International Open Access Journal of Prevention and Research in Medicine
Director Prof. Francesco Tomei

Nanotechnology, the great revolution of the twenty-first century, consists in the preparation of materials, the nanoparticles (NP), having at least one of the dimensions below 100 nm (i.e. less than 10^-9 meters). The drastic reduction in size confers to nanoparticles physico-chemical characteristics very different from those of the parent material since, decreasing the size, the surface to volume ratio considerably increases which, in turn, leads to a predominant distribution of the atoms at the surface of the nanoparticles, thus conferring them a high chemical and biological reactivity. In the context of  engineered nanoparticles, the single-walled carbon nanotubes (SWCNT) are considered as one  of the most promising materials in both biomedical and industrial fields applications . There are, however, indications that the SWCNT can be potentially toxic in some biological contexts. For example, we have recently shown that certain types of SWCNT, mainly produced for industrial applications, when administered to female mice at an early stage of pregnancy, are capable of inducing fetal malformations of varying severity, up to abortion, in case of their administration at high concentrations.
This observation raises the question of occupational safety of pregnant women exposed to this kind  of nanoparticles in the workplace.
In this work, we report some of our subsequent results showing that the addition to the carbon nanotubes of functional groups consisting of polyethylene glycol chains (PEG-SWCNT)  significantly reduces their embryotoxic effect and does not appear to cause harmful effects in maternal tissues. The functionalization with the polyethylene glycol is, in fact, one of the methods generally used to increase the biocompatibility of many types of nanoparticles. For our study, two different experimental protocols were adopted: in the first protocol, a group of  pregnant female mice (5.5 day of pregnancy) were exposed to the test material with a single dose; in a second group of experiments, females at the same stage
of gestation, received multiple doses up to day 15 of gestation. This second protocol was chosen in order to mimic the possible daily exposure that a pregnant woman may have in occupational setting. At the end of the experiments the effects observed both at the level of fetal development and health of the mother's tissues were evaluated for both groups. Our results showed that the functionalization is actually able to reduce the toxic effect on the fetus. However, we have observed the occasional appearance of embryos with obvious structural malformations. These observations leave open the question of safety of  exposure to carbon nanotubes in pregnancy, especially at the high doses that can accidentally occur in the workplace, or in the case  of biomedical use of these nanoparticles.

The use of nanoparticles for the development of innovative materials represents a real industrial revolution because at these dimensional levels behaviors and characteristics of matter change drastically and therefore nanotechnologies represent a radically new way to produce materials, structures and devices with properties and functionality greatly improved, or entirely new compared to those of the materials made up of the same molecules, but of higher dimensions.
A responsible development of new materials requires a careful assessment of the risks to health and the environment associated with the production, use and disposal of these materials.
As the production and applications of nanoparticles are increasing, it is becoming  important to determine the impact and the potential harmful effects of these nanomaterials on human health and the environment before their use becomes widespread.
Carbon is found in nature in a wide variety of allotropic forms: graphite, diamond, fullerenes, amorphous carbon and many others. This is due to the properties of the carbon to form different types of bonds, each one with a particular geometry.
Through oxidation and exfoliation, it is possible to obtain graphene, a material consisting of a layer of monoatomic carbon atoms, from graphite. The winding of graphene sheets upon themselves allows to obtain tubular structures of nanometric size called nanotubes: the winding of a single layer of graphene allows the formation of single-wall carbon nanotubes (SWCNTs), while the winding of more layers generates multi-walled nanotubes, MWCNTs.
Carbon nanotubes (CNTs) have an elongated cylindrical structure with diameters of the order of nanometers and lengths of the order of micrometers: they are considered as one of the most promising nanomaterial for different types of applications, from industrial to biomedical field, thanks to their particular physico-chemical characteristics. For example, the extremely high length/diameter ratio (in the order of 104) allows to consider them as virtually one-dimensional nanostructures; also, the CNTs are among the most rigid and strongest materials in terms of tensile strength and elastic modulus ever discovered before. From an electrical point of view, the CNT have electronic insulating properties, semiconductive or conductive according to their geometrical structure (1, 2).
Moreover, under certain conditions, electrons can pass inside a nanotube without heating, thus making CNTs much interesting, bearing in  mind the shift  from microelectronics to nanoelectronics; nanotubes are also excellent thermal conductors along the cylindrical structure, but good insulators laterally to 'axis of the cylinder. In the biomedical field, CNTs have been used as "drug carrier" for their high load capacity of biomolecules. For example, SWCNT conjugates with the chemotherapy agent paclitaxel have recently been used in vivo to block tumor growth in a mouse model of breast cancer (3).

In order to assess whether the conjugation with chains of polyethylene glycol (PEG) could be able to reduce or abolish the embryotoxic effect reported for some types of SWCNT (4), we decorated carbon nanotubes with PEG chains (5), and studied their ability to induce fetal malformations after intravenous administration to female mice during the first few days of pregnancy. For this study we used different dosages of nano material, between 10 and 30 m g / ml and tested a number of females equal to or greater than 10 for each group. The presence of fetal and placental abnormalities was evaluated shortly before delivery, when the process of organogenesis is complete and the fetus is in the growth phase.
We measured the length of the fetuses (crown-rump length, LVS) taken from each of the treated mothers and compared it with that of control fetuses taken from mothers which had received only vehicle (saline). The ability to induce fetal underdevelopment has been reported for some nanomaterials, such as nanoparticles of silicon and titanium (6). The administration of these nanoparticles, in a later stage of gestation compared to that provided by our protocol, causes a significant increase in the number of resorptions and fetal growth retardation and is not associated with changes in the weight of the placentas (6).
In our conditions, after administration of PEG-CNT, the comparison of the average values ​​of LVS showed no statistically significant differences between the fetuses of mothers exposed (1.58 ± 0.12) and control groups (1.50 ± 0, 15). Through use of a dissecting microscope, we assessed the presence of gross structural malformations and fetal placenta. In a small number of females who had received the highest concentrations of PEG-CNT we observed the presence of one or more fetuses with obvious abnormal development, associated with alterations of placental cytoarchitecture.
To assess whether the administration of this nanomaterial could affect the health of the mother and hence be reflected in the alteration observed in fetal development, we evaluated some biochemical parameters in maternal blood, such as ALT, AST, BUN, CREA, LDH and CHO. Comparing the values ​​obtained by mothers of control with those of mothers treated with the highest concentration of PEG-CNT, none of the analyzed parameters was found to be altered in a statistically significant manner. These results were further corroborated by histological analysis of the main maternal tissues (liver, spleen, kidney, lung), which did not reveal the presence of apparent structural alterations.

Discussion and Conclusions
In conclusion, the results of reports indicate that the administration of single-walled carbon nanotubes functionalized with PEG chain during the early stages of embryonic development is safe for the mother, but can pose a risk to the developing fetus, although with a low incidence. On the basis of these observations, exposure to PEG-CNT in the workplace and the environment, whether accidental or deliberate, must be strictly monitored. In addition, our  results demonstrate the need to establish, in the short term, measures to regulate occupational exposure of pregnant women.

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