In the previous study, we observed that after intramyocardial transplantation of SPION labeled MSCs, SPION, as foreign bodies, induced a large number of macrophages infiltration in peripheral zones of myocardial infarction, which potentially aggravated the inflammatory response of infarcted myocardium, thereby provoking a concern whether SPION could trigger a pro-inflammatory effect when applied in cardiovascular field . Macrophages at days 1–3 following myocardial infarction are dominated by classically activating pro-inflammatory M1 subtype, which secrete cytokines, chemokines, growth factors, and matrix metalloproteinase to clear up the cell debris and degrade the extracellular matrix. M1 macrophages polarize into alternative anti-inflammatory M2 subtype at days 5–7 post-myocardial infarction. In contrast, M2 macrophages are preparative, which produce anti-inflammatory, preparative and proangiogenic factors (examples, IL-10, TGF-β1 and vascular endothelial growth factor) and remove dead cells to promote neovascularization and scar repair [27, 28]. However, the persistence of M1 macrophages can result in an expansion of infarct size and hinder the resolution of inflammation and scar formation .
Disturbingly, SPION have been reported to inhibit tumor growth by inducing macrophage polarization to pro-inflammatory subtype in tumor tissues . Different morphology of SPION can induce pyroptosis, inflammasome activation and IL-1β secretion, especially the plate and octapod SPION demonstrating significantly higher activity than the sphere and cube SPION . The above results implied the possibility that the SPION, when employed as therapeutic substance carriers or magnetic resonance contrast agents in ischemic myocardium, may disturb the shift of macrophages from M1 to M2 by inducing the prolonged pro-inflammatory effect, thereby impeding the repair process after myocardial infarction.
Moreover, it has been proved that compared to normo-ferremic ApoE-/- mice, atherosclerosis is dramatically aggravated in iron-loaded ApoE-/- FPNwt/C326S mice, indicating that iron can promote the progression of atherosclerosis. Excess iron deposits in the arterial media layer, facilitating vascular oxidative stress, dysfunction and plaque formation. Atherosclerosis is aggravated by iron-provoked vascular permeabilization, persistent endothelial activation, increased pro-atherogenic inflammatory mediators, lipid profile alterations, and reduced nitric oxide availability. Correspondingly, iron chelation therapy and a low-iron diet significantly alleviated the severity of the disease in ApoE-/-FPNwt/C326S mice . Recently, SPION have been used as contrast agents to visualize atherosclerotic plaque, for they are easy to be internalized by macrophages in atherosclerotic plaque . Another concern is that SPION might induce iron overload at the cellular level after being internalized and degraded by macrophages in the plaque, aggravating plaque progression or promoting plaque instability through mediating oxidative stress, pro-inflammatory effect and endothelial cell dysfunction. Good safety and biocompatibility are the necessary prerequisites for the clinical transformation of SPION. Therefore, it is urgent to explore strategies to improve the biosafety of SPION.
SPION have been confirmed to damage a variety of organelles by inducing ROS generation, including Golgi stress, destruction of lysosome and mitochondria and over activated endoplasmic reticulum stress and autophagy [17, 33], especially mitochondria, as the energy factory of cells, is very crucial to maintain energy metabolism and physiological function. Furthermore, mitochondria are also the largest iron metabolism organelle and the main place for ROS production in cells. Thus, mitochondria are speculated to be the main target organelle of SPION [11, 34].
Mitochondrial targeted antioxidant peptides have been confirmed to play a strong protective role against ischemic brain injury and kidney ischemia-reperfusion injury [35, 36]. In particular, SS-31 (D-Arg-DMT-Lys-Phe-NH2) has been proved to be the most effective protective agent against ischemia-reperfusion injury in SS peptides [37, 38]. After entering mitochondria, tyrosine residues on SS-31 polypeptide scavenge ROS by forming inactive tyrosyl groups, significantly inhibit ROS production and reduce lipid peroxidation and cardiomyocyte death, eventually reducing infarct size [39, 40]. In view of this, SS-31 might bear the potential to improve the cardiovascular safety of SPION by protecting mitochondria.
We first prepared negatively charged SPION as previously described , and then successfully constructed [email protected] by PEI mediated adsorption (Fig. 1). Our data showed that compared with the control group, SPION induced loss of viability and increase of early apoptosis, up-regulated the expression of CD86 and CD80 (M1-like subtype markers) and down-regulated the expression of CD163 (M2-like subtype marker) in macrophages, accompanied by the increased secretion of TNF-α and IL-6 and the decreased secretion of TGF-β and IL-10, strongly suggesting that SPION can induce the polarization of macrophages into pro-inflammatory M1-like subtype in vitro study. Interestingly, compared with the control group, [email protected] only induced a slight loss of cell viability and early apoptosis of macrophages after 24 hours of treatment. Notably, [email protected] cells demonstrated that the expression of CD163 was up-regulated and the expression of CD86 and CD80 was down-regulated, along with the decreased secretion of TNF-α and increased secretion of TGF-β and IL-10. Meanwhile, we observed that the SPION induced a significant increase in the level of ROS and serious damage to the mitochondria, characterized by mitochondrial swelling, blurring of mitochondrial cristae and vacuolation, implying that oxidative stress and mitochondrial damage might be the main toxic mechanism of SPION. The level of cellular ROS and the mitochondrial damage in the [email protected] group were significantly lower than those in the SPION group. All these suggested that after internalized by macrophages, [email protected] can release the active form of SS-31 to the mitochondrial inner membrane, where it exerts a mitochondrial targeted antioxidant effect, protecting mitochondria from oxidative stress damage mediated by Fenton reaction catalyzed by SPION degradation to ferrous ion. Moreover, [email protected] exhibited a tendency to induce macrophages to polarize to M2-like subtype even though there is no significant difference in the expression of CD206 between the SPION and the [email protected] groups. At the present study we only detected mitochondrial structure and oxidative stress injury. Further detection of the downstream molecular mechanism of oxidative stress, including proteins related to iron metabolism, such as ferritin, iron exporter ferroprotein, transferrin, and signaling pathways of pyroptosis [30, 41] or ferroptosis [42, 43], which have been reported in other studies would be investigated in future work.
Previous studies have confirmed that the cytotoxicity of SPION is time and concentration dependent [44–46]. Here, we only detected the cellular events of a moderate concentration of 50µg/ml Fe3O4 at 24-hour observation. The time course of SS-31 releasing from the [email protected] exhibited that SS-31 has the potential to maintain the effective concentration for at least 96 hours, thereby it is possible to stably play the mitochondrial targeted antioxidant effect in higher concentration and longer observation.
Our previous study has confirmed that the [email protected] can attenuate cytotoxicity of iron oxide nanoparticles in hypoxia/reoxygenation cardiomyocytes. However, our in vivo experiments showed that [email protected] only mildly improved the negative left ventricular remodeling mediated by iron oxide nanoparticles in a rat model of myocardial ischemia-reperfusion (data not published). We speculated that the unsatisfactory result was attributed to the fact that NAC was rapidly released 72% within 2.5 hours, and only lasted until 48 hours , so it is difficult to play a long-term protective role in animal experiments; More importantly, we observed that the main target organelle of iron oxide nanoparticles is mitochondria, whereas NAC has no mitochondrial targeting function, so it failed to prevent mitochondria against ROS attack. Thus, we determined to employ mitochondrial targeted antioxidant SS-31 to modify SPION. The observation of 24 hours in vitro has been shown [email protected] can effectively inhibit the pro-inflammatory effect induced by SPION. Next, we will plan to construct a sustained release system of [email protected] by polyethylene glycol (PEG) modification, maintaining the long-term effective concentration of SS-31 in vivo study, in which PEG has been employed in preparing drug sustained-release delivery system with good biocompatibility [47, 48].
Our study demonstrated for the first time that the modification of mitochondrial targeted antioxidant SS-31 can significantly inhibit the pro-inflammatory effect mediated by SPION. Considering that the pro-inflammatory effect mediated by SPION may impede the repair of infarcted heart or induce plaque instability, it is particularly important to improve the cardiovascular safety of SPION. The SS-31 modification is expected to be a promising strategy to improve the cardiovascular safety of SPION, which needs to be further verified by animal experiments.