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Tamás Mészáros,1,2,* Gergely Tibor Kozma,2,* Taro Shimizu,3 Koga Miyahara,3 Keren Turjeman,4 Tatsuhiro Ishida,3 Yechezkel Barenholz,4 Rudolf Urbanics,1,2 János Szebeni1,2,5 1Nanomedicine Research and Education Center, Department of Pathophysiology, Semmelweis University, Budapest, Hungary; 2SeroScience Ltd, Budapest, Hungary; 3Department of Pharmacokinetics and Biopharmaceutics, Institute of Biomedical Sciences, Tokushima University, Tokushima, Japan; 4Laboratory of Membrane and Liposome Research, Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, Jerusalem, Israel; 5Department of Nanobiotechnology and Regenerative Medicine, Faculty of Health, Miskolc University, Miskolc, Hungary *These authors contributed equally to this work 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. Keywords: adverse drug reactions, immunotoxicity, nanoparticles, pseudoallergy, anaphylatoxins, PIM cells, phagocytosis |
