Nanogel Drug Carriers Presenting Platelet GPIbα Mimic and Enhance Platelet Adhesion

Lysozyme‐dextran nanogels are nanostructures composed of a hydrophobic crystalline lysozyme core embedded in a highly hydrophilic dextran polysaccharide network. As biomedical agents, nanogels have promising transport properties and a capacity for hydrophilic swelling that enables facile drug loading and delivery. The initial adherence of platelets under prothrombotic conditions is strongly dependent on the interaction between the GPIbα glycoprotein subunit on the platelet surface and von Willebrand Factor (vWF) multimers present at the site of vascular injuries. This work focuses on lysozyme‐dextran nanogels presenting human GPIbα, characterizing capacity to mimic the vWF targeting and pro‐hemostatic capacity of platelets. Lysozyme‐dextran nanogels were synthesized via Maillard conjugation followed by heat gelation. Periodate oxidation permitted addition of GPIbα to the nanogels via amine‐aldehyde conjugation. Nanogel hydrodynamic diameter was assessed before and after GPIbα addition. Nanogels were added to platelet rich plasma (PRP) to assess impact on platelet agglutination (via ristocetin stimulation) and aggregation (via phorbol 12‐myristate 13‐acetate). Binding of rhodamine‐labeled non‐functional and GPIbα nanogels in buffer to vWF‐coated slides was assessed under set shear rates. Nanogels were implemented in whole blood in endothelialized (HUVECs) microfluidics channels under arterial shear. Aggregation of calcein‐labeled platelets and rhodamine‐labeled nanogels was assessed following pro‐thrombotic hematoporphyrin injury. GPIbα nanogels manifest a 304nm hydrodynamic diameter (compared to 288nm before addition of GPIbα). GPIbα nanogels at .25mg/ml in PRP accelerate ristocetin‐induced agglutination (n=2) ( Figure 1), but have no effect on PMA‐induced aggregation (n=3). GPIbα nanogels bind to vWF‐coated plates in greater quantities than non‐functional nanogels at shear rates of 1000s −1 and 5000s −1 (n=3, 2). After a photochemical injury in endothelialized microfluidics, red fluorescent GPIbα nanogels co‐localize with aggregating platelets, whereas non‐functional control nanogels do not ( Figure 2). Quantifying the extent of platelet and nanogel accumulation following injury, GPIbα nanogels (n=6) accumulated in greater quantities coincident with greater quantities of platelet adhesion ( Figure 3A) as compared to control nanogels (n=7) ( Figure 3B). GPIbα nanogels show capacity for specific binding to vWF and participation in and enhancement of platelet adhesion and agglutination dependent on the platelet GPIbα‐vWF interaction. GPIbα nanogels have potential as pro‐hemostatic agents and as drug delivery agents targeted to sites of platelet adhesion. 1Platelet agglutination with and without GPIbα nanogels2Platelet (green) and nanogel (red) accumulation following prothrombotic injury in a microfluidic model. A‐GPIbαnanogels, B‐non‐functional control nanogels3Quantification of platelet (green) and nanogel (red) binding following prothrombotic injury. Nanogel binding not coincident with platelet binding is depicted in black. A‐GPIbα nanogels, B‐non‐functional control nanogels