Novel Extracellular Vesicle Enrichment Method and Their Function and Properties Modulated by Nanofibrillar-Based 3D Matrix Mechanical Stiffness

Abstract

Extracellular vesicles (EVs) are critical mediators of intercellular communication, transporting diverse molecular cargoes, including proteins and microRNAs, with significant potential for diagnostics, therapeutics, and drug delivery. However, the inherent heterogeneity and complexity of EVs pose considerable challenges for their efficient isolation and comprehensive analysis, limiting their clinical translation. My PhD research addressed these challenges through two major projects: the development of a novel EV enrichment method and the investigation of extracellular matrix (ECM) mechanical stiffness on EV properties and functions within a nanofiber-based three-dimensional (3D) culture system.

In the first project, a novel method was established for efficient EV enrichment by leveraging the high dispersibility and large surface area of nanocellulose fibers, functionalized via NHS/EDC chemistry to conjugate biotinylated CD63 antibodies using streptavidin. This resulted in the creation of TOCNF@CD63 nanomaterials for specific immunoaffinity-based EV capture. Key experimental parameters, including nanomaterial concentration, separation time, and antibody loading, were systematically optimized. Comparative analyses demonstrated that this method significantly outperformed conventional EV isolation techniques, such as ultracentrifugation, size-exclusion chromatography, and magnetic beads, in terms of recovery efficiency. The high performance of this method is further validated by the efficient capture of EVs from biological blood plasma, allowing the detection of bioactive markers from EV-derived miRNAs and proteins using LC-MS/MS and RT-qPCR. This approach not only enhances EV isolation efficiency but also has the potential to advance biomedical applications, particularly in diagnostics and therapeutic EV research.

The second project explored the effects of ECM mechanical stiffness on EV properties and functions in a nanofiber-based 3D culture environment. To closely mimic the fibrotic ECM in vivo, a hybrid hydrogel system comprising TOCNF and GelMA was developed and extensively characterized via SEM, FT-IR, swelling ratio, and degradation studies. Biocompatibility was confirmed through long-term culture of two cancer cell lines, demonstrating over 80% cell viability and the formation of robust tumor spheroids within the 3D matrix. Under varying stiffness conditions, EVs were isolated from the culture supernatant and subjected to proteomic and small RNA sequencing analyses. Results revealed that increased ECM stiffness significantly altered EV cargo, with enrichment of molecules associated with tumor progression and metastasis. Functional assays, including wound healing and CCK-8, showed that EVs derived from stiff matrices (StEVs) more effectively promoted recipient cell proliferation and migration compared to those derived from soft matrices (SoEVs). Further mechanistic investigations revealed that StEVs activated the MAPK signaling pathway in recipient cells, and pathway inhibition studies confirmed that MAPK activation was critical for the pro-tumor effects of StEVs. These findings highlight the importance of ECM stiffness in shaping EV cargo and function, offering a more physiologically relevant 3D tumor model compared to traditional 2D cultures. Furthermore, the 3D nanofiber-based matrix holds potential for studying fibrotic diseases, such as liver and lung fibrosis, and may provide novel therapeutic strategies by modulating EV-stiffness interactions.

In conclusion, my research introduces an innovative method for EV enrichment and reveals the critical role of ECM mechanical stiffness in regulating EV properties and functions within 3D culture systems. These findings advance our understanding of the tumor microenvironment, providing valuable insights into the interplay between physical cues and EV-mediated cancer progression, while also offering promising strategies for therapeutic interventions targeting the tumor ECM.

Date of Award	2 Jun 2025
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	M YANG (Supervisor)