Development and characterization of soluplus® nanomicelles associated to specific igg as an innovative strategy for the detection and neutralization of shiga toxin type 2

Abstract

Shiga toxin type 2 (Stx2) is the main virulence factor of Shiga toxinproducing Escherichia coli and is responsible for triggering HemolyticUremic Syndrome (HUS). We aimed to develop and characterizepolymeric nanomicelles (PN) with the amphiphilic polymerSoluplus® coupled to anti-Stx2 IgG in order to introduce innovativeproposals for the detection of Stx2 and treatment of HUS. PN ofSoluplus® were formulated in PBS and coupled with IgG anti Stx2from hyperimmune (PN-IgG-Stx2) or control bovine colostrum (PNIgG-Ctrl). The hydrodynamic size of PN, PN-IgG-Stx2 and PN-IgGCtrlwas evaluated by Dynamic Light Scattering. Morphology of PNor PN-IgG-Stx2 was analyzed by Transmission Electron Microscopy(TEM). The PN toxicity was evaluated on both Vero and Human GlomerularEndothelial cells (HGEC) and cell viability was determinedby neutral red uptake. After coupling PN with IgG, Stx2 neutralizationcapacity of PN-IgG-Stx2 or PN-IgG-Ctrl was evaluated on Vero andHGEC cells and the percentage of cell viability was analyzed. Thehydrodynamic size of the PN of Soluplus® and IgG-Stx2 showed anaverage diameter of 70.2 ± 1.5 nm and 40.9 ± 2 nm, respectively.When both components were coupled, a single peak with a similarhydrodynamic size of the PN was observed (70.4 ± 0.2 nm). TEManalysis revealed circular particles with a diameter corresponding to100 nm either in PN and PN-IgG-Stx2 particles. PN-IgG-Stx2 wereable to neutralize Stx2 on Vero and HGEC cells in a dose dependentmanner. When comparing the neutralization capacity of Stx2 by IgGStx2vs PN-IgG-Stx2 a significant improvement in the cell viabilityof Vero and HGEC was observed with the PN-IgG-Stx2 (p<0.001).The association between anti-Stx2 IgG from bovine colostrum andSoluplus® PN was optimized and characterized. Encouraging resultsof antibody functionality coupled with PN were registered.These results may open the perspective of the design of new nanoplatformsfor neutralization and/or detection of Stx2.