Gerald Alter (Committee Member), Steven Higgins (Committee Member), Barbara Hull (Committee Member), Mill Miller (Committee Member), Dawn Wooley (Advisor)
Doctor of Philosophy (PhD)
Silver nanoparticles (AgNPs) have received tremendous attention for their antimicrobial properties; however, many gaps in knowledge exist. To address these issues, three research objectives were examined. The first objective hypothesized AgNPs can be size selected and concentrated via tangential flow ultrafiltration. The second objective hypothesized a high-throughput method could be developed to screen nanoparticle antiviral-activity and cytotoxicity simultaneously. The third objective hypothesized AgNPs inhibit viruses by preventing viral entry.
For objective one, a tangential flow ultrafiltration scheme was tested on AgNPs synthesized via the Creighton Colloid method. AgNPs were analyzed via transmission electron microscopy. In objective two, an HIV-1 vector was adapted to 96-well format and modified to utilize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for simultaneous cytotoxicity and antiviral measurement. The third objective was investigated using Vaccinia virus (VACV). AgNP effects on VACV entry were monitored through β-galactosidase reporter assay and confocal microscopy. Western blots detected AgNP/VACV interaction. Plaque assays monitored AgNP inhibition over the entire VACV replication cycle. MTT and trypan blue exclusion measured AgNP cytotoxicity.
In objective one, tangential flow ultrafiltration demonstrated size selection and concentration of AgNPs. Filtered AgNPs were uniform and unaggregated with average 11 nm diameters. A high-throughput, standardized assay was developed in objective two. AgNPs had antiviral-activity at non-cytotoxic concentrations (IC50 = 16 μg mL-1). In the third objective, AgNPs prevented VACV entry in both cytoprotective and virucidal capacities at non-cytotoxic concentrations (IC50 = 48 μg mL-1). In the absence of macropinocytosis, AgNPs retained virucidal-activity but not cytoprotective effects. AgNPs bound to viral entry proteins. Plaque assays demonstrated that AgNPs inhibited the entire VACV replication cycle. Cytotoxicity assays demonstrated AgNPs were non-cytotoxic at antiviral concentrations.
Objective one's size selection and concentration method permits accurate investigation into nanoparticle antimicrobial activity by eliminating size and reagent incompatibility problems inherent to AgNP synthesis. In objective two, the traditional viral titering assay was replaced with a standardized assay measuring simultaneously antiviral activity and cytotoxicity, permitting faster and cheaper antiviral screening of nanoparticles. In the third objective, AgNP-mediated antiviral-activity was pinpointed to viral entry. Multiple mechanisms of entry inhibition were observed. These data suggest that AgNPs can be potent and broad-spectrum antiviral agents and therapeutics.
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