Contribution of CARPA to polystyrene NP effects in pigs

Background: It has been proposed that many hypersensitivity reactions to nanopharmaceuticals represent complement (C)-activation-related pseudoallergy (CARPA), and that pigs provide a sensitive animal model to study the phenomenon. However, a recent study suggested that pulmonary hypertension, the pivotal symptom of porcine CARPA, is not mediated by C in cases of polystyrene nanoparticle (PS-NP)-induced reactions. Goals: To characterize PS-NPs and reexamine the contribution of CARPA to their pulmonary reactivity in pigs. Study design: C activation by 200, 500, and 750 nm (diameter) PS-NPs and their opsonization were measured in human and pig sera, respectively, and correlated with hemodynamic effects of the same NPs in pigs in vivo. Methods: Physicochemical characterization of PS-NPs included size, ζ-potential, cryo-transmission electron microscopy, and hydrophobicity analyses. C activation in human serum was measured by ELISA and opsonization of PS-NPs in pig serum by Western blot and flow cytometry. Pulmonary vasoactivity of PS-NPs was quantified in the porcine CARPA model. Results: PS-NPs are monodisperse, highly hydrophobic spheres with strong negative surface charge. In human serum, they caused size-dependent, significant rises in C3a, Bb, and sC5b-9, but not C4d. Exposure to pig serum led within minutes to deposition of C5b-9 and opsonic iC3b on the NPs, and opsonic iC3b fragments (C3dg, C3d) also appeared in serum. PS-NPs caused major hemodynamic changes in pigs, primarily pulmonary hypertension, on the same time scale (minutes) as iC3b fragmentation and opsonization proceeded. There was significant correlation between C activation by different PS-NPs in human serum and pulmonary hypertension in pigs. Conclusion: PS-NPs have extreme surface properties with no relevance to clinically used nanomedicines. They can activate C via the alternative pathway, entailing instantaneous opsonization of NPs in pig serum. Therefore, rather than being solely C-independent reactivity, the mechanism of PS-NP-induced hypersensitivity in pigs may involve C activation. These data are consistent with the “double-hit” concept of nanoparticle-induced hypersensitivity reactions involving both CARPA and C-independent pseudoallergy

Human serum albumin nanoparticles loaded with phthalocyanine dyes for potential use in photodynamic therapy of atherosclerotic plaques

Diseases caused by obstruction or rupture of vulnerable plaques in the arterial walls such as cardiovascular infarction or stroke are the leading cause of death in the world. In the present work, we developed human serum albuminnanoparticles loaded by physisorption with zinc phthalocyanine, TT1, mainly used for industrial application as near-infrared photosensitizer and compared these to HSA NPsloaded with the well-known silicone phthalocyanine (Pc4). The use of NIR light allows for better tissue penetration, while the use of nanoparticles permitshigh local concentrations. The particles were characterized and tested for toxicity and stability as well as for their potential use as a contrast agent and NIR photosensitizer for photodynamic therapy in cardiovascular disease. We focused on the distribution of the nanoparticles in RAW264.7macrophage cells and atherosclerotic mice. The nanoparticles had an average size of 120 nm according todynamic light scattering, good loading capacity for zinc phthalocyanine,and satisfying stability in 50% (v/v) fetal bovine serum for 8 hours and in an aqueous environment at 4°C for 4–6 weeks. Under light irradiation we found a high production of singlet oxygen and the products showed no dark toxicity in vitro with macrophages(the target cells in vulnerable plaques),but at a low μg/mL nanoparticleconcentration killed efficiently the macrophagesupon LED illumination. Injection of the contrast agentin atherosclerotic mice led to a visible fluorescence signal of zinc phthalocyaninein the atherosclerotic plaque at 30 minutes and in the lungs with afast clearance of the nanoparticles. Zinc phthalocyanine loaded human serum albumin nanoparticles present an interesting candidate for the visualization and potentially photodynamictreatment of macrophages in atherosclerotic plaquesThe research leading to these results has received funding from FP7-NMP CosmoPHOS-Nano under grant agreement No. 310337. Additional funding was received by the Spanish groups from MINECO (CTQ2017-85393-P) and ERA-NET/MINECO EuroNanoMed2017-191 / PCIN-2017-04

Immunological response to nitroglycerin-loaded shear-responsive liposomes in vitro and in vivo

Liposomes formulated from the 1,3-diamidophospholipid Pad-PC-Pad are shear- responsive and thus promising nano-containers to specifically release a vasodilator at stenotic arteries. The recommended preclinical safety tests for therapeutic liposomes of nanometer size include the in vitro assessment of complement activation and the evaluation of the associated risk of complement activation-related pseudo-allergy (CARPA) in vivo. For this reason, we measured complement activation by Pad-PC- Pad formulations in human and porcine sera, along with the nanopharmaceutical- mediated cardiopulmonary responses in pigs. The evaluated formulations comprised of Pad-PC-Pad liposomes, with and without polyethylene glycol on the surface of the liposomes, and nitroglycerin as a model vasodilator. The nitroglycerin incorporation efficiency ranged from 25% to 50%. In human sera, liposome formulations with 20 mg/mL phospholipid gave rise to complement activation, mainly via the alternative pathway, as reflected by the rises in SC5b-9 and Bb protein complex concentrations. Formulations having a factor of ten lower phospholipid content did not result in measurable complement activation. The weak complement activation induced by Pad- PC-Pad liposomal formulations was confirmed by the results obtained by performing an in vivo study in a porcine model, where hemodynamic parameters were monitored continuously. Our study suggests that, compared to FDA-approved liposomal drugs, Pad-PC-Pad exhibits less or similar risks of CARPA

Pulmonary intravascular macrophages: Prime suspects as cellular mediators of porcine CARPA

Pigs provide a highly sensitive and quantitative in vivo model for complement (C) activation-related pseudoallergy (CARPA), a hypersensitivity reaction caused by some state-of-art nanomedicines. In an effort to understand the mechanism of the pigs' unique sensitivity for CARPA, this review focuses on pulmonary intravascular macrophages (PIMs), which are abundantly present in the lung of pigs. These cells represent a macrophage subpopulation whose unique qualities explain the characteristic symptoms of CARPA in this species, most importantly the rapidly (within minutes) developing pulmonary vasoconstriction, leading to elevation of pulmonary arterial pressure. The unique qualities of PIM cells include the following; 1) they are strongly adhered to the capillary walls via desmosome-like intercellular adhesion plaques, which secure stable and lasting direct exposition of the bulk of these cells to the blood stream; 2) their ruffled surface engaged in intense phagocytic activity ensures efficient binding and phagocytosis of nanoparticles; 3) PIM cells express anaphylatoxin receptors, this way C activation can trigger these cells, 4) they also express pattern recognition molecules on their surface, whose engagement with certain coated nanoparticles may also activate these cells or act in synergy with anaphylatoxins and, finally 5) their high metabolic activity and capability for immediate secretion of vasoactive mediators upon stimulation explain the circulatory blockage and other robust physiological effects that their stimulation may cause. These qualities taken together with reports on liposome uptake by PIM cells during CARPA and the possible presence of these cells in human lung suggests that PIM cells may be a potential therapeutic target against CARPA. © 2015 by De Gruyter

Liposome-induced complement activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome

Hypersensitivity reactions to liposomal drugs, often observed with Doxil and AmBisome, can arise from activation of the complement (C) system by phospholipid bilayers. To understand the mechanism of this adverse immune reaction called C activation-related pseudoallergy (CARPA), we analyzed the relationship among liposome features, C activation in human serum in vitro, and liposome-induced cardiovascular distress in pigs, a model for human CARPA. Among the structural variables (surface charge, presence of saturated, unsaturated, and PEGylated phospholipids, and cisplatin vs. doxorubicin inside liposomes), high negative surface charge and the presence of doxorubicin were significant contributors to reactogenicity both in vitro and in vivo. Morphological analysis suggested that the effect of doxorubicin might be indirect, via distorting the sphericity of liposomes and, if leaked, causing aggregation. The parallelism among C activation, cardiopulmonary reactions in pigs, and high rate of hypersensitivity reactions to Doxil and AmBisome in humans strengthens the utility of the applied tests in predicting the risk of CARPA

Complement activation in vitro and reactogenicity of low-molecular weight dextran-coated SPIONs in the pig CARPA model : Correlation with physicochemical features and clinical information

The unique magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) have led to their increasing use in drug delivery and imaging applications. Some polymer-coated SPIONs, however, share with many other nanoparticles the potential of causing hypersensitivity reactions known as complement (C) activation-related pseudoallergy (CARPA). In order to explore the roles of iron core composition and particle surface coating in SPION-induced CARPA, we measured C activation by 6 different SPIONs in a human serum that is known to react to nanoparticles (NPs) with strong C activation. Remarkably, only the carboxymethyldextran-coated (ferucarbotran, Resosvist®) and dextran-coated (ferumoxtran-10, Sinerem®) SPIONs caused significant C activation, while the citric acid, phosphatidylcholine, starch and chitosan-coated SPIONs had no such effect. Focusing on Resovist and Sinerem, we found Sinerem to be a stronger activator of C than Resovist, although the individual variation in 15 different human sera was substantial. Further analysis of C activation by Sinerem indicated biphasic dose dependence and significant production of C split product Bb but not C4d, attesting to alternative pathway C activation only at low doses. Consistent with the strong C activation by Sinerem and previous reports of HSRs in man, injection of Sinerem in a pig led to dose-dependent CARPA, while Resovist was reaction-free. Using nanoparticle tracking analysis, it was further determined that Sinerem, but not Resovist, displayed multimodal size distribution and significant fraction of aggregates – factors which are known to promote C activation and CARPA. Taken together, our findings offer physicochemical insight into how key compositional factors and nanoparticle size distribution affect SPION-induced CARPA, a knowledge that could lead to the development of SPIONs with improved safety profiles

Complement activation in vitro and reactogenicity of low-molecular weight dextran-coated SPIONs in the pig CARPA model: Correlation with physicochemical features and clinical information

The unique magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) have led to their increasing use in drug delivery and imaging applications. Some polymer-coated SPIONs, however, share with many other nanoparticles the potential of causing hypersensitivity reactions known as complement (C) activation-related pseudoallergy (CARPA). In order to explore the roles of iron core composition and particle surface coating in SPION-induced CARPA, we measured C activation by 6 different SPIONs in a human serum that is known to react to nanoparticles (NPs) with strong C activation. Remarkably, only the carboxymethyldextran-coated (ferucarbotran, Resosvist®) and dextran-coated (ferumoxtran-10, Sinerem®) SPIONs caused significant C activation, while the citric acid, phosphatidylcholine, starch and chitosan-coated SPIONs had no such effect. Focusing on Resovist and Sinerem, we found Sinerem to be a stronger activator of C than Resovist, although the individual variation in 15 different human sera was substantial. Further analysis of C activation by Sinerem indicated biphasic dose dependence and significant production of C split product Bb but not C4d, attesting to alternative pathway C activation only at low doses. Consistent with the strong C activation by Sinerem and previous reports of HSRs in man, injection of Sinerem in a pig led to dose-dependent CARPA, while Resovist was reaction-free. Using nanoparticle tracking analysis, it was further determined that Sinerem, but not Resovist, displayed multimodal size distribution and significant fraction of aggregates – factors which are known to promote C activation and CARPA. Taken together, our findings offer physicochemical insight into how key compositional factors and nanoparticle size distribution affect SPION-induced CARPA, a knowledge that could lead to the development of SPIONs with improved safety profiles

Liposome-induced hypersensitivity reactions : Risk reduction by design of safe infusion protocols in pigs

Intravenous administration of liposomal drugs can entail infusion reactions, also known as hypersensitivity reactions (HSRs), that can be severe and sometimes life-threatening in a small portion of patients. One empirical approach to prevent these reactions consists of lowering the infusion speed and extending the infusion time of the drug. However, different liposomal drugs have different levels of reactogenicity, which means that the optimal protocol for each liposomal drug may differ and should be identified and evaluated to make the treatment as safe and convenient as possible. The goal of the present study was to explore the use of pigs for the above purpose, using PEGylated liposomal prednisolone (PLP) as a model drug. We compared the reactogenicities of bolus versus infusion protocols involving 2-, 3- and 4-step dose escalations for a clinically relevant total dose, also varying the duration of infusions. The strength of HSRs was measured via continuous recording of hemodynamic parameters and blood thromboxane B2 levels. We showed that bolus administration or rapid infusion of PLP caused transient changes in systemic and pulmonary blood pressure and heart rate, most notably pulmonary hypertension with paralleling rises in plasma thromboxane B2. These adverse responses could be significantly reduced or eliminated by slow infusion of PLP, with the 3-h 3-step dose escalation protocol being the least reactogenic. These data suggest that the pig model enables the development of safe infusion protocols for reactogenic nanomedicines

Pseudo-anaphylaxis to polyethylene glycol (PEG)-coated liposomes: Roles of anti-PEG IgM and complement activation in a porcine model of human infusion reactions

Polyethylene glycol (PEG)-coated nanopharmaceuticals can cause mild to severe hypersensitivity reactions (HSRs), which can occasionally be life threatening or even lethal. The phenomenon represents an unsolved immune barrier to the use of these drugs, yet its mechanism is poorly understood. This study showed that a single i.v. injection in pigs of a low dose of PEGylated liposomes (Doxebo) induced a massive rise of anti-PEG IgM in blood, peaking at days 7–9 and declining over 6 weeks. Bolus injections of PEG-liposomes during seroconversion resulted in anaphylactoid shock (pseudo-anaphylaxis) within 2–3 min, although similar treatments of naı̈ve animals led to only mild hemodynamic disturbance. Parallel measurement of pulmonary arterial pressure (PAP) and sC5b-9 in blood, taken as measures of HSR and complement activation, respectively, showed a concordant rise of the two variables within 3 min and a decline within 15 min, suggesting a causal relationship between complement activation and pulmonary hypertension. We also observed a rapid decline of anti-PEG IgM in the blood within minutes, increased binding of PEGylated liposomes to IgM+ B cells in the spleen of immunized animals compared to control, and increased C3 conversion by PEGylated liposomes in the serum of immunized pigs. These observations taken together suggest rapid binding of anti-PEG IgM to PEGylated liposomes, leading to complement activation via the classical pathway, entailing anaphylactoid shock and accelerated blood clearance of liposome–IgM complexes. These data suggest that complement activation plays a causal role in severe HSRs to PEGylated nanomedicines and that pigs can be used as a hazard identification model to assess the risk of HSRs in preclinical safety studies

A porcine model of hemodialyzer reactions: roles of complement activation and rinsing back of extracorporeal blood

Hemodialysis reactions (HDRs) resemble complement-activation-related pseudoallergy (CARPA) to certain i.v. drugs, for which pigs provide a sensitive model. On this basis, to better understand the mechanism of human HDRs, we subjected pigs to hemodialysis using polysulfone (FX CorDiax 40, Fresenius) or cellulose triacetate (SureFlux-15UX, Nipro) dialyzers, or Dialysis exchange-set without membranes, as control. Experimental endpoints included typical biomarkers of porcine CARPA; pulmonary arterial pressure (PAP), blood cell counts, plasma sC5b-9 and thromboxane-B2 levels. Hemodialysis (60 min) was followed by reinfusion of extracorporeal blood into the circulation, and finally, an intravenous bolus injection of the complement activator zymosan. The data indicated low-extent steady rise of sC5b-9 along with transient leukopenia, secondary leukocytosis and thrombocytopenia in the two dialyzer groups, consistent with moderate complement activation. Surprisingly, small changes in baseline PAP and plasma thromboxane-B2 levels during hemodialysis switched into 30%–70% sharp rises in all three groups resulting in synchronous spikes within minutes after blood reinfusion. These observations suggest limited complement activation by dialyzer membranes, on which a membrane-independent second immune stimulus was superimposed, and caused pathophysiological changes also characteristic of HDRs. Thus, the porcine CARPA model raises the hypothesis that a second “hit” on anaphylatoxin-sensitized immune cells may be a key contributor to HDRs