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Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2024)

There are multiple challenges and limitations in the current separation methods for large biological complexes, including insufficient resolution, time-consuming pre-treatment processes and downstream analysis, potential analyte-column interactions leading to denaturation or adsorption, and the need for improving efficiency in separation and characterization when dealing with large complexes with small molecules simultaneously. In this dissertation, I aim to develop improved solutions and strategies to overcome these challenges, focusing on two powerful analytical separation techniques: Asymmetrical Flow Field-Flow Fractionation (AF4) and Capillary Electrophoresis (CE). AF4, a separation technique without a stationary phase, has proven valuable in separating large analytes with sizes ranging from nanometers to micrometers over the past three decades, offering an effective alternative to Size Exclusion Chromatography (SEC). Meanwhile, CE has matured into a robust analytical separation technique, featuring advantages like minimal sample consumption, rapid analysis, high resolution, and efficiency.In Chapter 2, the offline coupling of AF4 and CE is detailed for extracellular vesicle (EV) analysis. EVs, as important mediators of cell-to-cell communication capturing great research interests to study their functions and explore their utility in biomedical practices, pose challenges in purification due to their heterogeneity and low abundance in biological samples. AF4 provides rapid size-based separation, while CE resolves EVs from contaminants with similar sizes eluted from AF4. The coupled AF4-CE system demonstrates efficiency in monitoring EV secretion from cells in the cell culture media, and detecting serum EVs, offering rapid quantification in biological samples. Chapter 3 introduces the coupling system of AF4 with Large Volume Sample Stacking/CE (LVSS/CE) to obtain high-purity EVs for downstream protein profiling. This two-dimensional separation method first separates EVs from smaller serum proteins by AF4 and then resolves EVs from non-vesicular matrix components by CE following LVSS. The approach enhances EV isolation and collection from LVSS/CE, enabling EV immuno-fluorescence labeling and permitting the identification of EV proteins through proteomic analysis. Chapter 4 presents an online separation and characterization platform for mRNA and mRNA-loaded lipid nanoparticles (LNPs) using AF4 with multi-detectors (MD-AF4). LNPs, promising in drug delivery, present challenges in characterization. The MD-AF4 method achieves exceptional resolution between mRNA-LNPs and mRNAs, offering comprehensive and multi-attribute particle characterizations within a single injection, including size distribution, batch-to-batch variability, shape factor and encapsulation efficiency. The platform also proves potential for monitoring LNP stability under various stress conditions. This dissertation highlights the significance of AF4 and CE in advancing the analysis of large biological complexes. The novel methodologies introduced herein hold promise for consolidating our understanding of complex biological matrices, and advancing biomedical applications. 

    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2022)
    Development of new capillary electrophoresis (CE) methodology and instrumentation, as well as application of CE to solve new problems, remains an active research area because of the attractive features of CE compared to other separation techniques. In this review, we focus on the representative works about sample preconcentration, separation media or capillary coating development, detector construction, and multidimensional separation in CE, which are judiciously selected from the papers published in 2018-2020.
      Cover page: Recent (2018–2020) development in capillary electrophoresis
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2022)
      Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.
        Cover page: Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles.
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2024)
        Extracellular vesicles (EVs) play important roles in cell-cell communication but are highly heterogeneous, and each vesicle has dimensions smaller than 200 nm with very limited amounts of cargos encapsulated. The technique of NanOstirBar (NOB)-EnabLed Single Particle Analysis (NOBEL-SPA) reported in the present work permits rapid inspection of single EV with high confidence by confocal fluorescence microscopy, thus enables colocalization assessment for selected protein and microRNA (miRNA) markers in the EVs produced by various cell lines, or present in clinical sera samples. EV subpopulations marked by the colocalization of unique protein and miRNA combinations were discovered to be able to detect early-stage (stage I or II) breast cancer (BC). NOBEL-SPA can be adapted to analyze other types of cargo molecules or other small submicron biological particles. Study of the sorting of specific cargos to heterogeneous vesicles under different physiological conditions can help discover distinct vesicle subpopulations valuable in clinical examination and therapeutics development and gain better understanding of their biogenesis.
          Cover page: Colocalization of protein and microRNA markers reveals unique extracellular vesicle subpopulations for early cancer detection.
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2023)
          Flexible, water-soluble hosts are capable of selective molecular recognition in cellular environments and can detect neurotransmitters such as choline in cells. Both cationic and anionic water-soluble self-folded deep cavitands can recognize suitable styrylpyridinium dyes in cellular interiors. The dyes selectively accumulate in nucleotide-rich regions of the cell nucleus and cytoplasm. The hosts bind the dyes and promote their relocation to the outer cell membrane: the lipophilic cavitands predominantly reside in membrane environments but are still capable of binding suitable targets in other cellular organelles. Incubating the cells with structurally similar biomarkers such as choline, cholamine, betaine, or butyrylcholine illustrates the selective recognition. Choline and butyrylcholine can be bound by the hosts, but minimal binding is seen with betaine or cholamine. Varying the dye allows control of the optical detection method, and both "turn-on" sensing and "turn-off" sensing are possible.
            Cover page: Selective Molecular Recognition and Indicator Displacement Sensing of Neurotransmitters in Cellular EnvironmentsCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2022)
            Applications of reduced graphene oxide (rGO) in many different areas have been gradually increasing owing to its unique physicochemical characteristics, demanding more understanding of their biological impacts. Herein, we assessed the toxicological effects of rGO in mammary epithelial cells. Because the as-synthesized rGO was dissolved in sodium cholate to maintain a stable aqueous dispersion, we hypothesize that changing the cholate concentration in the dispersion may alter the surface property of rGO and subsequently affect its cellular toxicity. Thus, four types of rGO were prepared and compared: rGO dispersed in 4 and 2 mg/mL sodium cholate, labeled as rGO and concentrated-rGO (c-rGO), respectively, and rGO and c-rGO coated with a protein corona through 1 h incubation in culture media, correspondingly named pro-rGO and pro-c-rGO. Notably, c-rGO and pro-c-rGO exhibited higher toxicity than rGO and pro-rGO and also caused higher reactive oxygen species production, more lipid membrane peroxidation, and more significant disruption of mitochondrial-based ATP synthesis. In all toxicological assessments, pro-c-rGO induced more severe adverse impacts than c-rGO. Further examination of the material surface, protein adsorption, and cellular uptake showed that the surface of c-rGO was coated with a lower content of surfactant and adsorbed more proteins, which may result in the higher cellular uptake observed with pro-c-rGO than pro-rGO. Several proteins involved in cellular redox mediation were also more enriched in pro-c-rGO. These results support the strong correlation between dispersant coating and corona formation and their subsequent cellular impacts. Future studies in this direction could reveal a deeper understanding of the correlation and the specific cellular pathways involved and help gain knowledge on how the toxicity of rGO could be modulated through surface modification, guiding the sustainable applications of rGO.
              Cover page: Biological Impacts of Reduced Graphene Oxide Affected by Protein Corona Formation
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2022)
              Epidemiological studies demonstrate an association between breast cancer (BC) and systemic dysregulation of glucose metabolism. However, how BC influences glucose homeostasis remains unknown. We show that BC-derived extracellular vesicles (EVs) suppress pancreatic insulin secretion to impair glucose homeostasis. EV-encapsulated miR-122 targets PKM in β-cells to suppress glycolysis and ATP-dependent insulin exocytosis. Mice receiving high-miR-122 EVs or bearing BC tumours exhibit suppressed insulin secretion, enhanced endogenous glucose production, impaired glucose tolerance and fasting hyperglycaemia. These effects contribute to tumour growth and are abolished by inhibiting EV secretion or miR-122, restoring PKM in β-cells or supplementing insulin. Compared with non-cancer controls, patients with BC have higher levels of circulating EV-encapsulated miR-122 and fasting glucose concentrations but lower fasting insulin; miR-122 levels are positively associated with glucose and negatively associated with insulin. Therefore, EV-mediated impairment of whole-body glycaemic control may contribute to tumour progression and incidence of type 2 diabetes in some patients with BC.
                Cover page: Cancer-cell-secreted extracellular vesicles suppress insulin secretion through miR-122 to impair systemic glucose homeostasis and contribute to tumour growth
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