DNA Origami–Platelet Adducts: Nanoconstruct Binding without Platelet Activation

Abstract

Objective. Platelets are small, mechanosensitive blood cells responsible for maintaining vascular integrity and activatable on demand to limit bleeding and facilitate thrombosis. While circulating in the blood, platelets are exposed to a range of mechanical and chemical stimuli, with the platelet membrane being the primary interface and transducer of outside-in signaling. Sensing and modulating these interface signals would be useful to study mechanochemical interactions; yet, to date, no methods have been defined to attach adducts for sensor fabrication to platelets without triggering platelet activation. We hypothesized that DNA origami, and methods for its attachment, could be optimized to enable nonactivating instrumentation of the platelet membrane. Approach and Results. We designed and fabricated multivalent DNA origami nanotile constructs to investigate nanotile hybridization to membrane-embedded single-stranded DNA-tetraethylene glycol cholesteryl linkers. Two hybridization protocols were developed and validated (Methods I and II) for rendering high-density binding of DNA origami nanotiles to human platelets. Using quantitative flow cytometry, we showed that DNA origami binding efficacy was significantly improved when the number of binding overhangs was increased from two to six. However, no additional binding benefit was observed when increasing the number of nanotile overhangs further to 12. Using flow cytometry and transmission electron microscopy, we verified that hybridization with DNA origami constructs did not cause alterations in the platelet morphology, activation, aggregation, or generation of plateletderived microparticles. Conclusions. Herein, we demonstrate that platelets can be successfully instrumented with DNA origami constructs with no or minimal effect on the platelet morphology and function. Our protocol allows for efficient high-density binding of DNA origami to platelets using low quantities of the DNA material to label a large number of platelets in a timely manner. Nonactivating platelet−nanotile adducts afford a path for advancing the development of DNA origami nanoconstructs for celladherent mechanosensing and therapeutic agent delivery.

Shear-Mediated Platelet Microparticles Demonstrate Phenotypic Heterogeneity as to Morphology, Receptor Distribution, and Hemostatic Function

“…In sheared platelets, the number of secretory granules greatly decreased, with the remaining granules grouped together and closely colocalized with the open canalicular system, indicating ongoing degranulation. 98,102 It was previously reported that similar morphological changes accompanied PDMP formation in response to activation by strong biochemical agonists. 103 Microvesiculation induced by thrombin and calcium ionophore was associated with filopodia formation, dilatation of the open canalicular system, degranulation, and cell defragmentation.…”

“…The arbitrary density of receptor distribution on the platelet surface was calculated by normalizing the fluorescence intensity (MFI) of platelet or microparticle to their forward scatter (MFS), a well-known flow cytometric parameter indicating particle size. 37, 98 In Figures S2 & S3 , alterations of fluorescence intensity, size, and receptor density with shear exposure or sonication are reported. Shear stress did not largely affect CD41 and CD42a fluorescence intensity ( Fig.…”

“…96, 97 The arbitrary density of surface receptors on platelet and microparticle surfaces was calculated as the median fluorescence intensity (MFI) of the population normalized to the median FSC indicating platelet or microparticle size. 37, 98…”

“…96,97 The arbitrary density of surface receptors on platelet and microparticle surfaces was calculated as the median fluorescence intensity (MFI) of the population normalized to the median FSC indicating platelet or microparticle size. 37,98 Thrombin generation on platelets and microparticles. The effect of platelets and PDMPs on prothrombin activation by factor Xa was assessed using the modified thrombin generation assay.…”

Clinically Expired Platelet Concentrates as a Source of Extracellular Vesicles for Targeted Anti-Cancer Drug Delivery

“…Transmission electron microscopy (TEM) analysis confirmed that platelets displayed typical size heterogeneity and a discoid or spherical shape ( Figure 1 C). Platelets with elongated cell membrane projections and with a decrease in platelet size compared to resting platelets were also observed, which is typical of activated platelets [ 15 , 42 ].…”