Liangliang Liu

The use of human umbilical vein endothelial cells (HUVECs) as an in vitro model to assess the toxicity of nanoparticles to endothelium: a review

With the rapid development of nanotechnologies, nanoparticles (NPs) are increasingly produced and used in many commercial products, which could lead to the contact of human blood vessels with NPs. Thus, it is necessary to understand the adverse effects of NPs to relevant cells lining human blood vessels, especially endothelial cells (ECs) that cover the lumen of blood vessels. Human umbilical vein endothelial cells (HUVECs) are among one of the most popular models used for ECs in vitro. In the present review, we discussed studies that have used HUVECs as a model to investigate the EC–NP interactions, the toxic effects of NPs on ECs and the mechanisms. The results of these studies indicated that NPs could be internalized into HUVECs by the endocytosis pathway as well as transported across HUVECs by exocytosis and paracellular pathways. Exposure of HUVECs to NPs could induce cytotoxicity, genotoxicity, eNOS uncoupling and endothelial activation, which could be explained by NP‐induced oxidative stress, inflammatory response and dysfunction of organelles. In addition, some studies have also evaluated the influences of microenvironment (e.g. the presence of proteins and excessive nutrients), the physiological and/or pathological stimuli related to the diversity of ECs (e.g. shear stress, cyclic stretch and inflammatory stimuli), and the physicochemical properties of NPs on the responses of ECs to NP exposure. In conclusion, it has been suggested that HUVECs could be considered as a relatively reliable and simple in vitro model for ECs to predict and evaluate the toxicity of NPs to endothelium. Copyright

A review of endoplasmic reticulum (ER) stress and nanoparticle (NP) exposure

ABSTRACT Understanding the mechanism of nanoparticle (NP) induced toxicity is important for nanotoxicological and nanomedicinal studies. Endoplasmic reticulum (ER) is a crucial organelle involved in proper protein folding. High levels of misfolded proteins in the ER could lead to a condition termed as ER stress, which may ultimately influence the fate of cells and development of human diseases. In this review, we summarized studies about effects of NP exposure on ER stress. A variety of NPs, especially metal‐based NPs, could induce morphological changes of ER and activate ER stress pathway both in vivo and in vitro. In addition, modulation of ER stress by chemicals has been shown to alter the toxicity of NPs. These studies in combination suggested that ER stress could be the mechanism responsible for NP induced toxicity. Meanwhile, nanomedicinal studies also used ER stress inducing NPs or NPs loaded with ER stress inducer to selectively induce ER stress mediated apoptosis in cancer cells for cancer therapy. In contrast, alleviation of ER stress by NPs has also been shown as a strategy to cure metabolic diseases. In conclusion, exposure to NPs may modulate ER stress, which could be a target for future nanotoxicological and nanomedicinal studies.

The Interactions between ZnO Nanoparticles (NPs) and α-Linolenic Acid (LNA) Complexed to BSA Did Not Influence the Toxicity of ZnO NPs on HepG2 Cells

Background: Nanoparticles (NPs) entering the biological environment could interact with biomolecules, but little is known about the interaction between unsaturated fatty acids (UFA) and NPs. Methods: This study used α-linolenic acid (LNA) complexed to bovine serum albumin (BSA) for UFA and HepG2 cells for hepatocytes. The interactions between BSA or LNA and ZnO NPs were studied. Results: The presence of BSA or LNA affected the hydrodynamic size, zeta potential, UV-Vis, fluorescence, and synchronous fluorescence spectra of ZnO NPs, which indicated an interaction between BSA or LNA and NPs. Exposure to ZnO NPs with the presence of BSA significantly induced the damage to mitochondria and lysosomes in HepG2 cells, associated with an increase of intracellular Zn ions, but not intracellular superoxide. Paradoxically, the release of inflammatory cytokine interleukin-6 (IL-6) was decreased, which indicated the anti-inflammatory effects of ZnO NPs when BSA was present. The presence of LNA did not significantly affect all of these endpoints in HepG2 cells exposed to ZnO NPs and BSA. Conclusions: the results from the present study indicated that BSA-complexed LNA might modestly interact with ZnO NPs, but did not significantly affect ZnO NPs and BSA-induced biological effects in HepG2 cells.

Influence of phytochemicals on the biocompatibility of inorganic nanoparticles: a state-of-the-art review

Inorganic nanoparticles (NPs) are among the most produced NPs that could be used in consumer products and as healthcare materials, however, the intrinsic toxicity particularly through the mechanism associated oxidative stress raises the health concern about inorganic NP exposure. Phytochemicals are bioactive metabolites derived from plants as well as non-pathogenic microorganisms living within plants and have been shown to be beneficial to human health with their anti-aging, anti-cancer, anti-inflammation and anti-oxidant properties. In the present review, the influence of on the biocompatibility of inorganic NPs was discussed. It has been shown that phytochemicals could be used as bio-friendly capping agents for green synthesis of inorganic NPs, and phytochemical coated inorganic NPs were remarkable stable and biocompatible with high therapeutic efficiency. Meanwhile, the presence of phytochemicals was also able to reduce the side effects and enhance the therapeutic abilities of inorganic NPs, which is likely attributed to the anti-oxidative properties of phytochemicals. Thus, using phytochemicals could be a promising and plausible way to reduce side effects and increase the biocompatibility of inorganic NPs for biomedical applications.

The effects of baicalein or baicalin on the colloidal stability of ZnO nanoparticles (NPs) and toxicity of NPs to Caco-2 cells

Abstract Recent study suggested that the presence of phytochemicals in food could interact with nanoparticles (NPs) and consequently reduce the toxicity of NPs, which has been attributed to the antioxidant properties of phytochemicals. In this study, we investigated the interactions between ZnO NPs and two flavonoids baicalein (Ba) or baicalin (Bn) as well as the influence of the interactions on the toxicity of ZnO NPs to Caco-2 cells. The antioxidant properties of Ba and Bn were confirmed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, with Ba being stronger. However, the presence of Ba or Bn did not significantly affect cytotoxicity, intracellular superoxide or release of inflammatory cytokines of Caco-2 cells after ZnO NP exposure. When Ba was present, the cellular viability of Caco-2 cells after exposure to ZnO NPs was slightly increased, associated with a modest decrease of intracellular Zn ions, but these effects were not statistically different. Ba was more effective than Bn at changing the hydrodynamic sizes, Zeta potential and UV–Vis spectra of ZnO NPs, which indicated that Ba might increase the colloidal stability of NPs. Taken together, the results of the present study indicated that the anti-oxidative phytochemical Ba might only modestly protected Caco-2 cells from the exposure to ZnO NPs associated with an insignificant reduction of the accumulation of intracellular Zn ions. These results also indicated that when assessing the combined effects of NPs and phytochemicals to cells lining gastrointestinal tract, it might be necessary to evaluate the changes of colloidal stability of NPs altered by phytochemicals.

The adverse vascular effects of multi-walled carbon nanotubes (MWCNTs) to human vein endothelial cells (HUVECs) in vitro: role of length of MWCNTs

BackgroundIncreasing evidences indicate that exposure to multi-walled carbon nanotubes (MWCNTs) could induce adverse vascular effects, but the role of length of MWCNTs in determining the toxic effects is less studied. This study investigated the adverse effects of two well-characterized MWCNTs to human umbilical vein endothelial cells (HUVECs).MethodsThe internalization and localization of MWCNTs in HUVECs were examined by using transmission electron microscopy (TEM). The cytotoxicity of MWCNTs to HUVECs was assessed by water soluble tetrazolium-8 (WST-8), lactate dehydrogenase (LDH) and neutral red uptake assays. Oxidative stress was indicated by the measurement of intracellular glutathione (GSH) and reactive oxygen species (ROS). ELISA was used to determine the release of inflammatory cytokines. THP-1 monocyte adhesion to HUVECs was also measured. To indicate the activation of endoplasmic reticulum (ER) stress, the expression of ddit3 and xbp-1s was measured by RT-PCR, and BiP protein level was measured by Western blot.ResultsTransmission electron microscopy observation indicates the internalization of MWCNTs into HUVECs, with a localization in nuclei and mitochondria. The longer MWCNTs induced a higher level of cytotoxicity to HUVECs compared with the shorter ones. Neither of MWCNTs significantly promoted intracellular ROS, but the longer MWCNTs caused a higher depletion of GSH. Exposure to both types of MWCNTs significantly promoted THP-1 adhesion to HUVECs, accompanying with a significant increase of release of interleukin-6 (IL-6) but not tumor necrosis factor α (TNFα), soluble ICAM-1 (sICAM-1) or soluble VCAM-1 (sVCAM-1). Moreover, THP-1 adhesion and release of IL-6 and sVCAM-1 induced by the longer MWCNTs were significantly higher compared with the responses induced by the shorter ones. The biomarker of ER stress, ddit3 expression, but not xbp-1s expression or BiP protein level, was significantly induced by the exposure of longer MWCNTs.ConclusionsCombined, these results indicated length dependent toxic effects of MWCNTs to HUVECs in vitro, which might be associated with oxidative stress and activation of ER stress.

Chemical Structures of Polyphenols That Critically Influence the Toxicity of ZnO Nanoparticles

Recent studies suggested that phytochemicals as natural antioxidants in food could alleviate nanoparticle (NP) toxicity. This study investigated the combined toxicity of ZnO NPs and a panel of polyphenols. Surprisingly, polyphenols with both high and almost no radical scavenging activities could elicit cytoprotective effects against NP exposure in Caco-2 cells, which were primarily influenced by the positions of the hydroxyl group. Polyphenols with different chemical structures variously influenced the hydrodynamic size, zeta potential, and solubility of ZnO NPs as well as NP-induced intracellular superoxide and Zn ions, which could all contribute to the combined effects. Responses of human endothelial cells appeared to be different from the responses of Caco-2 cells, which may indicate cell-type dependent responses to combined exposure of NPs and phytochemicals. In conclusion, the data from this study suggested a pivotal role of chemical structures of phytochemicals in determining their capacity to affect ZnO NP toxicity.

Toxicity of ZnO nanoparticles (NPs) to A549 cells and A549 epithelium in vitro: Interactions with dipalmitoyl phosphatidylcholine (DPPC)

Once inhaled, nanoparticles (NPs) will first interact with lung surfactant system, which may influence the colloidal aspects of NPs and consequently the toxic potential of NPs to pulmonary cells. In this study, we investigated the effects of dipalmitoyl phosphatidylcholine (DPPC), the major component in lung surfactant, on stability and toxicity of ZnO NPs. The presence of DPPC increased the UV-vis spectra, hydrodynamic size, Zeta potential and dissolution rate of ZnO NPs, which indicates that DPPC might interact with NPs and affect the colloidal stability of NPs. Exposure to ZnO NPs induced cytotoxicity associated with increased intracellular Zn ions but not superoxide in A549 cells. In A549 epithelium model, exposure to ZnO NPs induced cytotoxicity and decreased the release of interleukin 6 (IL-6) without a significant effect on epithelial permeability rate. Co-exposure of A549 cells or A549 epithelium model to DPPC and ZnO NPs induced a higher release of lactate dehydrogenase (LDH) and interleukin-6 (IL-6) compared with the exposure of ZnO NPs alone. We concluded that the presence of DPPC could influence the colloidal stability of ZnO NPs and increase the damage of NPs to membrane probably due to the increased positive surface charge.

3-Hydroxyflavone enhances the toxicity of ZnO nanoparticles in vitro: 3-Hydroxyflavone enhances ZnO NP toxicity

It is recently shown that flavonoids might reduce the toxicity of nanoparticles (NPs) due to their antioxidative properties. In this study, the influence of 3‐hydroxyflavone (H3) on the toxicity of ZnO NPs was investigated. H3 increased hydrodynamic size, polydispersity index and absolute value of the zeta potential of ZnO NPs, which indicated that H3 could influence the colloidal aspects of NPs. Surprisingly, H3 markedly decreased the initial concentration of ZnO NPs required to induce cytotoxicity to Caco‐2, HepG2, THP‐1 and human umbilical vein endothelial cells, which suggested that H3 could promote the toxicity of ZnO NPs to both cancerous and normal cells. For comparison, 6‐hydroxyflavone did not show this effect. H3 remarkably increased cellular Zn elements and intracellular Zn ions in HepG2 cells following ZnO NP exposure, and co‐exposure to H3 and NPs induced a relatively higher intracellular reactive oxygen species. Exposure to ZnO NPs at 3 hours induced the expression of endoplasmic reticulum stress markers DDIT3 and XBP‐1 s, which was suppressed by H3. The expression of apoptotic genes BAX and CASP3 was significantly induced by ZnO NP exposure after 3 and 5 hours, respectively, and H3 further significantly promoted CASP3 expression at 5 hours. In combination, the results from this study suggested that H3 affected colloidal stability of ZnO NPs, promoted the interactions between NPs and cells, and altered the NP‐induced endoplasmic reticulum stress–apoptosis signaling pathway, which finally enhanced the cytotoxicity of ZnO NPs.

Influence of pristine and hydrophobic ZnO nanoparticles on cytotoxicity and endoplasmic reticulum (ER) stress-autophagy-apoptosis gene expression in A549-macrophage co-culture

Exposure to ZnO nanoparticles (NPs) might modulate endoplasmic reticulum (ER) stress-autophagy gene expression, but the possible influence of hydrophobic surface coating on these responses was less studied. This study used A549-macrophage co-culture as the in vitro model for lung barrier and investigated the toxicity of pristine and hydrophobic ZnO NPs. Pristine and hydrophobic NPs exhibited different Zeta potential and solubility in water, which suggested that hydrophobic surface coating might alter the colloidal aspects of ZnO NPs. However, pristine and hydrophobic ZnO NPs induced cytotoxicity and reduced the release of soluble monocyte chemotactic protein-1 (sMCP-1) in A549-macrophage co-culture to a similar extent. Exposure to pristine ZnO NPs significantly promoted the expression of ER stress-apoptosis genes, namely DDIT3, XBP-1s, CASP9, CASP12 and BAX (p < 0.05), but hydrophobic ZnO NPs only significantly promoted the expression of BAX (p < 0.05). Exposure to pristine ZnO NPs also significantly reduced the expression of autophagic gene BECN1 (p < 0.05) but not ATG7 (p > 0.05), whereas hydrophobic ZnO NPs significantly reduced the expression of ATG7 and BECN1 (p < 0.01). Moreover, the expression of XBP-1s, HSPA5, CASP9, CASP12, BAX and ATG7 in pristine ZnO NP-exposed co-culture was significantly lower than that in hydrophobic ZnO NP-exposed co-culture (p < 0.05). In conclusion, hydrophobic surface coating might influence the colloidal aspects of ZnO NPs and alter ER stress-apoptosis-autophagy gene expression pattern by pristine ZnO NPs in A549-macrophage co-culture.