Effect of dextran coated ferrite nanoparticles on the antioxidant defense mechanism of mice brain.
Nanotechnology is a rapidly expanding area with new applications invented almost every day. It has the potential to improve, even revolutionize, many technology and industry sectors: environmental science, medicine, food safety, energy, information technology and homeland security among others. Application of nanotechnology in the medical and biotechnology sector will make the technology cheaper, portable, personalized, safer and easier to administer.
Magnetic nanoparticles are one such particle which is exploited for their use in various biomedical and neurobiological applications. Their property of super paramagnetism makes them one of the most sort out particle in the area of targeted drug delivery and cancer therapy. The very property of paramagnetism makes them non magnetic in the absence of an external magnetic field which in turn renders them the advantage not getting aggregated unintentionally . Magnetic nanoparticles are composed of a core magnetic particle coated by some kind of ligand to improve its biocompatibility and reduce cytotoxicity. These magnetic nanoparticles can be manipulated in presence of an external magnetic field and thus can be used in diagnostic tools, imaging , tissue repair  hyperthermia, and targeted therapies like gene and drug delivery [4-6].
Magnetic nanoparticles pave way to new hopes in the field of brain targeted biomedical applications. The major obstruction to the diagnosis and treatment of brain associated problems are its high sensitivity and impermeability to majority of the drugs developed. It is reported that the magnetic nanoparticles can cross blood brain barrier and can be controlled and concentrated with in specific regions of the brain with the aid of external magnetic field. In this regard, the magnetic nanoparticles prove to be a promising agent for brain targeted application including diagnosis, drug delivery and cancer therapy [7-8].
Though the magnetic nanoparticles guarantee a wide variety of application the concerns about the toxicological aspects of these nanoparticles still remain. Nanoparticles toxicity is mainly due to its ability to produce reactive oxygen species (ROS) [9-10]. The ROS generation can lead to oxidative stress, lipid peroxidation, DNA damage, protein denaturation and ultimately cell death and tissue damage [11-12]. No sufficient data are available on the retention and degradation of the nanoparticles inside the body. However many studies carried out in vitro on brain cells like neurons , astrocytes  and microglia  demonstrate the nanoparticles mediated ROS generation and associated toxicity in these cells.
Body has different mechanism to tackle the ROS generated, there by protecting the tissues from oxidative damage. The responses of different tissues to ROS vary based on the nature of the tissue. However the body has different antioxidant molecule which either scavenge the ROS or convert them to less toxic products, there by protecting body from oxidative damage. The antioxidant defense system of the body include enzymes like super oxide dismutase (SOD), Glutathione peroxidase (GPx), Glutathione reductase (GR) and low molecular weight free radical scavengers like glutathione, ascorbate and alfa tocopherol. The alteration in the level of these antioxidants can be used as a perfect marker for oxidative stress
Brain in particular is susceptible to ROS mediated oxidative damage due to following reasons: high polyunsaturated fatty acid (PUFA) content and high amount of iron and copper containing proteins like Transferrin and Ceruloplasmin that can mediate Fenton reaction [16-18]. ROS can be generated in the body in conditions like stress, injury, exposure to chemicals, neurodegenerative diseases or invasion of pathogen. If brain encounters such sort of disturbance it will be reflected in the level of antioxidant produced [19-21].
The administered nanoparticles may accumulate in the non targeted locations in the body were it will degrade gradually to release it's by products to the system. It is feared that such by products will evoke ROS and oxidative stress there by causing unforeseen damages to the tissues and organs. So a thorough understanding of the behavior of nanoparticles in vivo must be carried out before they are used for any biomedical applications. In the present study in-house synthesized dextran coated ferrite nanomaterials (DFN) designed for targeted therapies are assessed for its effect on the mice brain. The brain antioxidant levels were assessed at different time periods after delivering the particle via IP injection.
Materials and Methods
Phosphate Buffered Saline (PBS) and bovine serum albumin (BSA) were purchased from Gibco (Grand Island NY, USA). thiobarbituric acid (TBA), reduced glutathione (GSH), oxidized glutathione (GSSG) and dithio-bis-2nitrobenzoic acid [DTNB] were purchased from Sigma Chemicals Co. Ltd. (St. Louis, MO USA). Ethylene triamine tetra acetic acid (EDTA), disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were obtained from (Merck, Germany). All other chemicals were purchased locally from India and were of analytical grade
Mice were procured from the Division of Laboratory Animal Sciences of Biomedical Technology Wing, SCTIMST. Swiss albino mice weighing 16- 20g were housed in individually ventilated cages. They were maintained in a 12h light/dark cycle under controlled environment of temperature (22 [+ or -] 2 [degrees]C) and humidity (30-70%). Animals were provided with commercially available food and distilled water ad libitum. They were handled humanly with out causing them pain or stress. The entire animal experiments were carried out according to institute animal ethics committee regulations approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Synthesis of dextran coated ferrite nanomaterials
In house synthesized Dextran-coated ferrite nanomaterials (DFN) synthesized by co-precipitation method were used for the entire study. In brief the stoichiometric mixtures of FeCl3 and FeCl2. 4H2O (Fe3+/Fe2+: 2:1) were heated at 70[degrees]C. Ferrite nanoparticles were precipitated by the addition of 3M NaOH drop wise for 1h followed by hot stirring for another 1 hr. The precipitate was then washed three times with de-ionized water to get uniformly dispersed spherical magnetite particles. The overall reaction was carried out in N2 atmosphere to prevent oxidation of magnetite to magnemite. Surface coating of ferrite nanoparticles with dextran was done by stirring ferrite nanomaterial in a solution of dextran of appropriate concentration for overnight (at 37[degrees]C). The precipitate was then washed and lyophilized to obtain DFN. Detailed characterization of the DFN was reported by our own group . The size of the DFN used for the study was <25 nm (Fig. 1).
Toxicity of DFN
Swiss albino mice weighing 16-23g were randomly divided in to three experimental groups, each containing 3 animals and three animals were kept as control. To assess the effect of DFN on the mice brain, the particle at a concentration of 100mg/ml were dissolved in de-ionised water. After through mixing DFN particles were administered to mice (100mg/ kg) via IP injection. All the experimental and control animals were observed periodically during the first 24h and there after daily until they were sacrificed. They were examined for any abnormality in the appearance of fur, skin, eyes, tail and body posture and for respiratory rate, pulse rate, body temperature, autonomic reflexes and behavior. Each group animals were sacrificed after 7, 14 and 21 day's interval.
Preparation of brain homogenate
The animals were sacrificed using cervical dislocation and the brain was immediately isolated, washed in cold phosphate buffered saline (PBS) and placed in an ice bath. 10% tissue homogenate was prepared in 0.1M phosphate buffer (pH 7.4) and subjected to centrifugation at 3500 rpm for 10minutes at 4[degrees]C. The supernatant obtained were maintained in an ice bath and used for the estimation of total protein, lipid peroxidation, glutathione reductase, reduced glutathione, glutathione peroxidase and superoxide dismutase. All the assays followed standard protocols with slight modification.
Total protein in the mice brain homogenate was estimated following the method of Lowry et al using bovine serum albumin (BSA) as standard .
Reduced glutathione (GSH)
The level of GSH in the brain homogenate was determined by the method of Moron et al with slight modification. Here the DTNB (5, 5'-dithiobis- (2- nitrobenzoic acid) react with GSH to form GSH-TNB that absorb at 412 nm. The change in absorbance gives the GSH concentration in the reaction sample. The amount of GSH was expressed as nmol/mg protein .
Lipid peroxidation (LPO)
The degree of lipid peroxidation in the homogenate was determined by adapting the protocol described by Okhawa et al. here the malondialdehyde (MDA) generated as a result of lipid peroxidation react with TBA to form a coloured product. This coloured product is measured spectrophotometrically at 532 nm .
Glutathione peroxidase (GPx)
GPx activity in the homogenate was estimated following the method of Rotruck et al. the GSH remaining after the enzyme catalyzed reaction complexes with DTNB in the reaction mixture. This has an absorption maximum of 412nm. GPx activity was expressed as ig of GSH consumed /min/mg protein .
Glutathione reductase (GR)
GR activity in liver homogenate was determined by measuring the reduction of GSSG in the presence of NADPH as described by Mize and Langdon . Briefly, this assay measures the rate of NADPH oxidation to NADP+, which is accompanied by a decrease in absorbance at 340nm. Thus, one GR unit is defined as the reduction of one iM of GSSG per minute at 25[degrees] C and pH 7.6.
Superoxide dismutase assay (SOD)
SOD in the brain homogenate was assessed using modified pyrogallol autooxidation method initially described by Marklund and Marklund .The colorimetric measurement is done at 420nm.
All the samples were run in triplicates. Values are expressed as mean [+ or -] SD. Statistical differences between the control and experimental values were compared by Student's't' test. For comparisons, p<0.05 was considered significant.
Animal health monitoring
No abnormality in the vishual signs was observed. Fur, skin, eyes and tail appeared normal. None of the animal exhibited abnormal body posture or movement. Feeding, water intake and sleep were normal in all the animals. Also no change in body temperature, pulse rate and respiratory rate were observed. Autonomic reflexes were normal and animals responded to the external stimuli normally. There was no loss in body weight of the animals. Gross examination of all the organs appeared normal when the animals were sacrificed at the end of the experimental time period.
The GSH level as shown in fig. 2 showed a slight depletion in the 7day treatment group but was not statistically significant. However the 14 and 21 day treatment group had GSH level comparable to that of control. The mean values of GSH for the control group was 2.13 [+ or -]0.06 and that of 7, 14 and 21 day experiment groups were 1.67 [+ or -] 0.59, 2.03 [+ or -] 0.04 and 2.09 [+ or -] 0.03 respectively.
The LPO level was more or less same in all the experimental group and control as evidenced from the fig. 3. The mean value of LPO was found to be 15.33 [+ or -] 0.03, 15.32 [+ or -] 0.008, 15.32 [+ or -] 0.01 respectively for 7th, 14th and 21st day treatment group. For control the mean LPO value was 15.32 [+ or -] .32.
An increase of only 0.01unit in the level of GPx was observed in the 7 and 14 day treatment group. It was not statistically significant. The mean values of GPx activity for the 7, 14 and 21 day treatment groups were 0.24 [+ or -] 0.01, 0.24 [+ or -] 0.01 and 0.22 [+ or -] .005 respectively. The control group had GPx mean value of 0.23 [+ or -] 0.005 units/mg proteins (fig. 4).
GR activity showed a slight increase of 0.08 unit in the 7 day experiment group. But this was not statistically significant. The 14 day and 21day treatment group showed no change in the GR activity in comparison to control (fig. 5). The mean values of the GR activity for the control and 7, 14, 21 day experiment groups were 1.60 [+ or -] 0.01, 1.68 [+ or -] 0.06, 1.61 [+ or -] 0.007 and 1.61 [+ or -] 0.005 respectively.
As indicated in the fig. 6, there was no marked change in the SOD activity for control and the experimental groups. The mean values for SOD activity for control group was 0.52 [+ or -] 0.009 were as that of 7, 14 and 21 day experimental groups were 0.52 [+ or -] 0.04, 0.52 [+ or -] 0.05 and 0.51 [+ or -] 0.03 respectively.
All measurements were carried out using UV Spectrophotometer-1601, Shimadzu, Japan.
Magnetic nanoparticles find a plethora of application in the biomedical sector. Biodisrtibution studies shows that the magnetic nanoparticles can cross blood brain barrier to reach brain . Despite the enormous data available on the application of magnetic nanoparticle their toxicity concerns persist because only a very few reports are available from the in vivo studies on the particle retention and clearance. Studies attribute the toxicity of magnetic nanoparticles to their ability to generate ROS [30- 31]. So assessing the antioxidant level in the tissue will give a clear cut idea about the biocompatibility and toxicity of the particle in the system.
Dextran coated nanoparticles are widely used in imaging by magnetic resonance (MRI), optical and positron emission tomography (PET) as contrast agents. These nanoparticles are proven to be non toxic with optimum bio degradability and vascular retention . Present study assesses the mice brain antioxidant level in presence of DFN administered via IP injection. Because IP injection delivers more particle to the blood and chances of some particle reaching and retaining in the brain are high.
GSH is an important low molecular weight antioxidant present in animals and plants. It acts as a cofactor and substrate for many antioxidant enzymes like GPx and detoxifying enzymes. GSH is an important regulator of nitric oxide cycle  and play a vital role in iron metabolism . Apart from that it can act independently as a reducing agent to protect molecules from oxidative damage. When the tissue is under oxidative stress the GSH level will drop significantly and its oxidized counterpart GSSG will be prominent in the tissue. Current study indicates that there is no significant drop in the GSH level in brain in any of the experimental group. There was a slight drop in the GSH in the 7 day experimental group but was not statistically significant. The GSH level in other too groups were same as that of control.
ROS generation can lead to LPO resulting in reduced membrane fluidity, altered permeability and inactivation of membranes bound enzymes and receptors. In tissues like brain with high amount of PUFA, LPO plays a major role in oxidative damage. The result of the present study invivo shows no noticeable change in the LPO of experimental group and control. LPO level of even the 7 day experimental group were same as that of control.
GPx, SOD and GR are the major antioxidant enzymes in the body. These enzymes get unregulated when ROS level increase above normal. GPx metabolize hydrogen peroxide and lipid hydroperoxides produced in the body by making use GSH as a cofactor. SOD is involved in the dismutation of super oxide radical to either molecular oxygen or to hydrogen peroxide. GR revert back the oxidized glutathione to GSH. In the present study none of these enzymes were up regulated in response to DFN. This observation is par with the unaltered level of GSH and LPO in the experimental group. There were no signs of oxidative stress in any of the experimental group.
The results published by Syama et al  showed no fluctuation in the haematological, biochemical, liver antioxidant defense mechanism and lipid peroxidation when in-house synthesized DFN were administered orally. That acute study also proves the DFN to be nontoxic even at very high concentrations (2000 mg/kg body weight). Also a study on dermal exposure of DFN states that the DFN do not have any significant influence on the antioxidant defense of rats and report them as non toxic . However the concerns of DFN being accumulated in the non targeted regions like brain and causing side effects in the long run persist. The present study is conducted to rule out such chances and the result indicates that the DFN administered via IP injection do not alter the brain antioxidant level in the 21 days of the study. It illustrate that the DFN do not induce any noticeable fluctuation in the brain antioxidants even after 7 days. The 14 day and 21 day treatment groups responded similarly to control group. This eliminates the chances of DFN being retained in the brain and getting degraded to produce byproducts that can elicit oxidative response.
None of the animals exhibited any signs of stress, pain or any kind of uneasiness and appeared healthy through out the course of the study. This study concludes that a single dose (100mg/kg body weight) of the in-house synthesized DFN administered via IP injection do not produce any oxidative response in mice brain after 7-21 days. The study rule out the possibility of particle administered through blood being translocated and retained at the unintended locations like brain and there by causing unanticipated side effects in the long run.
The authors thank the Director and the Head, BioMedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram for providing the infrastructure. The authors gratefully acknowledge G. Harikumar for the technical assistance. Sruthi. S thanks University Grants Commission, New Delhi for the Junior Research Fellowship. The work was financially supported by the Nanomission, Department of Science and Technology (Govt. of India), New Delhi, (Grant No. SR/NM/NS-90/2008).
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S. Sruthi, P.V. Mohanan *
Toxicology Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695 012, Kerala, India
* Corresponding Author: Dr. PV. Mohanan, email@example.com firstname.lastname@example.org
Received 5 December 2014; Accepted 6 December 2014; Available online 10 December 2014
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|Title Annotation:||Original Article|
|Author:||Sruthi, S.; Mohanan, P.V.|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Oct 1, 2014|
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