Preparation of Molecularly Imprinted Cryogel Membranes for Isolating Extracellular Vesicles

Abstract

Cancer, which unfortunately accounted for one in every six deaths worldwide, led to the deaths of ten million people in 2020. Breast cancer is particularly important because it constitutes one out of every eight cancers in women worldwide and is observed in one out of every four women of all ages in Türkiye. Early diagnosis is crucial to increase survival rates and improve the quality of life, but there is a delay in early diagnosis due to the lack of cost-effective traditional tests and technological/biological barriers. While it is important to capture cancer cells through liquid biopsies, their rarity poses a significant challenge. Extracellular vesicles (EVs) are vesicles that continuously facilitate information exchange among cancer cells. However, the lack of easy-to-use, reliable, and repeatable platforms for their isolation and detection is noticeable. In this thesis, the aim is to isolate EVs cultured and collected on a microfluidic chip mimicking the cancer microenvironment with the use of molecularly imprinted cryogel membranes, achieving high sensitivity and efficiency. After the synthesis of EVs-imprinted cryogel membranes, they were characterized by different methods and an imprinted polymer with high selectivity, chemical and mechanical durability was prepared for EVs purification. The optimum conditions for EVs adsorption of the prepared cryogel membrane were determined in a batch system with the help of a rotator. The maximum EVs adsorption capacity was found to be 1075 particles/g cryogel in aqueous solution prepared at room temperature in pH 5.0 buffer solution. The EVs adsorbed on the prepared cryogel membrane was desorbed using 0.1 M NaCl after each analysis and the reusability of the imprinted polymer was investigated. Finally, the adsorption isotherm model that the interaction between EVs and cryogel membrane fits was determined with mathematical calculations. The obtained results were validated with HPLC experiments. By imprinting the spatial architecture of EVs onto cryogel membranes, the goal is to minimize stability issues in immunological analyses. Consequently, capturing small-sized but significant EVs that play a crucial role in cancer will pave the way for breakthroughs in precision healthcare, accelerate cancer early diagnosis, reduce costs, and help expand the field of care while minimizing global inequalities.