RGDfK-functionalized gold nanorods bind only to activated platelets

Abstract

Integrin-targeting peptide RGDfK-labeled gold nanorods (GNR) seek to improve hyperthermia targeted to solid tumors by exploiting the known up-regulation of integrin αvβ3 cell membrane proteins on solid tumor vasculature surfaces. Tumor binding specificity might be expected since surrounding tissues and endothelial cells have limited numbers of these receptors. However, RGD peptide binding to many proteins is promiscuous, with known affinity to several families of cell integrin receptors, and also possible binding to platelets after intravenous infusion via a different integrin receptor, αIIbβ3, on platelets. Binding of RGDfK-targeted GNR could considerably impact platelet function, ultimately leading to increased risk of bleeding or thrombosis depending on the degree of interaction. We sought to determine if RGDfK-labeled GNR could interact with platelets and alter platelet function. Targeted and untargeted nanorods exhibited little interaction with resting platelets in platelet rich plasma (PRP) preparations. However, upon platelet activation, peptide-targeted nanorods bound actively to platelets. Addition of RGDfK-GNR to unactivated platelets had little effect on markers of platelet activation, indicating that RGDfK-nanorods were incapable of inducing platelet activation. We next tested whether activated platelet function was altered in the presence of peptide-targeted nanorods. Platelet aggregation in whole blood and PRP in the presence of targeted nanorods had no significant effect on platelet aggregation. These data suggest that RGDfK-GNR alone have little impact on platelet function in plasma. However, nonspecific nanorod binding may occur in vascular beds where activated platelets are normally cleared, such as the spleen and liver, producing a possible toxicity risk for these nanomaterials. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 209-217, 2017.

Keywords: activation; blood clot; coagulation; integrin; nanoparticles; peptide ligand.

FIGURE 1

RGDfK-labeled GNRs dβo not bind unactivated platelets. Purified platelets were pre-incubated with the indicated concentration of RGDfK-labeled GNRs or untargeted GNRs and then imaged using dark field microscopy (intrinsic GNR light scattering detected). The platelets were then imaged using dark field microscopy. Experiments were performed at least three times.

FIGURE 2

RGDfK-labeled GNRs bind activated platelets. Purified platelets were stimulated 0.1 U/mL thrombin in the presence of the indicated concentration of RGDfK-labeled GNRs or untargeted GNRs. Inset shows a higher magnification of unstimulated and thrombin stimulated platelets in the presence of 100 μg/mL RGDfK GNR. The platelets were then imaged using dark field microscopy. Experiments were performed at least three times.

FIGURE 3

Purified platelets in PRP are not activated by RGDfK-GNRs in a time-dependent manner. RGDfK-labeled GNRs at 10 μg/mL or 100 μg/mL were added to purified platelets for 30 minutes or 1 hour. After these incubation periods, platelets were stained with CD41 antibody, and activation was assessed by flow cytometry for CD62P expression and conformational changes in integrin αIIbβ3 through PAC-1 binding. Positive staining was confirmed by activating platelets with the PAR1 agonist, thrombin (0.1 U/mL, final) compared to nontreated purified platelets (NT). Averages± standard deviations are shown from an n ≥ 3. * indicates significantly different compared to other conditions tested (p>0.05).

FIGURE 4

RGDfK-labeled GNRs do not induce platelet activation in whole blood. RGDfK-labeled GNRs at 10 μg/mL or 100 μg/mL were added to healthy human citrated blood and incubated for 30 min. After 30 min, platelets were stained with a CD41 antibody and activation was assessed by flow cytometry for CD62P expression and conformational changes in integrin αIIbβ3 (PAC-1 binding). Positive staining was confirmed by activating platelets with the PAR1 agonist, TRAP (10 μM, final) compared to nontreated whole blood (NT). Averages±standard deviations are shown from an n ≥ 3. * indicates significantly different compared to other conditions tested (p>0.05).

FIGURE 5

RGDfK-labeled GNRs do not alter platelet aggregation. Purified platelets in PRP were left untreated or treated with 1 μg/mL RGDfK- or RGEfK-labeled (control) GNRs. The platelets were then stimulated with 2.5 μg/mL collagen and platelet aggregation was monitored using Chrono-log platelet aggregator. A representative tracing for purified platelet PRP treated with RGDfK (blue) and RGEfK control (black) are shown above (A). Citrated whole blood was left untreated or treated with 10 μg/mL RGDfK or RGEfK-labeled GNRs. Whole blood was then stimulated with 10 μg/mL collagen and platelet aggregation was monitored using Chrono-log whole blood aggregometry. A representative tracing for whole blood treated with RGDfK (blue) and RGEfK (black) are shown above (B). N>3 for each condition examined.