Opsonization Of Particles Research Articles

Nanometer- and angstrom-scale characteristics that modulate complement responses to nanoparticles.

The contribution of the complement system to non-specific host defence and maintenance of homeostasis is well appreciated. Many particulate systems trigger complement activation but the underlying mechanisms are still poorly understood. Activation of the complement cascade could lead to particle opsonisation by the cleavage products of the third complement protein and might promote inflammatory reactions. Antibody binding in a controlled manner and/or sensing of particles by the complement pattern-recognition molecules such as C1q and mannose-binding lectin can trigger complement activation. Particle curvature and spacing arrangement/periodicity of surface functional groups/ligands are two important parameters that modulate complement responses through multivalent engagement with and conformational regulation of surface-bound antibodies and complement pattern-recognition molecules. Thus, a better fundamental understanding of nanometer- and angstrom-scale parameters that modulate particle interaction with antibodies and complement proteins could portend new possibilities for engineering of particulate drug carriers and biomedical platforms with tuneable complement responses and is discussed here.

Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice

An estimated 1% or less of nanoparticles (NPs) deposited in the lungs translocate to systemic circulation and enter other organs; however, this estimation may not be accurate given the low sensitivity of existing in vivo NP detection methods. Moreover, the biological effects of such low levels of translocation are unclear. We employed a nano-scale hyperspectral microscope to spatially observe and spectrally profile NPs in tissues and blood following pulmonary deposition in mice. In addition, we characterized effects occurring in blood, liver and heart at the mRNA and protein level following translocation from the lungs. Adult female C57BL/6 mice were exposed via intratracheal instillation to 18 or 162 µg of industrially relevant titanium dioxide nanoparticles (nano-TiO2) alongside vehicle controls. Using the nano-scale hyperspectral microscope, translocation to heart and liver was confirmed at both doses, and to blood at the highest dose, in mice analyzed 24 h post-exposure. Global gene expression profiling and ELISA analysis revealed activation of complement cascade and inflammatory processes in heart and specific activation of complement factor 3 in blood, suggesting activation of an early innate immune response essential for particle opsonisation and clearance. The liver showed a subtle response with changes in the expression of genes associated with acute phase response. This study characterizes the subtle systemic effects that occur in liver and heart tissues following pulmonary exposure and low levels of translocation of nano-TiO2 from lungs.

Human plasma protein adsorption onto alumina nanoparticles relevant to orthopedic wear.

Wear of ceramic orthopedic devices generates nanoparticles in vivo that may present a different biological character from the monolithic ceramic from which they are formed. The current work investigated protein adsorption from human plasma on alumina nanoparticles and monolithic samples representative of both wear particles and the ceramic components as implanted. A physicochemical characterization of the particles and their dispersion state was carried out, and the protein adsorption profiles were analyzed using 1D SDS-PAGE and mass spectrometry. Significant differences in protein-binding profiles were identified where the nanoparticles selectively bound known transporter proteins rather than the more highly abundant serum proteins that were observed on the monoliths. Proteins associated with opsonization of particles were seen to be present in the protein corona of the nanoparticles, which raises questions regarding the role of wear particles in periprosthetic tissue inflammation and aseptic loosening.

Of Mice, Men and Nanoparticle Biocoronas: Are In Vitro to In Vivo Correlations and Interspecies Extrapolations Realistic?

A great deal of research on the potential bio­ logical effects and biodistribution of nano­ materials in humans are inferred from in vitro cell culture studies or in vivo rodent models. A common theme emerging in predicting nanoparticle biological effects is the role of the protein corona. However, to link these data to what occurs in humans requires the use of extrapolation techniques that take into account species­specific physiology, such as the basal metabolic rate that drives most biodis­ tribution and elimination processes. Forma­ tion and stabilization of nanoparticle protein coronas are independent of these physiological rates, making simple extrapolations theoreti­ cally problematic. Similarly, if nanoparticle biocorona complexes determine in vivo bio­ distribution, how does one extrapolate in vitro cell culture data, where coronas may be fun­ damentally different or even absent, to in vivo data, where they are deterministic. Currently, simple extrapolation strategies used in phar­ macology and toxicology studies cannot com­ pensate for these processes. The development of mechanistic physiologically based pharma­ cokinetic models could incorporate these pro­ cesses if appropriate data were available. These issues have not been adequately considered by the nanomedicine or nanotoxicology commu­ nity, and are even ignored in current risk assess­ ment paradigms, yet are crucial for making biologically meaningful extrapolations. Recent research has made the formation of nanoparticle protein coronas an essen­ tial component in describing nanoparticle biodistribution in the body [1–3]. This has long been recognized to occur with particle opsonization and has been a major factor driving pharmaceutical strategies to avoid this process (e.g., PEGylation) [4]. However, the latest studies have described a more complex

Differential uptake of functionalized polystyrene nanoparticles by human macrophages and monocytic cells

Tumor cell lines are frequently used as models to analyze the cellular uptake of nanoparticles. Here we show that carboxy‐ (PS‐COOH) and amino‐functionalized (PS‐NH2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, THP‐1 monocytic leukemia cells, and by PMA‐differentiated THP‐1 cells via different mechanisms. Macrophages and THP‐1 rapidly internalized PS‐COOH and to a lesser extent PS‐NH2 particles when incubated in buffer. The uptake of both nanoparticles was drastically reduced in media containing serum. Using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between the CD64 receptor and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization in THP‐1 cells occurred via dynamin II‐dependent endocytosis. PMA‐differentiated THP‐1 cells took up the particles via macropinocytosis. In line with the in vitro data, more PS‐COOH particles were accumulated in liver tissue, whereas PS‐NH2 particles were preferentially targeted to tumor tissue. These data show that the extent of the particle internalization, the uptake kinetics and its mechanism differ significantly among primary cells and model tumor cells, whether differentiated or not, and are further critically dependent on the particle opsonisation by serum proteins. Supported by the DFG, SPP1313.

Peritoneal macrophages from patients with cirrhotic ascites show impaired phagocytosis and vigorous respiratory burst

Cirrhotic patients (CPs) are susceptible to spontaneous bacterial peritonitis (SBP). Aim of this study was to examine if this susceptibility was related to peritoneal macrophages' (PMs) altered host defence. Absorbance of phagocytosed particles by PMs from CPs was lower than that of control (31.88% vs. 77.2%). Particle opsonisation increased the absorbance to 41% in CPs' PMs, and this value remains lower than the control; 77.2%. Respiratory burst (RB) was expressed as fluorescence index values, and these were higher in PMs from CPs than in controls (82 vs. 41, 73 vs. 26 and 71 vs. 26). IFN-γ made no further increase of RB values in PMs from CPs. CD14 expression was also higher in CPs' PMs. IFN-γ significantly downregulated CD14 expression in both CPs' PMs and control. Reduced phagocytosis by predominantly CD14-positive PMs from CPs could be related to intense RB. Findings suggest altered host defence that could contribute to susceptibility to SBP.

Use of Size and a Copolymer Design Feature To Improve the Biodistribution and the Enhanced Permeability and Retention Effect of Doxorubicin-Loaded Mesoporous Silica Nanoparticles in a Murine Xenograft Tumor Model

A key challenge for improving the efficacy of passive drug delivery to tumor sites by a nanocarrier is to limit reticuloendothelial system uptake and to maximize the enhanced permeability and retention effect. We demonstrate that size reduction and surface functionalization of mesoporous silica nanoparticles (MSNP) with a polyethyleneimine-polyethylene glycol copolymer reduces particle opsonization while enhancing the passive delivery of monodispersed, 50 nm doxorubicin-laden MSNP to a human squamous carcinoma xenograft in nude mice after intravenous injection. Using near-infrared fluorescence imaging and elemental Si analysis, we demonstrate passive accumulation of ∼12% of the tail vein-injected particle load at the tumor site, where there is effective cellular uptake and the delivery of doxorubicin to KB-31 cells. This was accompanied by the induction of apoptosis and an enhanced rate of tumor shrinking compared to free doxorubicin. The improved drug delivery was accompanied by a significant reduction in systemic side effects such as animal weight loss as well as reduced liver and renal injury. These results demonstrate that it is possible to achieve effective passive tumor targeting by MSNP size reduction as well as by introducing steric hindrance and electrostatic repulsion through coating with a copolymer. Further endowment of this multifunctional drug delivery platform with targeting ligands and nanovalves may further enhance cell-specific targeting and on-demand release.

177 Peptide Linkers Between Natural Antibodies and HIV Target

In human serum approximately 1 to 8% of the total IgM and 1 to 2,4% of the total IgG recognize the epitope gal(a1,3)gal, which is part of a penta-saccharide present mainly on endothelial cells of all mammals except humans and old world monkeys. These natural antibodies have been shown to participate in the antibody-mediated opsonization of particles and in the induction of the classical cascade of the complement. Moreover, these antibodies can trigger the activation of NK cells via CD16 recognition of the Fc fraction of the antibody and induce an antibody-dependent cellular cytotoxicity (ADCC). We thought it would be useful to temporarily be able to redirect this high level preexisting antibody pool to a new antigen, which rapidly would increase the pool of biologically active antibodies with a pre-determined specificity as an alternative to administering a monoclonal antibody. To explore this possibility we chose to target the receptor-binding region of the envelope protein (gp120) of the human immunodeficiency virus (HIV). The interaction between gp120 and its receptor the CD4 molecule is highly conserved and involves only a limited amount of residues. The envelope glycoprotein binds to 22 residues on the D1 region of the CD4 receptor located between amino acids 25 to 64 and the atomic interactions are well characterized. As previously published, we could show that CD4-derived, gp120-binding, synthetic peptides chemically linked to gal(α1,3)gal can redirect these natural antibodies and improve the HIV-1-neutralizing activity of the CD4-derived peptides in vitro and conferred antibody dependent cellular cytotoxicity after the addition of normal human sera. We have now extended these studies using a sterical mimetic of CD4 withou sequence hology and peptides derived from CCR5. I will also discuss how we now are employing a random RNA library to select peptides binding to new targets.

Differential Uptake of Functionalized Polystyrene Nanoparticles by Human Macrophages and a Monocytic Cell Line

Tumor cell lines are often used as models for the study of nanoparticle-cell interactions. Here we demonstrate that carboxy (PS-COOH) and amino functionalized (PS-NH2) polystyrene nanoparticles of ∼100 nm in diameter are internalized by human macrophages, by undifferentiated and by PMA-differentiated monocytic THP-1 cells via diverse mechanisms. The uptake mechanisms also differed for all cell types and particles when analyzed either in buffer or in medium containing human serum. Macrophages internalized ∼4 times more PS-COOH than THP-1 cells, when analyzed in serum-containing medium. By contrast, in either medium, THP-1 cells internalized PS-NH2 more rapidly than macrophages. Using pharmacological and antisense in vitro knockdown approaches, we showed that, in the presence of serum, the specific interaction between the CD64 receptor and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization in THP-1 cells occurred via dynamin II-dependent endocytosis. PMA-differentiated THP-1 cells differed in their uptake mechanism from macrophages and undifferentiated THP-1 cells by internalizing the particles via macropinocytosis. In line with our in vitro data, more intravenously applied PS-COOH particles accumulated in the liver, where macrophages of the reticuloendothelial system reside. By contrast, PS-NH2 particles were preferentially targeted to tumor xenografts grown on the chorioallantoic membrane of fertilized chicken eggs. Our data show that the amount of internalized nanoparticles, the uptake kinetics, and its mechanism may differ considerably between primary cells and a related tumor cell line, whether differentiated or not, and that particle uptake by these cells is critically dependent on particle opsonization by serum proteins.

Fexofenadine hydrochloride in the treatment of allergic disease: a review

Fexofenadine is a selective, non-sedating H1 receptor antagonist, marketed in the United States since 2000. The FDA approved an oral suspension in 2006, for the treatment of seasonal allergic rhinitis and chronic idiopathic urticaria in children. The tablet, capsule, and oral suspension are bioequivalent. Although fexofenadine does not use P450 CYP 3A4 it does interact with a number of drugs at P-glycoprotein and organic anion transporter polypeptides. The risk of toxicity from other drugs may increase with the administration of fexofenadine. Orange and grapefruit juices reduce the bioavailability of fexofenadine. Fexofenadine has been shown to have an impact on inflammatory mediators, other than histamine, such as decreasing the production of LTC(4), LTD(4), LTE(4), PGE(2), and PGF(2α); inhibiting cyclo-oxygenase 2, thromboxane; limiting iNOS generation of NO; decreasing cytokine levels (ICAM-1, ELAM-1, VCAM-1, RANTES, I-TAC, MDC, TARC, MMP-2, MMP-9, tryptase); and diminishing eosinophil adherence, chemotaxis, and opsonization of particles. These effects may provide benefit to some of the inflammatory responses of an acute allergic reaction and provide a basis for future development of H1 antagonists with stronger anti-inflammatory effects. These studies also support the contention that fexofenadine is effective for the treatment of allergic rhinits and chronic idiopathic urticaria.

Immunoglobulin G signaling activates lysosome/phagosome docking.

An important role of IgG antibodies in the defense against microbial infections is to promote the ingestion and killing of microbes by phagocytes. Here, we developed in vivo and in vitro approaches to ask whether opsonization of particles with IgG enhances intracellular targeting of lysosomes to phagosomes. To eliminate the effect of IgG on the ingestion process, cells were exposed to latex beads at 15-20 degrees C, which allows engulfment of both IgG-coated and uncoated beads but prevents the fusion of lysosomes with phagosomes. Upon shifting the temperature to 37 degrees C, phagosomes containing IgG beads matured significantly faster into phagolysosomes as judged by colocalization with lysosomal markers. The IgG effect was independent of other particle-associated antigens or serum factors. Lysosome/phagosome attachment was also quantified biochemically with a cytosol-dependent scintillation proximity assay. Interactions were enhanced significantly in reactions containing cytosol from mouse macrophages that had been exposed to IgG-coated beads, indicating that IgG signaling modulates the cytosolic-targeting machinery. Similar results were obtained with cytosol from primary human monocytes, human U-937 histiocytic lymphoma cells and from Chinese hamster ovary (CHO) cells transfected with a human IgG (Fcgamma) receptor. IgG-induced activation is shown to affect the actin-dependent tethering/docking stage of the targeting process and to proceed through a pathway involving protein kinase C. These results provide a rare example of an extracellular signal controlling membrane targeting on the level of tethering and docking. We propose that this pathway contributes to the role of antibodies in the protection against microbial infections.

The effect of taurolin on endogenous immunity and pathogen elimination in human peritonitis

The influence of taurolin on phagocytosis-related defense functions and on the elimination of bacteria has been investigated in a controlled study. Particle opsonisation, a prerequisite for sufficient phagocytosis, was undisturbed in presence of up to 0.1% taurolin. Intraabdominal taurolin application did not diminish the leukocyte influx into the abdominal cavity. Opsonic capacity of peritoneal exudate was low in the taurolin group. These exudates contained more severe bacterial contamination, suggesting that this deficit was due to enhanced specific consumption of opsonins. Taurolin treatment resulted in a marked reduction of bacterial growth. Thus the substance provides antiseptic capacities without afflicting the physiological defense systems.

Alternative complement pathway-dependent ingestion of fluolite particles by human granulocytes.

Fluorescent particles (Fluolite) with an average size of 0.1 micrometers were ingested by human granulocytes after incubation in fresh normal human serum (NHS). Ingestion was assessed by visual counting in a fluorescent microscope of cells containing particles. Ingestion required fresh normal serum and did not occur when serum was heated for 30 min at 50 degrees C or in the presence of ethylenediaminetetraacetic acid (EDTA). It did not occur in serum genetically deficient in C3b inactivator or in C3. Phagocytic activity was restored to C3-deficient serum by purified human C3 and to heat inactivated serum by purified factor B. Opsonic activity was present in NHS containing 5 mM Mg++ and 10 mM ethyleneglycoltetraacetic acid (EGTA) and in human serum genetically deficient in human C components C2 and C5. Agammaglobulinemic sera had normal opsonic activity. Opsonization of particles in this system is mediated through the alternative pathway of C activation, and its measurement serves as a simple quantitative functional assay for this system.

Salmonid macrophages: separation, in vitro culture and characterization

Abstract. A method is described for the separation of fish leucocytes and the establishment of pure monolayers of fish macrophages in vitro. The method makes it possible to study the important role of cellular immunity in fish. Fish leucocytes were obtained from the pronephros of rainbow trout, Salmo gairdneri Richardson, and compared to those obtained from the pronephros of Atlantic salmon, Salmo salar L. Single cell suspensions were separated by density gradient centrifugation and seeded on glass cover slips for maintenance in culture. After 20 h in culture a subpopulation of the cells had adhered and spread out on the cover slips and were macrophage‐like by morphological criteria. About 90–99% of these cells had the ability to phagocytose a variety of particles, including fixed sheep erythrocytes, latex, carbon particles, yeast and Vibrio anguillarum. Opsonization of particles with mammalian immunoglobulins and mammalian complement did not enhance the phagocytic activity.

Kinetics of phagocytosis of Staphylococcus aureus by alveolar and peritoneal macrophages.

The rate of uptake of radiolabeled Staphylococcus aureus by macrophages in vitro was studied by use of Lineweaver-Burk analysis. It was found that competition for ingestion by excess unlabeled particles, either staphylococci or unrelated particles, resulted in diminished uptake of the labeled particles and that opsonization of particles with specific antiserum enhanced that uptake solely by altering the maximum velocity of uptake (Vmax). Uptake of radiolabeled staphylococci opsonized with specific antiserum was not inhibited by excess numbers of unopsonized organisms; the ingestion was inhibited by excess numbers of opsonized unlabeled organisms, and that inhibition was characterized by depression of Vmax. Inhibition of phagocytosis by indoacetate and cytochalasin B resulted from depression in both Vmax and Michaelis constate (Km). In addition, the phagocytic function of macrophages improved during in vitro culture, a phenomenon which was particularly striking for alveolar macrophages. That enhancement of activity resulted from improvements in both Vmax and Km. Addition of opsonizing antibody at any stage of in vitro maturation resulted in further increases in phagocytic uptake, increases which affected only Vmax. The in vitro maturation of phagocytic function by alveolar macrophages could be inhibited by both 2-deoxy-D-glucose and cycloheximide, but not by culture in hypoxia. The data indicate that the terms of Lineweaver-Burk analysis cna be correlated with functional aspects of phagocytosis and that Vmax represents the avidity of the macrophage surface for the particle, whereas Km is an index of the capacity of the cell for ingestion.

The effect of tobacco smoke on the metabolism and function of rat alveolar macrophages

Alveolar macrophages harvested by bronchopulmonary lavage from rats exposed to tobacco smoke for 30 days ("smokers") showed alterations in oxidative metabolism, lactate production and phagocytosis of inert starch particles when compared with control macrophages. Phagocytosis of viable Staphylococcus aureus was unaffected by tobacco smoke. Glucose oxidation measured by conversion of glucose-1-14C to 14CO2 moderately affected while oxidation of glucose-6-14C to 14CO2 was not. Smokers routinely yielded fewer cells than controls, though these cells contained approximately 17% more protein than did controls. Opsonization of particles was not necessary for macrophages from either smoker or control animals to manifest a respiratory burst and increased superoxide and hydrogen peroxide release during phagocytosis. The glycolytic inhibitors, sodium fluoride and iodoacetamide, while effectively blocking glycolysis, did not inhibit phagocytosis by macrophages from either group. The results reported clearly distinguish alveolar macrophages from other phagocytic cells (peritoneal macrophages and polymorphonuclear leukocytes) and suggest a state of non-specific activation caused by exposure to tobacco smoke.