Can the biomolecular corona induce an allergic reaction?-A proof-of-concept study

Abstract

Ferumoxytol nanoparticles are being used clinically for the treatment of anemia and molecular imaging in patients. It is well documented that while most patients tolerate ferumoxytol well, a small percentage of patients (i.e., 0.01%) develop severe allergic reactions. The purpose of our proof-of-concept study was to determine whether patients with or without hypersensitivity reactions have specific protein corona profiles around ferumoxytol nanoparticles. In a retrospective, institutional review board approved pilot study, we enrolled 13 pediatric patients (5 girls, 8 boys, mean age 16.9 ± 8.2 years) who received a ferumoxytol-enhanced magnetic resonance imaging and who did (group 1, n = 5) or did not (group 2, n = 8) develop an allergic reaction. Blood samples of these patients were incubated with ferumoxytol, and the formation of a hard protein corona around ferumoxytol nanoparticles was measured by dynamic light scattering, zeta potential, and liquid chromatography-mass spectrometry. We also performed in vitro immune response analyses to randomly selected coronas from each group. Our results provide preliminary evidence that ex vivo analysis of the biomolecular corona may provide useful and predictive information on the possibility of severe allergic reactions to ferumoxytol nanoparticles. In the future, patients with predisposition of an allergic reaction to ferumoxytol may be diagnosed based on the proteomic patterns of the corona around ferumoxytol in their blood sample.

FIG. 1.

(a) Plasma protein NpSpC profile shows the similarities and differences across the allergic and healthy individuals. (b) 23 proteins are over- or under-represented in allergic vs healthy subjects (excluding allergic sample 5; the nonquantified proteins were given a value of 1e−6). (c) Stack plot shows the average NpSpC of the 23 proteins from volcano plot on (b), in protein coronas obtained from healthy vs allergic subjects. (d) Proteins found exclusively in the biomolecular/protein corona of allergic or healthy patients are shown in the pine plot. For instance, a percentage of 80 on the allergic side means that V2-7 is quantified in four out of five allergic samples and in none of the healthy samples.

FIG. 2.

Ferumoxytol does not activate healthy whole blood-derived basophils: Flow cytometry analysis of 300 ng/ml free iron (light blue), media alone (pink), 15 mg/ml carboxymethyldextran (blue), or 300 ng/ml (green), 1.5 mg/ml (red), or 15 mg/ml ferumoxytol (black), incubated with healthy control whole blood-derived basophils. Median fluorescence intensity of the CD63 marker (an activator of basophils) is graphed on the x axis; less than 10³ is negative.

FIG. 3.

Ferumoxytol does not cause an IgE-mediated immune response: Patient's plasma with ferumoxytol + protein corona (blue) was incubated with whole blood-derived basophils with (a) and without anti-IgE (b). Negative controls used were media alone (green), ferumoxytol alone (overlap with green), and plasma from healthy controls (overlap with green). Positive control used was polyclonal IgE (orange). Flow cytometry indicates that plasma proteins of patient's ferumoxytol corona can activate basophils; however, in the presence of anti-IgE, basophil response was significantly decreased as determined by CD63 log fold changes [CD63 expression via median fluorescence intensity (x axis) on gated basophils was detected by validated methods to indicate basophil reactivity].

FIG. 4.

Ferumoxytol + protein corona but not ferumoxytol alone induces proliferation of effector memory T cells, consistent with a type IV hypersensitivity reaction. CD4+CD3+ T cells from a patient with an allergic reaction against ferumoxytol (a) and an age-matched healthy control (b) were stained with CFSE and incubated for 3 days with ferumoxytol and patient's plasma (left panel), or ferumoxytol alone (right panel). Flow cytometry analysis showed that ferumoxytol with the protein corona (from the patient plasma) was associated with CD4+ T-cell proliferation, whereas ferumoxytol alone did not increase proliferation above background (background not shown). The y axis represents side scatter (SSC) indicating cell granularity (%) of proliferating populations. The x axis (CFSE) distinguishes proliferating vs nonproliferating populations.