical features of these fine particles. Composition, size, morphology and surface chemistry can now be enhanced by various physical as well as chemical processes to not only improve magnetic properties but also affect the behavior of nanoparticles in vivo [6]. In its simplest form, a biomedical MNP platform is comprised of an inorganic nanoparticle core and a biocompatible surface coating (mostly coating biopolymers) that provides stabilization at normal physiological conditions. This modular design enables MNPs to perform multiple functions simultaneously, such as in multimodal imaging [7], drug delivery and real-time monitoring, as well as combined therapeutic approaches. In recent years, there has been a growing interest in the design of colloidal with prolonged blood circulation time for drug delivery. Among them, the nanoparticles presenting poly (ethylene glycol) (PEG) [8] and polyvinyl alcohol (PVA) [6] chains at their surface have appeared to be a particularly promising system. An interesting *Address correspondence to this author at the Department of Biotechnology, Indian Institute of Technology Madras, Chennai-600 036, India; Tel: +914422574107; Fax: +914422574102; E.mail:
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property of these particles is their reduced uptake by the mononuclear phagocytic system (MPS). Poly(ethylene glycol) (PEG) is another widely used polymer for nanoparticle coating in biomedical applications [9]. The antifouling nature of PEG has been shown to reduce nanoparticle uptake by macrophages [10] and extend blood circulation time in vivo [11]. There are two pro-inflammatory cytokines, namely TNF- and IL6, which are involved in the response of the body or immune system towards foreign material and its increased upregulation. The extent of their upregulation is a good measure of inflammatory responses. Among various ferrites, CoFe2O4 is of particular interest because of its remarkable properties like tunable coercivity, large anisotropy, moderate saturation magnetization, unique lightinduced coercivity change, site-specific and strong binding to the serum albumin proteins etc. Exploiting the unique magnetic properties of CoFe2O4 nanoparticles, many interesting biomedical applications are developed e.g. drug delivery, DNA separation, Magnetic Resonance Image (MRI) contrast and hyperthermia [2, 12-18]. Besides, CoFe2O4 nanoparticles are widely used in magneto-optic recording medium, high density data storage devices, stress sensor and other high frequency applications. One of the important requirements of this nanoparticle is the tunability of magnetic properties. In general, magnetic nanoparticles are of relatively low toxicity and are ideal candidates for targeted drug delivery. Magnetic nanoparticles are coated with biocompatable biopolymers to reduce their cell cytotoxicity, for tagging or better binding of drugs. Further, these coatings provide steric stabilization to the nanoparticles from agglomeration. The main goal of this study is to synthesise cobalt ferrite nanoparticles and study their biocompatibility after coating them with different biocompatible polymers at different ratios. 2. MATERIALS AND METHODS 2.1. Preparation Method CoFe2O4 nanoparticles were prepared by the precipitation method. 2M FeCl3.6H2O and 1M CoCl2.6H2O salt solutions (Aldrich) were freshly prepared in water medium. These salt solutions © 2011 Bentham Science Publishers Ltd.
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were mixed in 1:1 ratio with continuous stirring speed of 1000 rpm at 90ºC, and at a pH of 1.5. CoFe2O4 nanoparticles were obtained by the addition of 6N NaOH in the above mentioned salt solution at the rate of 10 ml per sec. After the addition of alkali, the pH reaches a value of 12. At this stage, the solution turns black, indicating the formation of CoFe2O4 nanoparticles. The same pH, temperature and stirring speed were maintained for different digestion time (1, 2, 5, 15, 30, 60 and 120 minutes). The precipitated particles are waterwashed and dried at 35ºC for 24 hrs. in inert atmosphere. These dried powders were used for further characterization. 2.2. Characterization Methods The crystallographic properties and average particle size of the sample were determined by a MAC Science MXP18 X-ray diffractometer. 2 values were taken from 15 to 65o using CuK radiation ( value of 1.546 Å). The X-ray diffraction (XRD) patterns of the nanoparticles were verified by comparing with the JCPDS card no. 22-1086. High resolution transmission electron microscopy (HRTEM) investigations were carried out using a JEOL 2000 EX II (T) operated at 200 kV. One drop of colloidal suspension of CoFe2O4 in hexane was collected on to a carbon film coated copper grid, and dried overnight under a lamp. The magnetic properties of the CoFe2O4 nanoparticles are studied using Vibrating sample magnetometer (EG&G PRINCETON Model: 4500), at room temperature with applied magnetic field in the range of ±7 kOe. 2.3. Coating of Nanoparticles with PVA and PEG The polymer solutions were prepared on weight basis. For 100 mg of magnetite nanoparticles, quantity of the polymers taken were 100, 200, 300, & 400 mg of polyvinyl alcohol (Molecular weight: 15,000) and polyethylene glycol (Molecular weight: 20,000), thereby forming four batches at a ratio of 1:1, 1:2, 1:3, & 1:4 (on weight basis) respectively. The polymers were weighed in an electrical balance and added to different beakers containing 40ml of distilled water each. The content of beakers was stirred with a magnetic stirrer (bead) and was subjected to stirring in a magnetic spinot. PVA was hot water soluble; hence the temperature of the Spinot was set to 60C. The temperature is not allowed to exceed this value as the cloud point of PVA is 85C, beyond which the solution is bound to become turbid and evaporate due to the presence of water (Boiling Point: 100C). A PEG sample was also prepared in similar fashion. However, it was soluble at room temperature. Thus, the required amounts of polymer solutions were prepared. The prepared polymer solutions was mixed with 20 ml of freshly prepared magnetite nanoparticles solution and put for stirring in a digital stirrer fitted with a three headed glass rod propeller at 500 rpm continuously for 2 hrs. About 2 ml of each sample of the solutions were taken in culture tubes to measure the size and the zeta in the DLS Zetasizer. The remaining portion of the samples was centrifuged in a cooling centrifuge (Remi) at 8000 rpm for 10 minutes respectively to isolate the particles. The particles were separated by means of a strong magnet, then dried and collected in small ampoules for TG and biological analysis. 2.4. Cell Culture 3T3-L1 adipocytes, a mouse embryonic cell line with differentiation activity to adipocyte, were purchased from NCCS, Pune, India. The culture media DMEM (Dulbecco modified Eagle’s medium), FBS (Fetal bovine serum), trypsin, antibiotics (penicillin, streptomycin and amphotericin B) were purchased from PAN BIOTECH GmbH, Germany. DMSO was ordered from Merck, Mumbai, India. All other chemicals were purchased from SRL Mumbai, India. All the plasticwares were obtained from Tarson, Kolkata, India. Medox easy spin column total RNA Miniprep super kit was purchased from Medox Biotech, Chennai, India, and used for total
Prabhakar et al.
RNA extraction. RobusT I RTPCR kit from Finzymes, Finland, was used for RTPCR studies. 3.5105 3T3-L1 adipocytes were grown in each well of a 24 well plate in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) at 37°C in a humidified atmosphere composed of 5% CO2 and 95% air. The medium was changed every third day. For differentiation, the 3T3-L1 adipocytes were transferred to DMEM having 2% FBS for 4–6 days post-confluency. The extent of differentiation was established by observing the formation of elongated and multinucleate myotubes. These differentiated cells were used for further studies. 2.5. Biocompatibility of Coated Nanoparticles The cell cytotoxicity was measured using MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described in the literature [19]. This assay is based on the reduction of MTT into purple formazan pigment by the mitochondrial succinate-tetrazolium reductase system. The dark blue formazan dye generated by the live cells was proportional to the number of live cells and the absorbance at 595 nm was measured using a microplate reader [20]. Changes in MTT reductase activity is detected even before the membrane lysis, making this assay ideal for detecting cellular viability [21]. The cells are seeded at 5 104 cells/ml density in petri dishes and incubated with (5, 10, 25 and 50 μg/ml) uncoated and PVA & PEG coated CoFe2O4 nanoparticles for 24- h. After the specified time, the medium is replaced with MTT solution (0.5 mg/ml in PBS) for 4-h at 37°C. The formazan formed inside the cells is dissolved in 0.04N HCl taken in isopropanol and the absorbance is measured spectrophotometrically at 595 nm with a Spectramax Plus384® Spectrophotometer (Molecular Devices, CA, USA). The number of viable cells is directly proportional to the production of formazan. 2.6. Anti-inflammatery Studies on the Basis of RTPCR for TNF-alpha and IL-6 RTPCR was carried out as reported in literature [22]. The total RNA from 3T3-L1 adipocytes were isolated after incubation with uncoated and coated nanoparticles for 24 hours with the help of a total RNA Miniprep super kit as per the manufacturer’s instructions. Reverse transcription PCR was carried out to obtain cDNA with 5 U/l of AMV reverse transcriptase along with 20 pg of template RNA. The primers used were TNF sense, 5- ACCTTTC CAGATTCTTCCCTGAG-3; anti-sense 5- CCCGGCCTTC CAAATAAAT ACATT-3 [23], IL6 sense, 5- GAGGATACCACTCCCAACAGACC-3; and anti-sense, 5- AAGTGCATCATCGTTGTTCATACA-3 [23]. PCR reaction mix, consists of 10PCR buffer, 10 mM each dNTP, 10 pM of paired primers, two units of DNA polymerase and distilled water, to make up to a total volume of 50 ml. This mixture was placed in a PCR thermal cycler for 30 cyclic reactions in PTC-200 DNA Engine® thermal cycler (MJ Research. CA, USA). The products were run on 1.5% agarose gel, stained with ethidium bromide and photographed with a Gel DockTM system (Biorad laboratories Inc. UK) 2.7. Statistical Analysis Results were expressed as mean ± SD. Data were analysed using Student’s t-tests. A value of p
