Threshold-Switchable Particles to Treat Internal Hemorrhage
thesisposted on 31.10.2017, 00:00 authored by Alexander J Donovan
An artificial platelet nanotechnology with threshold-switchable procoagulant functionality is devised, employing one of the human body’s intrinsic hemostatic agents, inorganic polyphosphate (polyP). Inspired by the manner in which the anionic polyelectrolyte is stored in human platelets and how it exerts its hemostatic effects, polyP is nanoprecipitated in aqueous, polyvalent metal salt solutions. The particle formation is characterized by dynamic light scattering (DLS), and the particle morphology, structure, and elemental composition is determined by transmission electron microscopy (TEM) and energy-dispersive X-Ray spectroscopy (EDS). The ability for the polyP nanoparticles (NPs) to initiate blood coagulation in human plasma is accomplished by a standard turbidometric experiment assaying for contact pathway activation, validating that polyP NPs manifest robust procoagulant ability compared against the molecularly dissolved polymers of the same molecular weight. PolyP granules are stored in lipid bilayer shells approximately 250 nm across. These core-shell granular nanoparticles are referred to as dense granules in human platelets because of the presence of high molecular weight elements and their appearance under an electron microscope. A route to achieve a similar nanostructure is realized by brief ultrasonication of granular polyP NPs with sterically stabilized liposomes to give an Artificial Dense Granule (ADG). DLS was utilized to qualify colloidal stability and polyP encapsulation efficiency. High resolution imaging and two-dimensional spectroscopy are employed to verify the ADG core-shell structure and elemental distribution. A central design element of ADGs is to rapidly release the polyP cargo in the presence of high concentrations of phospholipase enzymes typically overexpressed in the blood stream adjacent to hemorrhagic bleeding sites. As a proof of concept, it is demonstrated that ADGs may initiate the contact pathway of blood coagulation in isolated protein assays and in human plasma only at above-threshold enzyme concentrations. In addition to its hemostatic functionality, polyP also serves as a generic molecular chaperone. Leveraging polyP’s role as a protein binder, polyP nanoprecipitation is further investigated for its ability to assist as a protein delivery vehicle using human factor VIII (FVIII) as a model protein. FVIII and polyP were co- nanoprecipitated in aqueous calcium and encapsulated in sterically stabilized liposomes. The ADG-FVIII NPs were subsequently assayed for their ability to clot FVIII-deficient plasma, a simplified model for Hemophilia A. However, further work is necessary to confirm the resulting particle’s morphology and the protein distribution. Although the groundwork has been created for a threshold-switchable procoagulant nanoparticle, additional ADG iterations must be created that are more structurally stable and more closely mimic actual human platelets. Further, these novel prototypes must be more rigorously tested in in vitro models of blood clotting to include the tissue factor and common pathways of coagulation and in in vivo models of hemorrhagic bleeding. A set of synthetic and experimental strategies will be presented in order to arrive at a bona fide, threshold-switchable nanoparticle hemostat.