Emerging micro-nanotechnologies for extracellular vesicles in immuno-oncology: from target specific isolations to immunomodulation

Abstract

Extracellular vesicles (EVs) have been hypothesized to incorporate a variety of crucial roles ranging from intercellular communication to tumor pathogenesis to cancer immunotherapy capabilities. Traditional EV isolation and characterization techniques cannot accurately and with specificity isolate subgroups of EVs, such as tumor-derived extracellular vesicles (TEVs) and immune-cell derived EVs, and are plagued with burdensome steps. To address these pivotal issues, multiplex microfluidic EV isolation/characterization and on-chip EV engineering may be imperative towards developing the next-generation EV-based immunotherapeutics. Henceforth, our aim is to expound the state of the art in EV isolation/characterization techniques and their limitations. Additionally, we seek to elucidate current work on total analytical system based technologies for simultaneous isolation and characterization and to summarize the immunogenic capabilities of EV subgroups, both innate and adaptive. In this review, we discuss recent state-of-art microfluidic/micro-nanotechnology based EV screening methods and EV engineering methods towards therapeutic use of EVs in immune-oncology. By venturing in this field of EV screening and immunotherapies, it is envisioned that transition into clinical settings can become less convoluted for clinicians.

Figure 1.. Potential applications of extracellular vesicles in immuno-oncology

Figure 2.. Conventional approaches to extracellular vesicle (EV) isolation and characterization

Figure 3.. Recent methods for EV isolation and characterization

(A) Schematic of the isolation and characterization strategy of CD63 expressing EVs using the immunoaffinity ExoChip. ^[1] (B) An CD63 aptamer coated nanofiltration membrane with a pore size of 20 nm. ^[2] (C) Schematic overview of Raman microscopy characterization of single optically trapped EVs. ^[3] (D) Schematic diagram of digital detection of BAM-DNA-Anchored EVs at the single-nanoparticle level, reliant on nucleic acid-based amplification. ^[4] (E) Schematic of fluorescence-based microfluidic diffusion sizing for the multiparametric characterization and quantification of EVs and images of the microfluidic device used for EV analysis. ^[5] (F) The hybrid macro- and nanomechanical oscillator-based exosome isolation system: EXO, photograph of the EXODUS station, on which an EXODUS device is installed. Scale bar, 1 cm. Chen ^[6]

Figure 4.. Immunomodulation mediated by EVs.

Schematic representation of the multiple potential pathways for EV uptake and interaction with T-cells.

Figure 5.. Microfluidics and nanotechnology based extracellular vesicle isolation for immuno-oncology:

(A-C) Tumor cell-derived EV isolation using conventional cancer-associated protein markers (A), new cancer-associated EV’s specific binding characteristic (B), and click-chemistry assisted TEV screening; (D) Immune cell derived EV isolation. Copyright 2014 by He ^[7], 2018 by Sharma ^[8], 2018 by Reátegui ^[9], 2019 by Kumar ^[10], 2019 by Kang ^[11], 2020 by Kang ^[12], 2020 by Dong ^[13], 2021 by Kang ^[14], 2020 by Pisano ^[15].

Figure 6.. Recent micro-nanotechnology approaches for immune-related extracellular vesicle (EV) modulation and therapeutics:

(A) hydrogel-based EV therapy via EV-prodrug loading (a) or mesenchymal stem cell-derived EVs loading (b). Copyright 2018 by Fuhrmann ^[16], 2021 by Zhu ^[17]; (B) microfluidics-based EV engineering via donor cell engineering and its EV screening on chip (c) or nanochannel based EV-cargo loading (d) or simple drug incubation on chip (e) or cell culture and EV surface engineering on chip (f). Copyright 2017 by Wang ^[18], 2021 by Hao ^[19], 2020 by Thakur ^[20], 2019 by Zhao ^[21]; (c) hybrid EV vesicle for cancer therapy (g) or neutrophil-derived EV mimetics-based therapy (h). Copyright 2020 by Zhu ^[22], 2022 by Zhang ^[23].

Figure 7.. Future directions for on-chip EV isolation in-situ EV engineering for translational applications of EVs in immuno-oncology