| Description |
The need for nanoparticle toxicity evaluations is well recognized as the number of applications for nanoparticles continues to grow. This dissertation seeks to reach beyond presently available assessments of nanoparticle-mediated toxicity and mortality to assess the consequences of intravenous nanoparticle injection upon the cellular and molecular participants in the hemostatic response. Based on published reports of severe in vivo coagulopathy, in vitro platelet aggregation and hemolysis for cationic poly(amido amine) (PAMAM) dendrimer nanoparticles, cationic generation-7 PAMAM dendrimers (G7-NH2) were hypothesized to possess strong and specific hemostatic properties and were utilized in the adaptation and development of assays evaluating nanoparticle procoagulant properties. The latter part of this dissertation utilizes the lipopolysaccharide (LPS)-sensitive limulus amoebocyte lysate (LAL) assay a surrogate to explore the hypothesis that intrinsically heightened nanoparticle properties of surface reactivity and specific surface area may disrupt endogenous biochemical cascades. The G7-NH2 were found to affect all key platelet functions, evidenced by severe morphological alteration, extensive aggregation and adhesion, release of alpha granule contents, and attenuation of thrombin generation. It was further demonstrated that extensive, direct, dendrimer-mediated aggregation of fibrinogen occurs via a thrombin-independent, electrostatic mechanism that also included G7-NH2 aggregation of bovine serum albumin and, by extension, the a majority of soluble plasma protein species due to their negative charge domains. Silica nanoparticles and two types of gold nanoparticles were demonstrated to increase the LAL assay response to LPS, while carboxy latex particles attenuated the LAL assay response. This apparent increase in the rate of production for the chromogenic LAL assay product for the gold and silica nanoparticles shows the potential for nanoparticles to exacerbate endogenous inflammatory responses to toxins co-presented in vivo. iv In summary, this dissertation demonstrates the potential for nanoparticle reactivity within or in concert with biological systems and specifically clarifies the mechanism of cationic dendrimer-induced hemostasis. Additionally, this work establishes additional models for the assessment of procoagulant properties of nanoparticles. The specific mechanistic findings presented herein represent an improvement upon the level of analysis usually performed for nanoparticles in the blood system and may guide rational design for safer nanoparticle-based, intravenous therapies. |
