Polyphosphate nanoparticles on the platelet surface trigger contact system activation.

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII.

Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement.

PEGylation of lipid-based nanoparticles and other nanocarriers is widely used to increase their stability and plasma half-life. However, either pre-existing or de novo formed anti-PEG antibodies can induce hypersensitivity reactions and accelerated blood clearance through binding to the nanoparticle surfaces, leading to activation of the complement system. In this study, we investigated the consequences and mechanisms of complement activation by anti-PEG antibodies interacting with different types of PEGylated lipid-based nanoparticles. By using both liposomes loaded with different (model) drugs and LNPs loaded with mRNA, we demonstrate that complement activation triggered by anti-PEG antibodies can compromise the bilayer/surface integrity, leading to premature drug release or exposure of their mRNA contents to serum proteins. Anti-PEG antibodies also can induce deposition of complement fragments onto the surface of PEGylated lipid-based nanoparticles and induce the release of fluid phase complement activation products. The role of the different complement pathways activated by lipid-based nanoparticles was studied using deficient sera and/or inhibitory antibodies. We identified a major role for the classical complement pathway in the early activation events leading to the activation of C3. Our data also confirm the essential role of amplification of C3 activation by alternative pathway components in the lysis of liposomes. Finally, the levels of pre-existing anti-PEG IgM antibodies in plasma of healthy donors correlated with the degree of complement activation (fixation and lysis) induced upon exposure to PEGylated liposomes and mRNA-LNPs. Taken together, anti-PEG antibodies trigger complement activation by PEGylated lipid-based nanoparticles, which can potentially compromise their integrity, leading to premature drug release or cargo exposure to serum proteins.

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow.

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions. A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

Iron nanomedicines induce Toll-like receptor activation, cytokine production and complement activation.

Approximately a dozen of intravenous iron nanomedicines gained marketing authorization in the last two decades. These products are generally considered as safe, but have been associated with an increased risk for hypersensitivity-like reactions of which the underlying mechanisms are unknown. We hypothesized that iron nanomedicines can trigger the innate immune system. We hereto investigated the physico-chemical properties of ferric gluconate, iron sucrose, ferric carboxymaltose and iron isomaltoside 1000 and comparatively studied their interaction with Toll-like receptors, the complement system and peripheral blood mononuclear cells. Two out of four formulations appeared as aggregates by Scanning Transmission Electron Microscopy analysis and were actively taken up by HEK293T- and peripheral blood mononuclear cells in a cholesterol-dependent manner. These formulations triggered in vitro activation of intracellular Toll-like receptors 3, -7 and -9 in a dose- and serum-dependent manner. In parallel experiments, we determined that these compounds activated the complement system. Finally, we found that uptake of aggregation-prone iron nanomedicines by peripheral blood mononuclear cells in whole blood induced production of the proinflammatory cytokine IL-1ß, but not IL-6.

A novel oral iron-complex formulation: Encapsulation of hemin in polymeric micelles and its in vitro absorption.

Anemia resulting from iron deficiency is one of the most prevalent diseases in the world. As iron has important roles in several biological processes such as oxygen transport, DNA synthesis and cell growth, there is a high need for iron therapies that result in high iron bioavailability with minimal toxic effects to treat patients suffering from anemia. This study aims to develop a novel oral iron-complex formulation based on hemin-loaded polymeric micelles composed of the biodegradable and thermosensitive polymer methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl)methacrylamide-dilactate], abbreviated as mPEG-b-p(HPMAm-Lac2). Hemin-loaded micelles were prepared by addition of hemin dissolved in DMSO:DMF (1:9, one volume) to an aqueous polymer solution (nine volumes) of mPEG-b-p(HPMAm-Lac2) followed by rapidly heating the mixture at 50°C to form hemin-loaded micelles that remain intact at room and physiological temperature. The highest loading capacity for hemin in mPEG-b-p(HPMAm-Lac2) micelles was 3.9%. The average particle diameter of the hemin-micelles ranged from 75 to 140nm, depending on the concentration of hemin solution that was used to prepare the micelles. The hemin-loaded micelles were stable at pH 2 for at least 3 h which covers the residence time of the formulation in the stomach after oral administration and up to 17 h at pH 7.4 which is sufficient time for uptake of the micelles by the enterocytes. Importantly, incubation of Caco-2 cells with hemin-micelles for 24 h at 37°C resulted in ferritin levels of 2500ng/mg protein which is about 10-fold higher than levels observed in cells incubated with iron sulfate under the same conditions. The hemin formulation also demonstrated superior cell viability compared to iron sulfate with and without ascorbic acid. The study presented here demonstrates the development of a promising novel iron complex for oral delivery.

Potential induction of anti-PEG antibodies and complement activation toward PEGylated therapeutics.

Conjugation of polyethylene glycol (PEG) to therapeutics has proven to be an effective approach to increase the serum half-life. However, the increased use of PEGylated therapeutics has resulted in unexpected immune-mediated side-effects. There are claims that these are caused by anti-PEG antibodies inducing rapid clearance. These claims are however hampered by the lack of standardized and well-validated antibody assays. PEGylation has also been associated with the activation of the complement system causing severe hypersensitivity reactions. Here, we critically review the clinical and analytical tools used. In addition, we propose an explanation of the immune-mediated side-effects of PEGylated products based on the haptogenic properties of PEG, responsible for complement activation and the induction of anti-PEG antibodies.

Questioning the Use of PEGylation for Drug Delivery.

Polyethylene glycol (PEG) is widely utilized in drug delivery and nanotechnology due to its reported "stealth" properties and biocompatibility. It is generally thought that PEGylation allows particulate delivery systems and biomaterials to evade the immune system and thereby prolong circulation lifetimes. However, numerous studies over the past decade have demonstrated that PEGylation causes significant reductions in drug delivery, including enhanced serum protein binding, reduced uptake by target cells, and the elicitation of an immune response that facilitates clearance in vivo. This report reviews some of the extensive literature documenting the detrimental effects of PEGylation, and thereby questions the wisdom behind employing this strategy in drug development.