Extracellular Vesicle Analysis and Engineering for Personalized Medicine

Abstract

Extracellular vesicles (EVs) have emerged as promising biomaterials for application in precision medicine, offering a potential breakthrough in the diagnosis and treatment of various diseases. This thesis mainly focuses on two critical aspects of utilizing EVs for diagnostics and nanomedicine. EVs are carrying various information from donor cells, and they exist in almost all of body fluids. Consequently, EVs are promising biomarker to detect disease and mutation through liquid biopsy, which allow to test in a less invasive way. Especially for the non-small cell lung cancer (NSCLC) patients, the epidermal growth factor receptor (EGFR) mutation has been widely used as a guideline for the prognosis and tyrosine kinase inhibitors (TKI) based drug selection in patients. In this thesis, we studied EGFR mutation analysis by using bronchial washing (BW)-derived EVs for lung cancer patients, which allow minimally invasive examination, compare to tissue biopsy. EVs are separated from BW sample by lab-on-a disc platform, Exo-Disc, and the EGFR mutation is detected by ddPCR. A total of 55 BW samples were analyzed, revealing detection sensitivities of 89.7% for extracellular vesicle- derived DNA (EV-DNA) and 31.0% for EV-excluded cell-free DNA (EV-X-cfDNA), both with a specificity of 100%. T790M detection rates in 13 matched samples from BW-derived EV-DNA, plasma-derived cell-free DNA (cfDNA), and tissue samples were 61.5%, 10.0%, and 30.8%, respectively. Notably, the acquisition of T790M mutation was identified earlier in BW-derived EVs compared to plasma or tissue samples. The longitudinal examination of BW-derived EVs exhibited a strong correlation with disease progression as observed through CT imaging. The EGFR mutations and analysis using BW-derived EVs from lung cancer patent, demonstrates the clinical potential of using them as liquid-biopsy to help precision medicine such as prognosis and drug resistance of lung cancer patients. Not only for the diagnosis, EVs have great potentials to be used as a nanomedicine. EVs have great potential as nanocarriers due to their innate characteristics such as inherent biocompatibility, appropriate size range, and cell communication capabilities. Recently, EV based delivery has been examined to deliver protein, nucleic acids (siRNA, mRNA) or chemotherapeutic agents. However, the current challenge in the use of extracellular vesicles (EVs) as drug delivery vehicles is loading cargoes into EV membrane without compromising their integrity or functionality. Intrinsic heterogeneity of EVs renders it difficult to evaluate encapsulation behavior and current loading procedures can harm the natural EV membrane or leave chemical residue. Here, we introduce a rapid, efficient loading method without causing damage to EVs by using tonicity control (TC). This method utilizes a lab-on-a-disc platform (ExoDisc), to change the tonicity rapidly. The fully automated enrichment of EVs from cell- culture supernatant and their tonicity is précised and rapidly controlled by tangential flow filtration of ExoDisc, integrated with nano membrane (20 nm) on a tabletop-sized centrifugal system. In this technique, a hypotonic solution was used for temporarily permeabilizing a membrane (“On” state), allowing the influx of molecules into EVs. The subsequent isotonic washing led to an impermeable membrane (“Off” state). The tonicity change allows the loading of different cargos into EVs by generating the osmotic gradient between inside and outside of membrane. In this study, we demonstrate multiple loading cargos such as doxorubicin hydrochloride (Dox), ssDNA, and miRNA. When comparing TC methods with conventional methods such as sonication or extrusion, it shows more effective in terms of loading yields, which were 4.3-folds and 7.2-folds greater, respectively. Finally, the intracellular assessments of miRNA-497-loaded EVs and doxorubicin-loaded EVs confirmed the superior performance of TC method, which demonstrated the possibility of using EV as a nanomedicine for therapeutic outcome, signifying potential opportunities for developing novel exosome-based therapeutic systems for clinical applications. In conclusion, EVs present a promising avenue for both diagnostics and therapeutic applications. We anticipate that this research contributes to unlocking the full potential of EVs, paving the way for their widespread use in healthcare.