Toxicity of PEG-Coated CoFe2O4 Nanoparticles with Treatment Effect of Curcumin
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In this work, CoFe2O4 nanoparticles coated with polyethylene glycol (PEG) were successfully synthesized via a hydrothermal technique. Morphological studies of the samples confirmed the formation of polycrystalline pure-phase PEG-CoFe2O4 nanoparticles with sizes of about 24 nm. Toxicity induced by CoFe2O4 nanoparticles was investigated, and biological assays were performed to check the toxicity effects of CoFe2O4 nanoparticles. Moreover, the healing effect of toxicity induced in living organisms was studied using curcumin and it was found that biochemical indexes detoxified and improved to reach its normal level after curcumin administration. Thus, PEG-coated CoFe2O4 synthesized through a hydrothermal method can be utilized in biomedical applications and curcumin, which is a natural chemical with no side effects, can be used for the treatment of toxicity induced by the nanoparticles in living organisms.
KeywordsNanoparticles Hydrothermal technique Annealing Toxicity PEG-coated CoFe2O4
Blood urea nitrogen
Fourier transform infrared spectroscopy
Nuclear factor erythroid 2-related factor 2
Transmission electron microscopy
Tumor necrosis factor
The use of nanoparticles (NPs) offers many advantages due to their unique chemical and physical properties which are substantially different from their bulk counterparts . Cobalt ferrite (CoFe2O4) as one of the most important magnetic materials has aroused immense interest at the nanoscale because of its various applications in recent technologies [2, 3, 4, 5]. It is considered as one of the competitive candidates for its wide range of applications, mostly in medical industry, due to its capability to possess the desired physical and chemical properties at the nanoscale. Moreover, CoFe2O4 is easy and cost-effective to be fabricated with controlled composition, shape, and size required for a particular application. In this regard, the diameter of CoFe2O4 nanoparticles for biological applications below 100 nm can greatly influence the physiochemical properties and pharmacokinetics in living organisms. Larger particles with a diameter greater than 100 nm are used as a contrast agent for magnetic resonance imaging of the gastrointestinal tract, while smaller particles, below ~ 20 nm, are used as carriers for tumor treatments. For clinical application of cobalt ferrite nanoparticles, it is very important to investigate the biosafety both in vivo and in vitro [6, 7]. Many nanoparticles taken via oral or intravenously into the body are mainly distributed in the liver, kidney, and lung so as to lead to various inflammations in these organs. Compared to other materials, cobalt ferrite has not been studied extensively to explore its toxicity in living organisms and then its healing effect using curcumin, although few other works have been reported on investigating the toxicity and biosafety of polyethylene glycol (PEG)-coated cobalt ferrite nanoparticles.
From the toxicity point of view, the main concern is the excessive exposure that requires elimination of accumulated nanoparticles from biological organs as well as it requires urgent treatment of inflammatory disorders. Some researchers have tried to study several anti-inflammatory drugs on the treatment of toxicity of nanoparticles in vivo, and they found that these anti-inflammatory drugs could promote the excretion of nanoparticles that are accumulated in the body to a certain extent in order to reduce or eliminate the tissue inflammatory effects [8, 9]. Curcuma longa (turmeric) is a traditional medicinal herb with a quite long history of its use as a treatment for inflammatory diseases in Southeast Asia. Numerous studies have been reported on the antioxidant properties, antimutation and antitumor effects, and carcinogenic characteristics of curcumin [10, 11]. Curcumin has the ability of healing wounds as well as treating the liver ailments, urinary tract diseases, and hepatitis . It alleviates oxidative stress and inflammation in chronic diseases through the Nrf2-keap1 pathway. Curcumin can suppress pro-inflammatory pathways related with most chronic diseases and blocks both the production of TNF and the cell signaling mediated by TNF in various types of cells. Moreover, curcumin may also act as a TNF blocker from in vitro and in vivo by binding to TNF directly .
In this study, we have successfully prepared PEG-coated CoFe2O4 nanoparticles with a controlled shape and size of about 25 nm using a hydrothermal technique. After giving different exposures (doses) of CoFe2O4 nanoparticles, we have examined blood analysis, HE staining, and biodistribution as well as the treatment effect of curcumin on the toxicity caused by PEG-CoFe2O4 nanoparticles. This study presents a new approach to investigate the toxicity effect of CoFe2O4 nanoparticles and then the treatment of the toxicity caused by PEG-CoFe2O4 nanoparticles in vivo using curcumin.
Preparation of Cobalt Ferrite Nanoparticles
Cobalt ferrite nanoparticles were synthesized using a hydrothermal technique. For this purpose, an adequate amount of ferric nitrate and cobalt chloride was dissolved in deionized water and then mixed with aqueous solutions of PEG and sodium hydroxide (NaOH). Double-distilled deionized water was used as the solvent to avoid the presence of any impurities in the final nanoparticles. The mixture was stirred for about 30 min using a magnetic stirrer and then poured into the autoclave and heated for 6 h at 180 °C to perform the hydrothermal reaction. After the reaction was completed, the product was cooled to room temperature and then washed twice with deionized water and then with ethanol to remove the excess PEG and other un-dissolved salts, if present in the solution. Finally, the product was dried at 80 °C overnight and then ground into powder to get the desired cobalt ferrite nanoparticles. In this stage, the nanoparticles were found amorphous which was confirmed by the XRD shown in Fig. 2a. To get the nanoparticles in crystalline form, the samples were then annealed at 500 °C for 6 h and the final product was obtained in the form of crystalline PEG-CoFe2O4 nanoparticles that was confirmed by the XRD shown in Fig. 2b.
99mTc Labeling of PEG-CoFe2O4 Nanoparticles
Radiolabeling of PEG-coated CoFe2O4 nanoparticles was performed with 99mTc using stannous chloride (SnCl2) as the reducing agent and dissolved the nanoparticles in deionized water under ultra-sonication conditions for about 0.5 h. SnCl2, ascorbic acid, and 99mTcO4 were then added into the nanoparticle suspension (with cobalt ferrite of ~ 0.4% by weight). For accurate data, the radioactive counts were measured within 24 h due to the short lifetime of 99mTc (~ 6 h). The pH of the mixture was adjusted in the range 5–10 using 1.0 M NaHCO3 solution; then, suspension of PEG-CoFe2O4 was added to it and the resultant mixture was then stirred at 10,000 for 25 min at 80 °C. After centrifugation, the supernatant was decanted, and the remaining material was identified to be 99mTc PEG-CoFe2O4. Paper chromatogram (under the chromatographic solutions of normal saline and acetone) was used to measure the yields of the labeled compounds. The radioactive labeling yield of the nanoparticles was found to be around 70% that reflects the real distribution and metabolism in vivo.
Biodistribution of PEG-CoFe2O4 Nanoparticles
Kunming mice weighing in the range 15–18 g were provided by the Laboratory Centre for Medical Science, Lanzhou University, Gansu, People’s Republic of China. All animals were housed in individual cages with a temperature-controlled system (21 to 22 °C), and lights were switched on from 08:00 to 20:00 hours. Proper food and water were given to the mice as recommended according to the animal protocols by the European Communities Council Directive of November 24, 1986 (86/609/EEC), and approved by Institutional Animal Care and Use Committees of Gansu Province Medical Animal Center and Lanzhou University Animal Committees Guideline (China). The mice were divided randomly into seven groups (five mice/group), injected intravenously with 99mTc-PEG-CoFe2O4 solution, and then killed at 1, 6, 16, and 24 h after the injection. Tissues from the heart, lung, liver, spleen, and kidney were immediately dissected, and then a substantial amount of blood was collected. Each tissue was wrapped in foil, properly weighed, and counted for 99mTc. Data points were corrected for physical decay of radioactivity. The distribution of the tissue was presented in percent injected dose per gram of wet tissue (%ID/g), which could be calculated by the percent injected (tissue activity/total activity dose) per gram of the wet tissue.
Dosage Effect on Toxicity of PEG-CoFe2O4 in Mice
In this experiment, 21 mice were divided into seven groups (three mice/group). PEG-CoFe2O4 nanoparticles were injected in mice intravenously at different doses of 125, 250, and 350 μg/mouse (0.2 ml) with the control group that was treated with normal saline of 0.9%. In the treatment group, different doses of 125, 250, and 350 μg/mouse of curcumin were also injected intravenously in mice. The damage groups were killed after 24 h while the treatment groups were killed after 3 days. Blood was collected from the mice and centrifuged for about 10 min to obtain the serum. The serum contents of total bilirubin (TB), alanine aminotransferase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine (CREA), and cystatin C (Cys-C) were measured. At the same time, the liver, the lung, the spleen, the kidney, and the heart were harvested immediately. These tissues were fixed in 10% buffered formalin and processed for the routine histology with hematoxylin and eosin. Microscopic observation of tissues was performed using an Olympus Microphot-CX41 microscope coupled with a digital camera.
Results and Discussion
TEM and XRD Analyses
Fourier Transform Infrared Spectroscopy, Raman, and TG Analyses
Dosage Effect of PEG-CoFe2O4 on Toxicity
Effect of Curcumin on Toxicity of PEG-CoFe2O4
In this work, we successfully fabricated 24-nm PEG-coated cobalt ferrite nanoparticles using a hydrothermal technique. Toxicity induced in various organs of mice using different dosages of PEG cobalt ferrite nanoparticles was explored in detail, and then its healing effect was studied using curcumin. Biological assays were performed to check the toxicity of CoFe2O4 nanoparticles. Positive changes were monitored in biochemical indexes after treatment with curcumin which either came to the normal level or decreased substantially. This study indicates that PEG-coated CoFe2O4 synthesized via a hydrothermal technique is a good model for a drug carrier and, curcumin, which is a natural chemical and possesses no side effects, could be utilized for the treatment of toxicity as well as for other diseases in living organisms.
Shahnaz Akhtar is thankful to the China Scholarship Council (CSC) for providing the fellowship for PhD research in Lanzhou University.
This work was also supported by the National Natural Science Foundation of China (Project No. 31471953).
SA, LG, and MM presented the idea of this work. WA, XN, and KL performed the experimental work. XN, KM, and SA prepared the nanoparticles. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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