21 research outputs found
Transferrin coated nanoparticles: Study of the bionano interface in human plasma
It is now well established that the surface of nanoparticles (NPs) in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC) and after washing (hard corona, HC) in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments
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Delivery of thrombolytic therapy using rod-shaped plant viral nanoparticles decreases the risk of hemorrhage
Cardiovascular thrombotic disease is an underlying cause of stroke, myocardial infarction and pulmonary embolism - some of the leading causes of death worldwide. Reperfusion therapy with anticoagulant, antiplatelet, and fibrinolytic agents has significantly reduced early mortality and morbidity from acute myocardial infarction and stroke. Nevertheless, bleeding side effects (e.g., intracranial hemorrhage) associated with the anti-thrombotic therapy can offset its benefits and limit its applicability to strictly defined short therapeutic windows. We have previously shown that elongated plant virus based nanoparticles can target cardiovascular thrombi and exhibited their utility for the delivery of streptokinase in an ex vivo model of thrombosis. Herein, we build upon our previous findings and demonstrate plant viral delivery of the current standard-of-care tissue plasminogen activator (tPA). Studies on a pre-clinical mouse model of arterial thrombosis indicate that while the therapeutic efficacy of free tPA and tPA-conjugated TMV are similar, the safety profile of the tPA-TMV formulation is improved, i.e. administration of the latter has less impact on hemostasis as demonstrated by decreased bleeding time. Thus, our data suggest that TMV-based delivery of thrombolytic therapies could be a promising and safer alternative to reperfusion therapy with the tPA
Reversible <i>versus</i> Irreversible Binding of Transferrin to Polystyrene Nanoparticles: Soft and Hard Corona
Protein adsorption to nanoparticles (NPs) is a key prerequisite to understand NP–cell interactions. While the layer thickness of the protein corona has been well characterized in many cases, the absolute number of bound proteins and their exchange dynamics in body fluids is difficult to assess. Here we measure the number of molecules adsorbed to sulfonate (PSOSO<sub>3</sub>H) and carboxyl-(PSCOOH) polystyrene NPs using fluorescence correlation spectroscopy. We find that the fraction of molecules bound to NPs falls onto a single, universal adsorption curve, if plotted as a function of molar protein-to-NP ratio. The adsorption curve shows the build-up of a strongly bound monolayer up to the point of monolayer saturation (at a geometrically defined protein-to-NP ratio), beyond which a secondary, weakly bound layer is formed. While the first layer is irreversibly bound (hard corona), the secondary layer (soft corona) exhibits dynamic exchange, if competing unlabeled is added. In the presence of plasma proteins, the hard corona is stable, while the soft corona is almost completely removed. The existence of two distinct time scales in the protein off-kinetics, for both NP types studied here, indicates the possibility of an exposure memory effect in the NP corona
DCS measurements of Tf coated NP-corona complexes before and after incubation in plasma.
<p>A–C, apparent size comparison between Tf coated NPs dispersed in PBS, NPs incubated in plasma and isolated by spinning (so called hard corona) and NPs incubated in plasma <i>“in situ”</i> (so called full corona); D–F, the particle-corona complexes were isolated after either 5 min or 1 hour of incubation in the biological fluid. DCS experiment for the same NPs in PBS is reported for comparison in all graphs.</p
Strategies of PS NPs functionalization with Tf.
<p>Cartoon representation of 100 nm polystyrene NPs modified with sulphonated groups (PSOSO<sub>3</sub>H, upper) and carboxylic groups (PSCOOH, down) coated with Tf by either physical adsorption (Tf@PSOSO<sub>3</sub>H and <a href="mailto:Tf@PSCOOH" target="_blank">Tf@PSCOOH</a>) or covalent coupling (Tf-PSCOOH): A, pristine NPs; B, Tf coated NPs; C, Tf coated NPs after incubation in human plasma. Both Tf@PSOSO<sub>3</sub>H and Tf-PSCOOH NPs are stable in human plasma, while Tf on <a href="mailto:Tf@PSCOOH" target="_blank">Tf@PSCOOH</a> NPs is easily replaced by environmental proteins.</p
Mass spectrometry data for human Tf amount on precoated PS nanoparticles before and after incubation in human plasma.
<p>Mass spectrometry data for human Tf amount on precoated PS nanoparticles before and after incubation in human plasma.</p
DCS characteristics of Tf precoated NPs; hard corona (HC) and full corona (FC).
<p>DCS characteristics of Tf precoated NPs; hard corona (HC) and full corona (FC).</p
Human protein arrays positive hits comparison.
<p>A, graph of total positive hits for bare and functionalized NPs in PBS and 10% human plasma solution; B, fluorescence intensity comparison for positive hits, between bare PSOSO<sub>3</sub>H NPs and Tf@PSOSO<sub>3</sub>H in PBS; C, fluorescence intensity comparison for positive hits, between PSOSO<sub>3</sub>H NPs and Tf@PSOSO<sub>3</sub>H in human plasma.</p
Characterization of Tf coated NPs in PBS at room temperature.
<p>Differential centrifugal sedimentation (DCS) results for: A, 100 nm PSOSO<sub>3</sub>H NPs and Tf@PSOSO<sub>3</sub>H NPs; B, 100 nm PSCOOH NPs and <a href="mailto:Tf@PSCOOH" target="_blank">Tf@PSCOOH</a> NPs and C, 100 nm PSCOOH NPs and Tf-PSCOOH. All size distributions show a shift towards larger apparent sizes in the presence of Tf coating. D, immuno detection of Tf coated NPs against monoclonal anti-Tf and anti-HAS.</p