Macroporous Epoxy-Based Monoliths Functionalized with Anti-CD63 Nanobodies for Effective Isolation of Extracellular Vesicles in Urine

Abstract

Extracellular vesicles (EVs) have enormous potential for the implementation of liquid biopsy and as effective drug delivery means, but the fulfilment of these expectations requires overcoming at least two bottlenecks relative to their purification, namely the finalization of reliable and affordable protocols for: (i) EV sub-population selective isolation and (ii) the scalability of their production/isolation from complex biological fluids. In this work, we demonstrated that these objectives can be achieved by a conceptually new affinity chromatography platform composed of a macroporous epoxy monolith matrix functionalized with anti-CD63 nanobodies with afflux of samples and buffers regulated through a pump. Such a system successfully captured and released integral EVs from urine samples and showed negligible unspecific binding for circulating proteins. Additionally, size discrimination of eluted EVs was achieved by different elution approaches (competitive versus pH-dependent). The physical characteristics of monolith material and the inexpensive production of recombinant nanobodies make scaling-up the capture unit feasible and affordable. Additionally, the availability of nanobodies for further specific EV biomarkers will allow for the preparation of monolithic affinity filters selective for different EV subclasses.

Kidney Cancer and Potential Use of Urinary Extracellular Vesicles

“…CD63 is found to be a representative exosomal marker for RCC cell lines, e.g., 786-O, 769-P, ACHN, Caki-2, Caki-1 and RCC53 due to its stable expression rather than other exosomal markers CD9 and CD81, which have variable expression among RCC cell lines [ 16 ]. Thus, anti-CD63 nanobodies have been applied for an efficient isolation of EVs from urine with high purification [ 38 ]. CANX is identified as a negative EV marker for RCC cell lines by spatial proteomics analysis [ 16 , 17 ].…”