Nanoparticle-cell interactions induced apoptosis: a case study with nanoconjugated epidermal growth factor.

In addition to the intrinsic toxicity associated with the chemical composition of nanoparticles (NP) and their ligands, biofunctionalized NP can perturb specific cellular processes through NP-cell interactions and induce programmed cell death (apoptosis). In the case of the epidermal growth factor (EGF), nanoconjugation has been shown to enhance the apoptotic efficacy of the ligand, but the critical aspects of the underlying mechanism and its dependence on the NP morphology remain unclear. In this manuscript we characterize the apoptotic efficacy of nanoconjugated EGF as a function of NP size (with sphere diameters in the range 20-80 nm), aspect ratio (A.R., in the range of 4.5 to 8.6), and EGF surface loading in EGFR overexpressing MDA-MB-468 cells. We demonstrate a significant size and morphology dependence in this relatively narrow parameter space with spherical NP with a diameter of approx. 80 nm being much more efficient in inducing apoptosis than smaller spherical NP or rod-shaped NP with comparable EGF loading. The nanoconjugated EGF is found to trigger an EGFR-dependent increase in cytoplasmic reactive oxygen species (ROS) levels but no indications of increased mitochondrial ROS levels or mitochondrial membrane damage are detected at early time points of the apoptosis induction. The increase in cytoplasmic ROS is accompanied by a perturbation of the intracellular glutathione homeostasis, which represents an important check-point for NP-EGF mediated apoptosis. Abrogation of the oxidative stress through the inhibition of EGFR signaling by the EGFR inhibitor AG1478 or addition of antioxidants N-acetyl cysteine (NAC) or tempol, but not trolox, successfully suppressed the apoptotic effect of nanoconjugated EGF. A model to account for the observed morphology dependence of EGF nanoconjugation enhanced apoptosis and the underlying NP-cell interactions is discussed.

Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells.

Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of human immunodeficiency virus-1 (HIV-1) viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

Nanoconjugation: A Materials Approach to Enhance Epidermal Growth Factor Induced Apoptosis.

Apoptosis evasion is a hallmark of cancer that motivates the development of novel strategies for inducing cell death in a controlled fashion. The size-compatibility of nanoparticles (NPs) with cellular components provides new opportunities for regulating cellular processes, potentially including apoptosis. We investigated the impact of the covalent attachment of epidermal growth factor (EGF) to 40 nm diameter Au NPs on cellular apoptosis levels, quantified as caspase-3 activity, in two in vitro cancer cell lines: A431 and HeLa. Our studies show that nanoconjugation enhances EGF-induced apoptosis in EGF receptor (EGFR) overexpressing A431 and triggers a quantifiable increase in apoptosis in HeLa. The latter has physiological receptor expression levels and does not show apoptosis in response to free EGF. Endocytosis and trafficking are involved in key EGFR regulation processes, most prominently signal termination. Our experimental findings indicate that these processes can be manipulated through nanoconjugation to induce apoptosis.

Probing subdiffraction limit separations with plasmon coupling microscopy: concepts and applications.

Due to their advantageous material properties, noble metal nanoparticles are versatile tools in biosensing and imaging. A characteristic feature of gold and silver nanoparticles is their ability to sustain localized surface plasmons that provide both large optical cross-sections and extraordinary photophysical stability. Plasmon coupling microscopy takes advantage of the beneficial optical properties and utilizes electromagnetic near-field coupling between individual noble metal nanoparticle labels to resolve subdiffraction limit separations in an all-optical fashion. This Tutorial provides an introduction into the physical concepts underlying distance dependent plasmon coupling, discusses potential experimental implementation of plasmon coupling microscopy, and reviews applications in the area of biosensing and imaging.

Scavenger receptor mediated endocytosis of silver nanoparticles into J774A.1 macrophages is heterogeneous.

We investigated the scavenger receptor mediated uptake and subsequent intracellular spatial distribution and clustering of 57.7 ± 6.9 nm diameter silver nanoparticles (zeta-potential = -28.4 mV) in the murine macrophage cell line J774A.1 through colorimetric imaging. The NPs exhibited an overall red-shift of the plasmon resonance wavelength in the cell ensemble as function of time and concentration, indicative of intracellular NP agglomeration. A detailed analysis of the NP clustering in individual cells revealed a strong phenotypic variability in the intracellular NP organization on the single cell level. Throughout the observation time of 24h cells containing non- or low-agglomerated NPs with a characteristic blue color coexisted with cells containing NPs with varying degrees of agglomeration, as evinced by distinct spectral shifts of their resonance wavelengths. Pharmacological inhibition studies indicated that the observed differences in intracellular NP organization resulted from coexisting actin- and clathrin-dependent endocytosis mechanisms in the macrophage population. Correlation of intracellular NP clustering with macrophage maturity marker (F4/80, CD14) expression revealed that differentiated J774A.1 cells preferentially contained compact NP agglomerates, whereas monocyte-like macrophages contained non-agglomerated NPs.

Diazoxide attenuates ischemia/reperfusion injury via upregulation of heme oxygenase-1 after liver transplantation in rats.

AIM: To evaluate the effects of diazoxide on ischemia/reperfusion (I/R)-injured hepatocytes and further elucidate its underlying mechanisms. METHODS: Male Sprague-Dawley rats were randomized (8 for donor and recipient per group) into five groups: I/R group (4 h of liver cold ischemia followed by 6 h of reperfusion); heme oxygenase-1 (HO-1) small interfering RNA (siRNA) group (injection of siRNA via donor portal vein 48 h prior to harvest); diazoxide (DZ) group (injection of DZ via donor portal vein 10 min prior to harvest); HO-1 siRNA + DZ group; and siRNA control group. Blood and liver samples were collected at 6 h after reperfusion. The mRNA expressions and protein levels of HO-1 were determined by reverse transcription polymerase chain reaction and Western blotting, and tissue morphology was examined by light and transmission electron microscopy. Serum transaminases level and cytokines concentration were also measured. RESULTS: We observed that a significant reduction of HO-1 mRNA and protein levels in HO-1 siRNA and HO-1 siRNA + DZ group when compared with I/R group, while the increases were prominent in the DZ group. Light and transmission electron microscopy indicated severe disruption of tissue with lobular distortion and mitochondrial cristae damage in the HO-1 siRNA and HO-1 siRNA + DZ groups compared with DZ group. Serum alanine aminotransferase, aspartate transaminase, tumor necrosis factor-α and interleukin-6 levels increased in the HO-1 siRNA and HO-1 siRNA + DZ groups, and decreased in the DZ group. CONCLUSION: The protective effect of DZ may be induced by upregulation of HO-1. By inhibiting expression of HO-1, this protection pretreated with DZ was abolished.