Daniela Segat

Procoagulant properties of bare and highly PEGylated vinyl-modified silica nanoparticles.

AIMS Undesired alterations of the blood clotting balance may follow the intravascular injection of nanotherapeutics/diagnostics. Here, we tested the procoagulant activity of synthetic amorphous silica (SAS) and organically modified silica (ORMOSIL) nanoparticles (NPs) and whether a high-density polyethylene glycol coating minimizes these effects. MATERIALS & METHODS Hageman factor- and tissue factor-dependent activation of human blood/plasma coagulation, and binding to human monocytes, endothelial cells and platelets were quantified in vitro using naked and PEGylated ORMOSIL-NPs. Their effects were compared with those of SAS-NPs, present in many industrial products, and of poly(lactic-co-glycolic acid)- and small unilamellar vesicles-NPs, already approved for use in humans. RESULTS Both SAS-NPs and ORMOSIL-NPS presented a significant procoagulant activity. However, highly PEGylated ORMOSIL-NPs were particularly averse to the interaction with the soluble factors and cellular elements that may lead to intravascular blood coagulation. CONCLUSION Stealth, highly PEGylated ORMOSIL-NPs with a poor procoagulant activity can be used as starting blocks to design hemocompatible nanomedical-devices.

Highly PEGylated silica nanoparticles: “ready to use” stealth functional nanocarriers

The peculiar properties of silica nanoparticles make them well suited to the development of smart nanomaterials for medicine. Here we report a new procedure to prepare doped and PEGylated silica-based nanoparticles. Thus, a complex multifunctional system is obtained simply by a one-pot reaction followed by a straightforward purification procedure. Control of the nanoparticles final size is obtained by carefully choosing reactants and conditions. Moreover, reactive functional groups necessary to allow subsequent bioconjugation are easily introduced in the PEG coating by the same procedure. Unprecedented high-density surface coating is obtained and this successfully stabilizes the nanoparticles against aggregation both in saline solution and in the presence of serum proteins. The absence of cytotoxicity and “stealth” behaviour toward phagocytic capture by human macrophages has been demonstrated and this enables such nanosystems as candidates for the development of drug delivery agents.

Proinflammatory effects of bare and PEGylated ORMOSIL-, PLGA- and SUV-NPs on monocytes and PMNs and their modulation by f-MLP

AIMS We wanted to test the proinflammatory effects of vinyltriethoxysilane-based organically modified silica nanoparticles (ORMOSIL-NPs) in vitro on blood leukocytes. MATERIALS & METHODS Cell selectivity, cytokines/chemokines and O(2) (-) production were analyzed using nonpolyethylene glycol (PEG)ylated and PEGylated ORMOSIL-NPs, poly(lactic-co-glycolic acid) (PLGA)-NPs and small unilamellar vesicles (SUV)-NPs. RESULTS ORMOSIL-NPs mostly bound to monocytes while other NPs to all leukocyte types similarly. Cell capture of PEGylated-NPs decreased strongly (ORMOSIL), moderately (PLGA) and weakly (SUV). Bare ORMOSIL-NPs effectively stimulated the production of IL-1β/IL-6/TNF-α/IL-8 by monocytes and of IL-8 by polymorphonuclear leukocytes (PMNs). NP PEGylation inhibited such effects only partially. Formyl-methionine-leucine phenylalanine (f-MLP) further increased the release of cytokines/chemokines by monocytes/PMNs primed with bare and PEGylated ORMOSIL-NPs. PEGylated SUV-NPs, bare and PEGylated ORMOSIL- and PLGA-NPs sensitize PMNs and monocytes to secrete O(2) (-) upon f-MLP stimulation. CONCLUSION ORMOSIL-NPs are preferentially captured by circulating monocytes but stimulate both monocytes and PMNs per se or by sensitizing them to another agonist (f-MLP). PEG-coating confers stealth effects but does not completely eliminate leukocyte activation. Safe nanomedical applications require the evaluation of both intrinsic and cooperative proinflammatory potential of NPs.

Helicobacter pylori‐derived neutrophil‐activating protein increases the lifespan of monocytes and neutrophils

An invariable feature of Helicobacter pylori‐infected gastric mucosa is the persistent infiltration of inflammatory cells. The neutrophil‐activating protein (HP‐NAP) has a pivotal role in triggering and orchestrating the phlogistic process associated with H. pylori infection. Aim of this study was to address whether HP‐NAP might further contribute to the inflammation by increasing the lifespan of inflammatory cells. We report that HP‐NAP is able to prolong the lifespan of monocytes, in parallel with the induction of the anti‐apoptotic proteins A1, Mcl‐1, Bcl‐2 and Bcl‐XL. This effect does not result from a direct action on the apoptotic machinery, but rather it requires the release of endogenous pro‐survival factors, such as interleukin‐1β, which probably acts in synergy with other unidentified mediators. We also report that HP‐NAP promotes the survival of Ficoll‐purified neutrophils in a monocyte‐dependent fashion: indeed, mononuclear cell depletion of Ficoll‐purified neutrophils completely abolished the pro‐survival effect by HP‐NAP. In conclusion, our data reinforce the notion that HP‐NAP has a pivotal role in sustaining a prolonged activation of myeloid cells.

Targeted delivery of photosensitizers: efficacy and selectivity issues revealed by multifunctional ORMOSIL nanovectors in cellular systems

PEGylated and non-PEGylated ORMOSIL nanoparticles prepared by microemulsion condensation of vinyltriethoxy-silane (VTES) were investigated in detail for their micro-structure and ability to deliver photoactive agents. With respect to pure silica nanoparticles, organic modification substantially changes the microstructure and the surface properties. This in turn leads to a modulation of both the photophysical properties of embedded photosensitizers and the interaction of the nanoparticles with biological entities such as serum proteins. The flexibility of the synthetic procedure allows the rapid preparation and screening of multifunctional nanosystems for photodynamic therapy (PDT). Selective targeting of model cancer cells was tested by using folate, an integrin specific RGD peptide and anti-EGFR antibodies. Data suggest the interference of the stealth-conferring layer (PEG) with small targeting agents, but not with bulky antibodies. Moreover, we showed that selective photokilling of tumour cells may be limited even in the case of efficient targeting because of intrinsic transport limitations of active cellular uptake mechanisms or suboptimum localization.

Selection of multipotent cells and enhanced muscle reconstruction by myogenic macrophage-secreted factors.

Skeletal muscle regeneration relies on satellite cells, a population of myogenic precursors. Inflammation also plays a determinant role in the process, as upon injury, macrophages are attracted by the damaged myofibers and the activated satellite cells and act as key elements of dynamic muscle supportive stroma. Yet, it is not known how macrophages interact with the more profound stem cells of the satellite cell niche. Here we show that in the presence of a murine macrophage conditioned medium (mMCM) a subpopulation of multipotent cells could be selected and expanded from adult rat muscle. These cells were small, round, poorly adhesive, slow-growing and showed mesenchymal differentiation plasticity. At the same time, mMCM showed clear myogenic capabilities, as experiments with satellite cells mechanically isolated from suspensions of single myofibers showed that the macrophagic factors inhibited their tendency to shift towards adipogenesis. In vivo, intramuscular administrations of concentrated mMCM in a rat model of extensive surgical ablation dramatically improved muscle regeneration. Altogether, these findings suggest that macrophagic factors could be of great help in developing therapeutic protocols with myogenic stem cells.

Mapping of the Neisseria meningitidis NadA Cell-Binding Site: Relevance of Predicted α-Helices in the NH2-Terminal and Dimeric Coiled-Coil Regions

NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH(2)-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH(2)-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH(2) globular head domain and the NH(2) dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.

The honeybee antimicrobial peptide apidaecin differentially immunomodulates human macrophages, monocytes and dendritic cells.

We show that apidaecin binds to human macrophages, monocytes and dendritic cells, displaying different intracellular distributions and inducing diversified effects. An apidaecin-cell association was detectable at concentrations as low as 5 µM and increased without saturation until 60 µM, was receptor independent and required a physiological temperature (37°C). For apidaecin, cytosolic localization was prevalent in macrophages and endosomal localization in monocytes, and associations with the plasma membrane were predominant in dendritic cells. Apidaecin upregulated T-lymphocyte co-stimulatory molecule CD80 and cytokine/chemokine production in macrophages, but not in monocytes and dendritic cells. Suboptimal stimulatory doses (5–10 µM) of apidaecin partially inhibited the lipopolysaccharide (LPS)-induced increase in major histocompatibility complex class II (MHCII) and CD86 in macrophages, and the release of selected cytokines/chemokines by both macrophages [interleukin (IL)-6 and tumor necrosis factor (TNF)-α] and monocytes [IL-6, TNF-α, basic fibroblast growth factor (FGF) and eotaxin]. Apidaecin had a double-edged effect: at low concentrations it partially antagonized LPS-stimulatory effects on both macrophages and monocytes while it stimulated pro-inflammatory and pro-immune functions of macrophages at higher concentrations.

CDX2 hox gene product in a rat model of esophageal cancer

BackgroundBarretts mucosa is the precursor of esophageal adenocarcinoma. The molecular mechanisms behind Barretts carcinogenesis are largely unknown. Experimental models of longstanding esophageal reflux of duodenal-gastric contents may provide important information on the biological sequence of the Barretts oncogenesis.MethodsThe expression of CDX2 hox-gene product was assessed in a rat model of Barretts carcinogenesis. Seventy-four rats underwent esophago-jejunostomy with gastric preservation. Excluding perisurgical deaths, the animals were sacrificed at various times after the surgical treatment (Group A: <10 weeks; Group B: 10–30 weeks; Group C: >30 weeks).ResultsNo Cdx2 expression was detected in either squamous epithelia of the proximal esophagus or squamous cell carcinomas. De novo Cdx2 expression was consistently documented in the proliferative zone of the squamous epithelium close to reflux ulcers (Group A: 68%; Group B: 64%; Group C: 80%), multilayered epithelium and intestinal metaplasia (Group A: 9%; Group B: 41%; Group C: 60%), and esophageal adenocarcinomas (Group B: 36%; Group C: 35%). A trend for increasing overall Cdx2 expression was documented during the course of the experiment (p = 0.001).ConclusionDe novo expression of Cdx2 is an early event in the spectrum of the lesions induced by experimental gastro-esophageal reflux and should be considered as a key step in the morphogenesis of esophageal adenocarcinoma.

Catastrophic inflammatory death of monocytes and macrophages by overtaking of a critical dose of endocytosed synthetic amorphous silica nanoparticles/serum protein complexes

AIMS We tested whether phagocytic monocytes/macrophages are more susceptible than nonphagocytes to nanoparticle (NP) toxicity. MATERIALS & METHODS We compared in vitro cell death and proinflammatory cytokine production in human monocytes, macrophages, lymphocytes and HeLa cells due to synthetic amorphous silica (SiO2)-NPs in different serum concentrations and correlated them with cellular uptake and distribution. RESULTS Phagocytes were approximately ten-times more sensitive than nonphagocytes to SiO2-NPs and more effectively endocytosed SiO2-NP-serum protein nanoagglomerates, so determining their accumulation in acidic endocytic compartments well beyond a critical/cytotoxic threshold. Monocyte/macrophage death was paralleled by cytokine secretion. CONCLUSION The physiological specialization of monocytes/macrophages to effectively capture NPs may expose them to the risk of catastrophic inflammatory death upon saturation of their maximal storage capacity.