The role of zinc nanoparticles in the initial events of olfaction, their characterization, preservation, and microenvironmental influence.
Type of DegreePhD Dissertation
DepartmentGeneral Veterinary Medicine
MetadataShow full item record
Olfactory responses are intensely enhanced with the addition of endogenous and engineered primarily-elemental small zinc nanoparticles (ZnNPs). The endogenous ZnNPs have been found as proteon nucleating centers (PNCs) present in human and animal blood along with other PNCs such as gold and copper nanoparticles. Engineered ZnNPs, mimicking the endogenous subset, which range in size from 1 to 2 nm, have been studied in their influence on olfaction. At low concentrations, ZnNPs can enhance electroolfactogram (EOG) or whole cell patch-clamp responses to odorants by about 3-fold, not seen with the other PNC identified metal nanoparticles or with zinc ions (Zn2+). To evaluate the role of ZnNPS in the initial events of olfaction, physiochemical characterization of the ZnNPs was performed. Particle size was determined by atomic force microscopy (AFM), crystallinity by transmission electron microscopy (TEM), oxidation by X-ray photoelectron spectroscopy (XPS), and zeta potential by laser Doppler velocimetry (LDV). Characterization by AFM revealed the zinc metal nanoparticles were in the size of 1-2 nm range of PNC’s at an average diameter of 1.2±0.3 nm. XPS demonstrated 94% of atoms in the zinc nanoparticle were non-oxidized with the shell atoms constituting 80% of the total atom number. Crystalline lattice fringes corresponding to elemental zinc were demonstrated on TEM. The zeta potential with LDV was found of -42.4±4.8 (SE) mV. The characteristics of the small Zn nanoparticles implied high stability and high potential chemical reactivity. Evaluation of the effects oxidation has on zinc nanoparticle function in olfaction was evaluated by EOG. ZnNPs were subjected to oxygen percolation and characterized in relation to bare ZnNPs. The oxidation levels of 12% was determined by the XPS spectra. This oxidation of bare ZnNPs resulted in halting the olfaction enhancement as measured by EOG. With aging throughout storage and at elevated temperatures, ZnNPs are also oxidized and no longer manifest the same degree of olfactory enhancement. The design of a polyethylene glycol coating to meet storage requirements of engineered zinc nanoparticles was evaluated in an effort to achieve maximal olfactory benefit. The zinc nanoparticles were covered with 1000 g/mol or 400 g/mol molecular weight polyethylene glycol (PEG). Non-PEGylated and PEGylated zinc nanoparticles were tested by EOG with isolated rat olfactory epithelium and odorant responses evoked by the mixture of eugenol, ethyl butyrate and (±) carvone after storage at 278 K (5 oC), 303 K (30 oC), and 323 K (50 oC). The particles were analyzed by AFM, TEM, XPS, and LDV. Our data indicates that stored ZnPEG400 nanoparticles maintain physiologically-consistent olfactory enhancement for over 300 days. These engineered nanoparticles support future applications in olfactory research, sensitive detection capabilities, and medicine. The isolation of PNCs from human and animal blood and the implication of zinc nanoparticle enhancement on olfaction support a biological role for ZnNPs. To evaluate the potential biological role ZnNPs have in the initial events of olfaction, the low molecular filtrate of olfactory mucosa was analyzed for its electrophysiological influence on olfactory neurons. Isolation of the low-molecular-weight filtrate from the olfactory mucosa demonstrated on EOG to provide ZnNP-like enhancement of olfaction. XPS analysis of the low-molecular-weight filtrate was inconclusive for positive identification of ZnNPs, though literature supports a zinc concentration of 14 µg/dl within the nasal mucus of mice. Commonly used testing methods for zinc are unable to differentiate ionic zinc from any range of zinc nanoparticles. Though limited to electrophysiological comparison, this data further supports a biological role of ZnNPs through indications of the endogenous presence of enhancement capable nanoparticles at the site of action. The perireceptor environment is mucus based with recent evidence supporting the presence of a microbial community. To evaluate the potential influence ZnNPs may have on the perireceptor environment, multiple bacterial taxa were screened for ZnNP induced growth effects. Bacterial species selected included gram-negative Escherichia coli M-17, Shigella flexneri and Salmonella typhimurium 52096 and gram-positive species Staphylococcus aureus ATCC 12600, MRSA-1, Lactobacillus acidophilus, Bacillus subtilis BSB3, Bacillus subtilis BS, and Bacillus licheniformis. The screening method involved co-culture with ZnNPs and comparative viability determined through spectrophotometric analysis. Results indicated a differential effect independent of bacterial cell wall structure. The effects on Staphylococcus species, with particular suppression on methicillin resistant Staphylococcus aureus (MRSA), and an apparent enhancement of growth in Bacillus subtilis, led to a selective study evaluating ZnNP antimicrobial effects in seven strains of MRSA and Bacillus subtilis. Results showed that ZnNPs were generally MRSA suppressive in a concentration-dependent manner, with an approximate 33.5% maximum reduction in viability (MRSA-45) (p<0.05). Conversely, a general trend of concentration-dependent bacterial growth enhancement is seen primarily in Bacillus subtilis at an approximate 18% maximum increase in viability (p<0.05). This differential pattern of activity between a bacterial species of clinical importance in infectivity and antibiotic resistance (MRSA), and a bacterial species utilized as a probiotic for its positive health benefits provides insight into a potential regulatory role that ZnNPs may play in the perireceptor environment. Understanding the molecular mechanism underlying odorant recognition in the olfactory system and the role that newly discovered ZnNPs may play in that dynamic interaction is important in the context of basic science and advancements in identification of therapeutic targets for application in clinical diseases and disorders associated with loss or dysfunction of smell. Continuing research in the neuroscience of olfaction and nanotechnology may secure future applications of ZnNPs in neurodegenerative medical conditions that involve a loss of smell such as Alzheimer’s and Parkinson’s, psychological conditions associated with loss of smell such as schizophrenia and depression, occupational health and safety, public health, agricultural production and processing, aromatic technologies related to food and perfume industries, and global security in detection enhancement.
- Dissertation Singletary, Melissa.pdf