(M-490) Development of a Next-Generation Nano-Pocket Membrane for the Isolation and Purification of Extracellular Vesicles

Introduction:: Extracellular vesicles (EVs) are heterogeneous nanoscale membrane vesicles secreted from most cells and biological fluids [1]. EVs are recognized as novel intercellular communications tools and biomarkers for several diseases including cancer, diabetes, and renal diseases [2]. EVs have three main subtypes: exosomes, microvesicles (MVs), and apoptotic bodies which share overlapping size ranges. The particle diameters are 30 to 150 nm for exosomes, 100 to 1000 nm for MVs, and 50 to 5000 nm for apoptotic bodies [2]. Isolating the EVs based on their size is a major obstacle for basic research and clinical translations. Various methods, such as ultracentrifugation, size exclusion chromatography, and ultrafiltration, have been employed for EV isolation based on size, but each approach has its drawbacks. Porous membranes have been used for size-based EV isolation. To capture specific size ranges of EVs, multiple membranes are required. Current filtration solutions fail to filter the EVs based on their specific size and some particles can become lodged deep within membrane pores. This flaw reduces the capturing efficiency due to the aggregation. In this research we present a novel nano-pocket geometry membrane to overcome the previous flaws. We have developed methods to create pores that are not cylindrical, but rather nano-pockets (conical) that taper from a wide opening to a narrower opening on the bottom side of the membrane. The membrane design helps capture EVs within a specific size range without allowing smaller or larger particles to block the pores in tangential flow filtration (TFF) (figure 1 a).


Materials and Methods:: The fabrication process was divided into six steps: sacrificial layer (SL), deposition I, pore patterning, deposition II, SL release, and etching. The first process involved spin coating the SL to a Si wafer. Next we deposited parylene C (PAR) to protect the SL from the next layer, the Photoresist (PR) layer. A layer of PR was deposited over the PAR layer I, and patterned into pores with conventional photolithography. A series of mask openings, exposure doses, and focus offsets, were tested to obtain the desired PR pore size and shape. A second deposition of parylene was applied to further shape and narrow pore sizes. The SL was dissolved in water, releasing the membrane, and precision etched with a reactive ion etcher, We used scanning electron microscopy to validate the actual size of the pores. To validate the parylene and photoresist deposition, a NanoSpec spectrophotometer was used to measure thickness. In order to be relevant for EV separations, the final target nano-pocket membrane shape has a wide opening (0.5 to 1 µm) at the top, narrows to a smaller opening (0.05 to 0.15 nm) at the bottom, and has a (1 to 2.5 µm) total thickness.


Results, Conclusions, and Discussions:: We successfully produced nano-pocket membranes featuring a wide opening at the top, measuring 600 nm, which narrows down to a smaller opening on the bottom side, ranging from 0.1 to 0.15 µm (figure 1b). The fabrication process involved determining the optimal amount of parylene coating required on top of the patterned PR layer, which was found to be 300 nm. Any increase or decrease in the parylene coating resulted in pore closure or widening, respectively, which would ultimately affect the EV isolation within the target size range.

Our investigation on the effects of different power levels on the reactive ion etching system (RIE-2000), while maintaining a gas flow rate of 30 sccm for O2 and 8 sccm for CHF3, showed that the etching rate was less stable than the parylene deposition process. We performed a comparative analysis of the effects of 50 W and 100 W on the RIE-2000 system and found 50 W etching was more consistent than 100 W etching. The uncertainty of the etch and deposition processes limit our ability to produce the controlled pore size and are therefore essential in the process optimization required to control pore size on these membranes.

We also noted that the time required to etch the membrane's bottom side depended on the parylene deposition layers and etching rate. To achieve the desired pore size range of 0.1 to 0.15 µm, the etching time should be around 9.5 to 10.5 min, with an etching rate of approximately 100 nm/min. These results demonstrate the importance of careful fabrication process optimization to achieve the desired nanostructure, which is crucial for the successful development of advanced membrane-based applications.

We are currently evaluating the ability of the membrane to capture and release particles of various sizes using polystyrene standards and liposomes, employing TFF. Results are being quantified with fluorescence and other detection methods.



It is anticipated that the nanopocket membranes developed and optimized through this work may be applied to achieve a better EV isolation. We successfully completed the fabrication process for our nano-pocket membranes for targeted pore geometries.




Acknowledgements (Optional): :

References (Optional): : [1] Théry, C., Witwer, K. W., Aikawa, E., Alcaraz, M. J., Anderson, J. D., Andriantsitohaina, R., ... & Jovanovic‐Talisman, T. (2018). Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of extracellular vesicles, 7(1), 1535750.

[2] Yáñez-Mó, M., Siljander, P. R. M., Andreu, Z., Bedina Zavec, A., Borràs, F. E., Buzas, E. I., ... & De Wever, O. (2015). Biological properties of extracellular vesicles and their physiological functions. Journal of extracellular vesicles, 4(1), 27066.