Due to the diverse commercial utility of MWCNTs and the limited knowledge about their potential health risks on the cardiovascular system, the effect of pulmonary MWCNT exposure on an imposed cardiac stress was evaluated. We report, for the first time, that oropharyngeal aspiration of three different forms of MWCNTs promotes an increased susceptibility of cardiac tissue to ischemic reperfusion (I/R) injury. These effects were observed at a concentration as low as 0.1 μg MWCNT per animal and persisted for 28 days following a single oropharyngeal aspiration. We suggest that a pulmonary exposure to MWCNTs may pose a significant risk to the injured heart, particularly at low doses where overt pulmonary toxicity is not observed.
The safety of MWCNTs is still in debate due to the lack of systematic and complete toxicity evaluation as well as a limited understanding of the human exposure levels in occupational settings . Current proposed guidelines by the National Institute for Occupational Safety and Health (NIOSH) limits exposure to 7 μg/m3, which is the lowest detectable level of airborne CNT by the latest analytical method . The present study involved delivering a single defined amount of MWCNTs in a log order by oropharyngeal aspiration and examining pulmonary and cardiovascular endpoints at multiple time points. The utilization of such acute high concentrations to probe potential effects is a standard approach in toxicology and experimental pathology for initially surveying adverse effects . While oropharyngeal aspiration provides a natural route of entry into the host and has been a method for the introduction of a variety of toxicants into the lungs  there is continued discussion regarding the real world relevance of this route of exposure. The pulmonary toxicity effects of CNT aspiration have shown to be comparable between inhalation and oropharyngeal aspiration . Even with these caveats the chosen oropharyngeal aspiration approach allows primary responses to substance exposure to be studied independent of the compensatory mechanisms that may come into play with chronic exposures.
Evidence accumulated from epidemiological studies confirms a significant pathogenic correlation between the inhalation of small and ultrafine sized particles, including nano-sized particles from ambient air and cardiovascular events, such as angina, arrhythmia, ischemic heart failure and sudden death . Epidemiologic and experimental studies have suggested an association between respiratory exposure to ambient ultrafine particles (including particles with a diameter < 100 nm) and the progression of cardiovascular disease [17, 28]. A number of studies have also shown that pulmonary inhalation of ultrafine particulate matter generated from synthetic nanoparticles resulted in development of vascular abnormalities, including atherosclerosis, increased vascular tone and impaired endothelial-dependent vascular dilation [14, 29–31]. While epidemiological and inhalation studies have provided indirect evidence that ambient particle inhalation exposure may be a risk factor for adverse cardiac events [12, 27, 32], no definitive study has identified a link between exposure to MWCNTs and a negative impact on the myocardium.
Various toxicological studies have demonstrated that pulmonary deposition of MWCNTs caused an acute and chronic pulmonary toxicity in animal models [7–9, 11]. Pulmonary responses to MWCNT exposure include an acute inflammatory phase, a progressive fibrotic response in the interstitium of the alveolar wall, and a granulomatous inflammation enveloping airspace deposits of MWCNT agglomerates [9, 33].
In our experiments we observed similar pulmonary inflammatory responses as reported in the literature following oropharyngeal aspiration of MWCNTs that peaked between 1 and 7 days and decreased by 28 days. This result is similar to that described in time of an acute inflammatory reaction which peaked at 7 days in the mouse and resolved over a 56 day period [7, 9, 34]. The pulmonary inflammatory response in our study may be related to how the MWCNTs were dispersed using a clinical grade animal-based lung surfactant. Previous reports have demonstrated that MWCNTs suspended in a natural lung surfactant or synthetic lung surfactants could influence both the lung dispersion and cytological effects of the CNTs [35–37]. In addition, the presence of proteins in the suspension that ultimately form a corona around the CNTs are believed to modulate MWCNT toxicity, phagocytosis and the inflammatory response [38, 39].
Lung histopathology showed persistence of MWCNTs within the tissue that ultimately resulted in granuloma-like lesion formation at both low and high concentration at 28 days following instillation with each MWCNT type. Our finding was consistent with chronic granuloma formation reported earlier by us and others in rodent models [10, 11].
Our study demonstrated a modest pulmonary inflammatory response but a more robust cardiac infarct exacerbation both in a time- and dose-dependent manner following oropharyngeal aspiration of MWCNTs. Infarct expansion remained evident at the highest concentration at 7 days post- oropharyngeal aspiration. Although we observed a diminution of the infarct size with all three forms of MWCNTs with time, the expanded infarct was still present as late as 28 days post- oropharyngeal aspiration. This type of result may underlie the importance of defining the endpoint of interest as to determining systemic toxicological response. Overall the results of our experiments contribute to the mounting evidence of the harmful effects of air pollution and engineered nanomaterials on the cardiovascular system [13, 17, 40]. Lack of blood supply or ischemia underlies many of the most important diseases encountered by physicians, including myocardial infarction, thrombotic stroke, embolic vascular occlusions, and peripheral vascular insufficiency. Evidence from animal studies suggests that reperfusion of ischemic areas may contribute to further tissue damage (I/R injury) . I/R injury plays a key role in the pathophysiology of myocardial tissue damage after thrombolysis, angioplasty, or coronary artery bypass surgery [42–44]. We chose to investigate the effects of MWCNTs on an acute I/R injury model to simulate the occurrence of the common cardiovascular events in an acute setting. The relative uniformity in anatomy and physiological process found in the mouse model allows experimental interpretations that can be translated to the clinical setting . As previously reported although the topographical left coronary artery in mice is not a functional left coronary artery as we understand it in humans,  and the mouse model as used to study the effects of ischemia reperfusion injury has been shown to be reproducible [45, 46]. Our experiments used a 20-min occlusion period. Thirty minutes is a more common occlusion period in this model, but those studies of I/R injury are focused on mechanisms to reduce injury, and the occlusion time is selected to produce a sufficiently large infarction to permit measurable decreases associated with treatment. In our experiments, we hypothesized that treatment would exacerbate injury, and the occlusion time was shortened to enable an accurate reflection of expanded injury. The twenty-minute ligation of the LAD has been reported previously in the murine model [47, 48].
Studies have investigated the dose- and time- dependent responses of pulmonary toxicity of MWCNTs after oropharyngeal aspiration in mice [9, 49]. Our experiments illustrate a concentration- and time- dependent response of cardiac toxicity after oropharyngeal aspiration of MWCNTs. We observed a concentration-dependent effect with the N-doped MWCNTs but not with either C-grade or COOH MWCNTs. In contrast, the C-grade and COOH MWCNTs exhibited a threshold response with 1 μg MWCNT associated with a significant expansion of the infarction. Surprisingly, at one day following oropharyngeal aspiration of the N-doped MWCNT, the infarct was significantly expanded at of 0.1 μg.
The infarct exacerbation that we observed with the N-doped MWCNTs may reflect its unique physicochemical properties notably their bamboo-like structure and surface charge distribution resulting in the augmentation of the cardiovascular response. The presence of nitrogen atoms during the nanotube growth process is known to promote the growth of inner caps to yield structures that resemble bamboo sticks . Previously, some studies found that N-doped MWCNTs exhibit different physiological response since the bamboo-like rough surfaces modify the van der Waal interactions between N-doped nanotubes [51, 52]. N-atoms often dope the carbon lattice in non-graphitic configuration via the removal of an electron (pyridinc, pyrrolic). Hence, a high doping percentage of N (~19% in our case; see Table 1) may lead to the formation of non-graphitic (pyridinc/pyrrolic) phases that may act as localized charge centers. Previously, several researchers have demonstrated differences in cytotoxicity, hemo- and biocompatibility between pristine and N-doped MWCNTs [51, 52]. However, the exact role of N atoms in the observation of such mitigated toxicity responses is yet to be understood. It is possible that the presence of large amount of N-atoms results in local charge centers. Such charge centers assist N-doped MWCNT interact more effectively with any polar entities compared to pristine or COOH functionalized forms. In fact, N-doped MWCNTs have been employed as electrodes in redox-active cells due to their unique charge distribution .
In addition to the potential role of nitrogen doping in contributing to a more robust cardiac response, surface area may also contribute to these differences. Physical characteristics of MWCNTs such as small size, large surface area and high reactivity, are cited as significant factors of their potential toxicity [12, 27, 32]. It has been postulated that the large surface area (250–300 mg/m2) of some MWCNTs may play a significant role in the development of pulmonary effects . In our results, it is interesting to note that the most extensive cardiac injury and dose sensitivity was associated with the N-doped MWCNT that had the highest measured surface area. However, there was no significant difference in the surface area or hydrodynamic size estimates for the COOH and C-grade MWCNTs but there were differences in the time course of the cardiac I/R response. We believe that surface area may be another contributing factor along with the surface modification.
The exact mechanism by which MWCNTs may exhibit cardiovascular toxicity is unknown. Mechanisms of nanoparticle cardiovascular toxicity postulated in the literature include oxidative stress, inflammation, emanating in part, from pulmonary inflammation leading to atherothrombosis ; translocation of inhaled particles to the vasculature , direct effects on endothelial cells and cardiovascular system [57, 58] and/or effects on the autonomic nervous system [55, 59].
Systemic cytokine response to MWCNT oropharyngeal aspiration has not been adequately investigated although studies have reported cytokine analysis of BALF after pulmonary exposure to MWCNTs [11, 36]. We therefore chose to study the circulating cytokine profile after oropharyngeal aspiration of MWCNTs. A close correlation between cytokines and I/R injury has been demonstrated . IL-6, IL-10, IL-12, KC have been implicated in I/R injury after myocardial infarction [21, 60, 61]. IL-6 has been shown to contribute to cardiovascular dysfunction induced by acute lung injury . MWCNTs and SWCNTs have been shown to provoke inflammation by induction of the pro-inflammatory genes IL-1β and IL-6 . Although not statistically significant, we also found elevated levels of IL-6, IL-10, IL-12, IL-13, KC and MIP1-α with the COOH and the N-doped MWCNTs. IL-1β did not reveal any statistically significant differences compared to the SS and across the three treatment groups.
Wang and co-workers demonstrated that pulmonary exposure to MWCNTs induces an inflammatory response marked by increased levels of Eotaxin (Ccl11) that promote inflammatory and fibrotic changes observed within the lung . We reported a statistically significant increase in the level of Eotaxin with the COOH exposed mice. Serum eotaxin levels have been associated with the etiologies of cardiac injury and heart failure as well as the regulation of inflammatory cell recruitment to infarcted myocardial tissue for wound repair [64–66]. The increase in the eosinophils with the COOH and the N-doped MWCNTs instillation in mice in the BALF observed in our experiments may suggest a possible link of Eotaxin to increased response of the myocardium to an imposed stress from I/R injury. The hematological assessment from these groups at 24 hours post oropharyngeal aspirations does not reflect a strong inflammatory either. As previously reported by Robertson there was no indication of systemic inflammation at 24 hours after diesel exhaust particle instillation although there were increased levels of inflammatory mediators at 6 hours . While there was significant elevation in a common marker of inflammation in the number of neutrophils from the COOH exposed mice but overall WBC numbers remained in the normal range and suggest there is no significant inflammatory state. Any conclusion drawn from these observations must be viewed with caution as the cytokines and blood cells counts are known to rapidly change following an acute pulmonary exposures and we do not provide direct evidence for the role of these cytokines exacerbating the I/R injury. It still remains a hypothesis that circulating cytokines may prime target organs to response more robustly to any imposed injury.
As previously demonstrated by Cozzi et al, oropharyngeal aspiration of mice with particulate matter increased cardiac oxidative stress within the myocardium after I/R injury . Furthermore, it has also been shown that carbon nanotubes in general can lead to reactive oxygen species (ROS) generation . Xia and co-workers have shown that redox cycling can contribute to ROS production. This can occur because of the presence of transition metals or redox cycling organic chemicals on the particle surface . Moreover, transition metals can generate hydroxyl radicals through the Fenton reaction . The Fenton reaction is one of the mechanisms by which metal impurities on or in the CNT may induce ROS production . Although we do not have any evidence, it is plausible that ROS production could contribute to some of the infarct exacerbation that we observed in our experiments.
Translocation and extrapulmonary toxicities of MWCNTs were demonstrated by Reddy et al  while Aiso and co-workers have shown translocation of intratracheally instilled MWCNTs to lung associated lymph nodes  such evidence may suggest a close association with the circulatory system following exposure. Although there is a lack of definitive studies on the translocation of MWCNTs leading to direct particle effect on the cardiac myocytes after oropharyngeal instillation, it could be hypothesized that MWCNTs could promote infarct exacerbation based on the underlying translocation mechanisms as demonstrated in the above mentioned studies.
In summary, our results show that oropharyngeal aspiration of MWCNTs in mice exacerbates myocardial infarction with an increase in infarct size in a concentration-, time- and surface modification-dependent manner. This study was the first to assess the impact of adverse outcomes of exposure to MWCNTs on the susceptibility of cardiac tissue to ischemic injury. However, the mechanisms by which MWCNTs exposure generates cardiac tissue that is susceptible to injury secondary to MWCNTs oropharyngeal aspiration remains to be determined. The extent of I/R injury seen at low doses, where there is a lack of robust pulmonary response, is significant and suggests that the cardiovascular impact should be considered when assessing the safety of engineered nanomaterials. Taken together, the findings are of sufficient significance to warrant continued studies to evaluate the systemic effects of MWCNTs associated with various exposure conditions.