New Approaches for Basophil Activation Tests Employing Dendrimeric Antigen-Silica Nanoparticle Composites

Abstract

In vitro cell activation through specific IgE bound to high-affinity receptors on the basophil surface is a widely used strategy for the evaluation of IgE-mediated immediate hypersensitivity reactions to betalactams. Cellular activation requires drug conjugation to a protein to form a large enough structure displaying a certain distance between haptens to allow the cross-linking of two IgE antibodies bound to the basophil's surface, triggering their degranulation. However, no information about the size and composition of these conjugates is available. Routine in vitro diagnosis using the basophil activation test uses free amoxicillin, which is assumed to conjugate to a carrier present in blood. To standardize the methodology, we propose the use of well-controlled and defined nanomaterials functionalized with amoxicilloyl. Silica nanoparticles decorated with PAMAM-dendrimer-amoxicilloyl conjugates (NpDeAXO) of different sizes and amoxicilloyl densities (50-300 µmol amoxicilloyl/gram nanoparticle) have been prepared and chemically characterized. Two methods of synthesis were performed to ensure reproducibility and stability. Their functional effect on basophils was measured using an in-house basophil activation test (BAT) that determines CD63⁺ or CD203c^high activation markers. It was observed that NpDeAXO nanocomposites are not only able to specifically activate basophils but also do so in a more effective way than free amoxicillin, pointing to a translational potential diagnosis.

Keywords: PAMAM dendrimers; basophil activation test; dendrimeric antigens; drug allergy diagnosis; silica nanoparticles.

Figure 1

General procedure for the chemical modification of ϕdNp dispersions and 50Nps with different DeAXO densities in their surface.

Figure 2

NMR spectra of (a) AX in basic D₂O and (b) 50NpDeAXO in D₂O suspensions.

Figure 3

Basophil activation test (BAT) dose–response curves of NpDeAXO of different sizes: 20 nm (A,B), 30 nm (C,D), and 50 nm (E,F) and (G,H). Np dispersions or solid-state Nps labels at the top of the figure only indicate the synthetic methodology used for Np preparation. Black lines represent healthy controls (HCs), and blue and green lines represent allergic patients (APs). Size sample included HCs (N = 10) and APs (N = 10) for Nps synthesized as dispersions (A–F), while HCs (N = 45) and APs (N = 54) were included in the study for the Nps synthesized as a solid state. * p < 0.05, ** p < 0.01.

Figure 4

BAT dose–response curves of Nps with different DeAXO surface densities: 300 µmol AXO/gNp (A,B); 130 µmolAXO/gNp (C,D); 100 µmol AXO/gNp (E,F); 80 µmol AXO/gNp (G,H); 50 µmol AXO/gNp (I,J). Black lines represent healthy controls (HCs), and blue and green lines represent allergic patients (APs). Size sample included HCs (N = 6) and APs (N = 4) (A–J). * p < 0.05, ** p < 0.01.

Figure 5

BAT dose–response curves using 50NpDeAXO and free AX at three different concentrations. APs (N = 54) are depicted by blue or green lines and HCs (N = 45) are depicted by black lines. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Percentage of positive cases in allergic patients (N = 54) in BAT using CD63 (A) and CD203c^high (B) as basophil activation markers. Positive cases were obtained after using the cut-offs described in Table S4 for AX and 50NpDeAXO at the different concentrations with both CD63 and CD203c.