Exosomes: natural nanoparticles as bio shuttles for RNAi delivery

Accepted Manuscript Exosomes: natural nanoparticles as bio shuttles for RNAi delivery

Saber Ghazizadeh Darband, Mohammad Mirza-AghazadehAttari, Mojtaba Kaviani, Ainaz Mihanfar, Shirin Sadighparvar, Bahman Yousefi, Maryam Majidinia PII: DOI: Reference:

S0168-3659(18)30572-8 doi:10.1016/j.jconrel.2018.10.001 COREL 9484

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

3 July 2018 30 September 2018 1 October 2018

Please cite this article as: Saber Ghazizadeh Darband, Mohammad Mirza-AghazadehAttari, Mojtaba Kaviani, Ainaz Mihanfar, Shirin Sadighparvar, Bahman Yousefi, Maryam Majidinia , Exosomes: natural nanoparticles as bio shuttles for RNAi delivery. Corel (2018), doi:10.1016/j.jconrel.2018.10.001

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ACCEPTED MANUSCRIPT Exosomes: natural nanoparticles as bio shuttles for RNAi delivery Saber Ghazizadeh Darbanda, Mohammad Mirza-Aghazadeh-Attarib,c, , Mojtaba Kavianid, Ainaz Mihanfare, Shirin Sadighparvarf, Bahman Yousefig,h,* [email protected], Maryam Majidiniai,j,**,1 [email protected] a

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Danesh Pey Hadi Co., Health Technology Development Center, Urmia University of Medical

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Sciences, Urmia, Iran; b

Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Aging Research Institute, Tabriz University of Medical Sciences, Tabriz, Iran;

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School of Nutrition and Dietetics, Acadia University, Wolfville, Nova Scotia, Canada;

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Department of Biochemistry, Faculty of Medicine, Urmia University of Medical Sciences,

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c

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Urmia, Iran;

Neurophysiology Research Center, Urmia University of Medical Sciences, Urmia, Iran;

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Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;

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Molecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;

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Solid Tumor Research Center, Urmia University of Medical Sciences, Urmia, Iran; j

*

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Student Research Committee, Urmia University of Medical Sciences, Urmia, Iran;

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Correspondence to: Bahman Yousefi, Immunology Research Center, Tabriz University of Medical

Sciences, Tabriz, Iran. **

Correspondence to: Maryam Majidinia, Solid Tumor Research Center, Urmia University of Medical Sciences, Urmia, Iran. 1

These authors contributed equally to this work

Abstract Application of exosomes, natural nanoscale vesicles, as specific delivery vehicles has received considerable attention in recent past years. The presence of various adhesive proteins on the

ACCEPTED MANUSCRIPT surface of these lipid bilayers, gives them the ability to interact with cellular membranes. Although the function of exosomes was not known for some time, further researches have suggested that they are effective in multiple cellular pathways, as well as pathogenesis of a broad range of diseases including neurodegenerative diseases, cardiovascular diseases, and more

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importantly, multiple human malignancies. As erstwhile research brought to light more aspects

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of exosomes’ biogenesis and functions, researchers sought to benefit from their effects in

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therapeutic and diagnostic purposes. Gene therapy is one of these fields that has seen many endeavors made. The present review article looks at gene therapy and its latest advancements,

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structure and function of exosomes and their role as bio shuttles in various clinical contexts

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relating to gene therapy.

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Keywords: Exosomes; miRNA, siRNA, gene transferring, vehicle

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Introduction The past century is hallmarked with giant steps taken towards diagnosis and management of infectious and non-infectious diseases, which led to increased quality and duration of life in virtually all societies. Notwithstanding, many diseases and pathologies such as cancers, chronic

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inflammatory diseases, single gene defects and degenerative diseases of organs have been largely

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immune to the mentioned effect, and classic therapies and diagnostic methods have limited

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application (1). Various new modalities have been introduced to further the treatment and

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diagnosis of these diseases. One of these relatively new concepts is gene therapy in which genetic material is transferred to where it is needed (2). One crucial step of gene therapy is the

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medium by which the load is delivered, or the so called vector. Finding an efficient vector is one

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of the main hurdles of development in gene therapy (3). Fortunately, progress in cellular biochemistry and physiology has resulted in better understanding of cellular mechanisms and

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discovery of various mediators. One of these mediators is exosomes, which was introduced in

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1983. The function of exosomes was not known for some time, but further research suggested that exosomes play roles in multiple cellular pathways and pathologies (4). As new functions of

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these lipid bilayers were unraveled, researchers sought to benefit from their application for

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therapeutic and diagnostic purposes, for example, gene therapy (5). Accordingly, the present review article explores gene therapy and its latest advancements. It further delineates the structure and function of exosomes, and, finally, investigates exosomes’ role as bio shuttles in various clinical contexts relating to gene therapy.

ACCEPTED MANUSCRIPT Gene therapy Gene therapy or the replacement of a defective gene with a corrected one, is an attractive and promising strategy for scientists, and more importantly patients (6). Many scholars believe that the foundations of gene therapy were upheld in 1928, when a British scientist called Griffith

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made a shocking observation-non-virulent bacteria had the capability to transform into virulent

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specimen. Future studies proved that the transforming agent in Griffith’s observation was part of the cell-free extract and would precipitate by adding alcohol, which would be called

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deoxyribonucleic acid (DNA). Over the years, other mechanisms of sharing DNA between bacteria were understood, and new principles emerged in the field of genetics. In the wake of

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these advances, the first case of gene therapy on a human was conducted on an adenosine

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deaminase (ADA) patient (7). Gene therapy can be divided to two distinct variants with the ability of passing the therapeutic effect on to the generations; one targeting somatic cells and the

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other targeting germ line cells,. Somatic gene therapy is the dominant form and is classified into

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three types of ex-vivo, in-situ and in-vivo delivery (8-10). One important obstacle in the way of introducing gene therapy into clinics is gene delivery, as many specialists express concerns about

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the safety and efficiency of the vectors used (11). Various vectors have been introduced to

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facilitate the entry of genes into cells, including viral and non-viral vectors. Viral vectors include retroviral, adenoviral, herpes simplex, lentivirus, poxvirus and Ebstein-Barr virus (EBV) vectors. Viral vectors are the most common vectors used, but have their share of limitations as causing immune responses, mutagenesis and limited capacity to transfer genes. The non-viral methods comprise a wide array of chemical and physical methods. Although they are not as efficient as viral counterparts, they do not elicit any immune response.(12). Since the first human gene therapy, various uses have been developed for gene therapy, such as cystic fibrosis and other

ACCEPTED MANUSCRIPT single gene diseases (13, 14), chronic neurologic diseases including Alzheimer’s and Parkinson’s (15), craniofacial regeneration (16), orthopedic applications (17) and the emergency field of gene therapy in cancer. Numerous clinical studies have studied the efficacy of gene therapy in cancer such as prostate cancer (18, 19), melanoma (20), glioblastoma (21), to name a few. Gene therapy

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in cancer is divided into three general subtypes: immunotherapy or enhancing the function of the

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native immune system to target cancer cells and eliminate them, has led to the emergence of

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vaccines against cancers, oncolytic agents, which are viruses that have been modified to target cancer cells, and gene transfer, which is the new concept of introducing specific genes into

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cancerous cells of their surrounding environment in order to destroy or incapacitate them (22).

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This third subtype can be further divided into three subtypes consisting of introduction of anti-

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angiogenic agents, mutation compensation and molecular chemotherapy (23).

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Recent progress in gene therapy

A lot has changed since the composition of the concept of gene therapy, around a century ago, to

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the first approved gene therapy in 1989, and the first approved clinical trial in the European

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Union in 2012. Despite limitation in the number of gene therapies available to the public, many clinical trials are being done annually in the context of gene therapy. One staggering study

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showed that from 1989 in which only one clinical trial was conducted, the number had increased to 163 trials in the year 2015 (24). Interestingly, originally the trials mainly dealt with single gene defect diseases currently, however, the majority of clinical trials performed are concerned with cancer therapy (more than 64%) and single gene disease accounts for only 10% of new trials (24, 25). A longstanding challenge facing gene therapy is gene delivery which has caused countless considerable achievements. Classically, viruses have been used as vectors, but gave their way to

ACCEPTED MANUSCRIPT newer ones owing to various toxic and immunological side-effects. One of the relatively newer vectors is the exosome (discussed in the next section). Other newly introduced vectors are bacteria. These have been used specially in tumor-targeting therapies, by mechanisms such as growth in hypoxic regions, in environment in which tumors are active, or the chemotaxis of

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bacteria to compounds found in necrosis (26). Recent studies have implicated various species of

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bacteria such as clostridium, salmonella, listeria, E-coli, Bifidobacterium in gene therapy via the

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aforementioned mechanisms of gene therapy in cancer. One of the uses of bacteria has been the advent of new imaging techniques such as optical imaging in which luciferase enzymes produce

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light, which is detected by cooled charged coupled device (CCD) cameras. These enzymes are

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mostly derived from a firefly called Photinus pyralis and a sea pansy, Renilla luciferase. Bacteria expressing these enzymes, such as Mycobacterium smegmatis or S. Typhimurium, can be used in

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designing a vectors to introduce the enzyme (27, 28). The other types of vectors studied

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extensively in the past years are mammalian mesenchymal stem cells. These cells have the advantage of eliciting no adverse immunologic response, as well as possessing easy expansion

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compared to other vectors. The downside of these cells, however, is that their kinetics and

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possibility to promote tumorigenesis is not well understood (29). Nanoparticles are the last major group of vectors introduced, which comprise four types of lipid-based, polymeric, inorganic and

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hybrid nanoparticles (30). While nanoparticles do not activate the immune system, as viruses and bacteria do and do not have the toxic potential of virulent bacteria and viruses, utilizing them, however, can be quite challenging as to overcoming natural barriers in the way of their absorption, targeting them to localize at wanted sites, for example the cancer environment, medicalizing the research, as many are performed on murine models, and finally, concerns regarding production in large scales (31, 32).

ACCEPTED MANUSCRIPT Current delivery methods for gene therapy Viral vectors Viruses are the result of millions of years of evolutionary progress, which has endowed them

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many acquired abilities such as entering into host cells, long term survival in the cells via

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genome integration and changes in the host immune system. The first use of viruses as vectors in

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gene therapy in clinical contexts dates back to the late 1990s and the early 2000s when retroviruses were used to treat adenosine deaminase deficiency in patients with severe combined

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immunodeficiency. The result of those study showed superior outcomes compared with the

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conventional immune boosting treatments, paving the way for further use of viral vectors (33). Since then multiple viral entities , other than retroviruses have gained attention in the field of

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gene therapy including Adenoviruses, Lentiviruses, pox viruses adeno-associated viruses and

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herpes viruses (34, 35). Retroviruses are RNA-bearing viruses which depend on reverse transcription for proliferation. These enable the transfer of a transgene up to 10 Kb with a

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lifelong expression, because of reverse transcription and the preferred route of delivery of these

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viruses has been ex vivo administration (33). Adenoviruses are medium-sized non-enveloped double stranded DNA viruses which are prepared for gene therapy with the removal of some of

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their genes encoding primary proteins, and can deliver genes up to 30 Kb with a short period of expression, in contrast to retroviruses (34). These viruses are mostly used in a local form in instances in which an immune response is needed and preferred, such as vaccine development or targeting malignant cells (36, 37). They Lentiviruses are a genus of retroviruses which came to light later. They resemble retroviruses in terms of application and characteristics, but gained popularity in treating the conditions of the hematopoietic system and hemophilia A and B (38, 39). Adeno-associated viruses (AAV) are a member of parvo viruses bearing a single stranded

ACCEPTED MANUSCRIPT DNA. They have a rather limited capacity of gene transfer (only up to 4 Kb) with relatively less safety concernsas they have low immunogenicity and yield a long period of gene expression comparable to retroviruses. Conversely, however, AAV do not integrate into the genome of the host cell, thus the amount of expression decreases with time (40). AAVs have been used in

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multiple trials in various clinical contexts such as hemophilia, cystic fibrosis, rheumatoid

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arthritis, macular dystrophies, Parkinson’s disease and more (41). Pox viruses are large double

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stranded DNA viruses with a famous application in producing vaccines. They can carry genes up to 20Kb and have a short period of gene transcription (42). Herpes viruses are the last group

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discussed in this article. They are a family of DNA viruses that have a unique ability to carry a

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comparatively larger load of genome than other viruses (30-40 kb – 152 kb in the HSV1amplicon) (43). In addition, characteristics such as a high ability in infecting cells, a genome

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with large segments of unnecessary DNA and easy recombinant generation make it a suitable

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target for gene therapy (44).

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Non-viral vectors

Other biologic vectors have been introduced in addition to viruses, which include bacteria,

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bacteriophages, virus like particles, erythrocyte ghosts and exosomes (47). The simplest way to

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transfer genes into a tissue has been the direct administration of the gene, usually via injection. This method has shown some success, but further studies have suggested alternative physical and chemical methods to improve the absorption of the nucleic acids. Some of the physical methods are ballistic DNA, electroporation, sonoporation, photoporation, magnetofection, hydroporation and mechanical massage. Chemical methods utilize inorganic particles, such as silica, calcium phosphate and gold, or synthetic biodegradable compounds such as cationic lipids, lipid nanoemulsions, solid lipid nano-particles, peptide based products, or polymer based vectors, such as

ACCEPTED MANUSCRIPT Polyethylenimine, chitosan, poly (DL- Lactide) (PLA) and poly ( DL-Lactide- co- glycoside) (PLGA), Dendrimers and Polymethacrylate (45, 46).

Challenges So far, multiple types of vectors with different properties have been introduced to the field of

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gene therapy, but still there are many challenges to be overcome concerning safe and efficient

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delivery of genetic material. Regarding safety, some vectors can cause severe immunologic reactions, even may lead to anaphylaxis and death. Vectors such as adenoviruses, pox viruses,

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bacteria, virus like particles and bacteriophages are among the most immunogenic of the vectors (47-49). In addition, the use of some vectors, especially those of viral origins, has the potential of

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causing malignancies. The first studies concerning the use of retroviruses for the treatment of

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severe combined immunodeficiency showed development of leukemia in some patients after the

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treatment. This was associated with the unwanted and unpredicted increase in the expression of some proto-oncogenes after viral treatment (50), a phenomenon commonly referred to as

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insertional mutagenesis, which still is a major challenge to the use of viral vectors (51). One other safety issue regarding vector selection is the possibility of systemic inflammatory

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responses resulting from serious infections seen in biologic vectors. The odds of this happening

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can be minimalized by attenuating the viruses or bacteria used in gene therapy, although it does not eradicate the risk (52). The next major challenge to gene therapy is the biological limitation of conventional vectors such as viruses in transferring genetic material. Most viruses used for this purpose can only transfer segment up to 40 Kb in size (53). Currently, some researchers are trying to use less investigated vectors such as lentiviruses, or using methods such as luminal charge alteration to increase the packaging capacity, but no series of methods is yet sufficiently studied (54, 55). Another major challenge to gene therapy is the specificity of transgene delivery.

ACCEPTED MANUSCRIPT As particles, vectors of gene therapy will most likely enter the cites proximal to the circulation, and not always the desired location. Multiple methods have been introduced to overcome this problem, specifically in viral vectors, including changes in the capsid proteins, using tissue specific anti-bodies, tissue‐specific promoters or serial testing of multiple serotypes, but the

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tissue specificity of other vectors and also ways to further make them more organ or tissue

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specific is less studied (56, 57).

Exosomes

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First described over 30 years ago, exosome are extracellular vesicles that are excreted to the

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extracellular space by the conjugation of intermediate endocytic bodies to the plasma membrane. The name of this intermediate structure is “Multivesicular body”, which can also connect to

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lysosomes (58). Multivesicular body releases intraluminal vesicles to the extracellular space.

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Intraluminal vesicles or exosomes are generated by two pathways, one dependent on the endosomal sorting complexes required for transport (ESCRT), which are complex of cytosolic

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proteins binding to membrane proteins marked, usually but not necessarily by ubiquitination

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(59), and a non-dependent pathway (60). Exosomes are excreted from a wide array of living mammalian cells, such as B cells, dendritic cells, T cells, mastocytes, platelets as well as

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reticulocytes and tumors, and proteins from various families are involved in their structure , including integrins, tetraspanins, MHC molecules, membrane fusion peptides such as RAB5 and 7, RAP1B and annexins, cytoskeleton associated molecules and many other undiscovered components (figure 1) (61). As noted, exosomes are present in various cell lines, and with diversity of origin comes diversity of function. One important function of exosomes concerns maturation of reticulocytes, more specifically, dumping the excess proteins in the process of maturation (62).. Exosomes are a key

ACCEPTED MANUSCRIPT effector of inflammation, and diseases such as multiple sclerosis, Parkinson’s and Alzheimer’s, signaling the future use of exosomes as biomarkers in these pathologies (64-67). Exosomes also have established roles in cancer, as they are frequently encountered in the micro-environment wherein the tumors are active(68), being effective in adaptive immunity towards malignant cells,

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possibly both as a pro and anti-cancer agent (69). In addition, they are an important mediator of

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angiogenesis and vascular integrity and permeability. Uptake of exosome secreted by cancers is

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effective in increasing angiogenesis, and promoting leakage of vessels, leading to more metastasis (70). The entities also contribute to the interaction of cancer-associated fibroblasts

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with neoplastic cells; Exosome secreted by fibroblasts adjacent to the tumoral tissue causes

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chemo-resistance in colorectal cancer cells (71). Further discussion is done in the next

cancer. advances

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Recent

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paragraphs regarding the function of exosomes in normal cells and human pathologies and

communication and human pathologies or treating and diagnosing human cancers and other

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diseases has compelled researchers to focus on finding techniques and modalities help isolate

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and enrich exosomes, such as ultracentrifugation, size based isolation like ultra-filtration, immune-affinity capture, precipitation and micro-fluid based isolation each with their specific

(73).

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shortcomings (72). Various commercial kits are available for exosome extraction in this regard

Exosomes formation As a subgroup of extracellular vesicles, exosomes have biogenesis similarities to microvesicles, and to some extend to apoptotic bodies. The formation of exosomes is hallmarked by the fusion of the endosome to the cellular membrane. But multiple steps are needed to be taken beforehand;

ACCEPTED MANUSCRIPT Genesis of exosomes starts with the emergence of intra-luminal vesicles dependent on a subset of molecules called endosomal sorting complex required for transport (ESCRT). Four major complexes of ESCRT associated with intra-luminal vesicles formation include ESCRT-0, which is responsible for clustering ubiquitinated proteins on the endosome, ESCRT-I with bridging

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roles between ESCRT 0 and 2, ESCRT2 with roles in budding, and ESCRT3 which recruits

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other proteins to the site of endosome formation and vesicle scission (74). The next major step in

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the formation of exosomes comprises the transport and fusion of multi-vesicle bodies with the cell membrane. This step is mainly mediated by RAB family of proteins, which control functions

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such as budding, mobility and interactions of the cytoskeleton (75, 76). The final step in the

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formation of the exosome is the fusion of the membranes of the multi-vesicle bodies and the cell membrane. This is regulated by soluble NSF-attachment protein receptor (SNARE) complexes,

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with considerable heterogeneity among various cell lines and conditions (77). This step results in

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Composition of exosomes

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the releasing of the exosomes into extracellular space.

Since the discovery of exosomes, the knowledge of their composition has grown significantly.

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Early studies of exosomes revealed that these entities were indeed a small space covered by a

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lipid membrane (4). More recent studies have established that exosomes are a complicated mixture of various lipids, proteins and RNAs. The abundance is so much that specific databases such as EXOCARTA have been created to list the latest molecules found in the structure of exosomes. Based on this data, over 1010 kinds of lipids, over 9690 kinds of proteins, more than 3300 kinds of mRNAs, over 1400 kinds of mi-RNAs, 18 kinds of r-RNAs, 60 kinds of t-RNAs, 110 kinds of sno-RNAs, 27 kinds of snRNAs, 6 kinds of Inc-RNAs, 3 kinds of Linc-RNAs, 5 kinds of nc-RNAs are present in the structure of the exosomes being made in different cell lines

ACCEPTED MANUSCRIPT in different species and under different conditions (78, 79). The components of exosomes significantly dictate the function and application that these entities have in multiple contexts. Probably the most important functional compartment consists of proteins (tetraspanins, Rab family proteins etc.) with vital roles in the creation of exosomes.); they can have enzymatic

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activities (such as peroxidase, dehydrogenase) or can induce functional overlaps between

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exosomes and some signaling pathways (such as NOTCH and WNT-b catenin pathway) (80). Of

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clinical interest, however, are some proteins of exosomes proposed to be tumor markers, such as CEA in colorectal cancer, or Survivin in breast cancer (81). Furthermore, exosomes have shown

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to have major histocompatibility complex class I and II, shown in studies focusing on

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bronchoalveolar lavage fluid showing that exosomes could have roles in the local immune

exosomes in the bone marrow (83).

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system of the respiratory system (82), and in a study which found the same function for

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In addition to proteins, exosomes have multiple RNA molecules, both functional m-RNAs with

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enriched 3′-untranslated regions and a wide array of non-coding RNAs (84). The diversity of various m-RNAs existent in exosomes led to the creation of a specific data base for this purpose,

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called the exoRBase (85). The study that found the abundance of m-RNAs in exosomes also

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noted that these m-RNAs could have regulatory functions in the host cells, because of their enriched 3′-untranslated regions (84). Exosome microRNAs are also important from a functional point of view as their role has been stablished in numerous human pathologies, including cancer. It has been shown that these micro-RNAs have key mediatory roles in promoting angiogenesis, facilitation of migration and metastasis as well as changing the tumor micro-environment (86).

ACCEPTED MANUSCRIPT Cellular uptake, and intracellular trafficking Though we are well on our way to understanding exosomes’ function and structure, our knowledge of how these entities are incorporated into cells is still young, only recently research initiatives have been able to describe to some extend how exosomes are taken by cells. After

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release, exosomes are freely, but temporarily, available in body fluids and inter-cellular spaces

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before being relocated in the reticolo-endothelial system. When an exosome comes into contact

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with a cell membrane, the interaction between receptor molecules on the two membranes is necessary to establish the function of exosomes. It is believed that exosomes tend to attach to

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cells with a suitable receptor profile for MHC classes 1 and 2 already present externally on

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exosomes, which is determined by the parent cell (87). Exosomes can internalize into cells in different methods, including fusion, probably mediated by integrins and tetraspanins (88, 89),

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phagocytosis which is dependent on the action of opsonins, actin cytoskeleton and dynamin2

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(90), Macropinocytosis dependent on phosphatidylinositol 3-kinase and Na+−H+ ion exchange (91, 92), Receptor- and Raft-mediated Endocytosis (Clathrin-mediated Endocytosis) which relies

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on a ligand-receptor interaction between the exosome and the cell or the presence of rich

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cholesterol- and sphingolipid domains on the cell membrane (91, 93), Heparan sulfate proteoglycan-dependent endocytosis which is dependent on Heparan sulfate proteoglycan, a

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macromolecule receptor (94). A study by Tian et al. found that four related steps distinguishable in the trafficking of exosomes. In the first step, which is probably dependent on actins, exosomes move in the periphery of cells, in the second step, exosomes moved from the periphery to the perinuclear region, the third step, is recognized by exosomes entering a random movement and in the last step, exosomes made a reversely move away from the central parts of the cell towards the periphery, mediated by kinesins (95).

ACCEPTED MANUSCRIPT Isolation and characterization of exosomes As tiny particles smaller than 100 nm, isolation and purification of exosomes may by elusive (96), as they vary greatly in size, shape, density, surface potential and surface molecules. However, advances in laboratory methods, have enabled researchers to utilize the unique

isolation,

ultrafiltration

techniques

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sieving

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ultracentrifugation-based

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characteristics of exosomes in order to isolate them. A few of these methods include and

exclusion

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chromatography methods, which sort out exosomes based on size; immune-affinity capture-

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based techniques, which utilize the existence of specific molecules on the membrane of the exosomes; precipitation, which uses solubility or dispensability of exosomes; and finally

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microfluidics-based isolation techniques, which separate exosomes according to their multiple physical and biologic characteristics (72). These methods have their own advantages and

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disadvantages with many factors affecting their efficiency based on the specimen, technical

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competency, number of specimens and the given time, so researchers should decide which

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method suits their needs better. The differential centrifugation is probably the most frequently used method used for exosome isolation (97). In addition to exosome isolation, various

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techniques are made available for the evaluation of different, which vary likewise based on

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characteristics such as shape, size, size distribution, quantity, surface charge, and biochemical composition of these nanoparticles. Some of techniques use super-high resolution electron microscopy (EM), cryo-EM and immuno-cryoEM to evaluate structure, size and surface molecules; low resolution nanoparticle tracking analysis (NTA) are used in the assessment of size and physical properties such as zeta potential; some of other important techniques include protein analysis via mass spectrometry, western blotting, immunofluorescence staining, and ELISA, RNA analysis via RNA sequencing, PCR and other platforms, and biochemical analyses

ACCEPTED MANUSCRIPT of lipids, sugar, and other components(80). Validation of the isolated exosomes can also be challenging, because of the similarities between exosomes and other cellular components and debris. Since exosomes are of endosomal origin, it would be practical to use molecules such as tetraspannins like CD9, 63, 81 and 82, Hsc70 and Hsp90, MVB proteins such as Alix and

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TSG101 as markers of detection (98).

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Labeling of Exosomes

The recently gained importance of exosomes has made their visualization and detection a

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necessity. Multiple protocols have been proposed for exosome labeling, with fluorescent staining paying a major role in most of them. A recent protocol suggested by Morales-Kastresana et al.

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recommended the use of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester as a

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fluorescent dye, coupled with nanoFACS a high end flow cytometric method, and a

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complementary size exclusion chromatography to undermine the unconjugated labels (99). Other labeling agents include such as PKH26 (lipophilic fluorescent dyes), 1, 1'-Dioctadecyl-3, 3, 3', (100),

PKH67

(101),

octadecyl rhodamine B

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3'-tetramethylindocarbocyanine perchlorate

chloride (102), SYTO RNA for exosomes with RNAs (103), BODIPY TR ceramide (104) and

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possibly more agents to be utilized in the future. Other methods of staining have also been

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suggested for exosome labeling such as immunostaining, negative staining and immuno-gold labelling (105) as well as radiolabeling of extracellular vesicles, a well-studied method which could be done with agents such as (99m)Tc used in in vivo studies (106), 111In-oxine (107) and Iodine 125 (108).

ACCEPTED MANUSCRIPT Drug Loading into Exosomes We previously noted that exosomes are composed of both hydrophilic and hydrophobic compounds, a fact that makes loading therapeutic agents into exosomes challenging, especially when the trend is to use exosomes to deliver various agents ranging from RNAs to proteins of

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various size. Various methods have been introduced in order to tackle the problem efficiently,

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which are classified into two major groups of active and passive. The passive methods refer to

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techniques in which mostly hydrophobic agents are incubated with exosomes or cells with the

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capacity of forming and excreting exosomes (109). These methods have gained significance as it was proposed that exosomes that were made by cancer contributed to tumor progression by

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effecting the tumor micro-environment, and that the same functions could be used to introduce miRNAs with anti-tumor effects (110, 111). The active ones, consisted of sonication, extrusion,

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freeze and thaw cycles, electroporation, incubation with membrane permeabilizers and click

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chemistry method for direct conjugation. In sonication, a mechanical force, for example sound

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energy, is used to temporarily disrupt the membrane of the exosome so that the therapeutic compound can enter the exosome. In a study by Haney et al. with the aim of introducing

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constructs to be used for the treatment of Parkinson’s disease, researchers sonicated the

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exosomes in a mixture containing catalase(112). Another study also used sonication to produce paclitaxel loaded exosomes, where researchers found that the use of sonication resulted in an optimal loading efficacy and sustainable release of paclitaxel (113). The next method of active loading which also disrupts the membrane of the exosome is extrusion. In this method sheer mechanical force is used to combine exosomes and drugs using a lipid extruder under controlled temperatures. One example is the hot melt extrusion process which uses high temperatures and pressures (114). This method was used in a study by Yang et al. where extrusion of MCF-10A

ACCEPTED MANUSCRIPT cells was done in order to deliver si-RNAs to cancerous tissues (115). A similar study by Jang et al. further evaluated the efficacy of using exclusion in creating exosome like structures to target cancer cells, and found that the exosome mimetics resulted from the extrusion process were able to induce TNF-α-stimulated cell death (116).

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Freeze and thaw cycles differs from the previously mentioned methods in that it does not require

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physical force for drug encapsulation. Instead, a mixture of exosomes and the drug is incubated and rapidly frozen, and then let to thaw, multiple times (117). This method tends to better

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preserve the integrity of the membrane of the exosome, but it may produce un-even sized

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exosomes due to the fusion of exosomes together (118).

Electroporation, or electropermeabilization, is a process in which an electrical field is used to

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create pores in the membranes of exosomes in order to facilitate the influx of multiple agents into

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the exosome (119). This method of introduction has been mainly used to deliver RNA products, such as miRNAs (120). A study by Liang et al. used electroporation- created exosomes

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containing miR-26ato suppress cell division and migration in HepG2 cells (121). Another study

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loaded exosomes with RNAs targeting c-MYC using this method (122). An alternative pathway to induce pore formation in cells is to use special compounds such as saponin, a toxic agent

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found in marine creatures and plants which tends to act as a soap (123). The study done by Haney et al. used this method to introduce catalase to in vivo and in vitro models of Parkinson’s disease (112). The theoretical limitation of saponin use is that it is a toxic agent and has the potential to induce autophagy via multiple apoptotic pathways (124). Two additional pathways exist for loading agents onto exosomes by cross linking exosomes to them. Chemistry click and antibody use are examples of these methods. These two methods have

ACCEPTED MANUSCRIPT not been studied as much as the others, but seem promising as being quick, specific and less invasive towards exosome structural integrity (125, 126). Bio-distribution of Exosomes Bio-distribution of exosomes is an important matter to consider in using them as a therapeutic

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strategy. In vivo studies have shown that it is dependent on the source cell, route of

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administration and various methods of targeting. A study by Morishita et al. looked into various studies on the topic of exosome pharmacokinetics and found that with regard to characteristics of

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exosomes, most of them would have the most concentration in the liver, spleen and the pancreas, alongside other organs of the abdomen and the lungs (127). In a study by Wiklander et al.

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multiple kinds of extracellular vesicles from cell lines of HEK293T, C2C12, B16-F10 and OLN-

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93 were extracted and administered in routes of I.V, I.P and S.C. It was shown that I.V administration caused a significant increase in levels of exosomes in the liver compared to the

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other two methods, but it led to a decreased accumulation in the pancreas and the gastrointestinal

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system as well. Cell lines also made different exosomes with different affinities, as ones made by the C2C12 cell line had increased accumulation in the liver, compared to ones from dendritic

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cells with the least accumulation (128). Distributions of exosomes has been shown to be

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dependent on tissue structure and the vasculature of the environment, as it was shown that I.V administrated exosomes were able to concentrate in xenograft tumors with leaky vasculature in less than an hour (129). As we already noted, swift clearance of natural exosomes by liver and spleen is one challenge of exosome use . Many efforts have been made to increase the stability of exosomes, either by modifying the surface molecules of exosomes or using polyethylene glycol to cover the exosomes. Kooijmans et al. even suggested using polyethylene glycol combined with nanobodies to confer specificity to exosomes. In their study, polyethylene glycol was

ACCEPTED MANUSCRIPT conjugated to nanobodies specific for epidermal growth factor receptor, which resulted in a significant increase in binding of exosomes to tumor cells over-expressing EGFR (130).

The exosome function in physiological and pathological conditions

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Exosomes released from the membrane are biologically active entities that mediate different and

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absolutely important physiological and biological processes attributed to them since their first

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introduction in reticulocytes (131, 132). Participating in immune response and facilitating antigen presenting are two most documented functions of exosomes (61). For example, it was reported

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that CD4+ T cells can be potently stimulated by exosomes released from Eppstein- Barr virus-

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transformed B cells (133). Therefore, activation of T cells is a part of exosomes-mediated stimulation of immune responses (133). Furthermore, tumor cells- derived exosomes have also

M

demonstrated to modulate immune reactions (134).

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In cancer, exosomes are major players of cell–cell communication between tumor cells through exchanging efficient information in order to induce a pro-tumoral milieu for tumorigenesis and

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control the immune system promoting tumor initiation/progression (68, 134). A cumulative body

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of studies have attributed a critical function to exosomes in angiogenesis, apoptosis, inflammation and coagulation (135). Promotion of pro-tumor micro-environment and harboring metastatic

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niches is another important function of exosomes (136). Additionally Exosomes may suppress the immune system and, thus mediate tumor escape from the immune attack. Exosomes also deliver mutant proteins such as KRAS and MET oncoprotein into tumor cells through endocytosis or other internalization strategies (81). In addition to proteins, onco-miRNA are also carried by exosomes to modulate the tumor cells function at post-transcriptional level (137-139). Besides the critical function of exosomes in the cancer progression, these carriers also play major function in the normal physiology and development of the nervous system, as well as regeneration of normal

ACCEPTED MANUSCRIPT neurons. In a study by Lachenal et al (140) it was reported that exosomes released from somatodendritic compartments are involved in the trans-synaptic diffusion of pathogenic molecules throughout the tissue. Additionally, the authors observed that glutamatergic synapses are also involved in the regulation of exosomal release as a part of normal synaptic physiology. More

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interestingly, Frühbeis et al (141) reported that exosomes are key players of local cell–cell

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communication between neurons and oligodendrocytes. Neuronal stress-induced exosomes

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derived from oligodendrocytes deliver protective cargos such as mRNA, miRNA, proteins, and glycolytic enzymes to axons, and exert neuroprotection (142). In addition to delivering

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neuroprotective cargos, exosomes are also associated with the transportation of misfolded

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proteins, hence initiation and progression of various neurodegenerative diseases (143). For example, in Parkinson’s disease, α-synuclein mutated proteins are transported from producing

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cells to the nearby cells via exosomes, whereby the disease spreads from cell to cell within the

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brain (144). In another neurodegenerative disorder, Alzheimer’s disease, exosomal surface was reported to function as a seed for the β-amyloid aggregation after protein conformational

and

Gerstmann–Sträussler–Scheinker

syndrome,

exosomes

mediate

the

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Creutzfeldt–Jakob

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modifications, and promotion of neurodegeneration (145-147). In the prion diseases such as

propagation of the disease-related infectious agent, PrPSc (148-150). Taking together, these

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findings implicate a role for exosomes in neurodegenerative diseases, making them an interesting biomarker for diagnostic and prognostic purposes thereof. Exosomes function is also frequently investigated in cardiovascular diseases because of their importance in the regulation of angiogenesis, coagulation, and inflammation (151, 152). The molecular components of exosomes may have a significant function in cell–cell communication and exerting cardioprotective effects in pathological conditions such as myocardial infarction, and ischemic reperfusion (153-155).

ACCEPTED MANUSCRIPT Exosomes as bio shuttle for gene therapy Observation of different contents of exosomes with different source, particular RNA and protein profiles, as well as exosome-mediated effects on other cells led to the hypothesis that exosomes might be good candidates for transferring gene in the gene therapy process (156). In an early

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study in this context, Eldh et al (157) demonstrated that the plausible mechanism that was

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involved in the cell- cell communication were actually RNA-contained exosomes. Individual cells exposed to oxidative stress release exosomes, which can affect target cells and mediate resistance

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to oxidative stress (157). Additionally, exosomes originated from EBV-infected nasopharyngeal carcinoma (NPC) cells carried signaling pathways-related particular molecules, as well as virus-

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coded microRNSs (miRNAs), which mediated intracellular communication and exerted a potent

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impact on the proliferation of other neighboring cells (158). Therefore, transferring of important stimulatory or inhibitory information between donor and recipient cells, as a key function of

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exosomes contributes to the various aspects of development, as well as pathogenesis of various

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diseases (159). Being a natural transporters, low homing in liver, low toxicity and immune reaction, easily crossing biological barriers, especially blood brain barrier, art among some

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important features rendering exosomes advantageous over other vectors (160).

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In gene therapy process, exosomes are used as carriers of a basic amount of therapeutic agents, such as small interfering RNAs (siRNAs) and miRNAs, in order to deliver them in more efficient and specific manner to the target tissues (161). Several recent studies have delivered therapeutic siRNA into the target tissues by using exosomes (table 1) (119, 162-164). SiRNAs-loaded exosomes are frequently shown to selectively silence gene expression. Low stability of siRNAs and rapid degradation in the systemic circulation are the two most important problems facing siRNA-based gene therapy, and are now resolved by the advent of exosomes delivering (165,

ACCEPTED MANUSCRIPT 166). Exosomes have amply shown to be good candidates for the efficient delivery of nucleotide sequences into recipient cells, some studies have evaluated shuttling potential of these natural nano-carriers as a therapeutic strategies in combating cancer, neurodegenerative disease, cardiovascular disorders and other diseases.

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Application in cancer

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Cancer is probably the single biggest challenge facing medicine and healing from cancer is

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regarded as the Holy Grail of medicine (167-170). Recent attention in cancer therapy has been

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directed to the use of exosomes as natural carriers for delivering small nucleotides, specifically transferring of multiple miRNA molecules such as let-7a, miR-143, miR-146b, and miR-223, into

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different cancer cell lines in order to halt the tumor growth, as well as suppressing the target

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genes (171-174). Banizs et al (175) showed that endothelial cells- derived exosomes have delivered siRNA against luciferase into primary endothelial cells. In addition, exosomes

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containing siRNA against GFP, or a c-Myc shRNA were efficiently transfected into recipient

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cells, and consequently resulted in suppression of target gene expression (122). Munoz et al (176)showed that exosome base delivery of anti-miR- 9 overcame the chemo

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resistance in glioblastoma. Zhang et al (177) demonstrated that exosomes derived from mouse fibroblast L929 cell line could be used as an ideal siRNA delivery tool for the transportation of

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siRNA against transforming growth factor (TGF) 1, thereby treatment of murine sarcomas tumor cells. Cancer cells treatment with exosomes containing TGF-1 siRNA significantly reduced the proliferation and invasion of cancer cells, as well as resulting in considerable increase in the apoptosis of tumor cells. In animal model of cancer, it was reported that TGF- 1 levels and more importantly the TGF- 1 signaling downstream were suppressed due to intravenous injection of designed exosome into tumor cells. Therefore, exosomes- mediated gene silencing effectively

ACCEPTED MANUSCRIPT inhibited the growth of cancer, as well as their metastases in lung (177). Shtam et al (178) transfected HeLa and HT1080 cell lines with exosomes containing siRNAs against RAD51 and RAD52 to evaluate the possible therapeutic application, where siRNAs caused an effective gene silencing at post-transcription level in receipt cells. In addition, S/G2 cell cycle arrest, as well as

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massive cell death was the significant result of the down-regulation of RAD51 but not RAD52

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protein by recipient cell treatment with exosomes containing the specific siRNA. siRNA

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mediated- suppression of RAD51 decreased the recruitment of RAD51 as a key player of homologous recombination at double-stranded breaks induced in HeLa cells by ionizing radiation

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(178). In another study, Yang et al (179) used a modified exosomes to deliver siRNA against

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Survivin into bladder cancer in vitro an in vivo. A tumor-penetrating peptide (iRGD peptide) was expressed on the membrane surface of the designed exosome, which facilitated the delivery of

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exosome cargo into tumor cells. The expression levels of Survivin was significantly suppressed in

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exosome-recipient bladder cancer cells. Likewise, these exosomes significantly suppressed the growth rate of tumor in animal models (179). In another study done also on the bladder cancer

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cell lines, the efficacy of the exosomes derived from human embryonic kidney 293 (HEK293)

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cell and mesenchymal stem cell (MSC) for the delivery of siRNA against polo-like kinase-1 (PLK-1) gene into recipient cells was evaluated (180). Successful suppression of the PLK-1

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expression mediated by exosome resulted in a significant decrease in cancer cells proliferation (180). Yang et al (115) designed a nanoscale exosome-mimics containing CDK4 siRNA and transfected it into breast cancer cell lines. The expression levels of CDK4 plummeted in exosome transfected cells and this inhibitory effect was accompanied by the induction of cell cycle arrest at G1. The authors also examined the anti-tumor impacts of exosomes in BALB/C-nu mice, a mouse model of breast cancer, and showed significant suppression of tumor growth (115). In addition to

ACCEPTED MANUSCRIPT application of exosomes as a delivery system for suppressing tumor growth, some recent studies have investigated the potential of exosomes to deliver nucleotide sequences for the inhibition of angiogenesis, metastasis, as well as overcoming multidrug resistance. For example, Gong et al (181) demonstrated that mesenchymal stem cells (MSCs)-derived exosomes containing pro-

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angiogenic miRNAs, such as miR-424, -30b, -30c, and Let-7, promoted tube-like structure

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formation and angiogenesis capacity of human umbilical vein endothelial cells (HUVECs). Cells

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treatment with the exosome secretion blocker GW4869 reduced the pro-angiomiRs containing exosomes and hence blocking their stimulatory effects (181). Liao et al (182). showed that

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transfection of esophageal cancer cell line with exosomes shuttling miR-21 resulted in significant

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promotion of the invasion and migration of cancer cells. It was also demonstrated that these exosomes targeted programmed cell death 4 (PDCD4) and activated c-Jun N-terminal kinase

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(JNK) signaling pathway downstream. Therefore, the authors reported exosome-containing miR-

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21a has a close association with esophageal cancer recurrence and distant metastasis (182). On the other hand, delivery of MSC derived-exosomes containing anti-mir-9 to glioblastoma

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multiform cells was shown to reverse multidrug resistance to temozolomide. The underlying

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mechanisms included the downregulation of multidrug resistance transporters such as Pglycoprotein and induction of apoptosis (176). Taking together, it has been indicated that

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exosomes could be an appropriate vehicle for the delivery of miRNAs and siRNAs to inhibit the various aspects of tumor progression (174). More importantly, delivery of tumor-suppressive miRNAs via exosomes into cancer cells could successfully inhibit cancer proliferation through the silencing of target genes (164). In addition to direct targeting of tumor cells by exosomes, it was reported that stimulation of dendritic cells by exosomes containing trans-activator gene enabled the immune system to interact and perish cancer cells (183).

ACCEPTED MANUSCRIPT Application in neurodegenerative and brain- related diseases In addition to human malignancies, exosomes have also been used as shuttling system todeliver siRNAs into neurons in order to affect the production rate of particular proteins implicated in neural diseases characterized by the accumulation of misfolded proteins or protein deficiencies

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(184). This application of exosomes is related to their capability in crossing the blood brain

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barrier (BBB) (137). Alvarez-Erviti et al (163). dendritic cells-derived exosomes could

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efficiently deliver siRNA against BACE1, as an important gene contributed in pathogenesis of

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Alzheimer’s disease to the brain in mice. Exosomes-containing siRNA are shown to specifically deliver their cargo to neurons, microglia, oligodendrocytes in the brain, and consequently

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knockdown target gene. Exosomes with some important features such as broad distribution and high efficacy for delivering siRNA to brain contribute to designing novel, specific and potent

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therapeutic strategies to combat Huntington’s and other neurodegenerative diseases. In a study

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by Didiot et al (185), exosomes containing hydrophobically modified siRNAs (hsiRNAs) that

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target Huntingtin mRNA were shown to efficiently become internalized by mouse primary cortical neurons, which resulted in downregulation of Huntingtin gene. Additionally, unilateral

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infusion of exosomes containing hsiRNA into mouse striatum promoted bilateral distribution of oligonucleotide and statistically significant bilateral downregulation of Huntingtin mRNA, in

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comparison to hsiRNAs alone. Suppressing the expression levels of Alpha-synuclein ( -Syn), whose aggregation characterizes Parkinson’s disease, is said to help attenuate the progression of the disease. Copper et al (186) designed an exosome containing siRNA against  -Syn to induce a significant reduction in the expression as well as the aggregated levels of -Syn in mouse brains. The expression levels of  -Syn was significantly decreased in the brain of treated mice. As a result, protein aggregates levels dropped in dopaminergic neurons of the substantia nigra.

ACCEPTED MANUSCRIPT Furthermore, it was reported that communication between mesenchymal stem cells (MSCs) and brain parenchymal cells, obtained by transferring miR-133b-loaded exosomes into neural cells, influences the regulation of neurite outgrowth (187). Following treatment of stroke, this communication acts to promote neural plasticity and functional remodeling. In other words,

and synaptophysin, hencestimulating neurite

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regulating the expression levels of NF-200

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MSCs-derived exosomes mediate the miR-133b transferring into astrocytes and neurons, hence

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remodeling and recovery after stroke (188). The beneficial effects of neurons transfection with exosomes carrying miRNA in functional recovery after stroke was also reported in a study by

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Xin et al (189). The authors observed that mice treatment with exosome containing miR-17–92

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cluster resulted in the significant recovery of neurological functions and increase in the proliferation of oligodendrocytes, as well as neurogenesis and neurite repairing/ neuronal

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dendrite plasticity in the ischemic sites. A recent study used exosomes-based gene delivery for

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the treatment of morphine relapse in vitro and in vivo (190). The authors indicated that siRNA against opioid receptor mu delivered by exosomes potently suppressed morphine relapse via

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decreasing expression levels of the opioid receptor mu (190).

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Application in cardiovascular and other diseases

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In recent years, a great progress has been made in the application of gene therapy for cardiovascular diseases (191, 192). Accumulating body of studies have demonstrated the critical role of exosomes and exosomes-mediated cross-talk between multiple cell types in the perpetuation of heart homeostasis, as well as development of cardiac disorders (193-196). Exosomes derived from cardiac progenitor cells (CPC) containing miR-210 and miR-132 could deliver their content into HL-1 cells (193). Under normal conditions, plasma-derived exosomes are reported to be involved in the protection of healthy cardiomyocytes from ischemia/

ACCEPTED MANUSCRIPT reperfusion

injury

(154,

197).

Following

myocardial infarction,

CPC-derived

exosomes

containing some protective factors such as clusterin, miR-21, and miR-210 were released and exerted potent protective effects as inhibition of apoptosis in cardiomyocytes, stimulation of angiogenesis, promotion of repair mechanisms, and, subsequently, improvement of cardiac

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function (155, 198, 199). Therefore, the exosome application as a gene delivery shuttle seems

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rational for cardiovascular diseases. Additionally, the beneficial effects of exosome-base gene

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therapy was reported in ischemic kidney injury (200). Endothelial colony forming cells (ECFCs)derived exosomes enriched with miR-486-5p caused significant upregulation and downregulation

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of miR-486-5p and PTEN, respectively, as well as stimulation of Akt phosphorylation in

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hypoxia-exposed endothelial cells. Injection of ECFC-derived exosomes into animal model of ischemic kidney injury strongly resulted in the induction of functional and histologic protection,

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such as reduced PTEN levels and activated Akt, all related to overexpression of miR-486-5p in

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the kidney. Thus, it was suggested that delivery of ECFC-derived exosomes can decrease ischemic kidney injury via transferring miR-486-5p targeting PTEN (200). plays

a

critical

role

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MiRNA-122

in

the

hepatocyte

damage

and

inflammation

in

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monocytes/macrophages, hence pathogenesis of alcoholic hepatitis (AH). Momen-Heravi et al (201) demonstrated that delivery of exosome-loaded miRNA-122 inhibitor suppress the

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inflammatory effects of exosomes from ethanol-treated hepatocytes. Alcohol binge drinking resulted in significant enhancement of the number of exosomes containing miRNA-122 in the healthy groups. In monocytes, exosome- mediated shuttling of miRNA-122 resulted in the significant suppression of heme oxygenase -1 (HO-1) pathway, sensitization of cells to liposaccharide stimulation, and overexpression of pro-inflammatory cytokines. Table 1: exosomes application for gene delivery in various pathological conditions

ACCEPTED MANUSCRIPT Exosome cargo

Exosome source

Results

Ref.

Breast cancer cell lines

let-7a miRNA

Human embryonic kidney cell line 293 (HEK293) cells HeLa and HT1080 human fibosarcoma cells non-cancerous cells

Suppression of tumor growth

(171)

M assive reproductive cell death Selective gene silencing

(162)

Inhibition of cell proliferation

(202)

M acrophages PLC-luc cells,

Invasion of breast cancer cells Protein expression

(173) (203)

miRNA released via ceramide-dependent (164) secretory mechanism

Promotion of angiogenesis Horizontal transfer of drug-resistant trait

Tumor-suppressive microRNAs miR-223 mRNA

Breast cancer cells Hepatocellular carcinoma cells M etastatic prostate siRNA cancer cell line

siRNA

non-small cell lung cancer cell lines M esenchymal stem cells chronic myeloid leukemia cells Immortalized hepatocyte

miRNA - 222-3p miR-30b miR-365

Endothelial cells Sensitive cancer cells

Esophageal cancer cells

miR-21

Breast cancer cells Bladder cancer cells M ouse xenograft tumor Xenografted nude mice

Non-coding RNAs siRNA

AN

M

(174)

Liver tissue

Promoting colon cancer progression

(209)

Regulation of gene expression, Promoting neurite remodeling, Improving functional recovery Specific gene knockdown

(188)

Enhancing neuroplasticity and functional recovery after stroke Reducing alpha-synuclein aggregates in brains

(189)

siRNA

M ultipotent mesenchymal stromal cells Self-derived dendritic cells M esenchymal stromal cell M urine dendritic cells harvested from bone marrow Peripheral blood

Selective gene silencing

(119)

siRNA

M odified exosomes

Treatment of morphine relapse

(190)

hsiRNAs

Glioblastoma cells

Huntingtin mRNA silencing

(185)

Anti-miR-9

M esenchymal Cell

AC

CE

Rat model of primary miR-146b brain tumor Colon carcinoma cell miR-193a lines Brain cells subjected to miR-133b cerebral artery occlusion

Brain cells in siRNA Alzheimer’s disease Rat brain cells subjected miR-17–92 Cluster to stoke Brain of transgenic mice siRNA

M onocytes and lymphocytes M ouse skeletal muscle cell lines M ouse primary cortical neurons Glioblastoma multiforme cells

Promotion of cell motility (207) Activation of PI3K/AKT and M APK signal transductions Enhanced secretion of M M P-2 and M MP-9 Esophageal cancer Promotion of cell migration and invasion (182) cell line EC9706 Stromal cells Regulation of therapy resistance pathways (208) M odified exosomes Inhibition of tumor growth (179) Human monocytic Inhibition of tumor growth leukemia THP-1 cells M arrow stromal cells Inhibition of glioma growth

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miR-143

(205) (206)

proteins Hepatocellular carcinoma cells

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Oncogenic and RNAs

Successful knockdown of target mRNA (180) and protein Transferring malignant phenotypic traits (204)

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Bladder cancer cell lines

HEK293 human embryonic kidney cell line COS-7 African green monkey kidney fibroblast like cell line HEK293 and M SC cell Sensitive cancer cells

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Cancerous cells

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HeLa and HT1080 siRNA human fibosarcoma cells

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Target tissue

Stem Reversing the chemoresistance

(172)

(163)

(186)

(176)

ACCEPTED MANUSCRIPT Neonatal cardiomyoctes Astrocytes

rat miR-146a Neuronal miR-124a

Effector T cells

miR-150

HSC or hepatocytes

miR -214

M ast cells

Exosomal RNA miRNAs

Cardiosphere-derived cells Neuron-conditioned medium Lymph node and spleen cells M ouse or human hepatic stellate cells

Cardiac regeneration

(210)

Astroglial GLT1 expression

(211)

Inhibition contact sensitivity

(212)

Suppression of connective tissue growth (213) factor (CCN2) 3′-UTR activity Suppression αSM A or collagen synthesis

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IP

M onocyte-derived dendritic cells Endothelial cell

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shuttle M ouse and a human Protein synthesis (214) mast cell line EBV-infected B cells Repression of (215) EBV Target Genes mRNA Adenocarcinoma Proliferation, migration, sprouting, and (216) model (AS-Tspan8) maturation of EC progenitors. cells miRNA T, B and dendritic M odulation of gene expression (217) immune cells Class II transactivator B16F1 murine anti-tumor effects in recipient cells (183) gene (CIITA) melanoma cell line

Antigen-presenting cells

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Dendritic cells

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Conclusion

Because of low toxicity and high functional efficiency, exosomes are new promising vectors for

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the gene therapy of various diseases. Nearly four decades after introduction of exosomes, major

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efforts have been made in order to determine their biological and functional features. Exosomes have opened a completely new avenue for the therapeutic delivery of small nucleic acids to

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specific tissues. Various studies have explored the potential clinical applications of exosome in

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various pathological conditions. However, this field is still in its infancy and needs further researches. Tests of therapeutic efficiency should be designed and conducted before entering the clinical application.

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Figure 1: Key components of a typical exosome.

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Figure 1