The increasing use of nanomaterials warrants a thorough understanding of the hazards of the environment and man 1. C₆₀ fullerenes are spherical molecules with a diameter of around 1 nm, comprising 60 carbon atoms arranged in a truncated icosahedron structure similar to a soccer ball. C₆₀ fullerenes have already been marketed in cosmetics, whereas their cage like structures are being developed e.g. for drug delivery with surface modification for targeting properties including cancer and antimicrobial activity. Multiple other applications can be envisaged, although widespread commercial use of C₆₀ fullerenes is still in the future 2.

In keeping with the notion that exposure to particulate matter in air pollution is associated with increased risk of cardiovascular diseases, engineered nanomaterials are expected to generate similar effects 3. The detrimental effects on the cardiovascular system of particulate matter in the small size range include altered activity of the fibrinolytic system, a procoagulant state, endothelial dysfunction, and cardiac effects 345. Of special interest is the studies showing that airway exposure to concentrated ambient particles and single wall carbon nanotubes can promote progression of the atherosclerosis process in apolipoprotein E knockout mice (apoE^-/-) that develop plaques in blood vessels at early age 678. Similarly, pulmonary exposure to nano-sized carbon black is associated with accelerated plaque area development in the aorta of low-density lipoprotein receptor knockout mice that are also predisposed to atherosclerosis 9. In addition, studies relating exposure to particulate matter by exposure to air pollution or diesel exhaust particles have shown increased area of atherosclerotic lesions in the aorta of Watanabe heritable hyperlipidemic rabbits 1011.

The endothelium plays an important role in maintaining the vascular homeostasis by producing vasoactive factors that regulate the tone of the vascular system in response to cell surface receptor stimulation or mechanical stress 12. In the functional endothelium, acetylcholine (ACh) binding to muscarinic receptors on the luminal surface is associated with the generation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). The NO subsequently diffuses into the smooth muscle cells and facilitates the relaxation of the vessel. In experimental settings, the endothelium-independent vasorelaxation can be studied by NO-donors such as sodium nitroprusside (SNP). In contrast, the phenylephrine (PE) mediated vasoconstriction is mediated by binding to α₁-adrenoceptors on the smooth muscle cells. Endothelial dysfunction is believed to be an event that leads to atherosclerosis as well as being a marker of the severity of atherosclerosis 13. In apoE^-/- mice, the vasomotor function is inversely correlated with the plaque-size, whereas it is not affected by hypercholesterolemia 14. In apoE^-/- mice, the acetylcholine-induced vasorelaxation was decreased and the PE-induced maximal vasoconstriction was enhanced after 5 months inhalation exposure to concentrated air pollution particles although this could be due to much further progressed aortic plaque development 68. A number of studies have shown acute effects of particle exposure on vascular functions. E.g. ultrafine TiO₂ particles were more potent in inducing systemic microvascular dysfunction in rats after exposure than fine TiO₂ particles after inhalation at doses with minimum pulmonary effects 15. We have shown that systemic exposure to diesel exhaust particles by intraperitoneal (i.p.) injection reduced acetylcholine-elicited vasorelaxation in apoE^-/- mice with minimum atherosclerosis, whereas opposite effects were seen in wild type mice 16.

Research concerning the effect of nanomaterials on the vascular function is still very sparse. The effect of C₆₀ fullerenes or derivatives on vascular function has only been investigated in a few studies and only in ex vivo exposure designs. It has been shown that the endothelium-dependent relaxation of rabbit thoracic aorta segments is affected by water-soluble monomalonic acid-modified C₆₀ fullerenes, whereas the PE-induced vasoconstriction was unaffected 1718. Water-soluble C₆₀(OH)₂₄ fullerenes have been shown to induce the endocytotic uptake of oxidized low-density-lipoproteins in macrophages and stimulate ADP-induced platelet aggregation 19. To the best of our knowledge no data on vasomotor function following in vivo exposure to C₆₀ fullerenes have been published.

The purpose of this study was to investigate the effect of systemic exposure to pristine C₆₀ fullerenes by i.p. injection on the vascular function in apoE^-/- mice with varying degree of atherosclerosis. I.p. injection of C₆₀ fullerenes has been used in some investigations as a route of exposure in order to focus on C₆₀ fullerene's ability to prevent toxic effects of carbon tetrachloride 20 and methamphetamine and morphine 21. We used the i.p. route of exposure and 1 hour post-treatment time similarly to our previous study on diesel exhaust particles in order to investigate direct systemic vascular effects unrelated to inflammatory responses 16. The i.p. route allows a relatively slow systemic dose rate which would be difficult to achieve with i.v. injection and it is also similar to intramuscular or subcutaneous administration or even dermal uptake or translocation from the airways. We hypothesized that exposure to C₆₀ fullerenes would affect the vasomotor function in aorta vessels with atherosclerosis in a similar way as diesel exhaust particles are associated with microvascular dysfunction.

In this study we showed that systemic treatment with C₆₀ fullerenes in apoE^-/- mice was associated with reduced vasorelaxation responses indicating that treatment with pristine C₆₀ fullerene nanoparticles alters the balance of the vasomotor functions.

Treatment with C₆₀ fullerenes at a dose of 0.05 mg/kg resulted in significantly lower E_max values of the acetylcholine-induced relaxation of aorta segments in both young and old apoE^-/- mice. A similar tendency was also observed for the greater dose (0.5 mg/kg), although it only reached statistical significance in the group of young apoE^-/- mice: this might be due to a slightly smaller variation in that group. The old male apoE^-/- mice were only exposed to 0.5 mg/kg of C₆₀ fullerenes as part of a pilot study to assess the reliability of vasomotor function tests. The assessment of the vasomotor function in aorta rings of the arch of the vessel of the male apoE^-/- mice were unsatisfactory because the results were rather variable and the endothelium-dependent vasorelaxation was severely affected, although a decrease in E_max of the endothelium-independent vasorelaxation was suggested for SNP. In addition, the results for the male mice are inconclusive because the few animals per group are associated with low statistical power. For the main experiment in female apoE^-/- mice we therefore used the more distal portion of the aorta for the investigation of the vasomotor function. This means that it is difficult to directly compare vasomotor response in the young and old female apoE^-/- mice because the aortic arch and the more distal part of the descending aorta have different vascular architecture and atherosclerotic progression. The data observed in this investigation are in keeping with our previous study on the effect of i.p. injection of diesel exhaust particles, which we found to deteriorate the endothelium-dependent acetylcholine-response, whereas the EC₅₀ and E_max values of the SNP-response could not be estimated because sigmoid concentration-response curves were not obtained 17. This was achieved in the present study possibly by adding two low concentrations of SNP and allowing more time for each response to reach a steady state. NO is released intracellularly by metabolism of SNP and it is thus difficult to assess whether the reduced relaxation is due to interference with the metabolism or downstream signalling. Comparison with other NO donors such as diethylamine NONOate or spermine NONOate, which release NO directly, together with NO-independent vasodilators (e.g. calcium channel blocker), would be required for further understanding. In contrast to the dysfunction of the vasorelaxation, vasoconstriction does not appear to be affected by C₆₀ treatment. The unaltered effect of the vasoconstriction is not likely to be due to insensitivity of the method because the order in which the segments were mounted in the myograph and used in the different concentration-response experiments was fixed with regard to distance from the heart in order to reduce the variation between experiments. It is possible that there may have been a difference in sensitivity to particle exposure along the length of the aorta, but as the segments used for the PE experiments were always dissected from a slightly more proximal part of the aorta than the segments used in the acetylcholine experiments, this should if anything have led to a higher sensitivity of the segments used in the PE experiments than in the segments used in the acetylcholine experiments. Generally, the data reported here indicate that the balance between vasorelaxation and vasoconstriction is shifted towards the latter. A direct consequence of this unbalance in the vasomotor function is increased stiffness of the vessels as has been shown in mesenteric arteriolar vessels of aged apoE^-/-mice 22.

Oxidative stress is considered to be an important mechanism of action describing the toxic effect of particulate matter 2324. Biomarkers of oxidative stress, including oxidation products of biomolecules such as oxidatively modified DNA base lesions have been found in target tissue of animals and in blood cells and urine of humans as well as in in vitro models in relation to particulate matter and especially nanoparticles 25. Particle-generated oxidative stress may arise as a consequence of direct generation of reactive oxygen species on the surface of the particles or indirectly by particle-elicited inflammatory reactions 23. It seems unlikely that the alteration in the vascular function in our study was elicited by C₆₀ fullerene-generated inflammatory reactions because the post-treatment period was only one hour and the inflammatory potential of C₆₀ fullerenes appears to be modest 26. However, we have shown that our batch of pristine C₆₀ fullerenes can generate reactive oxygen species in aqueous solution as well as within cultured cells, although the concentration-response relationship was bell-shaped 27. It is thus possible that the direct presence of C₆₀ fullerenes in the vessel wall is associated with increased generation of reactive oxygen species. This would imply translocation of C₆₀ fullerenes to the circulation. In a previous study on diesel exhaust particles we observed that the treated mice had particulate matter in lymph nodes of the head/neck region, which could have originated from both trapping of free particulate matter in the lymph node or migration of macrophages containing engulfed particulate matter 28. However, most studies have focussed on the translocation of particulate matter from the lungs to the circulation and the most recent, employing impeccable methodology, indicate very little translocation of particulate matter 29303132. Although we do not have final proof of any extent of C₆₀ fullerenes translocation, our data suggest that particulate matter in the peritoneum reaches the circulation and directly affects vascular function. Our results on the endothelium-dependent vasorelaxation are in accordance with a previous investigation on ex vivo treatment of rabbit thoracic aorta segments with water-soluble monomalonic acid derivative of C₆₀ fullerenes that was associated with a decrease in the endothelium-dependent vasorelaxation, whereas the SNP-induced endothelium-independent relaxation and PE-induced vasoconstriction was not affected in those studies 1718. The difference between this and our SNP-response may be due to a number of differences in the experimental setups, such as the use of ex vivo or in vivo exposure conditions, modified or pristine C₆₀ fullerenes and/or the use of aorta from normal rabbits or from mice with atherosclerosis. In the studies utilizing rabbit aorta, addition of superoxide dismutase alleviated the endothelium dysfunction caused by the monomalonic derivative of C₆₀ fullerenes, which indicates that the mechanism of action involves the generation of superoxide anions 17. It is well-known that superoxide anions react with NO leading to the formation of peroxynitrite, a highly oxidizing species that forms 3-nitrotyrosine in a reaction with proteins 33. This product was observed in aorta from apoE^-/- mice following exposure to air pollution particles, where the exposure was associated with increased PE-induced vasoconstriction and lower responsiveness of acetylcholine-induced vasorelaxation 8. However, besides the generation of superoxide anions other scavenging of NO could also impair vasorelaxation, and C₆₀ fullerenes may have more complicated mechanisms of action in this respect. For instance, it has been reported that hexasulfobutylated C₆₀ fullerenes potentiated the endothelium-dependent vasorelaxation in aorta segments from guinea pigs 34. In addition, it has been reported that other derivatives of C₆₀ fullerenes (called C₃- or D₃-tris-malonyl-C₆₀-fullerene) inhibited the activity of eNOS and C₃-tris-malonyl C₆₀ fullerenes, which had the largest hydrophobic surface area, inhibited eNOS the most 35. This is an effect which could be predicted to inhibit the acetylcholine-dependent vasorelaxation of the aorta. Collectively, the data obtained so far from miscellaneous C₆₀ fullerenes suggest that it is difficult to draw general conclusions based on extrapolation from a single type of particle. In addition, it is likely that the dose of C₆₀ fullerenes is an important issue to consider.

Our data fit with a general picture observed following treatment with air pollution particles in mice where it has been shown that intratracheal instillation of ambient particulate matter was associated with reduced maximal acetylcholine-response and there was no change in vasoconstriction induced by PE 36. In contrast, it has been shown that intratracheal instillation of ambient particulate matter in spontaneous hypertensive rats was associated with increased acetylcholine-dependent vasorelaxation and no effect related to PE-induced vasoconstriction 37. It is possible that differences in the preexisting conditions and/or species can explain the discrepancy in the acetylcholine-induced vasorelaxation. In addition, long-term exposure to concentrated ambient particles was associated with altered vasomotor function in terms of both vasorelaxation and vasoconstriction, although this might partly be explained by particle-induced acceleration of atherosclerosis because the mice exposed to particulate matter exhibited a larger area of aorta covered with plaques 8. In healthy humans, short bouts of exposure to air pollution particulate matter was associated with vascular impairment of vasorelaxation 3839404142. However, it is interesting that among patients with prior myocardial infarction, exposure to diesel exhaust did not aggravate pre-existing vasomotor dysfunction of endothelium-dependent or endothelium-independent vasorelaxation 43. The results from our investigation calls attention to the possible detrimental effect of low doses of C₆₀ fullerenes in humans considering that exposure to the C₆₀ particles elicits a similar pattern of vascular motor-function effect related mainly to the endothelial cells whereas a direct effect on the smooth muscle cells is less pronounced.

The exposure concentrations of pristine C₆₀ fullerenes in the environment, consumer products or drugs are still largely unknown. The highest dose of C₆₀ fullerenes (0.5 mg/kg bodyweight) in the present experiment was chosen because we have previously found this dose to be associated with the largest effect in terms of endothelial dysfunction by exposure to diesel exhaust particles by the same route of exposure 16. The maximum effect of the C₆₀ fullerenes appeared to be attained already at 0.05 mg/kg in the present investigation. However, the effect was only a 15–30% reduction in the maximal endothelium-dependent relaxation and a 50–60% increase in EC₅₀ of the SNP response, which might have limited impact on the risk of development of cardiovascular disease. In a recent study on inhalation exposure in rats with the cumulated dose of about 0.3 mg/kg (assuming the rats weighed 250 g), there was minimal pulmonary toxicity, whereas the C₆₀ fullerene treated group showed weak signs of hepatic effects and the creatine kinase concentration in serum was increased suggesting subtle toxic effects to the myocardium 44. We believe that the lowest dose in the present experiment (0.05 mg/kg bodyweight) is rather modest, although it is difficult to provide a reasonable dose-justification since we have no reliable information about exposure to humans. It appears that the doses of C₆₀ fullerenes, which have been used in other in vivo studies by the same route of exposure, have been one (3.5 and 7.0 mg/kg bodyweight; 45 and two (30 and 100 mg/kg bodyweight; 21 orders of magnitude larger than the doses used in this experiment.

Our study indicate that systemic exposure to C₆₀ fullerenes by intraperitoneal injection at low doses can disturb the vasomotor balance toward a reduced vasorelaxation in apoE^-/- mice, which are hyperlipidemic animals prone to develop atherosclerotic lesions resembling those observed in humans. We therefore advise that caution should be exercised when these and similar nanomaterials are used in nanomedicine and other parental administrations.

apoE: apolipoprotein E; ACh: acetylcholine; CI: confidence interval; eNOS: endothelial nitric oxide synthetase; NO: nitric oxide; PE: phenylephrine; PSS: physiological saline solution; SEM: standard error of the mean; SNP: sodium nitroprusside.

The authors declare that they have no competing interests.

HWA, SL and PM conceived the study. LKV, HWA, SL and PM designed the experiments. LKV and JKF made the experiments on vasomotor function, assisted and supervised by MS. The characterization of the particulate matter and supervision of the animal experiments were carried out by NRC. LKV made the draft of the manuscript, which was revised critically by SL and PM. All authors have read and approved the manuscript.