Elsevier

Biomaterials

Volume 31, Issue 32, November 2010, Pages 8198-8209
Biomaterials

Endothelial cells dysfunction induced by silica nanoparticles through oxidative stress via JNK/P53 and NF-κB pathways

https://doi.org/10.1016/j.biomaterials.2010.07.069Get rights and content

Abstract

Drug carriers are generally introduced into the body intravenously and directly exposed to endothelial cells. Silica nanoparticles could be promising delivery vehicles for drug targeting or gene therapy. However, few studies have been undertaken to determine the biological behavior of silica nanoparticles on endothelial cells. Here we measured reactive oxygen species (ROS) generation, apoptosis and necrosis, proinflammatory and prothrombic properties and the levels of the apoptotic signaling proteins and the transcription factors in human umbilical vein endothelial cells (HUVECs) after exposure to silica nanoparticles of different concentrations (25, 50, 100, and 200 μg/mL) for 24 h. The results showed that silica nanoparticles, ranging from 50 μg/mL to 200 μg/mL, markedly induced ROS production, mitochondrial depolarization and apoptosis in HUVECs. At the highest concentration, the necrotic rate, LDH leakage, the expression of CD54 and CD62E, and the release of TF, IL-6, IL-8 and MCP-1 were significantly increased. Silica nanoparticles also activated c-Jun N-terminal kinase (JNK), c-Jun, p53, caspase-3 and NF-κB, increased Bax expression and suppressed Bcl-2 protein. Moreover, inhibition of ROS attenuated silica nanoparticles-induced apoptosis and inflammation and the activation of JNK, c-Jun, p53 and NF-κB. In summary, our findings demonstrated that silica nanoparticles could induce dysfunction of endothelial cells through oxidative stress via JNK, p53 and NF-κB pathways, suggesting that exposure to silica nanoparticles may be a significant risk for the development of cardiovascular diseases such as atherosclerosis and thrombus.

Introduction

Nanomaterials, defined as particles with diameters of less than 100 nm, have been rapidly applied in technical and medicinal fields due to their unique physiochemical properties and tunable characteristics. One of the most important members of this class of materials is silica nanoparticles, which have been found extensive applications in biomedical and biotechnological fields, such as drug carrier, gene therapy, and molecular imaging [1], [2], [3]. Thus, possible health impact of silica nanoparticles upon introduction into the body is of great interest.

Once silica nanoparticles as intravascular carriers have entered the bloodstream by intravenous administration, endothelial cells lining the lumen of all blood vessels and the heart will have direct contact with them. The single layer of endothelial cells is not only just a biological barrier which mediating clearance of nanoparticles but also critical for the maintenance of vascular function and homeostasis [4], [5]. Moreover, endothelium also serves as an important target for cell or gene therapies as they are involved in various pathophysiologic conditions such as atherosclerosis, myocardial infarction and restenosis [6]. Therefore, it is important to understand the interaction between nanoparticles and endothelial cells. However, in spite of their wide use as drug carriers or for gene therapy, there are only a few studies investigating adverse effects of silica nanoparticles on endothelial cells in vitro. Studies by Dorota Napierska demonstrated that smaller silica nanoparticles induced a higher cytotoxic response and affected exposed endothelial cells faster with cell death (by necrosis) [7]. Peters [8] also found that IL-8 release could be enhanced in endothelial cells by exposure to high dosage of silica nanoparticles. However, significant knowledge gaps currently exist concerning precise mechanisms of endothelial injury induced by silica nanoparticles.

Recently, oxidative stress has often been reported as toxic mechanism of silica nanoparticles-induced apoptosis and inflammation in various cell types including macrophages, embryonic kidney cells and epithelial cells [9], [10], [11], [12], [13], suggesting a potential role of reactive oxygen species (ROS) in changes of biological behavior in endothelial cells exposed to silica nanoparticles. However, the involvement of the oxidative stress responding signal transduction pathway in the toxicity of silica nanoparticles is poorly understood. ROS causing oxidative stress are known to activate members of the mitogen activated protein kinase (MAPK) family. The latter ones are important mediators of signal transduction, and play a key role in the regulation of many cellular processes, such as cell growth and proliferation, differentiation, and apoptosis [14]. The MAPK cascades are composed of three distinct signaling modules, the c-Jun N-terminal kinase (JNK) cascade, the p38MAPK cascade, and the extracellular signal-regulated kinase (ERK) cascade. In particular, JNK can induce apoptosis and is a key regulator of the transmission of pro-apoptotic signals [15]. The activation of MAPK pathway was also observed in endothelial cells exposed to nano-size copper (II) oxide or ambient ultrafine particles [16], [17]. ROS could also induce p53 activation, followed cell-cycle arrest or apoptosis [18]. Our previous studies have reported that p53 activation was involved in hydroxyapatite nanoparticles-mediated apoptosis in macrophages [19]. Moreover, redox-sensitive transcription factor, such as nuclear factor kappaB (NF-κB) was recently investigated as target transcription factors of nanoparticles-mediated inflammation response [12], [20]. Based on these evidences, it seems that the activation of JNK, p53 and NF-κB due to oxidative stress plays essential roles in silica nanoparticles-induced endothelial cells apoptosis and inflammation response.

In the present study, to investigate the basic mechanism underlying biological behavior of silica nanoparticles on endothelial cells, we conducted a battery of evaluations including oxidative stress, proinflammatory state, prothrombic properties and apoptosis response in human umbilical vein endothelial cells (HUVECs) after exposure to silica nanoparticles for 24 h. Moreover, we also measured the expression of p-JNK, p-p53, NF-κB and apoptosis-related protein to determine whether silica nanoparticles induce endothelial dysfunction through oxidative stress via JNK, p53 and NF-κB pathways.

Section snippets

Silica nanoparticles synthesis, characterization and preparation

The silica nanoparticles were synthesized using a sol–gel process according to previously published procedures [21]. Briefly, one solution composed of ammonium hydroxide, deionized water and ethanol was slowly added to another solution containing tetraethylorthosilicate (TEOS) (Sigma, St. Louis, MO, USA) and ethanol, with stirring, and the final mixture was stirred overnight to allow the silica nanoparticles to coarsen. After two alcohol washes, silica nanoparticles were obtained by vacuum

Characterization of silica nanoparticles

Prior to the study of impact of silica nanoparticles on HUVECs, characterization of synthesized silica nanoparticles was performed using TEM and SEM methods. As shown in Fig. 1, the TEM and SEM images of silica nanoparticles from the test medium confirmed that the nanoparticles types were near-spherical and the sizes approximately matched the sizes measured after synthesis (20 nm).

Uptake of silica nanoparticles by HUVECs

To understand the interactions of silica nanoparticles with HUVECs, the uptake of silica nanoparticles by HUVECs was

Discussion

Silica nanoparticles represent a promising class of nanomaterials that offers several attractive features for diagnostics and targeting cells in drug delivery [27]. A key administration route of silica nanoparticles is intravenous injection, thus endothelial cells will be the primary cells exposed to these nanoparticles. To date, a complete understanding of how silica nanoparticles interact with endothelial cells, especially at the molecular level, is still unclear. Our findings demonstrated

Conclusion

In summary, data from the current study show that exposure to silica nanoparticles causes endothelial cells ROS generation, which induces apoptosis via JNK/p53 dependent mitochondrial pathways. Exposure to silica nanoparticles at high concentrations also causes activation of NF-κB due to oxidative stress in endothelial cells, which results in the upregulation of CD54, CD62E, TF, IL-6, IL-8 and MCP-1. Moreover, our overall findings suggest that exposure to silica nanoparticles is possibly a

Acknowledgment

This work was supported by grants from Natural Science Foundation of China (no. 30670556, no. 30470479), Shanghai Sci-Tech Committee Foundation (0752nm026) and Shanghai Leading Academic Discipline Project (no. S30206).

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