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Doctoral Exit Seminar

Doctoral Exit Seminar: Real-time In Vivo Tracking of Nanocarriers in the Cerebrovasculature by Fluorescence Correlation Spectroscopy

Photo of doctoral candidate Xiaojin Wang sitting in a wooded area.Cerebrovasculature refers to the network of blood vessels in the brain, and its coupling with neurons plays a critical role in regulating ion exchange, molecule transport, nutrient and oxygen delivery, and waste removal in the brain. Abnormalities in cerebrovasculature and disruptions of the blood supply are associated with a variety of cerebrovascular and neurodegenerative disorders. Nanocarriers, a nano-sized drug delivery system synthesized from various materials, have been designed to encapsulate therapeutic agents and overcome delivery challenges in crossing the blood-brain barrier (BBB) to achieve targeted and enhanced therapy for these diseases. Unraveling the transport of drugs and nanocarriers in the cerebrovasculature is important for pharmacokinetic and hemodynamic studies but is challenging due to difficulties in detecting these particles within the circulatory system of a live animal. In this dissertation, we developed a technique to achieve real-time in vivo tracking of nanocarriers in the cerebrovasculature using fluorescence correlation spectroscopy (FCS), which has great potential for determining the pharmacokinetics of drugs and nanocarriers, as well as for studying disease-related connections between the cerebrovascular and neurodegenerative diseases.

Animated graphic illustrating the effects of disease-related conditions on vasoconstriction and vasodilation.

We utilized novel fluorescent probes composed of DNA-stabilized silver nanocluster (DNA-Ag16NC), that emit in the first near-infrared window (NIR-I) upon two-photon excitation in the second NIR window (NIR-II), encapsulated in liposomes, which were then used to measure cerebral blood flow rates in live mice with high spatiotemporal resolution by two-photon in vivo FCS. Liposome encapsulation concentrated and protected DNA-Ag16NCs from in vivo degradation, enabling the quantification of cerebral blood flow velocity within individual capillaries of a living mouse. We also loaded another DNA-stabilized silver nanocluster (DNA640), which exhibited higher quantum yield and anti-Stokes fluorescence upon upconversion absorption, into cationic mesoporous silica nanoparticles (CMSNs) and successfully coated them with liposomes. The cerebrovasculature was chronically labeled using an adeno-associated viral (AAV) vector encoding Alb-mNG secretion into the bloodstream, combined with FCS under upconversion excitation, enabling real-time observation of the flow velocity and particle number of DNA640-CMSN-liposomes within the capillaries. We applied our proposed techniques to study the cerebrovascular structure and blood flow velocity in Alzheimer's disease mouse models and to explore the effects of disease-related conditions on vasoconstriction and vasodilation.

KEYWORDS: Cerebrovascular, nanocarrier, FCS, NIR fluorescence, DNA-AgNC, in vivo

Zoom link: https://uky.zoom.us/j/5984755867?omn=87194697892

Meeting ID: 598 475 5867.

Date:
Location:
Zoom

Doctoral Exit Seminar: Real-time In Vivo Tracking of Nanocarriers in the Cerebrovasculature by Fluorescence Correlation Spectroscopy

Photo of doctoral candidate Xiaojin Wang sitting in a wooded area.Cerebrovasculature refers to the network of blood vessels in the brain, and its coupling with neurons plays a critical role in regulating ion exchange, molecule transport, nutrient and oxygen delivery, and waste removal in the brain. Abnormalities in cerebrovasculature and disruptions of the blood supply are associated with a variety of cerebrovascular and neurodegenerative disorders. Nanocarriers, a nano-sized drug delivery system synthesized from various materials, have been designed to encapsulate therapeutic agents and overcome delivery challenges in crossing the blood-brain barrier (BBB) to achieve targeted and enhanced therapy for these diseases. Unraveling the transport of drugs and nanocarriers in the cerebrovasculature is important for pharmacokinetic and hemodynamic studies but is challenging due to difficulties in detecting these particles within the circulatory system of a live animal. In this dissertation, we developed a technique to achieve real-time in vivo tracking of nanocarriers in the cerebrovasculature using fluorescence correlation spectroscopy (FCS), which has great potential for determining the pharmacokinetics of drugs and nanocarriers, as well as for studying disease-related connections between the cerebrovascular and neurodegenerative diseases.

Animated graphic illustrating the effects of disease-related conditions on vasoconstriction and vasodilation.

We utilized novel fluorescent probes composed of DNA-stabilized silver nanocluster (DNA-Ag16NC), that emit in the first near-infrared window (NIR-I) upon two-photon excitation in the second NIR window (NIR-II), encapsulated in liposomes, which were then used to measure cerebral blood flow rates in live mice with high spatiotemporal resolution by two-photon in vivo FCS. Liposome encapsulation concentrated and protected DNA-Ag16NCs from in vivo degradation, enabling the quantification of cerebral blood flow velocity within individual capillaries of a living mouse. We also loaded another DNA-stabilized silver nanocluster (DNA640), which exhibited higher quantum yield and anti-Stokes fluorescence upon upconversion absorption, into cationic mesoporous silica nanoparticles (CMSNs) and successfully coated them with liposomes. The cerebrovasculature was chronically labeled using an adeno-associated viral (AAV) vector encoding Alb-mNG secretion into the bloodstream, combined with FCS under upconversion excitation, enabling real-time observation of the flow velocity and particle number of DNA640-CMSN-liposomes within the capillaries. We applied our proposed techniques to study the cerebrovascular structure and blood flow velocity in Alzheimer's disease mouse models and to explore the effects of disease-related conditions on vasoconstriction and vasodilation.

KEYWORDS: Cerebrovascular, nanocarrier, FCS, NIR fluorescence, DNA-AgNC, in vivo

Zoom link: https://uky.zoom.us/j/5984755867?omn=87194697892

Meeting ID: 598 475 5867.

Date:
Location:
Zoom

Advancing Waste Management: Biomass Ozonolysis, Wastewater Chlorination, and Coal Ash in Landfills

Reagan WittThe health of our communities depends on the effective treatment of both solid and liquid waste to eradicate hazardous pollutants before they can interact with living organisms or contaminate the environment. Daily, society generates solid waste (commonly destined for landfills) and liquid waste, (commonly discharged into wastewater systems) and without proper treatment, these wastes can release hazardous primary secondary pollutants. Industries producing wastewater with high pollutant concentrations, especially those utilizing lignin-based biomass, face complex challenges because each facility may require a tailored treatment approach. In response, this work investigates the use of ozonolysis to transform lignin monomers into smaller, less hazardous components that can be more efficiently managed by public wastewater systems. Furthermore, while conventional wastewater treatment systems are effective for common water quality issues, they can inadvertently allow complex compounds, such pollutants from hospital effluent, to pass through. Under simulated treatment conditions incorporating sunlight and chlorination, a pollutant released from medical facilities is degraded, but this process may also lead to the formation of carcinogenic disinfection by-products (DBPs) that pose direct toxicological risks to nearby communities. The implications extend to solid waste management as well. Chemical phenomena, such as those occurring in poorly understood elevated temperature landfills (ETLFs), can compromise treatment methods and increase community exposure to harmful pollutants. By monitoring hazardous components, such as volatile organic compounds (VOCs), over time, this work aims to elucidate the chemical reactions occurring both during treatment and in the environment thereafter. Ultimately, this research underscores the need for fundamental, innovative approaches to pollution transformation. Bridging the gap between existing practices for solid and liquid waste treatment will be critical to safeguarding environmental and public health.

Witt - graphic

Date:
Location:
CP 114

Advancing Waste Management: Biomass Ozonolysis, Wastewater Chlorination, and Coal Ash in Landfills

Reagan WittThe health of our communities depends on the effective treatment of both solid and liquid waste to eradicate hazardous pollutants before they can interact with living organisms or contaminate the environment. Daily, society generates solid waste (commonly destined for landfills) and liquid waste, (commonly discharged into wastewater systems) and without proper treatment, these wastes can release hazardous primary secondary pollutants. Industries producing wastewater with high pollutant concentrations, especially those utilizing lignin-based biomass, face complex challenges because each facility may require a tailored treatment approach. In response, this work investigates the use of ozonolysis to transform lignin monomers into smaller, less hazardous components that can be more efficiently managed by public wastewater systems. Furthermore, while conventional wastewater treatment systems are effective for common water quality issues, they can inadvertently allow complex compounds, such pollutants from hospital effluent, to pass through. Under simulated treatment conditions incorporating sunlight and chlorination, a pollutant released from medical facilities is degraded, but this process may also lead to the formation of carcinogenic disinfection by-products (DBPs) that pose direct toxicological risks to nearby communities. The implications extend to solid waste management as well. Chemical phenomena, such as those occurring in poorly understood elevated temperature landfills (ETLFs), can compromise treatment methods and increase community exposure to harmful pollutants. By monitoring hazardous components, such as volatile organic compounds (VOCs), over time, this work aims to elucidate the chemical reactions occurring both during treatment and in the environment thereafter. Ultimately, this research underscores the need for fundamental, innovative approaches to pollution transformation. Bridging the gap between existing practices for solid and liquid waste treatment will be critical to safeguarding environmental and public health.

Witt - graphic

Date:
Location:
CP 114

Exit Seminar: Chromatographic and Electrophoretic Analysis of Lignin-Derived Monomers and Dimers: Extraction Method Development and Evaluation of Cyclodextrins for Recovery

Biorefineries play a vital role in reducing dependence on fossil fuels and addressing global warming concerns. Unfortunately, investment in biorefineries remains limited due to economic sustainability concerns. One approach to enhance the economic viability of biorefineries is by fully utilizing the potential of feedstocks, particularly lignocellulosic biomass, which is commonly used in these processes. Traditional processing methods often alter the lignin portion of biomass, making it unsuitable for further utilization. However, when retained in its native form, lignin can serve as a valuable source of chemicals that contribute to the economic sustainability of biorefineries.

The lignin-first approach, which removes lignin from the cellulosic portion before biofuel conversion, allows for the recovery of value-added chemicals from lignin and enhances the sustainability of biorefineries. One common method for implementing this approach is organosolv, which removes the lignin from biomass. This research developed an organosolv pretreatment method to isolate syringaresinol (S-β-β'-S), a lignin-derived dimer with various biomedical and industrial applications, from oak sawdust as the source biomass. The method incorporated a heat treatment step to increase syringaresinol yields, and key treatment parameters were optimized for maximum output. This approach was then applied to other biomass types: hardwood, softwood, and grass. Poplar and hemp were identified as alternative sources of syringaresinol. The method also revealed the presence of several other potential value-added compounds in the biomass types investigated.

Additionally, this research established a linear retention index (LRI) for the gas chromatographic (GC) analysis of 25 common lignin-derived monomers and dimers, which are frequently detected in biomass chromatograms after pretreatments like organosolv. LRI values are independent of column characteristics, enabling unambiguous identification of analytes and ensuring reproducibility in experimental results. These calculated values were validated with a second GC system. The LRI facilitates the identification and confirmation of compounds without the need for mass spectrometric (MS) analysis, allowing for comparison of chromatographic results across multiple GC systems and aiding in the prediction of retention times for these compounds.

While extracting value-added compounds like syringaresinol from biomass is the first step, recovering them from complex reaction mixtures presents additional challenges. This study explored the use of cyclodextrins (CDs) as potential recovery materials for syringaresinol. CDs are conical molecules with a hydrophilic exterior and a hydrophobic cavity, which can selectively capture lignans through guest-host complex interactions. Initial high-performance liquid chromatography (HPLC) analysis with a commercial β-CD column revealed that lignans, particularly syringaresinol and pinoresinol interact more strongly with β-CD than with most other monomeric lignin decomposition products. Furthermore, the study suggested that γ-CD might be a better recovery material for syringaresinol compared to β-CD. To test this hypothesis, a modified frontal analysis continuous capillary electrophoresis (FACCE) method was developed and validated as an inexpensive, simple approach to estimate the binding constants of lignans to CDs. The FACCE method provided insights into the interactions between lignans and CDs, facilitating the selection of the potentially suitable CD-type for recovery.

graphic

Date:
Location:
CP 114

Exit Seminar: Chromatographic and Electrophoretic Analysis of Lignin-Derived Monomers and Dimers: Extraction Method Development and Evaluation of Cyclodextrins for Recovery

Biorefineries play a vital role in reducing dependence on fossil fuels and addressing global warming concerns. Unfortunately, investment in biorefineries remains limited due to economic sustainability concerns. One approach to enhance the economic viability of biorefineries is by fully utilizing the potential of feedstocks, particularly lignocellulosic biomass, which is commonly used in these processes. Traditional processing methods often alter the lignin portion of biomass, making it unsuitable for further utilization. However, when retained in its native form, lignin can serve as a valuable source of chemicals that contribute to the economic sustainability of biorefineries.

The lignin-first approach, which removes lignin from the cellulosic portion before biofuel conversion, allows for the recovery of value-added chemicals from lignin and enhances the sustainability of biorefineries. One common method for implementing this approach is organosolv, which removes the lignin from biomass. This research developed an organosolv pretreatment method to isolate syringaresinol (S-β-β'-S), a lignin-derived dimer with various biomedical and industrial applications, from oak sawdust as the source biomass. The method incorporated a heat treatment step to increase syringaresinol yields, and key treatment parameters were optimized for maximum output. This approach was then applied to other biomass types: hardwood, softwood, and grass. Poplar and hemp were identified as alternative sources of syringaresinol. The method also revealed the presence of several other potential value-added compounds in the biomass types investigated.

Additionally, this research established a linear retention index (LRI) for the gas chromatographic (GC) analysis of 25 common lignin-derived monomers and dimers, which are frequently detected in biomass chromatograms after pretreatments like organosolv. LRI values are independent of column characteristics, enabling unambiguous identification of analytes and ensuring reproducibility in experimental results. These calculated values were validated with a second GC system. The LRI facilitates the identification and confirmation of compounds without the need for mass spectrometric (MS) analysis, allowing for comparison of chromatographic results across multiple GC systems and aiding in the prediction of retention times for these compounds.

While extracting value-added compounds like syringaresinol from biomass is the first step, recovering them from complex reaction mixtures presents additional challenges. This study explored the use of cyclodextrins (CDs) as potential recovery materials for syringaresinol. CDs are conical molecules with a hydrophilic exterior and a hydrophobic cavity, which can selectively capture lignans through guest-host complex interactions. Initial high-performance liquid chromatography (HPLC) analysis with a commercial β-CD column revealed that lignans, particularly syringaresinol and pinoresinol interact more strongly with β-CD than with most other monomeric lignin decomposition products. Furthermore, the study suggested that γ-CD might be a better recovery material for syringaresinol compared to β-CD. To test this hypothesis, a modified frontal analysis continuous capillary electrophoresis (FACCE) method was developed and validated as an inexpensive, simple approach to estimate the binding constants of lignans to CDs. The FACCE method provided insights into the interactions between lignans and CDs, facilitating the selection of the potentially suitable CD-type for recovery.

graphic

Date:
Location:
CP 114

Exit seminar: Developing Synthetic Strategies for Three-Coordinate Gold(I) Complexes as Anticancer Agents

headshotGold(I) complexes typically bond in a linear fashion; however, an increased valence can be achieved via ligand modulation. The most prevalent therapeutic gold complex, auranofin, contains a linear Au(I) center and has shown great potential in several diseases and conditions. On the other hand, the potential of three-coordinate Au(I) complexes have scarcely been probed as therapeutics. Reported here are the synthesis, characterization, and applications of novel three-coordinate Au(I) complexes. The degree of asymmetry varies between complexes depending on the Au-X ancillary ligands. This insight suggests that the degree of asymmetry influences the potency when incubated in various cancer cell lines. In addition, the coordination of bidentate phenanthroline ligand derivatives effect the symmetry by inducing varying degrees of distortion in the crystal structure. When the center Au(I) is bound to an N-Heterocyclic Carbene (NHC), the compound shifts from a distorted trigonal planar geometry to a distorted linear geometry. These complexes were used to probe glioblastoma, an aggressive head-and-neck cancer. When the center Au(I) is bound to biaryl dialkyl phosphine ligands, the geometry varies in symmetry, but the distorted trigonal planar geometry remains intact. Structure activity relationship studies were performed on these complexes in triple negative breast cancer cell lines. Previous research shows a disruption of mitochondrial dynamics when cancer cells were treated with three-coordinate Au(I) complexes, and the novel Au(I)-NHC library indeed disrupts mitochondrial dynamics. Mitochondria are the main energy production centers in the cell and are desirable therapeutic targets due to their implication in aging, inflammation, and cancer. The Au(I)-P library shows little mitochondrial disruption; instead, these complexes induce significant stress in the endoplasmic reticulum. The endoplasmic reticulum transports and folds proteins that allow the cell to function properly and synthesizes lipids and cholesterols. When the endoplasmic reticulum undergoes stress, the several signaling pathways, known as the unfolded protein response, activate, which can lead to lipid accumulation. Both a disruption of mitochondrial dynamics and an induction of endoplasmic reticulum stress can lead to apoptotic cell death. These effects were characterized by several in vitro and in vivo experiments. 

1

Carboranes are electron-delocalized clusters containing as few as five and as many as fourteen boron and carbon atoms, the majority of which contain two cage carbons. The carbons in the cluster can be easily functionalized with alkyl and aryl phosphines for coordination to metal complexes. Described here is the synthesis of phosphine-functionalized carborane (DPPCb) containing three-coordinate Au(I) complexes. Taken as a whole, this work expands on the current three-coordinate gold(I) libraries and evaluates their in vitro and in vivo biological efficacy.

Date:
Location:
CP 114

Exit seminar: Developing Synthetic Strategies for Three-Coordinate Gold(I) Complexes as Anticancer Agents

headshotGold(I) complexes typically bond in a linear fashion; however, an increased valence can be achieved via ligand modulation. The most prevalent therapeutic gold complex, auranofin, contains a linear Au(I) center and has shown great potential in several diseases and conditions. On the other hand, the potential of three-coordinate Au(I) complexes have scarcely been probed as therapeutics. Reported here are the synthesis, characterization, and applications of novel three-coordinate Au(I) complexes. The degree of asymmetry varies between complexes depending on the Au-X ancillary ligands. This insight suggests that the degree of asymmetry influences the potency when incubated in various cancer cell lines. In addition, the coordination of bidentate phenanthroline ligand derivatives effect the symmetry by inducing varying degrees of distortion in the crystal structure. When the center Au(I) is bound to an N-Heterocyclic Carbene (NHC), the compound shifts from a distorted trigonal planar geometry to a distorted linear geometry. These complexes were used to probe glioblastoma, an aggressive head-and-neck cancer. When the center Au(I) is bound to biaryl dialkyl phosphine ligands, the geometry varies in symmetry, but the distorted trigonal planar geometry remains intact. Structure activity relationship studies were performed on these complexes in triple negative breast cancer cell lines. Previous research shows a disruption of mitochondrial dynamics when cancer cells were treated with three-coordinate Au(I) complexes, and the novel Au(I)-NHC library indeed disrupts mitochondrial dynamics. Mitochondria are the main energy production centers in the cell and are desirable therapeutic targets due to their implication in aging, inflammation, and cancer. The Au(I)-P library shows little mitochondrial disruption; instead, these complexes induce significant stress in the endoplasmic reticulum. The endoplasmic reticulum transports and folds proteins that allow the cell to function properly and synthesizes lipids and cholesterols. When the endoplasmic reticulum undergoes stress, the several signaling pathways, known as the unfolded protein response, activate, which can lead to lipid accumulation. Both a disruption of mitochondrial dynamics and an induction of endoplasmic reticulum stress can lead to apoptotic cell death. These effects were characterized by several in vitro and in vivo experiments. 

1

Carboranes are electron-delocalized clusters containing as few as five and as many as fourteen boron and carbon atoms, the majority of which contain two cage carbons. The carbons in the cluster can be easily functionalized with alkyl and aryl phosphines for coordination to metal complexes. Described here is the synthesis of phosphine-functionalized carborane (DPPCb) containing three-coordinate Au(I) complexes. Taken as a whole, this work expands on the current three-coordinate gold(I) libraries and evaluates their in vitro and in vivo biological efficacy.

Date:
Location:
CP 114