RESUMO
Ultrasmall gold nanoparticles (usNPs) and nanoclusters are an emerging class of nanomaterials exhibiting distinctive physicochemical properties and in vivo behaviors. Although understanding the interactions of usNPs with blood components is of fundamental importance to advance their clinical translation, currently, little is known about the way that usNPs interact with the hemostatic system. This study describes the effects of a model anionic p-mercaptobenzoic acid-coated usNP on the coagulation cascade, with particular emphasis on the contact pathway. It is found that in a purified system, the anionic usNPs bind to and activate factor XII (FXII). The formed usNP-FXII complexes are short-lived (residence time of â¼10 s) and characterized by an affinity constant of â¼200 nM. In human plasma, the anionic usNPs activate the contact pathway and promote coagulation. The usNPs also exhibit anticoagulant activity in plasma by interfering with the thrombin-mediated cleavage of fibrinogen. Taken together, these findings establish that anionic usNPs can disturb the normal hemostatic balance, which in turn may hinder their clinical translation. Finally, it is shown that usNPs can be designed to be nearly inert in plasma by surface coating with the natural peptide glutathione.
Assuntos
Hemostáticos , Nanopartículas Metálicas , Anticoagulantes/farmacologia , Fator XII/química , Fator XII/metabolismo , Fibrinogênio , Glutationa , Ouro/química , Ouro/farmacologia , Humanos , Nanopartículas Metálicas/química , Trombina/metabolismoRESUMO
To date, extensive effort has been devoted toward the characterization of protein interactions with synthetic nanostructures. However, much remains to be understood, particularly concerning microscopic mechanisms of interactions. Here, we have conducted a detailed investigation of the kinetics of nanoparticle-protein complexation to gain deeper insights into the elementary steps and molecular events along the pathway for complex formation. Toward that end, the binding kinetics between p-mercaptobenzoic acid-coated ultrasmall gold nanoparticles (AuMBA) and fluorescently-labeled ubiquitin was investigated at millisecond time resolution using stopped-flow spectroscopy. It was found that both the association and dissociation kinetics consisted of multiple exponential phases, hence suggesting a complex, multi-step reaction mechanism. The results fit into a picture where complexation proceeds through the formation of a weakly-bound first-encounter complex with an apparent binding affinity (KD) of â¼9 µM. Encounter complex formation is followed by unimolecular tightening steps of partial desolvation/ion removal and conformational rearrangement, which, collectively, achieve an almost 100-fold increase in affinity of the final bound state (apparent KD â¼0.1 µM). The final state is found to be weakly stabilized, displaying an average lifetime in the range of seconds. Screening of the electrostatic forces at high ionic strength weakens the AuMBA-ubiquitin interactions by destabilizing the encounter complex, whereas the average lifetime of the final bound state remains largely unchanged. Overall, our rapid kinetics investigation has revealed novel quantitative insights into the molecular-level mechanisms of ultrasmall nanoparticle-protein interactions.
Assuntos
Ouro , Nanopartículas Metálicas , Cinética , Concentração Osmolar , Eletricidade EstáticaRESUMO
Nanomaterials displaying well-tailored sizes and surface chemistries can provide novel ways with which to modulate the structure and function of enzymes. Recently, we showed that gold nanoparticles (AuNPs) in the ultrasmall size regime could perform as allosteric effectors inducing partial inhibition of thrombin activity. We now find that the nature of the AuNP surface chemistry controls the interactions to the anion-binding exosites 1 and 2 on the surface of thrombin, the allosterically induced changes to the active-site conformation, and, by extension, the enzymatic activity. Ultrasmall AuNPs passivated with p-mercaptobenzoic acid ligands (AuMBA) and a peptide-based (Ac-ECYN) biomimetic coat (AuECYN) were utilized in our investigations. Remarkably, we found that while AuMBA binds to exosites 1 and 2, AuECYN interacts primarily with exosite 2. It was further established that AuMBA behaves as a "mild denaturant" of thrombin leading to catalytic dysfunction over time. Conversely, AuECYN resembles a proper allosteric effector leading to partial and reversible inhibition of the activity. Collectively, our findings reveal how the distinct binding modes of different AuNP types may uniquely influence thrombin structure and catalysis. The present study further contributes to our understanding of how synthetic nanomaterials could be exploited in the allosteric regulation of enzymes.
Assuntos
Nanopartículas Metálicas , Trombina , Regulação Alostérica , Sítios de Ligação , Ouro , LigantesRESUMO
Synthetic ultrasmall nanoparticles (NPs) can be designed to interact with biologically active proteins in a controlled manner. However, the rational design of NPs requires a clear understanding of their interactions with proteins and the precise molecular mechanisms that lead to association/dissociation in biological media. Although much effort has been devoted to the study of the kinetics mechanism of protein corona formation on large NPs, the nature of NP-protein interactions in the ultrasmall regime is radically different and poorly understood. Using a combination of experimental and computational approaches, we studied the interactions of a model protein, CrataBL, with ultrasmall gold NPs passivated with p-mercaptobenzoic acid (AuMBA) and glutathione (AuGSH). We have identified this system as an ideal in vitro platform to understand the dependence of binding affinity and kinetics on NP surface chemistry. We found that the structural and chemical complexity of the passivating NP layer leads to quite different association kinetics, from slow and reaction-limited (AuGSH) to fast and diffusion-limited (AuMBA). We also found that the otherwise weak and slow AuGSH-protein interactions measured in buffer solution are enhanced in macromolecular crowded solutions. These findings advance our mechanistic understanding of biomimetic NP-protein interactions in the ultrasmall regime and have implications for the design and use of NPs in the crowded conditions common to all biological media.