Opsonins and Dysopsonins of Nanoparticles: Facts, Concepts, and Methodological Guidelines.

Understanding the effects mediated by a set of nanoparticle (NP)-bound host biomolecules, often indicated with the umbrella term of NP corona, is essential in nanomedicine, nanopharmacology, and nanotoxicology. Among the NP-adsorbed proteome, some factors mediate cell binding, endocytosis, and clearing by macrophages and other phagocytes (opsonins), while some others display few affinities for the cell surface (dysopsonins). The functional mapping of opsonins and dysopsonins is instrumental to design long-circulating and nanotoxicologically safe next-generation nanotheranostics. In this review, we critically analyze functional data identifying specific proteins with opsonin or dysopsonin properties. Special attention is dedicated to the following: (1) the simplicity or complexity of the NP proteome and its modulation, (2) the role of specific host proteins in mediating the stealth properties of uncoated or polymer-coated NPs, and (3) the ability of the innate immune system, and, in particular, of the complement proteins, to mediate NP clearance by phagocytes. Emerging species-specific peculiarities, differentiating humans from preclinical animal models (the murine especially), are highlighted throughout this overview. The operative definition of opsonin and dysopsonin and the measurement schemes to assess their in vitro efficacy is critically re-examined. This provides a shared and unbiased approach useful for NP opsonin and dysopsonin systematic identification.

Anti-Pseudomonas aeruginosa IgY antibodies promote bacterial opsonization and augment the phagocytic activity of polymorphonuclear neutrophils.

Moderation of polymorphonuclear neutrophils (PMNs) as part of a critical defense against invading pathogens may offer a promising therapeutic approach to supplement the antibiotic eradication of Pseudomonas aeruginosa infection in non-chronically infected cystic fibrosis (CF) patients. We have observed that egg yolk antibodies (IgY) harvested from White leghorn chickens that target P. aeruginosa opsonize the pathogen and enhance the PMN-mediated respiratory burst and subsequent bacterial killing in vitro. The effects on PMN phagocytic activity were observed in different Pseudomonas aeruginosa strains, including clinical isolates from non-chronically infected CF patients. Thus, oral prophylaxis with anti-Pseudomonas aeruginosa IgY may boost the innate immunity against Pseudomonas aeruginosa in the CF setting by facilitating a rapid and prompt bacterial clearance by PMNs.

Inspired by nature: fundamentals in nanotechnology design to overcome biological barriers.

Synergy between nanotechnology and drug delivery has created a multitude of novel drug-delivery systems with great therapeutic potential. However, directing these systems across the biological barriers to the target site has proven difficult. Nanotechnology is looking for inspiration in natural systems that have evolved to overcome such barriers. Here, we review nature-inspired strategies and fundamental features common to successful drug-delivery systems across biological barriers.

Biomimetic delivery with micro- and nanoparticles.

The nascent field of biomimetic delivery with micro- and nanoparticles (MNP) has advanced considerably in recent years. Drawing inspiration from the ways that cells communicate in the body, several different modes of "delivery" (i.e., temporospatial presentation of biological signals) have been investigated in a number of therapeutic contexts. In particular, this review focuses on (1) controlled release formulations that deliver natural soluble factors with physiologically relevant temporal context, (2) presentation of surface-bound ligands to cells, with spatial organization of ligands ranging from isotropic to dynamically anisotropic, and (3) physical properties of particles, including size, shape and mechanical stiffness, which mimic those of natural cells. Importantly, the context provided by multimodal, or multifactor delivery represents a key element of most biomimetic MNP systems, a concept illustrated by an analogy to human interpersonal communication. Regulatory implications of increasingly sophisticated and "cell-like" biomimetic MNP systems are also discussed.

Time-dependent changes in opsonin amount associated on nanoparticles alter their hepatic uptake characteristics.

The relationship between the time-dependent change in serum proteins adsorbed on nanoparticles and their disposition to the liver was investigated by employing lecithin-coated polystyrene nanosphere with a size of 50 nm (LNS-50) as a model nanoparticle in rats. The total amount of proteins adsorbed on LNS-50 increased and the qualitative profile of serum proteins adsorbed on LNS-50 changed during the incubation with serum up to 360 min. The liver perfusion study indicated that the hepatic uptake of LNS-50 incubated with serum for 360 min was significantly larger than those of LNS-50 incubated for shorter period. It was suggested that the increase in the hepatic uptake of LNS-50 with the increase in incubation time would be ascribed mainly to the increase in the opsonin-mediated uptake by Kupffer cells. Semi-quantification of major opsonins, complement C3 (C3) and immunoglobulin G (IgG), and in vitro uptake study in primary cultured Kupffer cells demonstrated that the increase in C3 and IgG amounts adsorbed on LNS-50 was directly reflected in the increased disposition of LNS-50 to Kupffer cells. These results indicate that the amounts of opsonins associated on nanoparticles would change over time and this process would be substantially reflected in the alteration of their hepatic disposition characteristics.

Helicobacter pylori disrupts NADPH oxidase targeting in human neutrophils to induce extracellular superoxide release.

Helicobacter pylori (Hp) infection triggers a chronic influx of polymorphonuclear leukocyte neutrophils (PMNs) into the gastric mucosa. Although Hp reside in a neutrophil-rich environment, how these organisms evade phagocytic killing is largely unexplored. We now show that live Hp (strains 11637, 60190, DT61A, and 11916) are readily ingested by PMNs and induce a rapid and strong respiratory burst that is comparable to PMA. Relative to other particulate stimuli, Hp are more potent activators of PMNs than opsonized zymosan, Staphylococcus aureus, or Salmonella. Strikingly, biochemical and microscopic analyses demonstrate that Hp disrupt NADPH oxidase targeting such that superoxide anions are released into the extracellular milieu and do not accumulate inside Hp phagosomes. Specifically, nascent Hp phagosomes acquire flavocytochrome b558 but do not efficiently recruit or retain p47phox or p67phox. Superoxide release peaks at 16 min coincident with the appearance of assembled oxidase complexes in patches at the cell surface. Oxidant release is regulated by formalin-resistant and heat-sensitive bacterial surface factors distinct from urease and Hp(2-20). Following opsonization with fresh serum, Hp triggers a modest respiratory burst that is confined to the phagosome, and ingested bacteria are eliminated. We conclude that disruption of NADPH oxidase targeting allows unopsonized Hp to escape phagocytic killing, and our findings support the hypothesis that bacteria and PMNs act in concert to damage the gastric mucosa.

Combined therapy of granulocyte transfusions, intravenous opsonins and antibiotics for gram-negative pneumonia in neutropenic cancer patients.

Four patients with hematological malignancies, following bone marrow transplantation, who developed documented gram-negative [Klebsiella pneumoniae (2), Pseudomonas aeruginosa or Acinetobacter calcoaceticus] pneumonia during absolute neutropenia, were treated with a combination of antimicrobial therapy, granulocyte transfusions, and high-dose intravenous immunoglobulin. The patients recovered following this regimen, including 2 who had septic shock and respiratory failure, necessitating intubation and mechanical ventilation. These data suggest that therapy combining antimicrobial agents, granulocyte transfusions and opsonins may be effective in neutropenic patients who develop gram-negative pneumonia.

Antibacterial effect of bovine milk antibody against Escherichia coli in a mouse indigenous infection model.

A skim-milk fraction and a whey-protein concentrate (WPC) fraction were prepared from the cows that had been immunized with E. coli isolated from the mouse intestine. The antibacterial effect of these fractions against E. coli was examined. They contained antibody with a high affinity for E. coli strain 48, a representative strain in the mouse intestine, which is composed of a large amount of IgG and smaller amounts of IgA and IgM. Although these fractions showed no bactericidal or bacteriostatic activity against E. coli 48 directly in vitro, they exhibited strong agglutination and opsonization activities against the bacteria in vitro. The bacteria opsonized with the WPC fraction were taken up more effectively by liver macrophages in vivo, compared with unopsonized E. coli, after an intravenous injection into mice. Oral administration of the skim-milk fraction to mice significantly reduced the susceptibility to the lethal toxicity of 5-fluorouracil (5 FU). The increase in the population levels of E. coli in the intestinal tract after administration of 5 FU was inhibited by oral administration of the skim-milk fraction. These results strongly suggest that specific antibody may be effective in the prophylaxis against the indigenous infection with gram-negative bacteria such as E. coli after a period of chemotherapy in cancer patients.

Ontogeny of fetal sheep polymorphonuclear leukocyte phagocytosis.

Premature infants and neonates are vulnerable to bacterial sepsis. This susceptibility may be due to the relative immaturity of their immune systems. To determine if neonates and, in particular, premature infants have decreased polymorphonuclear leukocyte (PMN) phagocytosis, we tested PMN phagocytosis of Staphylococcus aureus as a function of gestational age in the fetal lamb model. Because phagocytosis is made more efficient by the presence of opsonins in plasma, fetal and postnatal PMN phagocytosis were also measured after exposure to fetal and adult plasma. PMNs were isolated from fetal lambs at 104, 114, 124, and 141 days' gestation (term gestation for the fetal lamb is 145 days), as well as from 10-day-old neonatal sheep and adult sheep. Labeled S aureus were opsonized by incubation in either fetal or adult plasma, or left unopsonized for baseline values. Phagocytosis was measured as a percent of adult PMN phagocytosis after adult plasma opsonization. It was found that fetal PMN function is limited by two factors during the early third trimester: a primary defect in the ability of the PMN to phagocytose S aureus despite adequate opsonization, and the diminished ability of autologous fetal plasma to opsonize bacteria. The defect in PMN phagocytosis disappears late in the third trimester, but the inability of the fetal plasma to opsonize effectively continues until after birth.

Stimulation of erythrophagocytosis in mouse peritoneal macrophages by chondroitin sulfates: correlation with effect of phorbol esters.

Resident macrophages which were harvested from the mouse peritoneal cavity showed the attachment activity to opsonized erythrocytes (OE) without the treatment of chondroitin sulfates (CSA) or phorbol esters. Phorbol ester (phorbol 12-myristate 13-acetate or phorbol 12,13-diacetate) rapidly activated an opsonin-dependent erythrophagocytosis in resident macrophages, whereas CSA slowly activated it in vivo and in vitro. An additive effect of phorbol esters was observed in macrophages which were cultured with CSA in vitro or stimulated by the intraperitoneal injection of CSA for 1 or 2 day(s). In the case of macrophages stimulated by the intraperitoneal injection of CSA for 3 or 4 days the erythrophagocytic activity was at very high level and the additive effect of phorbol esters vanished. These results indicate that CSA plays a role in the induction of opsonin-dependent ingestion activity of resident macrophages.

Quantitative assessment of human neutrophil chemiluminescence induced by opsonized Escherichia coli K-12.

The interaction of opsonized E. coli K-12 bacteria and polymorphonuclear leukocytes (PMN) was quantified, using luminol-enhanced chemiluminescence (CL) as a parameter of PMN stimulation. On a double-logarithmic scale light emission depended on the opsonin concentration used during pre-opsonisation. The most potent CL-inducing agent was fresh human serum, and its stimulatory activity depended on an intact complement (C) system. Both inactivation of C by heating or blocking the classical C pathway with EGTD decreased the CL-inducing potency by a factor of 8 to 16. Hypogammaglobulinemic heated serum mediated little CL. IgG for intravenous use mediated CL generation, but reduction/alkylation and sulphitolysis reduced the stimulatory power. Evidence is presented that the anti-K-12 antibodies within commercial IgG and IgM used for substitution do not improve the stimulatory power of IgG-deficient, IgM- and C-sufficient serum, unless very high Ig concentrations are substituted.

Immunoglobulin therapy of neonatal group B streptococcal infections: an overview.

Group B streptococci (GBS) are a major cause of sepsis and meningitis in newborn babies. Neonatal GBS infections are often rapidly progressive, suggesting that the immunity to GBS is deficient. Studies have shown that opsonic antibody is required for efficient phagocytosis and killing of GBS, and neonatal GBS infections have been associated with diminished levels of anti-GBS antibody. Intravenous immunoglobulin (IVIG) has been shown to provide protective immunity in experimental GBS infection models. However, lot to lot variation in opsonic antibody levels occurs in standard IVIG preparations. Recently hyperimmune anti-GBS IVIG has been prepared with high levels of opsonic and protective antibody to GBS. Hyperimmune IVIG preparations will allow physicians to give higher quantities of specific anti-GBS antibody without having to administer large fluid volumes or large amounts of nonspecific immunoglobulin. In addition specific immunoglobulin preparations will ensure that the IVIG contains reliable antibacterial activity. Although human studies are limited they suggest that IVIG therapy in neonates may be safe and effective in treating neonatal sepsis. However, further studies are necessary to determine the role of IVIG in preventing or treating neonatal infections.

Respiratory burst of normal human eosinophils.

Oxidative metabolism of eosinophils has generally been studied in cells from patients with eosinophilia. We isolated eosinophils with purity of greater than 95% from normal donors. Oxygen metabolism of eosinophils and neutrophils was compared using O2-. production as a measure of the stimulus-induced respiratory burst. During 10 min of stimulation, using phorbol myristate acetate (PMA) or opsonized zymosan (OPZ), eosinophils produced two to three times as much O2-. as did neutrophils. Continuous kinetic analysis was used to measure the latency period and maximal rate of O2-. production. Eosinophils has a shorter latency period and greater rate of O2-. production than did neutrophils at all concentrations of PMA. With OPZ stimulation, O2-. production rates by both cells were similar. The latency periods were similar with high concentrations of OPZ but at limiting amounts of OPZ eosinophils had a longer latency period than did neutrophils. In addition, the kinetic assay demonstrated that the respiratory burst in eosinophils was more sustained than in neutrophils. These results indicate substantial differences in the oxidative burst of eosinophils and neutrophils with respect to activation, capacity, and regulation. These distinctive features of O2-. production by eosinophils may be important in host defense against metazoan parasites or in tissue injury during inflammation.

Opsonic and protective activity of immunoglobulin, modified immunoglobulin, and serum against neonatal Escherichia coli K1 infection.

Because modified immune serum globulin (M-ISG) has been proposed for therapy in neonatal bacterial sepsis, we evaluated it in a suckling rat model of Escherichia coli K1 sepsis. We compared a M-ISG preparation (lot 2581), which was protective against group B streptococcal (GBS) sepsis, with other M-ISG, standard ISG preparations and with adult and cord serum. All immune serum preparations and sera demonstrated opsonic activity against E. coli K1 and were superior to saline in protecting against death due to E. coli K1 sepsis. Survival rates were higher for one M-ISG preparation (lot 2581) than for randomly selected standard immune serum globulin and cord sera but were similar to adult sera in these protection studies. Therapeutic and opsonic activity of standard immune serum globulin and M-ISG prepared from the same donors were similar, suggesting that the different processes used for their manufacture did not affect these activities. Because the M-ISG preparation studied showed protective activity to both GBS and E. coli K1, we studied it further by adsorbing the preparation with GBS and E. coli K1. Opsonic activity to E. coli K1 was removed by E. coli adsorption but not by adsorption with GBS, indicating that this activity was not due to a single cross-reacting antibody. Empirical therapy with M-ISG prescreened for opsonic activity to both E. coli K1 and GBS may be of clinical value. Trials appear warranted to study the pharmacology and efficacy of M-ISG in septic newborns.

Superoxide-dependent chemotactic activity for PMNs derived from opsonized zymosan-stimulated human platelets.

Previous studies have shown that platelets exhibit a H2O2 producing, NADH-dependent system that is activated by interaction with particulate material. Current evidence suggests that this system could be critically involved in th generation of chemotactic factor(s). In the present studies, chemotactic activity for polymorphonuclear leukocytes of supernatants derived from zymosan-stimulated human platelets has been evaluated using an agarose gel technique. Supernatants of opsonized zymosan-stimulated platelets showed significant chemotactic activity (migration index = 300 +/- 50), in comparison with supernatants prepared from platelet suspensions stimulated with nonopsonized-zymosan (migration index = 10 +/- 15) or resting platelet supernatants (migration index = 15 +/- 15). Furthermore, a marked increase in chemotactic activity of the opsonized zymosan-treated platelet supernatants was demonstrated after the addition of NADH (migration index = 525 +/- 100). The inclusion of specific inhibitors of the cycloxygenase and lipoxygenase pathways resulted in a marked reduction of chemotactic activity, which was restored in the presence of NADH. Further, the addition of superoxide dismutase completely abolished the chemotactic response induced by NADH. These data suggest that platelets are the source of chemotactic factor(s) derived from the activation of a superoxide generating system.

Functional leukocyte administration in protection against experimental neonatal infection.

Studies have shown that human neonates who develop group B streptococcal sepsis usually lack opsonic antibody (Ab) to their infecting strain and that these neonates may also have impaired polymorphonuclear leukocyte (PMN) function. The present study was designed to determine the efficacy of administration of PMNs or opsonic Ab-containing serum in protecting against group B streptococcal infection in a newborn rat model. After intraperitoneal (IP) injection of congruent to 5 X 10(6) streptococci, animals received separate IP injections of saline, serum lacking opsonic antibody (Ab negative), Ab positive serum or washed adult human PMNs (2 X 10(6)). The mortality rate in 55 neonatal rats infected with group B streptococci who received saline or Ab negative serum was 91%. In contrast, 40 animals who received adult human PMNs at the time of inoculation had a survival rate of 50% (P less than 0.001). Human serum containing opsonic antibody also provided significant protection against mortality in this model (survival rate 51%, P less than 0.001). Leukocytes from normal term neonates, stressed neonates, or ones pretreated with cytochalasin B offered les protection than did functional adult human cell (P less than 0.001).

Prevention of liver reticuloendothelial systemic host defense failure after surgery by intravenous opsonic glycoprotein therapy.

Depression of the reticuloendothelial system (RES) was observed in rats following operative trauma consisting of laparotomy and jejunal enterotomy. This RES depression was manifested as a significant impairment in the phagocytic clearance of intravenously injected blood-borne test colloid mediated by a decline in hepatic Kupffer cell phagocytosis. Reticuloendothelial systemic host defense depression was correlated with a significant decline in bioassayable and immunoreactive opsonic alpha(2)SB glycoprotein concentration over a 1-3 hr postoperative period with rebound elevation in opsonic activity by 24 hr postsurgery. Intravenous administration of purified opsonic alpha(2)SB glycoprotein at the end of the operation prevented postoperative opsonic deficiency and restored normal hepatic RES phagocytic function. These studies coupled with previous observations in patients following surgery or after severe multiple trauma suggest that reticuloendothelial depression during and after operation mediated by opsonic deficiency may lead to a precarious imbalance between systemic host defense and the dissemination of blood-borne foreign and effete particulate matter such as injured platelets, fibrin microaggregates and immune complexes. Immunoreactive serum opsonic alpha(2)SB glycoprotein determinations may serve as a valuable index of hepatic RE clearance capacity and opsonin therapy may potentially be a selective means to augment systemic host defense.