Our study employs a combination of experimental techniques—fluorescence and UV spectroscopy, isothermal titration calorimetry (ITC), TEM microscopy, dynamic light scattering and circular dichroism (CD)—alongside molecular dynamics simulations to systematically investigate binding affinities, thermodynamic parameters, kinetics and structural implications of interactions with key plasma proteins, such as human serum albumin (HSA) and immunoglobulin G (IgG). The integrative nature of this approach ensures a thorough understanding of the molecular mechanisms governing these interactions, which is essential for optimizing drug and gene delivery systems.
We focused on the interaction between HSA or IgG and a self-assembling amphiphilic dendrimer (AD), often studied for its ability to efficiently transport both genes (like small-interfering RNA) and drugs, to explore protein corona formation. Our findings reveal that dendrimer binding to both proteins is moderate and primarily driven by electrostatic interactions. Structural analysis via circular dichroism demonstrated that HSA experiences minimal conformational changes upon complexation, maintaining its secondary structure integrity. Complementary molecular dynamics simulations confirmed stable interactions at the atomistic level, providing insight into the physicochemical stability and compatibility of dendrimers as nanocarriers. These results support the potential utility of amphiphilic dendrimers in drug and gene delivery applications by minimizing undesired protein conformational alterations while maintaining stable complex formation.
In parallel, we investigated the interactions of small-molecule inhibitors used in melanoma therapy, particularly BRAF and MEK inhibitors, with HSA to assess their pharmacokinetic profiles. The study examined Vemurafenib, Dabrafenib, Encorafenib (BRAFi) and Binimetinib (MEKi), where fluorescence quenching, ITC, and molecular modeling were employed to elucidate binding mechanisms. Results indicate that first two BRAFi and the couple Encorafenib and Binimetinib binds respectively Sudlow’s site II and site I, without significantly altering HSA's secondary structure. Both couples have a similar affinity for HSA but guided by a different binding mechanism: entropy-driven and enthalpic-driven, respectively. Specifically for the couple Vemurafenib and Dabrafenib kinetic parameter like the residence time has been computationally calculated: Vemurafenib, due to its specific binding mechanism, exhibits a longer residence time, suggesting enhanced stability and prolonged circulation time.
Beyond protein interactions, the study extended this methodology to evaluate possible off-target effects by analyzing the interaction of Vemurafenib with calf thymus DNA (ctDNA). Spectroscopic and calorimetric data, combined with molecular dynamics simulations, identified a minor groove-binding mechanism, characterized by minimal structural perturbation of the DNA double helix. This insight is pivotal in understanding the potential genotoxic effects of small-molecule inhibitors, ensuring the safety and efficacy.
The multidisciplinary framework employed in this study provides a robust and reproducible methodology for systematically evaluating the interaction profiles of both nanocarrier-based and small-molecule drug systems. By integrating diverse experimental techniques with computational insights, this work advances the understanding of how molecular interactions impact the pharmacokinetic behavior and efficacy of drug and gene delivery platforms. Ultimately, these findings contribute to the rational design and optimization of more effective and stable delivery systems, addressing critical challenges in cancer therapy and beyond.