Elsevier

Current Opinion in Biotechnology

Volume 60, December 2019, Pages 89-98
Current Opinion in Biotechnology

Extracellular vesicles: exosomes, microparticles, their parts, and their targets to enable their biomanufacturing and clinical applications

https://doi.org/10.1016/j.copbio.2019.01.005Get rights and content

Highlights

  • There are two types of extracellular vesicles (EVs): the submicron-size microparticles and the nanometer-size exosomes.

  • EVs are generated from mammalian cells under activation or stress, carry RNAs, proteins and lipids from their parent cells.

  • In addition to diagnostic applications, EVs are excellent candidates for enabling safe and potent cell and gene therapies.

  • Whole EVs or their membranes can be used as natively produced or engineered to enhance targeting and the biological effects.

  • Clinical applications and biomanufacturing of EVs are at an early stage of development.

Extracellular vesicles (EVs) are membrane vesicles, the submicron-size microparticles and the nanometer-size exosomes, that carry RNAs, proteins and lipids from their parent cells. EV generation takes place under cellular activation or stress. Cells use EVs to communicate with other cells by delivering signals through their content and surface proteins. Beyond diagnostic and discovery applications, EVs are excellent candidates for enabling safe and potent cell and gene therapies, especially those requiring strong target specificity. Here we examine EVs, their engineering and applications by dissecting mechanistic and engineering aspects of their components that endow them with their unique capabilities: their cargo and membranes proteins. Both EV cargo and membranes can be independently engineered and used for various applications. We review early efforts for their biomanufacturing.

Introduction

Extracellular vesicles (EVs) are generated by most if not all mammalian cells [1, 2, 3] and carry RNAs, proteins, and lipids from their parent cells during EV generation, which takes place frequently under cellular activation or stress [2]. Among EVs, the submicron-size microparticles/microvesicles (MPs/MVs; also known as ectosomes) are the larger ones ranging from 100 to 1000 nm in size. They bud off the cytoplasmic membrane of the parent cell under normal physiological or pathophysiological conditions, including coagulation, inflammation, tumorigenesis, and differentiation [2]. Exosomes (Exos), distinct from MPs, are nano-size particle (<100 nm) which originate from multivesicular bodies through exocytosis [2,4]. Besides mammalian cells, outer membrane vesicles (OMVs), derived from bacteria (especially Gram-negative bacteria) are involved in stress response, promoting survival, pathogenesis, and interaction between bacteria in a community [5].

Cells use EVs to communicate with other cells by delivering signals through their content [2]. As reviewed (e.g. Refs. [6, 7, 8]), over the last few years, EVs have emerged as important mediators of intercellular communication regulating an ever-expanding range of biological processes, both on normophysiology and pathophysiology. The former includes enhancing and accelerating native developmental programs in immunology, vascular repair, and angiogenesis, while the latter includes carcinogenesis and cancer metastasis, neurodegenerative disorders, and infectious and cardiovascular diseases. As such, EVs are suitable for a broad range of applications, from minimally invasive diagnostic applications to therapeutic interventions, including cell therapies and macromolecular drug delivery. In order to pursue such applications involving EVs, better EV characterization, as well as better understanding of the mechanisms of cell targeting and methods for EV biomanufacturing are needed (Figure 1).

Section snippets

How do EVs recognize and deliver cargo to target cells?

Understanding how do EVs target and are taken up by cells is crucial for their applications [9,10]. The interaction typically starts with a ligand–receptor mediated binding, adhesion, or docking of EVs to target cells (Figure 1). The ligands and receptors involved are EV and target-cell specific, and, in some cases, this ligand–receptor recognition step is sufficient to alter the fate of target cells [11, 12, 13]. Yet, in most cases, EVs exert their biological effect through transferring of

EVs alter the biology and fate of target cells through diverse mechanisms

Uptake of EVs enables delivery of EV cargo to recipient cells, thus triggering a broad spectrum of biological phenotypes. Tumor-derived EVs regulate the tumor microenvironment and impart an invasive effect in cancer progression and angiogenesis [24,25]. Exosomes derived from hepatocellular carcinoma cells delivered both SMAD Family Member 3 (SMAD3) protein and mRNA to circulating hepatocellular carcinoma cells, enhanced their adhesive ability, and supported their metastasis [26••]. EVs also

Translational applications of native EVs

As discussed, native EVs (MP and exosomes) have a good potential as therapeutic agents in translational applications. Below, we review the target specificity and the nature of the native cargo of EVs derived from a few select cell types. These and additional reports are summarized in Table 1.

Engineering using EVs or their components for cargo delivery

EVs exhibit desirable native characteristics that makes them suitable as vehicles for cargo delivery (Figure 3). As summarized in Figure 3, all EVs components (their native cargo, surface proteins, and membranes), can be engineered for various applications. Loading of synthetic cargo is an important first engineering goal. EVs can be also engineered to enhance target specificity. Finally, their native cargo can be used for diagnostic, discovery or therapeutic applications.

Mechanisms of EV biogenesis

Key to the biomanufacturing process is the mechanism by which one can induce EV formation from various cell types. EVs are produced typically under physiological or pathophysiological stress or stimulation. Biogenesis of two major type of EVs (exosomes or MPs) is quite distinct. For exosome generation, several stimuli such as cellular stress, irradiation, hypoxia, or starvation have been shown to increase exosome production. Details regarding the mechanisms of exosomes biogenesis at a molecular

Biomanufacturing of EVs: exosomes and MPs

EVs are currently pursued by several startup and larger companies for a broad range of applications, from minimally invasive diagnostic applications to therapeutic interventions, including cell therapies and cargo delivery. To achieve large-scale EV production, it is necessary to develop EV manufacturing using Good Manufacturing Practices (GMP). Currently, several approaches have been reported for GMP-grade manufacturing of exosomes either from MSCs [58••,72] or from cardiac progenitor cells [57

Future developments

Understanding the mechanisms of EV-to-target recognition and its specificity is crucial for developing more effective technologies for EV-based therapies or for using parts of the EVs or their parent cells to construct semi-synthetic delivery systems. Are there any reasons as to why some EV has exquisite target specificity while other do not? Can we determine if a cell’s EVs will have some specific targets? Can we use modular engineering of one EV type to specifically target different cell

Conflict of interest statement

The two authors are listed as inventors on a pending US/PCT patent application (publication US20170058262A1, Application No. 15/308,221, PCT No. PCT/US15/31388) on the MkMP-based technologies. The rights to the pending patent belong to the three inventors of the patent.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgement

This work was supported by a grant (CBET-1804741) by the US National Science Foundation.

References (80)

  • A. Mizrak et al.

    Genetically engineered microvesicles carrying suicide mRNA/protein inhibit schwannoma tumor growth

    Mol Ther

    (2013)
  • G. Fuhrmann et al.

    Active loading into extracellular vesicles significantly improves the cellular uptake and photodynamic effect of porphyrins

    J Controlled Release

    (2015)
  • K. Tang et al.

    Delivery of chemotherapeutic drugs in tumour cell-derived microparticles

    Nat Commun

    (2012)
  • F. Momen-Heravi et al.

    Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages

    Nanomedicine

    (2014)
  • L. Alvarez-Erviti et al.

    Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes

    Nat Biotechnol

    (2011)
  • C.Y. Kao et al.

    Engineering human megakaryocytic microparticles for targeted delivery of nucleic acids to hematopoietic stem and progenitor cells

    Sci Adv

    (2018)
  • M. Kanada et al.

    Differential fates of biomolecules delivered to target cells via extracellular vesicles

    Proc Natl Acad Sci U S A

    (2015)
  • Y. Tian et al.

    A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy

    Biomaterials

    (2014)
  • Y. Yano et al.

    The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane

    Biochem J

    (1994)
  • H. Xiao et al.

    Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets

    Cryobiology

    (2002)
  • K. Pachler et al.

    A good manufacturing practice-grade standard protocol for exclusively human mesenchymal stromal cell-derived extracellular vesicles

    Cytotherapy

    (2017)
  • T. Lopatina et al.

    Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential

    Cell Commun Signal

    (2014)
  • R.S. Cherry et al.

    Physical mechanisms of cell damage in microcarrier cell culture bioreactors

    Biotechnol Bioeng

    (1988)
  • M. Straat et al.

    Monocyte-mediated activation of endothelial cells occurs only after binding to extracellular vesicles from red blood cell products, a process mediated by beta-integrin

    Transfusion

    (2016)
  • X. Tan et al.

    Mesenchymal stem cell-derived microparticles: a promising therapeutic strategy

    Int J Mol Sci

    (2014)
  • G. Raposo et al.

    Extracellular vesicles: exosomes, microvesicles, and friends

    J Cell Biol

    (2013)
  • A.M. Curtis et al.

    Endothelial microparticles: sophisticated vesicles modulating vascular function

    Vasc Med

    (2013)
  • B. Gyorgy et al.

    Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles

    Cell Mol Life Sci

    (2011)
  • C. Schwechheimer et al.

    Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions

    Nat Rev Microbiol

    (2015)
  • S. El-Andaloussi et al.

    Extracellular vesicles: biology and emerging therapeutic opportunities

    Nat Rev Drug Discov

    (2013)
  • H.M. van Dongen et al.

    Extracellular vesicles exploit viral entry routes for cargo delivery

    Microbiol Mol Biol Rev

    (2016)
  • S. Fais et al.

    Evidence-based clinical use of nanoscale extracellular vesicles in nanomedicine

    ACS Nano

    (2016)
  • K.J. McKelvey et al.

    Exosomes: mechanisms of uptake

    J Circ Biomark

    (2015)
  • L.A. Mulcahy et al.

    Routes and mechanisms of extracellular vesicle uptake

    J Extracell Vesicles

    (2014)
  • A. Ghosh et al.

    Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice

    J Clin Invest

    (2008)
  • G. Turturici et al.

    Extracellular membrane vesicles as a mechanism of cell-to-cell communication: advantages and disadvantages

    Am J Physiol Cell Physiol

    (2014)
  • D. Faille et al.

    Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells

    J Cell Mol Med

    (2012)
  • S. Horibe et al.

    Mechanism of recipient cell-dependent differences in exosome uptake

    BMC Cancer

    (2018)
  • H. Costa Verdera et al.

    Cellular uptake of extracellular vesicles is mediated by clathrin-independent endocytosis and macropinocytosis

    J Controlled Release

    (2017)
  • T. Tian et al.

    Exosome uptake through clathrin-mediated endocytosis and macropinocytosis and mediating miR-21 delivery

    J Biol Chem

    (2014)
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