Evaluation of the Antibacterial Activity and Biocompatibility for Silver Nanoparticles Immobilized on Nano Silicate Platelets

Silver nanoparticles (AgNPs) are known for their bactericidal abilities. The antibacterial potency is dependent on the particle size and dispersion status. In this study, we synthesized AgNP/NSP nanohybrids in two different weight ratios (1/99 and 8/92) using the fully exfoliated clay, i.e., nanosilicate platelets (NSP), as a dispersing agent and carrier for AgNPs. Due to the size of NSP, the immobilized AgNPs do not enter cells readily, which may lower the risk associated with the cellular uptake of AgNPs. The biocompatibility, immunological response, and antimicrobial activities of AgNP/NSP hybrids were evaluated. The results revealed that AgNP/NSP hybrids elicited merely mild inflammatory response and retained the outstanding antibacterial activity. The hybrids were further embedded in poly­(ether)­urethane (PEU) to increase the biocompatibility. At the same silver content (20 ppm), the PEU-AgNP/NSP nanocomposites were nontoxic to mouse skin fibroblasts, while simultaneously exhibiting nearly complete bacterial growth reduction (99.9%). PEU containing the same amount of free AgNPs did not display such an effect. Our results verify the better biosafety of the AgNPs/NSP hybrids and their polymer nanocomposites for further clinical use

GroEL1, from <em>Chlamydia pneumoniae</em>, Induces Vascular Adhesion Molecule 1 Expression by p37^AUF1 in Endothelial Cells and Hypercholesterolemic Rabbit

<div><p>The expression of vascular adhesion molecule-1 (VCAM-1) by endothelial cells may play a major role in atherogenesis. The actual mechanisms <em>of chlamydia pneumoniae</em> (<em>C. pneumoniae</em>) relate to atherogenesis are unclear. We investigate the influence of VCAM-1 expression in the GroEL1 from <em>C. pneumoniae</em>-administered human coronary artery endothelial cells (HCAECs) and hypercholesterolemic rabbits. In this study, we constructed the recombinant GroEL1 from <em>C. pneumoniae</em>. The HCAECs/THP-1 adhesion assay, tube formation assay, western blotting, enzyme-linked immunosorbent assay, actinomycin D chase experiment, luciferase reporter assay, and immunohistochemical stainings were performed. The results show that GroEL1 increased both VCAM-1expression and THP-1 cell adhesives, and impaired tube-formation capacity in the HCAECs. GroEL1 significantly increased the VCAM-1 mRNA stability and cytosolic AU-binding factor 1 (AUF1) level. Overexpression of the p37^AUF1 significantly increased VCAM-1 gene expression in GroEL1-induced bovine aortic endothelial cells (BAECs). GroEL1 prolonged the stability of VCAM-1 mRNA by increasing both p37^AUF1 and the regulation of the 5′ untranslated region (UTR) of the VCAM-1 mRNA in BAECs. In hypercholesterolemic rabbits, GroEL1 administration enhanced fatty-streak and macrophage infiltration in atherosclerotic lesions, which may be mediated by elevated VCAM-1 expression. In conclusion, GroEL1 induces VCAM-1 expression by p37^AUF1 in endothelial cells and enhances atherogenesis in hypercholesterolemic rabbits.</p> </div

GroEL1 induces VCAM-1 and AUF1 expression in rabbits.

<p>(A) Immunohistochemistry to assess the VCAM-1 and ICAM-1 expression in the rabbit abdominal aorta. Corresponding hematoxylin staining was used for nucleus identification. The intima was markedly thickened in the GroEL1+HC diet treatment groups compared with the control, GroEL1 treatment, and HC diet treatment groups. The graphs show 100× magnification of the slide. Quantifications of immunohistochemical positively stained cells were shown in the lower panel. (B) Immunohistochemistry to assess AUF1 expression in the rabbit abdominal aorta. Compared with the control and HC diet treatment groups, AUF1 staining was observed on the luminal surface in the 4 µg/kg BW GroEL treatment group. Positive AUF1 staining in the thickened intima was observed in the 2 µg/kg BW GroEL1+HC diet and 4 µg/kg BW GroEL1+HC diet treatment groups. The graphs show a 100× magnification of the slides. The lumen is uppermost in all sections, and the internal elastic laminae is indicated by the arrows.</p

The 5′ UTR flanking sequence of VCAM-1 mRNA conferred P37^AUF1-responsiveness in the BAECs.

<p>(A) The BAECs were transfected with the 4His-A-AUF1-p37 plasmid (p37^AUF1), 4His-A-AUF1-p40 plasmid (p40^AUF1), 4His-A-AUF1-p42 plasmid (p42^AUF1), or 4His-A-AUF1-p45 plasmid (p45^AUF1). The level of VCAM-1 mRNA were analyzed using real-time PCR after transfectiuon for 24 hours. (B) The VCAM-1 mRNA stability was analyzed using an actinomycin D chase experiment in the AUF1-transfected BAECs. (C) Schematic representation of the various plasmids containing the luciferase and UTR of the VCAM-1 mRNA. Control plasmid: pcDNA™ 3.1 plasmid; construct A, CMV-Luciferase plasmid; construct B, CMV-Luciferase-VCAM1 5′UTR (sense) plasmid; construct C, CMV-Luciferase-VCAM1 5′UTR (antisense) plasmid; construct D, CMV-Luciferase-VCAM1 3′UTR (sense) plasmid; construct E, CMV-Luciferase-VCAM1 3′UTR (antisense) plasmid. (D), BAECs were co-transfected with the CMV-Luciferase-VCAM1 UTR plasmid, the β-galactosidase reporter plasmid, and the 4His-A-AUF1 plasmid. Uniform transfection efficiencies were confirmed using a β-galactosidase reporter plasmid. The luciferase activity was quantified by luminometry. Data are expressed as relative luciferase units, presented as the mean ± SEM and represent the results of three independent experiments (*<i>P</i><0.05 was considered significant and n = 3).</p

GroEL1 induces VCAM-1 expression and increases VCAM-1 mRNA stability on the HCAECs.

<p>Cells were treated for 8 or 24 h with 25–100 ng/mL of GroEL1. (A and B) Expression of VCAM-1 and ICAM-1 were detected by cell ELISA. All data represent the results of three independent experiments (mean ±SD; *<i>P</i><0.05 was considered significant and n = 3). (C) Cells were stimulated for 24 h with 25–100 ng/mL of GroEL1. Western blot analyses of VCAM-1 and ICAM-1 proteins were performed. The total β-actin protein was used as loading control. The bar graph shows the quantified results using densitometry. (D and E) Actinomycin D chase experiment was performed to evaluate the stability of the VCAM-1 mRNA and ICAM-1 mRNA. Cells were treated with 25 ng/mL (○) or 100 ng/mL (•) of GroEL1 before actinomycin D treatment for 40 minutes. (▾) The stability of VCAM-1 mRNA was demonstrated in naïve cells. Total RNA was extracted at various time points and quantitative real-time PCR was performed. All data represent the results of three independent experiments (mean ±SD; *<i>P</i><0.05 was considered significant compared to control and n = 3).</p

GroEL1 treatment induces AUF1 activation.

<p>(A) HCAECs were treated with 100 ng/mL of GroEL1 for 60 minutes. The total mRNA was extracted from cells. Traditional RT-PCR was performed for the four subunits of AUF1 expression. The β-actin mRNA was used as a internal control. (B) HCAECs were treated with 100 ng/mL of GroEL1 for 60 minutes. The total protein was extracted from cells. Western blot analysis was performed for the four subunits of AUF1 expression. The β-actin protein was used as a loading control. The endogenous total mRNA and protein extracted from the Hela or THP-1 cells was used to confirm the efficiency of the primer and antibodies. (C) HCAECs were treated with 100 ng/mL of GroEL1 for 15–120 minutes. The cytosolic level of AUF1 was analyzed using western blotting. The β-actin and hnRNP C1/C2 protein was used as a loading control. The density of band was quantified using densitometry and showed as bar graph. Data are expressed as % of control, presented as the mean ± SEM and represent the results of three independent experiments (n = 3, *<i>P</i><0.05 was considered significant compared to control at the same group). (D) The intracellular AUF1 was identified using immunocytofluorescence and observed with a fluorescent microscope. DAPI was used to stain the nuclei of the HCAECs.</p

GroEL1 induces the binding of HCAECs/THP-1 cells and impairs the tube formation capacity of the HCAECs.

<p>(A) HCAECs were pretreated with 25–100 ng/mL of GroEL1 for 24 h and then co-cultured with THP-1 cells for 1 h. (B) Cells were pretreated with anti-hVCAM-1 or anti-hICAM-1 antibodies for 30 min, followed by GroEL1 treatment for 24 h. HCAECs and THP-1 cells were co-cultured for 1 h. The degree of THP-1 adhesion to the HCAECs was counted using a Multilabel Counter Victor². The isotype IgG was as a negative control. (C) HCAECs were pretreated with 1–100 ng/mL of GroEL1 for 48 h. An <i>in vitro</i> tube formation assay was performed using ECMatrix gel to investigate the effect of GroEL1 on the HCAECs’ lining function. Representative photos for <i>in vitro</i> tube formation are shown. (D) The bar graph demonstrates the tube formation capacity of the GroEL1-treated HCAECs. All data represent the results of three independent experiments (mean ±SD; *<i>P</i><0.05 was considered significant and n = 3).</p

GroEL1 decreases EPC number and function.

<p>(A) The collected-human MNCs in growth medium were incubated with different concentrations of GroEL1 (1-100 ng/mL) for 4 days. Early EPCs are indicated with arrows. The numbers of EPCs were counted under microscope in a high-power field (HPF). (B) ECMatrix gel was used to assay the <i>in </i><i>vitro</i> angiogenesis capacity of late EPCs. Representative photos of <i>in </i><i>vitro</i> angiogenesis are shown. The mean total area of complete tubes formed by each GroEL1-treated group was calculated and compared with the non-GroEL1-treated group. (C) A modified Boyden chamber assay was used with VEGF as chemoattractive factor to evaluate late EPC migratory function. Representative photos are shown; the migrated cells were stained with hematoxylin and counted under microscope. (D) Late EPCs were treated with GroEL1 (1-100 ng/mL) for 24 hours. MTT assay was also performed to evaluate late EPC proliferation activity. (E and F) Late EPCs were preincubated for 24 hours with or without GroEL1, then labeled with BCECF/AM and attached to fibronectin/collagen-coated plates or HCAEC-cultured dishes for 1 hour. The attached EPCs were lysed using DMSO, and the fluorescence was quantified by fluorimetry. All data are expressed as the mean ± SEM (*<i>p</i> < 0.05 compared with untreated group). </p

GroEL1 decreases integrin and E-selectin expression and induces adhesion molecule production in EPCs.

<p>(A) Late EPCs were treated with GroEL1 (100 ng/mL) for 4-24 hours. Quantitative real-time PCR was also performed for integrin α1, -α2, -β1, -β3, and E-selectin. (B) Late EPCs were treated for 4 hours with 25-100 ng/mL GroEL1. eNOS activation was analyzed by western blotting. The total eNOS and β-actin levels were used as loading controls. (C) EPCs were pretreated with 10 μM SNAP or SNOC for 1 hour followed by 100 ng/mL GroEL1 treatment. Quantitative real-time PCR was performed for integrin α1, -β1, -β3, and E-selectin. All data represent the results of three independent experiments and are expressed as the mean±SEM (*<i>p</i> < 0.05 compared with untreated group, †<i>p</i> < 0.05 compared with GroEL1-only-treated group).</p

Effect of GroEL1 on blood flow recovery after hind limb ischemia in C57BL/B6 and C57BL/10ScNJ

<p><b>mice</b>. (A) Upper, representative results of laser Doppler measurements before operation (control) and 1 day after hind limb ischemia surgery in C57BL/B6 mice. Lower, representative results of laser Doppler measurements 8 weeks after hind limb ischemia surgery in C57BL/B6 and C57BL/10ScNJ mice treated with 0-4 μg/kg BW of GroEL1. Color scale illustrates blood flow variations from minimal (dark blue) to maximal (red). Arrows indicate the ischemic (right) limb after hind limb ischemia surgery. (B) Doppler perfusion ratios (ischemic/non-ischemic hind limb) over time in the different groups. In C57BL/B6 mice, administration of 4 μg/kg BW (△) of GroEL1 or 2 μg/kg BW (▼) of GroEL1 impaired beneficial blood flow recovery compared with the non-administered group (●) 6 or 8 weeks after hind limb ischemia surgery. There was no significant difference in blood flow in the limb in the 1 μg/kg BW GroEL1-treated C57BL/B6 mice (○) or the 4 μg/kg BW GroEL1-treated C57BL/10ScNJ mice (█) compared with the non-administered C57BL/B6 mice (●). The results are expressed as the mean ± SEM (*<i>p</i> < 0.05 compared with non-GroEL1-treated C57BL/B6 mice at the same time point after ischemic surgery). (C) Eight weeks after ischemic surgery, the ischemia/normal perfusion ratio in the GroEL1-treated C57BL/B6 mice, but not GroEL1-treated C57BL/10ScNJ mice, was lower than that in the non-GroEL1-treated C57BL/B6 mice. The results are expressed as the mean ± SEM (n=6, *<i>p</i> < 0.05 compared with non-GroEL1-treated C57BL/B6 mice; †<i>p</i> < 0.05 compared with 4 μg/kg BW of GroEL1-treated C57BL/B6 mice). (D) Representative results of immunohistochemistry before operation (naïve) in C57BL/B6 mice. Mice were sacrificed 8 weeks after surgery, and capillaries (white arrow) in the ischemic muscles were visualized by anti-vWF immunostaining (original magnification x400). Hoechst dye (blue) was used to counterstain the nucleus. The graph shows the quantification of capillary density in hind limb-ischemic and GroEL1-administered C57BL/B6 and C57BL/10ScNJ mice. The results are expressed as the mean ± SEM (n=6, *<i>p</i> < 0.05 compared with hind limb ischemia+ non-GroEL1-treated C57BL/B6 mice; †<i>p</i> < 0.05 compared with hind limb ischemia+ 4 μg/kg BW of GroEL1-treated C57BL/B6 mice).</p