Involvement of complement activation in the pulmonary vasoactivity of polystyrene nanoparticles in pigs: unique surface properties underlying alternative pathway activation and instant opsonization

Abstract

BackgroundIt 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.GoalsTo characterize PS-NPs and reexamine the contribution of CARPA to their pulmonary reactivity in pigs.Study designC 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.MethodsPhysicochemical 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.ResultsPS-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.ConclusionPS-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.

<p>Acute physiological changes caused by complement activators and amphotericin B-containing liposomes in mice</p>

“…Anaphylatoxins and secondary mediators can all contribute to both effects, one example being the C5aR mediated hypotension in rodents. 3 However, in the absence of C5a measurement we cannot confirm or contradict a role of C5a in these changes, and the relative contributions of different activation mechanisms [28][29][30] (Figure 7) need to be explored in further studies.…”

“…Furthermore, in mice, the initial hemodynamic change is hypertension, which is followed (or not followed) by hypotension, while in pigs and rats the primary response is often hypotension. 3,20,28 In pigs, the initial hypotension may split the pulmonary hypertension to become biphasic. 20,28 Mechanism of hemodynamic changes: the puzzle of liposome reactions…”

Human Clinical Relevance of the Porcine Model of Pseudoallergic Infusion Reactions

“…Table 1 lists 30 experimental studies [15, which utilized the pig model to analyze the cardiopulmonary adverse effects of different nanoparticles (NPs) or other agents. Some of these studies highlighted the concordance of HSR symptoms in pigs and hypersensitive patients [25][26][27][28][29]32,33,[46][47][48], others addressed the mechanism of HSRs [15,[21][22][23][24][25][26][27][28][29][30][31][32][33]36,[41][42][43][44][45][46][47], and yet others focused on the prevention of HSRs by pharmacological intervention [15], or by optimizing the structure [24,44] or administration protocol [24,37,48] of NPs. Importantly, many of these studies were initiated mainly for preclinical safety evaluation of nanomedicines [15,22,23,30,31,35,…”

“…PS-NPs were opsonized in pig serum by C3 derivatives, indicating C activation. [45] Hemostatic NPs based on PEG-PLGA-PLL-PEG-cRGD copolymers, developed to control traumatic blood loss…”

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

“…Other details of surgery, instrumentation and hemodynamic analysis were described in the original studies. 51,52 Testing procedure Baseline blood samples were taken, and the physiological parameters were recorded for 15 minutes before the first injection of the test material. Initially pig 1 and pig 2 were injected with previously prepared TT1-HSA-NPs (2.5 mg in 20 mL (=0.125 mg NP/mL for 0.1 mg/kg) and 25 mg in 20 mL (1.25 mg NP/mL for 1 mg/kg).…”