011 | Physicochemical and biological evaluation of IgG binding to self-assembling dendrimer nanomicelles

Gabriele Cavalieri, Anna Laura La Monaca, Erik Laurini, Sabrina Pricl | Molecular Biology and Nanotechnology Laboratory - MolBNL@UniTS, Department of Engineering and Architecture - DEA, University of Trieste, Italy.

Self-assembling nanomaterials are emerging as versatile platforms in cancer research and therapeutics; however, their clinical translation critically depends on how they interact with serum proteins and blood components, which collectively define their biological identity. In particular, immunoglobulin G (IgG), one of the most abundant circulating antibodies, plays a key role in immune recognition, complement activation, and nanoparticle clearance. In this study, we present a comprehensive physicochemical and biological investigation of the interaction between a self-assembling amphiphilic dendrimer (AD) and human IgG, with the aim of correlating specific protein binding mechanisms to in vitro safety outcomes. The interaction between AD nanomicelles and IgG was characterized using UV–visible spectroscopy, fluorescence spectroscopy, circular dichroism (CD), and isothermal titration calorimetry. UV–vis absorption measurements revealed a concentration-dependent hypochromic effect without spectral shifts, indicating complex formation without significant perturbation of IgG chromophores. Steady-state fluorescence spectroscopy showed weak and reversible binding, in the micromolar-to-millimolar range. ITC measurements confirmed a spontaneous and exothermic interaction primarily driven by enthalpic contributions, consistent with electrostatic interactions and hydrogen bonding. Structural analyses by far-UV CD, supported by three-dimensional and synchronous fluorescence spectroscopy, demonstrated that IgG retains its β-sheet-rich secondary structure, thermal stability, and local aromatic microenvironment upon binding, suggesting surface-level interactions that do not induce antibody denaturation. Building on this molecular-level understanding, the impact of AD-IgG interactions was examined in the context of blood compatibility and immune-related safety. Notably, despite measurable IgG association, CH50 assays revealed no significant activation or depletion of the complement system, indicating that AD binding does not promote IgG-mediated complement triggering. This observation is consistent with the preserved structural integrity of IgG and suggests that antibody adsorption alone is insufficient to induce immune effector functions. Similarly, plasma clotting assays showed no procoagulant effects, further supporting the limited involvement of IgG-dependent pathways in coagulation activation. In contrast, hemolysis assays revealed a pronounced and concentration-dependent erythrocyte lysis, highlighting a distinct safety concern that appears decoupled from IgG interaction and immune activation. The strong hemolytic activity is instead attributed to direct physicochemical interactions between the amphiphilic dendrimer and erythrocyte membranes, reflecting the intrinsic membranolytic potential of the nanocarrier rather than an antibody- or complement-mediated mechanism. These findings emphasize the importance of integrating protein interaction studies with functional safety assays and underscore the need for surface engineering and dose optimization strategies in the development of dendrimer-based nanocarriers for cancer therapeutics.