Poster Y11 - Engineering Peptide-Imprinted Nanopolymers for Astrocyte-specific Extracellular Vesicles Isolation and Analysis in Neurological disorders

Introduction: Neurological disorders require early detection, yet traditional diagnostic methods lack specificity and sensitivity. In particular, Neuromyelitis optica spectrum disorder (NMOSD), a chronic inflammatory disease of the central nervous system, Identifying blood biomarkers to track and predict the disease trajectory could significantly improve the clinical management of NMOSD. Blood-derived extracellular vesicles (EVs) provide a promising, non-invasive alternative for biomarker discovery, despite challenges in cell-specific isolating them from brain related EVs due to their complexity and scarcity. Brain-derived extracellular vesicles (BDEVs) offer a promising avenue for neurological disorder diagnosis. These small particles, particularly exosomes, carry biomolecules across the blood-brain barrier, providing molecular insights into brain tissue. BDEV-based diagnostics offer repeated, minimally invasive testing, reducing physical and economic burdens.
In this study, we introduce Engineered Polymer-Inorganic Nanocomposites (EPIN) as an innovative platform for isolating cell-specific EVs from biological matrices, overcoming the limitations of conventional methods. Utilizing magnetic nanoparticles, we developed EPIN with peptide templates that bind specifically to EV surface proteins, enhancing the capture efficiency. We analyzed miRNA expressions level in NMOSD patient serum during relapse and remission phases, marking a significant step forward in EV-based diagnostics. Our findings highlight the potential of EPIN-GLAST in elucidating disease dynamics and underscore the value of targeted EV analysis in neurological disorder research, providing a new avenue for biomarker discovery.

Materials and

Methods: The study outlines a comprehensive approach for the development and characterization of Engineering Peptide-Imprinted Nanopolymers (EPINs) for the isolation of brain-derived extracellular vesicles (BDEVs) from biological samples such as cell culture media and clinical serum samples.
The materials and reagents used in the study include iron oxide, ammonia solution, glutaraldehyde, functional monomers, antibodies, and commercial kits for protein and miRNA isolation. The synthesis process involves selection of specific peptide sequences, coating magnetic nanoparticles with amino groups, and synthesizing EPINs using protein and peptide templates. Characterization techniques include scanning electron microscopy (SEM), nanoparticle tracking analysis (NTA), zeta potential analysis, Western blotting, and RT-qPCR.
The study focuses on the isolation and characterization of BDEVs for potential use as biomarkers in neurological disorders like neuromyelitis optica spectrum disorder (NMOSD). Clinical serum samples from NMOSD patients and healthy controls are collected and processed for exosome isolation using the developed EPINs. The isolated EVs are characterized for morphology, concentration, surface charge, protein constituents, and miRNA content using SEM/TEM, NTA, zeta potential analysis, Western blotting, and RT-qPCR.

Results, Conclusions, and Discussions: In this study, we developed Engineered Polymer-Inorganic Nanocomposites (EPIN) as an innovative platform for isolating cell-specific EVs from biological matrices, overcoming the limitations of conventional methods. Conventional ultracentrifugation method and antibody-conjugated magnetic particles take a lot of time to produce primary antibodies and incur high costs. In addition to the technology to imprint protein particles like MIP, which has been proposed as an alternative, here we have developed a technology to imprint a partial sequence of a protein. Targeting exosomal membrane proteins CD63, CD9, and CD81, we designed specific peptide sequences for synthesizing EPINs. Also, utilizing magnetic nanoparticles, we developed EPIN, which imprinted specific peptide templates that induce binding specifically to EV surface proteins, enhancing the capture efficiency. EPIN showed high specificity in isolating EVs in cell culture media, confirmed through SEM and TEM imaging. The EPIN-glutamate aspartate transporter (GLAST) in particular, effectively isolated astrocyte-derived EVs, indicating significant promise for neuromyelitis optica spectrum disorder (NMOSD) diagnosis, where astrocytopathy is a key feature. We demonstrated the ability of EPIN for diagnosis by analyzing miRNA expressions in NMOSD patient serum during relapse and remission phases, marking a significant step forward in BDEV-based diagnostics. Our findings highlight the potential of EPIN-GLAST in elucidating disease dynamics and underscore the value of targeted EV analysis in neurological disorder research, providing a new avenue for biomarker discovery.

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