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Method of Large-Scale Exosome Purification and Its Use for Pharmaceutical Applications
Published in Peixuan Guo, Kirill A. Afonin, RNA Nanotechnology and Therapeutics, 2022
Fengmei Pi, Yangwu Fang, Dongsheng Li, Peixuan Guo
Exosomes are a class of extracellular vesicles (EVs) that originated from inward budding of endosome membranes into the multivesicular bodies. They contain endosomal trafficking markers such as tumor susceptibility gene 101 (TSG101), apoptosis linked gene-2 interacting protein (Alix), tetraspanin (CD63) and flotillins (Raab-Traub and Dittmer, 2017). Originally, exosomes were thought to act as cellular garbage disposals. Recent studies have found exosomes are natural carriers for mRNA, lncRNA, miRNA, siRNA, protein, DNA and peptide for long- distance intercellular communication. EVs also involve in regulating the cell differentiation, angiogenesis, metabolic reprogramming, tumor progression, immune modulation and pathogen challenges (Willms et al., 2018). Substantial studies have demonstrated that EVs are a potential source of cancer biomarkers. There has been a great progress from discovery of these biomarkers to validation and clinical application as cancer liquid biopsy (Zhao et al., 2019). EVs have also been studied as a natural delivery vehicle for small RNA therapeutics. Exosomes isolated from dendritic cells reengineered with RVG peptide can systemically deliver GAPDH siRNA to the brain after systemic injection into mice (Alvarez-Erviti et al., 2011). EVs can be also post genesis decorated with artificial RNA nanoparticle-based ligand to enhance its targeting selectively to cancer cells, and it has been used for delivery of siRNA for cancer regression in prostate cancer, breast cancer and colorectal cancer mice model (Pi et al., 2018).
The Emerging Role of Exosome Nanoparticles in Regenerative Medicine
Published in Harishkumar Madhyastha, Durgesh Nandini Chauhan, Nanopharmaceuticals in Regenerative Medicine, 2022
Zahra Sadat Hashemi, Mahlegha Ghavami, Saeed Khalili, Seyed Morteza Naghib
Other than EM, which meanly is used for direct observation of EVs morphology, different methods are used for their characterisation. As explained, exosomes can contain markers on their surface as their parent cells. The immuno-affinity-based approaches such as Enzyme-Linked Immunosorbent Assays (ELISA), Flow Cytometry, and antibody-coated magnetic beads rely on surface markers of EVs for their immuno-isolation and characterisation. Amongst these, the ELISA method has most commonly been applied for detection of EVs. The plates of ELISA kit could be coated by pan-exosome antibodies (anti-CD63 and anti-caveolin-1 melanoma-derived exosomes) to capture the exosomes existing in the samples (Logozzi et al. 2009; Khodashenas et al. 2019). As a high throughput approach of label-free protein analysis, the electrochemical sensing platforms and the plasmonic were combined with immuno-affinity methods (Contreras-Naranjo et al. 2017). However, EVs can be collected by their protein contents and applying conventional detection method, such as total protein analysis and western blots (Bradford assays, bicinchoninic acid (BCA)) (Khodashenas et al. 2019). Aside from the protein content, the nucleic acid contents (DNA, RNA, or miRNA) can be searched by PCR (polymerase chain reaction) or DNA sequencing methods (Eldh et al. 2012; Li et al. 2014; Zarovni et al. 2015).
Exosomes in Cancer Disease, Progression, and Drug Resistance
Published in Vladimir Torchilin, Handbook of Materials for Nanomedicine, 2020
Taraka Sai Pavan Grandhi, Rajeshwar Nitiyanandan, Kaushal Rege
Tetraspanins (CD63, CD9, CD81, and CD82) are integral membrane protein components that are highly enriched in exosomes. They organize into tetraspanin-enriched domains by interacting with lipids, other cytosolic and transmembrane proteins [33, 38]. Cytoplasmic and transmembrane protein interactome of tetraspanins is considered another major source of protein loading into the exosomes [33]. Sphingosine and fatty acid containing ceramides have been shown to be essential in exosome biogenesis [39]. It is well known that ceramides can induce stable and spontaneous curvature in lipid bilayer membranes (liposomes), and play an essential role in inward budding of late endosomal limiting membrane for ILV generation [39]. Kajimoto et al. [34] showed that continuous metabolization of ceramide into sphingosine 1-phosphate (S1P) activates inhibitory G protein-coupled S1P receptors, essential for successful cargo sorting into ILVs in HeLa cells; S1P signaling was shown to play a role in the sorting of the tetraspanin CD63 protein into exosomes [34].
Latest advances in extracellular vesicles: from bench to bedside
Published in Science and Technology of Advanced Materials, 2019
Tomofumi Yamamoto, Nobuyoshi Kosaka, Takahiro Ochiya
EVs have been observed in all body fluids, such as blood, urine, saliva, sputum, breast milk, semen, and cerebrospinal fluid [13–19]. EVs have been shown to contain miRNAs, mRNA, DNAs, and proteins within the lipid bilayer [11]. This bilayer construction, which is very stable, enables EVs to circulate intact through body fluids, and thus, EVs can transfer their components to distant recipient cells. The accumulating data have indicated that the contents, size, and membrane composition of EVs are highly heterogeneous, dynamic and depend on the cellular source, state, and environmental conditions. EVs have some exosomal markers, including members of the tetraspanin family (CD9, CD63, CD81), members of the endosomal sorting complex required for transport (ESCRT) (TSG101, Alix), heat shock proteins (Hsp60, Hsp70, Hsp90), and RAB proteins (RAB27 A/B) [20,21]. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments and EVs of various sizes budding from the plasma membrane [22]. Differential separation by immuno-isolation using either CD63, CD81, or CD9 was proposed. Several classically used exosome markers, like major histocompatibility complex (MHC), flotillin, and Hsp70 proteins, are similarly present in all EVs, and they also identified proteins specifically enriched in small EVs, and defined a set of five protein categories displaying different relative abundance in distinct EV populations. In this way, understanding the heterogeneity of EVs is important for EV-based therapy, since each EV has difference of their biological function on their membrane surface.
No-stain protein labeling as a potential normalization marker for small extracellular vesicle proteins
Published in Preparative Biochemistry & Biotechnology, 2023
Anjugam Paramanantham, Rahmat Asfiya, Siddharth Das, Grace McCully, Akhil Srivastava
UC and SEC are two gold standard methods frequently used in sEV based studies. We employed both isolation methods for each H1299 cell culture set to avoid any batch-to-batch bias in the yield. A comparable yield of sEVs was observed from both methods analyzed through the NTA system. The UC method was able to pellet ∼5 × 106 sEVs, while ∼3.68 × 106 sEVs were collected from the Izon® SEC method. The NTA determines the size distribution of purified sEVs over the population. Figure 2A implies the mean diameter size of sEVs purified by UC and Izon® SEC remained in the same range with peaked at ∼150 and ∼160 nm, respectively. The TEM images also suggested no change in morphological features of the sEVs isolated from either method. sEVs purified from both methods show an intact bilayer membrane and a nearly circular shape (Figure 2A-inset). Western blot assay of sEVs isolated from both methods resulted in a positive detection of sEV marker CD63 and the negative signal was observed for cytosolic protein HSP90B1 (Grp94). The absence of Grp94 bands in sEV lanes but not in cell lysate indicates the purity of sEVs negating any contamination from cytosolic proteins (Figure 2B). Grp94 is a suggested negative control for sEVs by International Society for Extracellular Vesicles (ISEV) in the Minimal Information for Studies of Extracellular Vesicles position paper.[50,51] In summary, three different characterization methods confirmed the presence of pure sEVs with comparable characteristics in the isolations done through UC and Izon® SEC methods.
Advances of engineered extracellular vesicles-based therapeutics strategy
Published in Science and Technology of Advanced Materials, 2022
Hiroaki Komuro, Shakhlo Aminova, Katherine Lauro, Masako Harada
Since all types of cells secrete EVs, their recovery has been reported in all bodily fluids [20], including blood [21], urine [22], saliva [23], breast milk [24], semen [25], bronchoalveolar lavage fluid [26], synovial fluid [27], cerebrospinal fluid [28], and amniotic fluid [29]. EVs are broadly classified into three types: exosomes, microvesicles, and apoptotic bodies (Figure 1). The classification of EV can be associated with their biogenesis. Exosomes (50–150 nm) derive from the inward budding of the endosomal compartment, multivesicular bodies (MVBs). Upon fusion with the cell membrane, exosomes are released to the extracellular space and mediate intercellular communication by transferring bioactive molecules such as RNAs, proteins, and lipids between cells in living organisms [30]. Tetraspanins belong to a cell surface protein family with four transmembrane domains required for intraluminal vesicle formation [31]. CD9, CD63, and CD81 are tetraspanin proteins found enriched in exosomes and often used as EV surface markers [12]. Microvesicles (100–1000 nm), on the other hand, are secreted directly from the plasma membrane. Microvesicles are not as well studied or defined as the other EV types, resulting in more broad characteristics. Apoptotic bodies (100–5000 nm) are produced from apoptotic cells when they undergo programmed cell death. Numerous studies illustrate the heterogeneity of these EVs and missing pieces of information left uncovered, implicating the change in descriptions in the future [12]. For example, there is no defined method or unique maker to separate one EV type from another. A report suggested that 100,000 g ultracentrifugation (UC) co-isolates two EV subtypes defined by specific proteins [32], proposing the importance of considering heterogeneity and diversity among EV types in the study design. Furthermore, some EV subtypes are named after the cell from which they are secreted, such as cardiosome (cardiovascular-origin) and oncosome (oncology-origin) [33–36], though ISEV strongly suggests that the generic term EV be widely adopted [37]. Moreover, the guidelines published by ISEV in 2018 recommend referring to small EVs (sEVs) (<100 nm or <200 nm) or >200 nm as medium/large EVs (m/lEVs) EVs based on their physical characteristic, size and density [12]. This article will adhere to the term ‘EV’ in place of exosome or the names used in the original literature to avoid terminology ambiguity.