The Health Implication and Application of Silver and Iron Oxide Nanoparticles

Nanoscience and nanotechnology have successfully created different nanomaterials, which are defined as materials with at least one dimension between 1-100 nm. This small size gives nanomaterials unique and novel properties compared with bulk material, such as a high surface area, specific mechanical, optical and magnetic properties. These novel properties have stimulated the interest of using nanoparticles in a wide range of consumer products and applications including biomedical, pharmaceutical and drug delivery. Silver and iron oxide nanoparticles attracted more attention commercially due to their antibacterial and magnetic properties, respectively. So, the overall objective of this dissertation was: 1) to examine the implication of well-characterized polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) on human peripheral blood mononuclear cells (PBMCs) at environmentally relevant concentrations, and 2) to investigate the application of iron oxide nanoparticles in fat removal using high-fat diet-fed mice. AgNPs biological behavior, such as bio-uptake and accumulation, may be affected by their novel properties and they might show different behavior than bulk material and free Ag ions. There is discrepancy in the results as to whether Ag toxicity is caused by nanoparticulate or ions from NP dissolution. In most nanotoxicology studies lack of NP characterization in the relevant exposure media biased the NP dose and toxicity outcome. This study aimed to use in house synthesized and well- characterized PVP-coated AgNPs for investigation of their bio-uptake and toxicity in PBMCs. Synthesis protocol was successful in generating spherical and monodisperse PVP-coated AgNPs. PVP-AgNPs showed more stability in the cell exposure media and no aggregation was observed at any exposed concentrations. AgNPs and silver nitrate (which was used as Ag ion control) indicated the same overall toxicity. Although, after normalizing toxicity to the amount of Ag that was taken up by cells, AgNPs seemed to be more toxic to cells than Ag ions. Our results emphasize the importance of sufficient characterization of nanoparticles in proper exposure media over the exposure time to have a better understanding of nanoparticle behavior and interaction in biological systems for evaluating risk assessment in human exposure. Iron oxide NPs which are frequently used in nanomedicine, have recently been investigated for oil removal purposes [1]. However, in vitro and in vivo studies on iron oxide NPs toxicity have revealed little consistency of result due to the usage of different NP characterizations and concentrations. In this study we used PVP-coated iron oxide NPs (that were used successfully before for oil removal from aqueous solutions) to remove dietary fat from mice fed with a high-fat diet. Our animal data demonstrated that oral exposure of PVP-coated iron oxide nanoparticles at concentrations up to 20 mg/kg mouse body weight did not cause any significant decrease in the body weight and fat percentage of high-fat diet-fed mice. Although, iron uptake by liver cells and some effects on glucose metabolism in HNP treatment was detected compared to controls. No increase in inflammatory markers were observed using high doses of nanoparticle treated mice compared with high-fat diet. Further studies using different concentrations of NPs with modified surface coatings should be performed to validate our findings. Given the increasing demand for obesity treatments, nanomedicine approaches, coupled with our results, may provide substantial contributions toward progress in this area

Upregulation of MIR21 and Repression of GRHL3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4-5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH

Upregulation of miR21 and Repression of Grhl3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

<div><p>Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4–5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH.</p></div

Sinusoidal endothelial dysfunction (SED) in NASH progression is mediated by leptin.

<p><i>A</i>. mRNA expression of sinusoidal endothelial dysfunction biomarkers (VEGFR-2, ICAM-1, E-selectin, VCAM-1, Cadherin 5 (Cdh5), VEGF-α and CD34) as measured by quantitative real-time PCR in DIO+BDCM (1w), ob/ob gene deleted mice (Lep KO (1w)) and db/db gene deleted mice (Lepr KO (1w)). Y-axis shows fold of mRNA expression of SED biomarkers normalized against DIO only groups. <i>B</i>. Immunofluorescence images for localization of SED biomarkers (VEGFR-2, ICAM-1 and E-selectin) from liver sections of both toxin model (DIO, DIO+BDCM (1w), Lep KO (1w), Lepr KO (1w)) and dietary model (MCS (4w) and MCD (4w)) of NASH.</p

miR21 play a key role in leptin signaling of sinusoidal endothelial dysfunction.

<p><i>A</i> and <i>C</i>. Phosphorylation of endothelial nitric oxide synthase (NOS3) is the key event in endothelial function. To access levels of NOS3 phosphorylation in liver homogenate, western blot analysis was carried out for phosphorylated NOS3 (NOS3-p) and native NOS3 protein. The mice groups for toxin model (fig. A) are DIO+BDCM (1w), DIO+BDCM (4w) and 3 individual miR21 KO mice (M1, M2 and M3) and the mice groups for dietary model (fig. C) are MCS (4w), miR21 KO fed with MCS diet (miR21 KO+MCS (4w)), MCD (4w) and miR21 KO mice fed with MCD diet (miR21 KO+MCD (4w)). The corresponding β-actin levels are shown in the lower panel. <i>B</i> and <i>D</i>. Levels of phosphorylated NOS3 (NOS3-p) protein normalized against respective NOS3 levels and β-actin levels (NOS3-p/NOS3 ratio) were plotted. Y-axis represent arbitrary unit of NOS3-p/NOS3 ratio of mice groups from both BDCM (fig. B) and diet model (fig. D) of NASH. P<0.05 is considered statistically significant (*).</p

Increased hepatic leptin is associated with NASH progression in obesity.

<p>qRTPCR analysis of hepatic leptin mRNA expression in two toxin model of NASH. <i>A</i>. Bromodichloromethane (BDCM) model: Y-axis represents fold of leptin mRNA expression in DIO, DIO mice exposed with BDCM for 24h, for 48h, for 1week and for 4 weeks post BDCM exposure. <i>B</i>. Carbon tetrachloride (CCl₄) model: Y-axis represents fold of leptin mRNA expression in DIO and DIO mice exposed with CCl₄ for 1w. n = 3, P<0.05 is considered statistically significant (*). <i>C</i>. Picro-sirius red (PSR) staining of liver sections of DIO, DIO+BDCM at 24h, DIO+BDCM at 48h, DIO+BDCM at 1w and DIO+BDCM at 4w post BDCM exposure; 20× images (n = 3). Black arrowhead depicts macro and micro vesicular fibrosis. <i>D</i>. Stages of fibrosis of stained liver sections from two different model of NASH (toxin and dietary model) were reviewed using the criteria of the NIH Non Alcoholic Steatohepatitis Clinical Research Network (NIH NASH CRN). Table depicts the NASH CRN scores for DIO, DIO+BDCM (toxin model) and MCS, MCD (Dietary model).</p

Increased miR21 expression, subsequent decreased expression of Grhl3 protein and NOS3 phosphorylation is mediated by leptin.

<p><i>A</i>. miR21 expression as measured by quantitative real-time PCR in DIO mice exposed with BDCM (DIO+BDCM (1w)), ob/ob gene deficient mice exposed with BDCM (Lep KO (1w)) and ob/ob gene deficient mice supplemented with leptin exposed with BDCM (Lep KO+Leptin (1w)). P<0.05 is considered statistically significant (*) <i>B</i>. Western blot analysis of Grhl3 in liver homogenates of BDCM exposed DIO mice (DIO+BDCM (1w)), mice that lacked the ob/ob gene and exposed with BDCM (Lep KO (1w)) and Lep KO supplemented with leptin and exposed with BDCM (Lep KO+Leptin (1w)). <i>C</i>. Column graph depict the band quantification analysis of Grhl3 protein with corresponding β-actin as shown in lower levels of fig B. <i>D</i>. Western blot analysis of phosphorylated NOS3 (NOS3-p) and NOS3 in DIO+BDCM (1w), Lep KO (1w) and Lep KO+Leptin (1w) mice group. <i>E</i>. NOS3-p/NOS3 ratio (indication of vascular endothelium function) was plotted after band quantification and normalization against respective β-actin. Y-axis represent arbitrary unit of NOS3-p/NOS3 ratio of DIO+BDCM (1w), Lep KO (1w) and Lep KO+Leptin (1w) mice groups. P<0.05 is considered statistically significant (*). #Band image of DIO+BDCM (1w) has been cropped from the same immunoblot image and placed separately in both fig B and D due to presence of other mouse group in between the DIO+BDCM (1w) and Lep KO (1w) lane which is not explained in this manuscript. The cropped images of the blot are separated by a distinct blank space to show the non-continuity of the image.</p

miR21 mediated sinusoidal endothelial dysfunction is the early event in NASH progression.

<p><i>A</i> and <i>B</i>. Immunofluorescence images for localization of sinusoidal endothelial dysfunction biomarkers (VEGFR-2, ICAM-1 and E-selectin) from liver sections of toxin model groups (fig. A) DIO+ BDCM (1w) and miR21 KO+BDCM (1w) and dietary model (fig. B) MCD (4w) and miR21 KO+MCD (4w). Immunoreactivity with red dots shows the localization of SED biomarkers.</p