Studies on nanotoxicity focused on GNPs as a model because they have the highest biocompatibility and the lowest toxicity. Furthermore, they have a high potential for surface modifications and the synthesis of this type of nanoparticles is easy and inexpensive. (Pissuwan et al. 2006).
There are several reports about higher toxicity of the nanoparticles than their bulk materials due to their smaller dimension and larger surface area. Physical dimensions such as surface chemistry, composition, size, shape and the type of the nanoparticles are some important factors which may have impact on their toxicity in vivo. It was reported that nanoparticles cause neurotoxicity, hepatotoxicity, renal toxicity and testis toxicity (Sun et al. 2013).
One factor which influences the toxicity of the nanoparticles is the method of their production. As it was mentioned previously, there are three main types of nanoparticles production which are named chemical, physical and biological techniques. By literature reviewing, it can be distinguished that most of the researches focused on the chemical and physical methods of nanoparticles production and a few papers are available about the in vivo toxic effects of the nanoparticles that are produced by the biological technique.
At the cellular level, it was shown that GNPs uptake occurred through receptor-mediated endocytosis and maximum uptake occurred when the sizes of the used nanoparticles were around 50 nm (Chithrani and Chan 2007). Hence, the size of the nanoparticles influence their cytotoxicity and cellular uptake. Some studies showed that the chemically and physically produced GNPs had low toxic effects in the cell culture. In our previous research, we have used GNPs with sizes of about 50–70 nm as the model of the biologically produced nanoparticles in the cell culture and showed that unlike the technique of production which is known as the safest one, the obtained GNPs had some dose dependent toxicity. But an important factor which must be considered is that the two dimensional cell cultures cannot resemble the complexity of the animal body. For example, in two different researches which analyzed the toxicity of the carbon nanotubes in vitro and in vivo, one report showed that the carbon nanotubes had toxicity in the cell culture and the other showed that the carbon nanotubes had no toxic effects in the animal model (Manna et al. 2005). Parameters such as the used dose, immune response to the nanoparticles, route of exposure in addition to the chemical and physical properties of the nanoparticles may be involved in their toxic effects in vivo (Zhang et al. 2010).
In the present research, we tried to analyze the toxic effects of the GNPs in their determined toxic and non-toxic doses in the animal model which were administered through intraperitoneal injection. This type of administration was chosen on the basis of the previous research which showed that tail vein injection had the lowest and intraperitoneal injection or oral administration of nanoparticles had the highest toxicity in the rat model (Chen et al. 2009; Zhang et al. 2010). We have assayed the differences between the influence of the GNPs that were biologically produced in vitro and in vivo.
There are some information about the elimination of the chemically produced GNPs in the animal blood which is due to their sizes. For example, it was reported that GNPs with the sizes of about 18 nm were eliminated from the blood stream and collected in the spleen and liver (Semmler-Behnke et al. 2008). In another research, it was reported that smaller GNPs (5–15 nm) can easily be distributed in the rat organs in contrast to the bigger ones (50–100 nm) (De Jong et al. 2008). Thus, as GNPs are good candidate for biomedical applications, before any usage of the GNPs with different sizes, shapes and nature for human applications, it is important to evaluate their behavior in the animal model.
In the first step of the examinations, GNPs were produced using F. oxysporum that is a well-known organism in the intra and extracellular production of nanoparticles. In the intracellular method of nanoparticles production, the nanoparticles accumulate within the microbial cells where the active components for bio-reduction are placed in the cells, and in the extracellular method of nanoparticles production, the nanoparticles will be produced out of the microbial cells. It was proven that F. oxysporum had strong secretion systems and is a good candidate for the extracellular production of nanoparticles. Furthermore, this fungal strain is nonpathogenic for human and its culture is safe, inexpensive and easy. After culturing of the fungal strain, the supernatant of the culture medium was used for extracellular production of GNPs, because the extraction of the nanoparticles is more facile than that of the intracellular technique.
The production of GNPs was confirmed by color changing and the use of spectrophotometer, TEM and XRD. The color of the reaction changed from yellow to pink due to the existence of the GNPs. Based on the shape and size of the GNPs, the obtained color will be altered. Appearance of pink to red color is the sign of the existence of the spherical GNPs in the culture medium (Burda et al. 2005).
The spectrophotometer results revealed that the obtained GNPs showed maximum absorbance peak about 525 nm. This phenomenon was due to the SPR of the GNPs which is the resonant oscillation of conduction electrons after stimulation by light. TEM results indicated that GNPs had sizes of about 50 nm with round and hexagonal shapes. Different magnifications of TEM showed that GNPs had uniform sizes. Finally XRD results revealed the existence of the elemental gold in the reaction mixture. As shown in Fig. 4, there are extra peaks in the XRD spectrum, which are due to the presence of the other materials and impurities in the microbial culture supernatant.
Prior to in vivo analysis, MTT assay was done, and as it was previously shown, the GNPs had toxicity which depended on the used doses. Based on the calculated IC50, the toxic and non-toxic doses of the GNPs were calculated. Because the animal blood will dilute the nanoparticles after injection, thus the nanoparticles were injected in the doses which after dilution in the animal’s body, toxic and non-toxic doses of them would be achieved.
It was reported that after injection of the GNPs, plasma proteins will be adsorbed on the surfaces of the nanoparticles. This may help the opsonisation and accumulation of the nanoparticles which entered into the blood stream. It was shown that the preferred organ in which nanoparticles accumulation occurs is the liver. The accumulation in the other organs is dependent on the type of the nanoparticles, size, shapes and other characteristics (Cardoso et al. 2014). Hence, the recent research tried to find the effects of GNPs on the liver and kidney of the animal model after 3 continuous days of exposure. Our investigation showed that short time exposure to GNPs had low impacts on the liver and kidney. Previous study showed that the short duration exposure to the chemically produced GNPs (50 nm) had minimum effects on the liver enzymes and had no effects on kidney enzymes (Abdelhalim and Moussa 2013) which, agrees with our histological results.
Our study showed that short time exposure to the non-toxic dose of GNPs induced mild changes on hepatocytes and lobular central vein and toxic dose of GNPs induced mild changes on hepatocytes and sinusoidal space which in long term exposure may cause infiltration and disability of bile and blood transferring and finally liver dysfunction and degeneration process. Furthermore, these two different doses had the same effects (i.e. mild changes) on the glomeruli of the kidney which in long term exposure may cause low filtration rate and kidney dysfunction. Overall, it is important to note that the changes in some parts of the mentioned organs are classified to the mild changes and this data again shows that the results that were obtained from in vivo studies are different from the in vitro ones.
Another organ that was analyzed was the testis. Testis was chosen as the organ in which the blood-testis barrier acts as the filter. In mammalians, an example of one of the tightest junction is blood-testis barrier which is made from the sertoli cells. The purpose of the presence of this barrier is to protect the meiosis process from any harmful agents that may exist in the animal’s blood (Mruk and Cheng 2015). Therefore, we tried to evaluate the toxic effects and penetration of the GNPs in this organ as well. This barrier acts like the blood–brain barrier which guards the brain from unfavourable materials.
Previous research demonstrated that small sizes of GNPs could penetrate the blood–brain barrier, and by the use of the ion channel blockers, this penetration can be under control. They showed that there were no adverse effects on the animals’ brain and suggested the use of the GNPs for drug delivery aims (Hainfeld et al. 2013). Although it was shown that nanoparticles have less effects on the blood–brain barrier, Prakash et al. showed that silver nanoparticles (SNPs) with sizes around 20–100 nm could penetrate blood–brain and blood-testis barriers and induce impairments in the function of the central nervous system (CNS) and teratogenic outcomes in the fetus of the animals which were treated by SNPs (Prakash et al. 2018).
The present study showed that the blood-testis barrier has some effects in the penetration of the GNPs to the testis. GNPs had no toxicity on the sertoli cells that are involved in the production of blood-testis barrier but by the administration of the toxic dose of GNPs, they had mild changes on the seminiferous tubules, which in long term exposure may have impact on the meiosis and therefore act on the spermatozoa. Moreover, toxic dose of GNPs administration had mild changes on leydig cells which in long term exposure may affect the production of testosterone.
Recent research showed that the produced GNPs even in their toxic doses had mild changes in different organs of the rat. This may be due to the use of the biologically produced GNPs, their chemistry, shapes and sizes. This research tried to analyze the toxicity of two different doses of the GNPs on the liver, kidney and testis of the rat model. Analysis of the three other important organs, heart, spleen and brain, are under research and in future we will publish the results.
The aim of this research was to evaluate the toxic effects and distribution of the 50–70 nm GNPs in the animal organs after 3 continuous days of administration. Results from the present study showed that the in vitro and in vivo behaviors of the GNPs are different. Overall, our results showed that the GNPs have easy access to the blood and all the three tested organs but firstly, the non-toxic dose of GNPs had little effects on the tested organs and in the case of the testis, it imposes no changes. Secondly, if administrations of the toxic and non-toxic doses of GNPs had effects, their effects were somewhat similar to each other in the tested organs. Thirdly, the used route of administration is known as the most toxic way of entrance of GNPs to the animal model, which should be compared with the other routes of administration in future. Furthermore, it is recommended to compare the toxic effects of the GNPs that will be produced by the chemical and physical techniques with the biological ones.