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ORIGINAL ARTICLE
Year : 2018  |  Volume : 2  |  Issue : 1  |  Page : 68-73

Preparation of chitosan-coated Fe3O4 nanoparticles and assessment of their effects on enzymatic antioxidant system as well as high-density lipoprotein/low-density lipoprotein lipoproteins on wistar rat


1 Department of Biochemistry, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
2 Young Researchers and Elite Club, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran

Date of Web Publication5-Mar-2018

Correspondence Address:
Dr. Kahin Shahanipour
Department of Biochemistry, Falavarjan Branch, Islamic Azad University, Isfahan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_98_17

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  Abstract 


Background: Recently, the nanoparticle (NP) application in many fields of medicine due to their specific physical and chemical properties has been developed. Therefore, especially in vivo evaluation of their toxicity is necessary. The aim of this study was to compare the toxicity of Fe3O4NPs coated with biocompatible compounds and uncoated NPs. Methods: Wetted chemical method (or wet chemical method) was used to synthesize Fe3O4NPs. The synthesized NPs were coated with chitosan and the coating interactions were investigated by Fourier-transform infrared spectroscopy. The magnetic and structural properties of Fe3O4and coated Fe3O4NPs were evaluated by transmission electron microscope and X-ray diffraction. The toxicity assessment of Fe3O4and coated Fe3O4NPs was studied in mice by intraperitoneal injections during the 1-month period. Antioxidant enzyme glutathione peroxidase (GPX), malondialdehyde, and low-density lipoprotein/high-density lipoprotein were measured 15 and 30 days after injection. Results: The synthesized NPs have a single phase and spinal structure and their size distribution in the net form is 5–10 nm. Some factors were changed due to the injection of both uncoated and coated NPs. The all-used concentration of chitosan-coated Fe3O4NPs could increase the GPX enzyme activity. The Fe3O4NPs can reduce the GPX enzyme activity in high concentration (50 mg/kg, 100 mg/kg, and 150 mg/kg). Conclusion: The results indicated that NPs based on their dosage and body condition can induce toxicity effects in the body. It should be mentioned that the chitosan-coated ones can decrease their effects.

Keywords: Nanoparticles, oxidative stress, toxicity


How to cite this article:
Khedri B, Shahanipour K, Fatahian S, Jafary F. Preparation of chitosan-coated Fe3O4 nanoparticles and assessment of their effects on enzymatic antioxidant system as well as high-density lipoprotein/low-density lipoprotein lipoproteins on wistar rat. Biomed Biotechnol Res J 2018;2:68-73

How to cite this URL:
Khedri B, Shahanipour K, Fatahian S, Jafary F. Preparation of chitosan-coated Fe3O4 nanoparticles and assessment of their effects on enzymatic antioxidant system as well as high-density lipoprotein/low-density lipoprotein lipoproteins on wistar rat. Biomed Biotechnol Res J [serial online] 2018 [cited 2019 Oct 20];2:68-73. Available from: http://www.bmbtrj.org/text.asp?2018/2/1/68/226585




  Introduction Top


Nanotechnology has found applications in industry, pharmaceuticals, medicine, electronics, robotics, and tissue engineering. Nanoparticles (NPs) are nanostructures, with at least one dimension being <100 nm.[1] The consumption of NP materials has many advantages because of their unique physical properties such as unique size. For example, guiding magnetic iron oxide nanoparticles with the help of an external magnetic field to specific target was the principle behind the development of superparamagnetic iron oxide nanoparticles (SPIONs). The reasons of using SPION is various as they are their unique in their chemical, thermal, and mechanical properties. Therefore, the SPIONs have a great potential [2],[3] for many biomedical uses such as cellular therapy, tissue repair,[4] magnetic resonance imaging,[5] tumor hyperthermia,[6] and gene and drug delivery.[7] The iron oxide NPs such as magnetite (Fe3O4) or its oxidized form magnetite (γ-Fe2O4) are by far the most commonly consumed in biomedical fields.[ 6] For biological and biomedical approaches, magnetic iron oxide NPs are the primary choice because of their biocompatibility, superparamagnetic behavior, and chemical stability.[3] The nanostructure is based on an inorganic core of iron oxide, such as Fe3O4 and γ-Fe2O4, coated with a polymer such as dextran, chitosan, polyethylenimine, and polyethylene glycol (PEG).[8],[9] The chitosan, poly-β-(1 → 4)-2-amino-2-deoxy-D-glucose, being deacetylated chitin, is currently obtained from the outer shell of crustaceans.[10],[11] The positive charge of chitosan provides various and distinctive physiological and biological properties with great potential application in a wide range of industries including agricultural, food, cosmetic, and pharmaceutical. The cationic nature of chitosan has been considered for the development of particulate drug delivery systems.[12] Chitosan has many significant biological reactive chemical groups including OH and NH2.[13] Therefore, chitosan and its derivatives have been widely used in the fields of pharmacy and biotechnology. Chitosan-coated magnetic NPs contain a core of magnetic material usually a mixture of Fe3O4 and γ-Fe2O4.[14] The origin of NPs toxicity for the viable cells could be mainly due to the toxicity of heavy ions atoms which can impress and penetrate into the macromolecules, organelles, and other parts of viable cells.[15] The most researches up to now have indicated and suggested that reactive oxygen species (ROS) generation and consequent oxidative stress are frequently observed with NP toxicity. The physicochemical properties of NP including particle size, surface charge, and chemical composition are the key indicators for the ROS response result. According to the present information, some of the NPs are able to activate inflammatory cells such as macrophages and neutrophils which can result in the increased production of ROS.[16]

The oxidative stress has been implicated in the pathogenesis of many disease states, such as aging, atherosclerosis, carcinogenesis, ischemia–reperfusion tissue injury, rheumatoid arthritis, and chronic inflammatory disorders.[17] The oxidative stress can be defined as the imbalance between the production of cellular oxidant species and antioxidant capability.[18] The cellular defense systems of cells include nonenzymatic and enzymatic substances, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) that are scavengers of radicals and protectors of major cellular biomolecules from oxidative damage.[19] When the cellular defense mechanisms are unable to cope with excessive generation of ROS, oxidative stress will happen subsequently.[16]

Objective

In this study, an attempt was made to evaluate the effect of chitosan (as a coating on the surface of Fe3O4 NPs) on the toxicity effects of Fe3O4 NPs.


  Methods Top


Synthesis of the Fe3O4 nanoparticles

In this study, wetted chemical method was used for the synthesis of Fe3O4 NPs. Three solutions of the FeCl2 (0.01 M equals to 1.98 g), FeCl3 (0.02 M equals to 5.41 g), and NaOH (0.08 M equals to 3.2 g) (all from the Merck Company) were needed and prepared in distilled deionized water. Synthesis was started by by pouring FeCl2 solution into a triple neck round balloon. Then, FeCl3 solution and NaOH solution were added to the same balloon, under vigorous magnetic string respectively. At finally, the obtained solution was washed by deionized water and then centrifuged for removing any aggregate as impurity. All processes were performed at room temperature.[20],[21]

FeCl2+2 FeCl3+8 NaOH →Fe3O4+4 H2O + 8 NaCl

Coating of Fe3O4 nanoparticles with chitosan

First, 20 mg of high-molecular-weight chitosan was dissolved in 1 M acetic acid solution with final volume of 100 ml. Then, 70 mg of Fe3O4 was added to the pervious solution and the mixture was mixed overnight for 18 h. During this process, the chitosan molecules were adsorbed on the surface of the NPs and a homogeneous dark brown suspension was obtained.[8]

Quality assurance and measurement of samples properties

XRD (X-ray diffraction) (Burker D and ADVANCE) and TEM (transmission electron microscopy) (Philips CM12 TEM, operated at 120 Kv) were used to evaluate the crystalline structure and size distribution, respectively. The coating chemical interactions were measured by FTIR (Fourier-transform infrared spectroscopy) (JASCO FT/IR-6300, Japan).

Nanoparticle injection to mice

For this step, 60 rats of Wistar strain were obtained from the Falavarjan University laboratory. They were kept in natural light and humidity at 22–24°C. They were divided into 10 equal groups (each group contained six mice). One group was injected with normal saline and served as the control group and the nine remaining groups received Fe3O4, chitosan-coated Fe3O4, and pure chitosan. The rats received intraperitoneal injection of different concentrations (50, 100, and 150 mg/kg according to the rats weight) for 1 month.

Measurement of factors

The blood samples of ketamine-anesthetized rats were taken directly from the heart in 15 and 30 days after injection. Then, principle factors such as GPX, malondialdehyde (MDA), HDL (high-density lipoprotein), and LDL (low-density lipoprotein) were measured. The GPX enzyme activity was measured with Rat Glutathione Peroxidase (GSH-PX) Elisa kit from EASTBIOPHARM Company.

Statistical analysis

The mean values of factors in all groups were compared by the ANOVA test (analysis of variance) and t-test using the SPSS (Statistics software package that used for the social sciences; version 20) computer program. P ≤ 0.05 was considered as statistical significant level.


  Results Top


Physical properties and quality assurance of the coated Fe3O4 nanoparticles

XRD was used to consider the structure of samples. XRD pattern of uncoated, chitosan-coated Fe3O4 NPs and chitosan is shown in [Figure 1]. According to the obtained picture, all samples have a single phase. The mean size of the particles was determined by the Debye–Scherer formula, and it was calculated as 12 nm for the uncoated Fe3O4 NPs.
Figure 1: Transmission electron microscope photograph of the uncoated Fe3O4 nanoparticles

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The TEM photograph of the uncoated Fe3O4 NPs is shown in [Figure 1]. This photograph indicates that the sizes of the particles are around 5–10 nm with approximately uniform size distribution and that is compatible with the results of the XRD patterns [Figure 2] because the particle size increases with the coating process.
Figure 2: X--ray diffraction pattern for Fe3O4 and Chitosan--coated Fe3O4

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The FTIR curves of the Fe3O4, chitosan-coated nanoparticles and chitosan are demonstrated in [Figure 3].
Figure 3: Fourier--transform infrared spectroscopy curve

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It can be observed that two peaks in the Fe3O4 curve (1628 cm −1 and 3419 cm −1) are related to OH junctions, and it can be concluded that there are water molecules in the material structure. Furthermore, it can be observed that another peak in the chitosan curve (3430 cm −1) is related to O-H and N-H junctions. The maximum attraction/absorbance in 1629 cm −1 and 1397 cm −1 showed that chitosan could coat the Fe3O4 NPs.

Antioxidant system enzyme measurement

The GPX enzyme activity was measured (standard curve showed in [Figure 4]) in 15 and 30 days postinjection in all 10 groups and [Figure 5] and [Figure 6] show the GPX measurement results in these times. The Fe3O4 NPs can reduce the GPX enzyme activity in high concentration. All the concentrations of chitosan-coated Fe3O4 NPs could increase the GPX enzyme activity. The groups that received all concentrations (50 mg/kg, 100 mg/kg, and 150 mg/kg) of chitosan had an increase in the GPX activity in 15 and 30 days postinjection.
Figure 4: Glutathione peroxidase standard curve

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Figure 5: Glutathione peroxidase measurement results 15 days postinjection

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Figure 6: Glutathione peroxidase measurement results 30 days postinjection

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Results indicated that Fe3O4 NPs at concentrations of 100 mg/kg and 150 mg/kg 15 days postinjection can decrease GPX enzyme activity significantly, but decrease of activity at a concentration of 50 mg/kg is not meaningful.

On the other hand, it was observed that 30 days postinjection of Fe3O4 NPs only at concentration of 50 mg/kg decreases GPX enzyme activity and at concentrations of 100 mg/kg and 150 mg/kg the decrease of enzyme activity is not significant [Figure 5] and [Figure 6].

The MDA concentration was measured 15 and 30 days postinjection in all groups. [Figure 7] and [Figure 8] indicate that the Fe3O4 NPs at 15 days postinjection reduce the MDA concentration at 50 and 100 mg/kg concentration and increase the MDA concentration at 150 mg/kg concentration. We can observe the increase of MDA at all concentrations at 30 days postinjection. It should be mentioned that all concentrations of chitosan-coated Fe3O4 NPs could increase the MDA at 15 and 30 days postinjection [Figure 7] and [Figure 8].
Figure 7: Malondialdehyde concentration results 15 days postinjection

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Figure 8: Malondialdehyde concentration results 30 days postinjection

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The amounts of LDL of all groups at 15 and 30 days after injection are showed in [Figure 9] and [Figure 10]. These changes showed that LDL concentration in groups that received the Fe3O4 NPs for 15 days after injection could not change the LDL concentration, but the concentrations of 50 and 100 mg/kg could increase after 30 days injection [Figure 9] and [Figure 10].
Figure 9: Low--density lipoprotein concentration results 15 days postinjection

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Figure 10: Low-density lipoprotein concentration results 30 days after injection

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The HDL concentrations were measured 15 and 30 days after injection and they are showed in [Figure 11] and [Figure 12]. These figures indicate that HDL concentrations were decreased at all concentrations of Fe3O4 NPs groups in 15 days, but in 30 days, only the concentrations of 100 and 150 mg/kg could decrease this parameter. Furthermore, all concentrations of chitosan-coated Fe3O4 NPs in 15 days reduced HDL, but at 30 days postinjection, the only concentrations that reduced this parameter were 50 and 100 mg/kg [Figure 11] and [Figure 12].
Figure 11: High--density lipoprotein concentration results 15 days postinjection

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Figure 12: High--density lipoprotein concentration results 30 days postinjection

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  Discussion Top


The results indicate that NPs had greater effects on biomarkers in comparison with uncoated iron oxide NPs. This probably occurred due to their greater stability in blood circulation and consequently better penetration in different organs and cells. Therefore, it seems that the use of such coating materials (chitosan) on the surface of the Fe3O4 NPs increases their stability and side effects on liver enzymes. In this work, we focused on the toxicity of metal oxide NP to the oxidative stress paradigm. Manke et al. reported that most work to date has suggested that ROS generation and consequent oxidative stress are frequently observed with NP toxicity.[16] The physicochemical properties of NP including particle size, surface charge, and chemical composition are key indicators for the resulting ROS response. Free radicals are generated from the surface of NP when both oxidants and free radicals bound to the particle surface. Therefore, the metallic NP (Zn, Ti, Si, and Fe) toxicity indicators are increasing ROS generation and oxidative stress and apoptosis. An example of the metabolism of NP including oxidative stress and resulting toxicity is shown in [Figure 13]. Furthermore, Lin et al. results showed that NP induced oxidative stress because of reducing the SOD and GPX levels and increasing the MDA.[22] Salata in 2004 reported that the chitosan coated polymeric can stay longer time in in bloodstream, biological solution, with reduce toxicity.[9] Hence, this NP can increase MDA concentration and reduce the catalase activity. By the way, chitosan can reduce the free fatty acid and MDA and increase the antioxidant enzyme activities such as SOD, GPX, and CAT. Therefore, the chitosan-coated Fe3O4 has a high antioxidant activity rather than chitosan.
Figure 13: Metabolisms of nanoparticle include oxidative stress and resulting toxicity

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In the other investigation, they conclude that the aqueous extract of leaves of Limonia acidissima can be used for the synthesis of zinc oxide NPs. These NPs control the growth of Mycobacterium tuberculosis, and this was confirmed with the microplate Alamar blue method. The potential of biogenic zinc oxide NPs may be harnessed as a novel medicine ingredient to combat tuberculosis disease.[23]

Our results showed a correlation of lecithin concentration with size, zeta potential, and loading capacity of rifampicin (RIF) RIF-NPs. Increases in lecithin concentration (0.2–2.0 g) could cause a significant size reduction in NPs (250–150 nm); the amount of zeta potential (from 14 to 49 mV; P < 0.05) and loading capacity increases from 8% to 20% (P < 0.05). Designed NPs had slow drug release profile which was influenced by pH and lecithin concentration. The cumulative percentage of RIF released at pH 7.4 was approximately 93% up to 12 h. Overall, the release profile was better than the standard drug, even in various pH conditions (pH = 1, 3.4, and 7.4). In another investigation, they concluded that the Chg/L-RIF NPs may be considered as a promising drug nanocarrier. These NPs release RIF at a slow and constant rate, which effectively might eliminate the bacilli and prevent the formation of RIF-resistant bacilli.[24]

Ag NPs in the range of concentrations exhibited no anti-Mtb effects, whereas ZnO NPs showed potent antibacterial activity at 1/128 of the initial concentration. ZnO NPs at all concentrations showed cytotoxic activity, whereas 100% of THP-1 (human monocytic cell line derived from an acute monocytic leukemia patient) cells remained viable in the presence of Ag NPs at 1/32 and 1/64 of the initial concentrations. However, at ratios of 8ZnO/2Ag, 39.94% of the cells at 1/16 of the initial concentration remained viable, with 100% of THP-1 cells at 1/32 of the initial concentration remaining viable.[25]

In another study, PEG was used as curcumin (CUR) solvent. They showed 2000-fold increases in solubility of CUR in PEG in comparison to water. The loading capacity of CUR in NPs was successfully increased up to 15%. This procedure represents a mild and safety process for synthesizing efficient CUR-NPs, as we have not used any organic or toxic solvents.[26]

Financial support and sponsorship

This study was funded by Flavarjan Branch, Islamic Azad University.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13]



 

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