Potential of Exosomes for Diagnosis and Treatment of Joint Disease: Towards a Point-of-Care Therapy for Osteoarthritis of the Knee

In the knee joint, articular cartilage injury can often lead to osteoarthritis of the knee (OAK). Currently, no point-of-care treatment can completely address OAK symptoms and regenerate articular cartilage to restore original functions. While various cell-based therapies are being developed to address OAK, exosomes containing various components derived from their cells of origin have attracted attention as a cell-free alternative. The potential for exosomes as a novel point-of-care treatment for OAK has been studied extensively, especially in the context of intra-articular treatments. Specific exosomal microRNAs have been identified as possibly effective in treating cartilage defects. Additionally, exosomes have been studied as biomarkers through their differences in body fluid composition between joint disease patients and healthy subjects. Exosomes themselves can be utilized as a drug delivery system through their manipulation and encapsulation of specific contents to be delivered to specific cells. Through the combination of exosomes with tissue engineering, novel sustained release drug delivery systems are being developed. On the other hand, many of the functions and activities of exosomes are unknown and challenges remain for clinical applications. In this review, the possibilities of intra-articular treatments utilizing exosomes and the challenges in using exosomes in therapy are discussed.     

Isolation of Platelet-Derived Exosomes from Human Platelet-Rich Plasma: Biochemical and Morphological Characterization

Platelet-Rich Plasma (PRP) is enriched in molecular messengers with restorative effects on altered tissue environments. Upon activation, platelets release a plethora of growth factors and cytokines, either in free form or encapsulated in exosomes, which have been proven to promote tissue repair and regeneration. Translational research on the potential of exosomes as a safe nanosystem for therapeutic cargo delivery requires standardizing exosome isolation methods along with their molecular and morphological characterization. With this aim, we isolated and characterized the exosomes released by human PRP platelets. Western blot analysis revealed that CaCl₂-activated platelets (PLT-Exos-Ca²⁺) released more exosomes than non-activated ones (PLT-Exos). Moreover, PLT-Exos-Ca²⁺ exhibited a molecular signature that meets the most up-to-date biochemical criteria for platelet-derived exosomes and possessed morphological features typical of exosomes as assessed by transmission electron microscopy. Array analysis of 105 analytes including growth factors and cytokines showed that PLT-Exos-Ca²⁺ exhibited lower levels of most analytes compared to PLT-Exos, but relatively higher levels of those consistently validated as components of the protein cargo of platelet exosomes. In summary, the present study provides new insights into the molecular composition of human platelet-derived exosomes and validates a method for isolating highly pure platelet exosomes as a basis for future preclinical studies in regenerative medicine and drug delivery.     

The Role of Exosomes and Their Applications in Cancer

Exosomes are very small extracellular vesicles secreted by multiple cell types and are extensively distributed in various biological fluids. Recent research indicated that exosomes can participate in regulating the tumor microenvironment and impacting tumor proliferation and progression. Due to the extensive enrollment in cancer development, exosomes have become a focus of the search for a new therapeutic method for cancer. Exosomes can be utilized for the therapeutic delivery of small molecules, proteins and RNAs to target cancer cells with a high efficiency. Exosome-carried proteins, lipids and nucleic acids are being tested as promising biomarkers for cancer diagnosis and prognosis, even as potential treatment targets for cancer. Moreover, different sources of exosomes exhibit multiple performances in cancer applications. In this review, we elaborate on the specific mechanism by which exosomes affect the communication between tumors and the microenvironment and state the therapeutic and diagnostic applications of exosomes in cancers.     

Utilizing Exosomal-EPHs/Ephrins as Biomarkers and as a Potential Platform for Targeted Delivery of Therapeutic Exosomes

Exosomes are cell-secreted nanoparticles containing various molecules including small vesicles, microRNAs (miRNAs), messenger RNAs or bioactive proteins which are thought to be of paramount importance for intercellular communication. The unique effects of exosomes in terms of cell penetration capacity, decreased immunogenicity and inherent stability, along with their key role in mediating information exchange among tumor cells and their surrounding tumor microenvironment (TME), render them a promising platform for drug targeted delivery. Compared to synthetic drugs, exosomes boast a plethora of advantages, including higher biocompatibility, lower toxicity and increased ability of tissue infiltration. Nevertheless, the use of artificial exosomes can be limited in practice, partly due to their poor targeting ability and partly due to their limited efficacy. Therefore, efforts have been made to engineer stem cell-derived exosomes in order to increase selectiveness and effectivity, which can then become loaded with various active substances depending on the therapeutic approach followed. Erythropoietin-producing human hepatocellular receptors (EPHs), along with their ligands, the EPH family receptor interacting proteins (ephrins), have been extensively investigated for their key roles in both physiology and cancer pathogenesis. EPHs/ephrins exhibit both tumorigenic and tumor suppressing properties, with their targeting representing a promising, novel therapeutic approach in cancer patients’ management. In our review, the use of ephrin-loaded exosomes as a potential therapeutic targeted delivery system in cancer will be discussed.     

The Role of Melanoma Cell-Derived Exosomes (MTEX) and Photodynamic Therapy (PDT) within a Tumor Microenvironment

Photodynamic Therapy (PDT), an unconventional cancer therapy with optimistic desirable effects, utilizes the delivery of a photosensitizer (PS) that is activated by light at a particular wavelength and inducing oxidative cytotoxic damage of a tumor and its surrounding vasculature. Deeper seated tumors such as internally metastasized melanomas are more difficult to treat with PDT as the penetration of laser light to those sites is less. Limitations in targeting melanomas can also be attributed to melanin pigments that hinder laser light from reaching targeted sites. Exosomes serve as naturally occurring nanoparticles that can be re-assembled with PSs, improving targeted cellular absorption of photosensitizing agents during PDT. Additionally, studies indicate that exosomes released from PDT-treated tumor cells play a critical role in mediating anti-tumor immune responses. This review collates the role of Melanoma Cell-Derived Exosomes (MTEX) in immune response mediation and metastasis. Tumor Cell-Derived Exosomes (TEX) post PDT treatment are also reviewed, as well as the effects of exosomes as carriers of photosensitizers and delivery systems for PDT. The understanding and research on the role of melanoma exosomes induced by Photodynamic Therapy and their tumor microenvironment will assist in future research in treatment prospects and implications.     

Synthetic Biology: Engineering Mammalian Cells To Control Cell-to-Cell Communication at Will

Cell-to-cell communication plays a key role in the regulation of many natural biological processes. Recent advances in mammalian synthetic biology are making it possible to rationally engineer cell-to-cell communication for therapeutic and other purposes. Here, we review state-of-the-art engineering principles to control cell-to-cell communication, focusing on communication between mammalian cells with diffusible factors (e.g., small molecules or exosomes) or direct cell contact, and on interkingdom communication between mammalian cells and bacteria. Potential applications include construction of artificial tissues able to perform complex computations, sophisticated cell-based cancer therapies, use of mammalian cells as a new class of cargo delivery modality, development of design principles to control pattern formation of cell populations, and treatment of infectious diseases. We also discuss the challenges facing practical applications, and possible enabling technologies to overcome them.     

A High Throughput Approach Based on Dynamic High Pressure for the Encapsulation of Active Compounds in Exosomes for Precision Medicine

In recent decades, endogenous nanocarrier-exosomes have received considerable scientific interest as drug delivery systems. The unique proteo-lipid architecture allows the crossing of various natural barriers and protects exosomes cargo from degradation in the bloodstream. However, the presence of this bilayer membrane as well as their endogenous content make loading of exogenous molecules challenging. In the present work, we will investigate how to promote the manipulation of vesicles curvature by a high-pressure microfluidic system as a ground-breaking method for exosomes encapsulation. Exosomes isolated from Uppsala 87 Malignant Glioma (U87-MG) cell culture media were characterized before and after the treatment with high-pressure homogenization. Once their structural and biological stability were validated, we applied this novel method for the encapsulation in the lipidic exosomal bilayer of the chemotherapeutic Irinotecan HCl Trihydrate-CPT 11. Finally, we performed in vitro preliminary test to validate the nanobiointeraction of exosomes, uptake mechanisms, and cytotoxic effect in cell culture model.     

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.     

Treatment of Oxidative Stress with Exosomes in Myocardial Ischemia

A thrombus in a coronary artery causes ischemia, which eventually leads to myocardial infarction (MI) if not removed. However, removal generates reactive oxygen species (ROS), which causes ischemia–reperfusion (I/R) injury that damages the tissue and exacerbates the resulting MI. The mechanism of I/R injury is currently extensively understood. However, supplementation of exogenous antioxidants is ineffective against oxidative stress (OS). Enhancing the ability of endogenous antioxidants may be a more effective way to treat OS, and exosomes may play a role as targeted carriers. Exosomes are nanosized vesicles wrapped in biofilms which contain various complex RNAs and proteins. They are important intermediate carriers of intercellular communication and material exchange. In recent years, diagnosis and treatment with exosomes in cardiovascular diseases have gained considerable attention. Herein, we review the new findings of exosomes in the regulation of OS in coronary heart disease, discuss the possibility of exosomes as carriers for the targeted regulation of endogenous ROS generation, and compare the advantages of exosome therapy with those of stem-cell therapy. Finally, we explore several miRNAs found in exosomes against OS.     

Exosomes as Targeted Delivery Platform of CRISPR/Cas9 for Therapeutic Genome Editing

Therapeutic genome editing harnesses the power of genome editing tools to correct erroneous genes associated with disease pathology. To bring the CRISPR/Cas9 tool from the bench to the bedside, a critical hurdle is the safe and efficient delivery of this nucleic acid tool to the desired type of cells in patients. This review discusses the use of natural carriers, extracellular vesicles (EVs), in particular exosomes, to fill the gap. Exosomes are lipid-containing nanovesicle released by various types of cells to mediate cell-cell communications. Their inherent long-distance transportation capability, biocompatibility, and engineerability have made EVs potential vehicles for delivering therapeutic drugs. We summarize the recent progress of harnessing exosomes as delivery vehicles for the CRISPR/Cas system to achieve therapeutic gene editing for disease treatment, with a focus on various strategies to achieve selective delivery to a particular type of cell and efficient packaging of the genome editing tools in the vesicles. Critical issues and possible solutions in the design and engineering of the targeting vehicles are highlighted. Taken together, we demonstrate EV/exosome-mediated packaging of the nucleic acid/protein tools and the cell/tissue-targeted delivery to be a viable way towards the clinical translation of the CRISPR/Cas9 technology.     

Nucleic acid delivery: the missing pieces of the puzzle?

The delivery of genes or RNA interference (RNAi) agents can increase or decrease the expression of virtually any protein in a cell, and this process opens the path for cures to most diseases that afflict humans. However, the high molecular weight, anionic nature, and instability of nucleic acids in the presence of enzymes pose major obstacles to their delivery and frustrates their use as human therapies. This Account describes current ideas about the mechanisms in nonviral nucleic acid delivery and how lipidic and polymeric carriers can overcome some of the critical barriers to delivery. Over the last 20 years, researchers have developed a multitude of polymeric and lipidic vectors, but only a small fraction of these have progressed into clinical trials. None of these vectors has received FDA approval, which indicates that the current vectors do not yet have suitable properties for effective in vivo nucleic acid delivery. Nucleic acid delivery is a multistep process and inefficiencies at any stage result in a dramatic decrease in gene delivery or gene silencing. However, the majority of studies investigating synthetic vectors focus solely on optimization of endosomal escape. A small number of studies address how to improve uptake via targeted delivery, and an even smaller fraction examine the intracellular fate of the delivery systems and nucleic acid cargo. The internalization of genes into the cell nucleus remains an inefficient and mysterious process. In the case of DNA delivery, strategies are needed to increase and accelerate the migration of DNA through the cytoplasm and transport it through the nuclear membrane. siRNA delivery involves fewer barriers. siRNA is more readily released from the carrier and more resistant to enzymatic degradation, and its target is in the cytoplasm; hence, siRNA delivery systems are becoming a clinical reality. With regard to siRNA therapy, the exact cytoplasmic location of RNA-induced silencing complex (RISC) formation and activity is unknown, which makes specific targeting of the RISC for more efficient delivery difficult. Furthermore, we would like to identify the factors that favor the binding of siRNA to Ago-2. If we could understand how the half-life of siRNA and Ago-2/siRNA complex in the cytoplasm can be modulated without interfering with RISC functions that are essential for normal cell activity, we could increase siRNA delivery efficiency. In this Account, we review the current synthetic vectors and propose alternative strategies in a few cases. We also suggest how certain cellular mechanisms might be exploited to improve gene transfection and silencing. Finally, we discuss whether some carriers that deliver the siRNA to cells could also repackage the siRNA into exosomes. The exosomes would then transport the siRNA into a subsequent population of cells that manifest the siRNA effect. This piggy-back mechanism may be responsible for reported deep tissue siRNA effects using certain carriers.     

Human Induced Pluripotent Stem Cell-Derived Exosomes as a New Therapeutic Strategy for Various Diseases

Recently, an increasing number of studies have demonstrated that induced pluripotent stem cells (iPSCs) and iPSC-derived cells display therapeutic effects, mainly via the paracrine mechanism in addition to their transdifferentiation ability. Exosomes have emerged as an important paracrine factor for iPSCs to repair injured cells through the delivery of bioactive components. Animal reports of iPSC-derived exosomes on various disease models are increasing, such as in heart, limb, liver, skin, bone, eye and neurological disease and so forth. This review aims to summarize the therapeutic effects of iPSC-derived exosomes on various disease models and their properties, such as angiogenesis, cell proliferation and anti-apoptosis, with the hopes of improving their potential role in clinical applications and functional restoration.     

The Role of Exosomes in Cancer Progression

Early detection, characterization and monitoring of cancer are possible by using extracellular vesicles (EVs) isolated from non-invasively obtained liquid biopsy samples. They play a role in intercellular communication contributing to cell growth, differentiation and survival, thereby affecting the formation of tumor microenvironments and causing metastases. EVs were discovered more than seventy years ago. They have been tested recently as tools of drug delivery to treat cancer. Here we give a brief review on extracellular vesicles, exosomes, microvesicles and apoptotic bodies. Exosomes play an important role by carrying extracellular nucleic acids (DNA, RNA) in cell-to-cell communication causing tumor and metastasis development. We discuss the role of extracellular vesicles in the pathogenesis of cancer and their practical application in the early diagnosis, follow up, and next-generation treatment of cancer patients.     

The Therapeutic Potential of Exosomes in Soft Tissue Repair and Regeneration

Soft tissue defects are common following trauma and tumor extirpation. These injuries can result in poor functional recovery and lead to a diminished quality of life. The healing of skin and muscle is a complex process that, at present, leads to incomplete recovery and scarring. Regenerative medicine may offer the opportunity to improve the healing process and functional outcomes. Barriers to regenerative strategies have included cost, regulatory hurdles, and the need for cell-based therapies. In recent years, exosomes, or extracellular vesicles, have gained tremendous attention in the field of soft tissue repair and regeneration. These nanosized extracellular particles (30–140 nm) can break the cellular boundaries, as well as facilitate intracellular signal delivery in various regenerative physiologic and pathologic processes. Existing studies have established the potential of exosomes in regenerating tendons, skeletal muscles, and peripheral nerves through different mechanisms, including promoting myogenesis, increasing tenocyte differentiation and enhancing neurite outgrowth, and the proliferation of Schwann cells. These exosomes can be stored for immediate use in the operating room, and can be produced cost efficiently. In this article, we critically review the current advances of exosomes in soft tissue (tendons, skeletal muscles, and peripheral nerves) healing. Additionally, new directions for clinical applications in the future will be discussed.     

Tumor-Derived Exosomes in Tumor-Induced Immune Suppression

Exosomes are a class of small membrane-bound extracellular vesicles released by almost all cell types and present in all body fluids. Based on the studies of exosome content and their interactions with recipient cells, exosomes are now thought to mediate “targeted” information transfer. Tumor-derived exosomes (TEX) carry a cargo of molecules different from that of normal cell-derived exosomes. TEX functions to mediate distinct biological effects such as receptor discharge and intercellular cross-talk. The immune system defenses, which may initially restrict tumor progression, are progressively blunted by the broad array of TEX molecules that activate suppressive pathways in different immune cells. Herein, we provide a review of the latest research progress on TEX in the context of tumor-mediated immune suppression and discuss the potential as well as challenges of TEX as a target of immunotherapy.     

Molecular Insight into the Therapeutic Effects of Stem Cell-Derived Exosomes in Respiratory Diseases and the Potential for Pulmonary Delivery

Respiratory diseases are the cause of millions of deaths annually around the world. Despite the recent growth of our understanding of underlying mechanisms contributing to the pathogenesis of lung diseases, most therapeutic approaches are still limited to symptomatic treatments and therapies that only delay disease progression. Several clinical and preclinical studies have suggested stem cell (SC) therapy as a promising approach for treating various lung diseases. However, challenges such as the potential tumorigenicity, the low survival rate of the SCs in the recipient body, and difficulties in cell culturing and storage have limited the applicability of SC therapy. SC-derived extracellular vesicles (SC-EVs), particularly SC-derived exosomes (SC-Exos), exhibit most therapeutic properties of stem cells without their potential drawbacks. Similar to SCs, SC-Exos exhibit immunomodulatory, anti-inflammatory, and antifibrotic properties with the potential to be employed in the treatment of various inflammatory and chronic respiratory diseases. Furthermore, recent studies have demonstrated that the microRNA (miRNA) content of SC-Exos may play a crucial role in the therapeutic potential of these exosomes. Several studies have investigated the administration of SC-Exos via the pulmonary route, and techniques for SCs and SC-Exos delivery to the lungs by intratracheal instillation or inhalation have been developed. Here, we review the literature discussing the therapeutic effects of SC-Exos against respiratory diseases and advances in the pulmonary route of delivery of these exosomes to the damaged tissues.     

Exosomes Derived from Mesenchymal Stem Cells

The functional mechanisms of mesenchymal stem cells (MSCs) have become a research focus in recent years. Accumulating evidence supports the notion that MSCs act in a paracrine manner. Therefore, the biological factors in conditioned medium, including exosomes and soluble factors, derived from MSC cultures are being explored extensively. The results from most investigations show that MSC-conditioned medium or its components mediate some biological functions of MSCs. Several studies have reported that MSC-derived exosomes have functions similar to those of MSCs, such as repairing tissue damage, suppressing inflammatory responses, and modulating the immune system. However, the mechanisms are still not fully understood and the results remain controversial. Compared with cells, exosomes are more stable and reservable, have no risk of aneuploidy, a lower possibility of immune rejection following in vivo allogeneic administration, and may provide an alternative therapy for various diseases. In this review, we summarize the properties and biological functions of MSC-derived exosomes and discuss the related mechanisms.     

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs

RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.     

Role of Exosomes in Prostate Cancer Metastasis

Prostate cancer remains a life-threatening disease among men worldwide. The majority of PCa-related mortality results from metastatic disease that is characterized by metastasis of prostate tumor cells to various distant organs, such as lung, liver, and bone. Bone metastasis is most common in prostate cancer with osteoblastic and osteolytic lesions. The precise mechanisms underlying PCa metastasis are still being delineated. Intercellular communication is a key feature underlying prostate cancer progression and metastasis. There exists local signaling between prostate cancer cells and cells within the primary tumor microenvironment (TME), in addition to long range signaling wherein tumor cells communicate with sites of future metastases to promote the formation of pre-metastatic niches (PMN) to augment the growth of disseminated tumor cells upon metastasis. Over the last decade, exosomes/ extracellular vesicles have been demonstrated to be involved in such signaling. Exosomes are nanosized extracellular vesicles (EVs), between 30 and 150 nm in thickness, that originate and are released from cells after multivesicular bodies (MVB) fuse with the plasma membrane. These vesicles consist of lipid bilayer membrane enclosing a cargo of biomolecules, including proteins, lipids, RNA, and DNA. Exosomes mediate intercellular communication by transferring their cargo to recipient cells to modulate target cellular functions. In this review, we discuss the contribution of exosomes/extracellular vesicles in prostate cancer progression, in pre-metastatic niche establishment, and in organ-specific metastases. In addition, we briefly discuss the clinical significance of exosomes as biomarkers and therapeutic agents.