Preface

Extracellular vesicles (EVs) are attracting attention in the scientific community due to the growing number of clinical investigations assessing their potential for diagnostic and therapeutic applications. EVs are lipid bilayer-enclosed, microscopic vesicles that transport a variety of substances (e.g., lipids, proteins, nucleic acids, and metabolites) and are secreted by most cells. EVs enable the interaction and communication between cells in both normal and abnormal settings, and they are critical mediators in the development of many disorders. This book thoroughly examines the latest developments in EV biology and the therapeutic applications of EVs. Due to their ability to transport bioactive compounds and traverse biological barriers, EVs are being extensively investigated as possible therapeutic agents. The book also examines the role of EVs in the context of biomaterial science and EV engineering. An exciting chapter further discusses the role of EVs in cardiac regeneration. Another chapter explores the role of EVs in understanding estrus physiology and the possibility of using EV-based miRNA as potential biomarkers. The central function of EVs in kidney physiology and pathology has been established by accumulating evidence, which the book also discusses. Another chapter delves into novel alternative biological sources for EV production. Thus, this edited volume is a collection of reviewed and relevant research chapters concerning the applications and therapeutic potential of EVs. The book includes scholarly contributions written by various authors and edited by experts. It is a valuable reference for biologists, physicians, and translational scientists.

Chapters in this book include:

Chapter 1: "Extracellular Vesicles for Therapeutic Applications"

Chapter 2: "Applications and Future Trends of Extracellular Vesicles in Biomaterials Science and Engineering"

Chapter 3: "Role of Extracellular Vesicles in Cardiac Regeneration"

Chapter 4: "Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical Review"

Chapter 5: "Extracellular Vesicles in Kidney Disease"

Chapter 6: "BP-EVs: A Novel Source of EVs in the Nanocarrier Field"

#### **Manash K. Paul, Ph.D.**

Associate Professor, Manipal School of Life Sciences, Manipal Academy of Higher Education (MAHE), Manipal, India

Formerly, Scientist (Group ID: Faculty), Department of Pulmonary and Critical Care Medicine, University of California, Los Angeles (UCLA), Los Angeles, USA

#### **Chapter 1**

## Extracellular Vesicles for Therapeutic Applications

*Jianbin Xu, Liwei Wang, Di Wang, Kaicheng Xu, Liang Chen, Minjun Yao and Zhaoming Ye*

#### **Abstract**

Extracellular vesicles (EVs) are cell-derived nanoparticles containing endogenous bioactivators or loading exogenously therapeutics, which serve as "messengers" in intercellular and inter-organismal communication, in both normal and pathological processes. EVs are reshaping our perspective on life science and public health. They are tools for mediating information exchange between cells and are unique in protecting and delivering their internal cargo to target cells through ligand-receptor interactions. Therefore, EVs are one of the most potential delivery systems for treating various diseases. This chapter summarizes the recent progress made in EV-based delivery systems applications, including cancer, cardiovascular diseases, liver, kidney, nervous system diseases, and COVID-19.

**Keywords:** extracellular vesicles, drug delivery, nanocarrier, targeted therapy, therapeutic applications

#### **1. Introduction**

Cells participate in the exchange of information between cells through a variety of biomolecules, which can be cytokines, chemokines, and metabolites. It has been found that cell-cell communication happens through extracellular vesicles (EVs). EVs are lipid bilayer vesicles with a diameter of 30–150 nm secreted by most cells, which carry a variety of biological molecules, including nucleic acids, proteins, lipids, and metabolites. When EVs are ingested by other cells, these goods are transferred and affect the biological behavior of receptor cells.. Recent studies have shown that EVs carry many important signaling molecules and are the "messengers" between intercellular communications [1]. EVs are widely distributed in all body fluids, such as blood, brain effusion, saliva, amniotic fluid, and urine. When the EVs bind to the recipient cells, they transmit the "cargo" into the recipient cells, thereby mediating the signal communication and substance exchange between the cells to adjust or change the function of the recipient cells [2]. In recent years, many studies have shown that EVs are involved in the development and metastasis of tumors and play an important role in immune response and inflammation [3]. In addition, compared to normal cell-derived EVs, "cargo" packaged in diseased cell-derived EVs is different in abundance and variety and could be a biomarker for diagnosis. In particular, EVs have been used as nanocarriers for drug delivery and have great potential in the field of disease treatment [4].

EVs have some advantages as a nanocarrier for precision therapy due to their low immunogenicity and biocompatibility, among other features. As its biological function in the human body, the membrane of EVs can protect its "cargo", and the inherent or artificially modified biomacromolecules expressed on the surface of EVs can help to recognize targeting cells. Moreover, natural EVs are safer than artificial nanocarriers such as liposomes. Therefore, EVs provide a yet source of delivery systems, and even treatment means to a great extent [5].

#### **2. EVs biology**

EVs are divided into three types, including exosomes, microvesicles, and apoptotic vesicles, which originates from the endosomal system and whose formation is associated with multivesicular bodies (MVBs) and intraluminal vesicles (ILVs). The ILVs are generated from the inward budding of the endosomal membrane during the maturation of MVBs. Ultimately, ILVs are secreted as exosomes with a diameter range of 40–160 nm when MVBs fuse with the endosomal membrane [6] (**Figure 1**).

EVs were first identified in the reticulocytes of sheep and were considered involved in removing unwanted proteins [7, 8]. Subsequent studies have, in turn, shown that EVs have an important regulatory role in the immune system [9–11]. At the beginning of the twenty-first century, researchers also discovered that EVs could transmit biological information [12] between cells depending on RNA [13, 14], lipids,

#### **Figure 1.**

*The biogenesis of EVs originates from the endocytic pathway, where the invagination of the inner membrane leads to the formation of luminal vesicles (ILVs) contained within the inner body. The resulting compartment is called multivesicular vesicles (MVB), which fuse with the plasma membrane and release in the form of EVs outside the cell.*

*Extracellular Vesicles for Therapeutic Applications DOI: http://dx.doi.org/10.5772/intechopen.113969*

and proteins [15, 16]. In the past few years, the significance of EVs in endo-environmental homeostasis [17], inflammation regulation, and tumor metastasis [18] have been elucidated. Recent studies suggest that EVs also play an increasingly irreplaceable role in the biomedical field as nano-vesicular carrier systems.

Almost all living cells can produce EVs, which on the one hand, can be modified by molecular engineering techniques to load exogenous cargo into EVs for exogenous loading. On the other hand, they can also be loaded by endogenous means, using cell sorting mechanisms to sort cargo into EVs. EVs can be loaded with therapeutic drugs, RNAs, and proteins for delivery to the interior of recipient cells. At the same time, therapeutic EVs' lipid bilayer membrane structure can also be modified to express specific surface molecules and thus mediate specific biological functions or target specific recipient cell types. In this chapter, we will focus on the recent years of EVs as carriers for the delivery of endogenous or exogenous cargoes for the treatment of relevant diseases, as well as discuss the latest relevant methods for EV isolation, loading, and storage, and provide thoughts on their breakthrough in the medical field.

#### **3. EVs as potential delivery vehicles**

EVs are stable in biological fluids and organisms, and they can circulate/travel over short and long distances and even penetrate biological barriers [19]. The unique feature of EVs is that they can protect their internal cargo and transport it to target cells through ligand-receptor interactions. Previous studies have shown that proteins on the surface of EVs promote the delivery of goods by promoting membrane fusion with target cells, inhibiting CD47-mediated phagocytosis clearance, and increasing the half-life in circulation, thereby enhancing the pharmacological properties of EVs. The uptake of EVs depends on their surface ligands, such as HSPG, or receptor cell surface receptors, such as SR-B1. Recent studies have found that EVs are vulnerable to the influence of specific organs. Based on this, we can load goods on EVs and deliver them to targeted receptor cells. Other issues need to be considered; for example, the size of the engineered EVs should be small enough to avoid uptake by the reticuloendothelial system (RES) and large enough to avoid rapid renal clearance [20]. As nano-sized particles, EVs can be easily transported through body fluids and biological barriers. If this specific method can be efficiently and accurately controlled, EVs will be an effective tool for transferring therapeutic components. Some biological molecules, such as miRNA, siRNA, and complex recombinant proteins or molecules, are challenging to deliver within cells without carriers, but engineered EVs can load specific goods into target cells and induce gene modification through endocytosis [21].

When developing EV-based therapeutics, the primary consideration is to select donor cells for the exocrine body. According to GMP principles, to ensure that EVs secreted by donor cells do not cause serious adverse effects such as proinflammatory, teratogenic, and carcinogenic effects in humans, the choice should then be made in relation to the therapeutic target, as EVs from different sources generally retain the characteristics of the donor cells. Currently, immature DC-derived EVs, mesenchymal stem cell-derived EVs, or HEK293-derived EVs are used as nanocarriers for drug delivery. Among them, the EVs from immature DC sources have lower toxicity and a weaker ability to induce immune responses, but the disadvantage is that the amount of EVs that can be collected is relatively small. Furthermore, compared to EVs derived from tumors, EVs derived from DC can induce more effective anti-tumor immunity. Mesenchymal stem cells (MSCs) and HEK293 cells secrete a large number of EVs.

MSCs have a wide range of cell sources and exhibit good stability and sustainability in human plasma at −20°C. Human bone marrow MHCs incubated with drugs have antitumor effects. In addition, the immortalization of cells does not affect the quantity and quality of EVs produced, thus ensuring the possibility of a continuous supply of EVs. However, it has been found that tumor-derived EVs may cause malignant changes in target cells due to related miRNAs. In order to overcome the problems related to mammalian EVs, many researchers have also started loading cargosinto EVs from plants [22], bacteria [23], and milk [24, 25] to study the regulatory treatment effect. For example, milk-derived EVs have been found to promote the healing of ulcer wounds in diabetes patients and can also be used to load nucleic acid drugs such as siRNA and miRNA. In addition, milk-derived EVs have been proven to exist under the degradation conditions of strong acidic gastric juice and the presence of digestive enzymes in the intestine. As oral drugs, they can greatly reduce costs and related inconveniences for intravenous treatment. Although milk-derived EVs are safe, stable, and cost-effective, there is a lack of data comparing their effectiveness with other mammalian cell-derived EVs.

#### **4. Therapeutic applications of EVs as delivery systems**

#### **4.1 The treatment of cancer**

Cancer is considered a significant threat to human health and ranks as the second leading cause of death globally. Intercellular communication can also contribute to changes in the tumor microenvironment, influencing the occurrence, development, and metastasis of cancer cells [26]. This signaling can occur through EVs. EVs play diverse roles in cancer progression, possibly due to the heterogeneity in their origin and composition. The release of EVs is crucial for the maintenance of pluripotency in embryonic stem cells (ESCs) activated by focal adhesion kinase (FAK), which may be one of the conserved mechanisms maintaining the stemness of both ESCs and tumor stem cells. Meanwhile, the tumor microenvironment (TME) plays a significant role in promoting or inhibiting cancer progression and treatment of resistance. The contribution of EVs from different cell subpopulations within the TME remains unclear and requires further investigation to elucidate the roles of distinct EVs in cancer progression.

During cancer metastasis, locally invasive cancer cell-derived EVs exhibit increased secretion, promoting cancer cell adhesion and directional migration. This is because EVs from cancer cells exchange mRNA with neighboring tissues, potentially leading to the transfer of cancer cells. Live imaging of zebrafish embryos demonstrates that EVs released into circulation by cancer cell sources can enter endothelial cells and macrophages, activating the latter to promote metastatic growth. At future metastatic sites, EVs can reshape the microenvironment, creating a new TME that supports tumor growth and metastasis. EVs not only influence the angiogenesis of bone marrow-derived cells (BMDCs) to facilitate vascular metastasis but also promote lymphatic metastasis to sentinel lymph nodes by enhancing extracellular matrix (ECM) deposition and vascular generation. Therefore, bidirectional communication between cancer cells and the microenvironment is likely a key factor in promoting cancer metastasis [26, 27] (**Figure 2**).

Due to the significant role of EVs in the occurrence, development, and metastasis of cancer, they also hold great potential in cancer treatment [28]. In addition to utilizing

#### **Figure 2.**

*Comparison of exosomes from normal cell and tumor cell. Compared with normal cells, the expression of rho-ROCK, RalB, YKT6, EARK 1/2, MAPK, and PI3K/AKT signaling pathway in tumor cells is increased, which leads to angiogenesis and over-expression of ESCRT complexes, syntenin, and heparinase in the tumor, thereby secreting more exosomes.*

endogenous components of EVs from different sources, EVs are also considered as delivery vehicles capable of packaging therapeutic molecules such as small molecules, nucleic acids, and proteins and protecting cargo inside the membrane, thereby prolonging the circulation time of the therapeutic molecules in the body [29]. It has been demonstrated that chemotherapeutic drugs such as doxorubicin, paclitaxel, and gemcitabine when packaged in EVs, exhibit significant inhibition of tumor growth [28]. Furthermore, for tumors with different gene mutation types, siRNA can be loaded into EVs for specific gene therapy. Research has also found that a combination of EVs and synthetic materials can further enhance the drug-loading capacity and biocompatibility of drug-delivery systems [30]. Currently, there are new therapies targeting cancer cells, including anticancer drugs and immunotherapy drugs. As excellent nanocarriers, EVs have attracted attention due to their efficient and selective drug delivery, providing promising possibilities for modern drug administration. Zhu's team designed an EV system with two positive charges, which can rapidly enter the lipid bilayer. Simultaneously, the loading of AIEgens and PPI, combined with glutamine inhibition therapy and photodynamic therapy, effectively inhibits tumor growth in a subcutaneous cancer model.

Cancer cachexia is a multifactorial syndrome characterized by significant skeletal muscle loss, which significantly negatively impacts the patient's quality of life. The negative regulator of muscle growth, myostatin (Mstn), is potentially effective in preventing muscle wasting in cancer cachexia. However, existing administration methods have low delivery efficiency and high toxicity, leading to repeated failures of Mstn inhibitors in clinical trials. Li discovered that red blood cell-derived EVs (RBCEVs) could target and deliver Mstn siRNA to skeletal muscles and validated it in a mouse model of cancer cachexia [31]. Through muscle injection, RBCEVs-Mstn siRNA specifically and efficiently inhibited the expression of Mstn, alleviated skeletal muscle loss in cachexia mice, prolonged their survival, and had no significant systemic toxicity, reducing the mortality rate of cancer cachexia patients and providing a safe and efficient potential treatment method for muscle degenerative diseases.

In colorectal cancer patients, microsomal triglyceride transfer protein (MTTP) expression in plasma EVs is increased, acting as an inhibitor of ferroptosis. In response to this new mechanism, EVs loaded with oxaliplatin may reverse chemoresistance in chemotherapy. Index enrichment systems have been developed, generating ligands with high affinity (Kd = 3.41 nM) for EVs derived from colorectal cancer (CRC). The data show that the ligands and CRC-EVs exhibit high affinity, with a detection limit of 1.0\*103 particles/μL, and the ligands significantly inhibit the EV-induced transfer process [32].

Gu et al. overcame the poor solubility and high liver and kidney toxicity of triptolide (TPL) by using hUCMSCs-derived EVs loaded with TPL and engineered with cyclic peptides, establishing a biomimetic targeted drug delivery system. This system demonstrated good tumor targeting, prolonged the half-life of TPL, significantly inhibited tumor growth through caspase cascade and mitochondrial pathways, and prolonged the survival time of a malignant melanoma mouse model.

To address the problem of heterogeneity in hepatocellular carcinoma (HCC) that makes the identification of new antigens difficult, Zhou and his colleagues designed a specific vaccine called DEXP&A2&N by combining the functional domains of DC-derived EVs (DEX), high mobility group nucleosome-binding protein 1 (HMGN1), and an immune adjuvant. The vaccine promoted the recruitment and activation of DCs by targeting A2 and HCC-specific peptide P47 (P) and α-fetoprotein epitope (AFP212-A2). Significant tumor delay was observed in HCC mice through the recruitment and activation of cross-presenting CD103+ CD11+ and CD8α<sup>+</sup> CD11c+ CD cells in the tumor upon intravenous injection of CD11c+ CD cells.

In head and neck squamous cell carcinoma (HNSCC), EVs expressing CD73 were found to promote malignant progression and activate the NF-κB pathway in tumorassociated macrophages (TAMs), leading to immune evasion and increased secretion of cytokines such as IL-6, IL-10, TNF-α, and TGF-β1, thereby inhibiting the immune system. Additionally, previous studies have shown a correlation between EVs enriched with HAX1 and metastasis in nasopharyngeal carcinoma (NPC), which can now be explained by the increased presence of ITGB6 in HAX1-enriched EVs.

In a xenograft model of chronic myeloid leukemia (CML), tumors in mice treated with EVs from CML cells were larger compared to the control group treated with PBS. It was found that anti-apoptotic molecules such as BCL-w and BCL-xl increased, while pro-apoptotic molecules BAD, BAX, and PUMA decreased in both in vitro and in vivo samples. Furthermore, TGF-β1 was enriched in CML cell-derived EVs, and it stimulated CML cell proliferation by activating the ERK, AKT, and anti-apoptotic pathways. EVs play a role in tumor initiation, development, immunity, and drug resistance, providing new targets for improving chemotherapy efficacy and offering new insights into precision therapy.

#### **4.2 Treatment of kidney diseases**

There is increasing evidence confirming the central role of EVs in kidney physiology and pathology. EVs present in urine or circulation may participate in the regulation of renal function and communication between glomeruli and renal tubules. Potential biomarkers related to kidney diseases can be detected in EVs isolated from urine, such as AQP1, for assessing water balance in acute kidney injury. The

#### *Extracellular Vesicles for Therapeutic Applications DOI: http://dx.doi.org/10.5772/intechopen.113969*

therapeutic potential of EVs has been demonstrated, particularly those derived from mesenchymal stem cells (MSCs), which have therapeutic properties that accelerate kidney recovery. This may be attributed to the high C-C motif chemokine receptor-2 (CCR2) expression that can bind with its ligand CCL2. Studies have shown using a mouse model that CCR2-overexpressing EVs reduce CCL2 concentration, inhibit macrophage activation, and provide superior rescue of renal function in early-stage acute kidney injury (AKI). Tang reported a method using EVs to deliver interleukin-10 (IL-10) for alleviating AKI by engineering macrophages. This approach not only enhances the stability of IL-10 but also enhances its targeting to the kidneys, significantly improving tubular injury, and effectively preventing progression to chronic kidney disease. MSC-derived EVs have also been considered as a promising cell-free therapeutic approach for AKI. MSC-derived EVs have been regarded as a promising cell-free therapeutic approach for acute kidney injury (AKI). A supramolecular hydrogel containing Arg-Gly-Asp (RGD) peptides has been developed to enhance the therapeutic efficacy of MSC-derived EVs in AKI treatment. Data has shown that RGD EV hydrogel, through the interaction between RGD and integrins, provides superior rescue of renal function in the early stage of AKI, reduces tubular injury, and promotes cell proliferation.

Chronic kidney disease (CKD) is a global public health issue, and tubulointerstitial inflammation (TII) is a common pathological feature of CKD, leading to progressive renal fibrosis and driving CKD progression. The interplay between renal tubular epithelial cells and macrophages mediated by EVs is considered an important mechanism in the development of inflammation, with heat shock proteins (Hsp) 70 and Hsp90 playing crucial regulatory roles in increasing EV release. Thus, previous studies have demonstrated that the administration of the Hsp inhibitor quercetin can reduce EV release in a TII mouse model, thereby alleviating tubulointerstitial inflammation and fibrosis. Additionally, it has been found that EVs derived from HK-2 cells induced by an epidermal growth factor receptor (EGFR) mimetic peptide significantly inhibit macrophage viability and promote macrophage apoptosis, with a potential molecular mechanism involving an elevation in MHC-1B concentration. Further experimental evidence has confirmed that the injection of EVs derived from EGFR-induced HK-2 cells can significantly alleviate collagen deposition and macrophage infiltration in renal tissue, thereby ameliorating kidney fibrosis [33].

#### **4.3 Treatment of liver diseases**

In pathological conditions of liver cells, cellular stress leads to the activation of various signaling pathways, resulting in diverse biological effects. EVs have been widely accepted as carriers of signals and cargo, making them a subject of significant interest in the treatment of liver diseases. Liver cell-derived EVs directly fuse with target liver cells and transfer neutral sphingomyelinase and sphingosine kinase 2 (SK2), leading to increased synthesis of sphingosine-1-phosphate (S1P) in the target liver cells, thereby mediating liver repair and regeneration. Furthermore, systemically administered EVs exhibit significant accumulation in the liver with minimal reduction in renal clearance, making them a suitable therapeutic approach for liver diseases. Studies on hepatitis, liver failure, or cancer have found that EVs play an important role in their treatment. Mice treated with conditioned medium from mesenchymal stem cells (MSCs), which primarily consists of EVs, showed lower levels of serum INF-γ, IL-1β, and IL-6 and higher levels of serum IL-10 in an acute liver failure (ALF) model after 48 hours. Additionally, Zhao et al. demonstrated that treatment with

bone marrow mesenchymal stem cell (BMSC)-derived EVs reduced the expression levels of pro-apoptotic proteins Bax and caspase-3 and increased the expression level of anti-apoptotic protein Bcl-2 in ALF mice. Therefore, it is hypothesized that BMSCderived EVs prevent liver cell apoptosis through autophagy in ALF. EVs derived from the livers of mice infected with Schistosoma japonicum deliver miR-142a-3p to target WASL, inducing the release of neutrophil extracellular traps (NETs) and inhibiting the development of Schistosoma japonicum. miR-142a-3p and NETs upregulate the expression of CCL2, which activates the immune system and recruits macrophages to suppress the development of Schistosoma japonicum. Furthermore, knocking out WASL accelerates the formation of NETs, suggesting that WASL may be a potential therapeutic target and that the delivery of miR-142a-3p via EVs can effectively treat Schistosoma japonicum. With increasing research on EVs, it is believed that their application in liver diseases will have a higher significance in the future.

#### **4.4 Treatment of orthopedic diseases**

With the global aging population, orthopedic diseases are becoming an increasingly significant social issue that threatens human health. Bone undergoes constant remodeling through the balance of osteoblast-mediated bone formation and osteoclast-mediated bone resorption. In the bone metabolism microenvironment, EVs also participate in the regulation of bone formation, bone resorption, and bone remodeling. Both endothelial cell-derived EVs and mesenchymal stem cellderived EVs have been shown to promote bone regeneration. Cartilage, a dense avascular connective tissue, presents a major challenge in delivering drugs to treat osteoarthritis. It has been reported [34] that cartilage-affinity peptide (CAP) and lysosome-associated membrane protein 2b (Lamp-2b) can be incorporated on the surface of EVs and loaded with miR-140 to target chondrocytes, effectively alleviating the progression of osteoarthritis in rats. Osteoporosis is a systemic disease characterized by progressive loss of bone mass. Xu's team [35] developed a drug delivery system targeting osteoclasts called OT-RBCEVs, which can deliver miRNA therapeutics to osteoclasts, thereby inhibiting osteoclast activity, improving bone density, and treating osteoporosis. In another study, Luo et al. conjugated the surface of BMSCs-EVs with BMSC-specific ligands and found that intravenous injection of these EVs in ovariectomized mice increased their bone content. Rheumatoid arthritis is a chronic autoimmune disease characterized by dysregulated macrophage activity, leading to joint inflammation, destruction of bone and cartilage, and loss of function. Drug-loaded EVs can reduce the systemic toxicity of drugs and effectively modulate the activity of pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. Research has shown that MSC-EVs can inhibit rheumatoid arthritis, which may be related to the ability of MSC-EVs to inhibit the proliferation and maturation of T cells and B cells in a dose-dependent manner. These findings demonstrate the tremendous potential of EVs in the treatment of orthopedic diseases, and their underlying mechanisms are being extensively explored.

#### **4.5 Treatment of pulmonary diseases**

Acute respiratory distress syndrome (ARDS) is the most severe form of acute lung injury caused by direct or indirect lung damage, requiring intensive care and prolonged hospitalization. Once the lungs are damaged, pro-inflammatory signaling pathways are upregulated, leading to the recruitment of large numbers of neutrophils *Extracellular Vesicles for Therapeutic Applications DOI: http://dx.doi.org/10.5772/intechopen.113969*

and the secretion of pro-inflammatory cytokines. Excessive inflammatory damage to the pulmonary capillaries further disrupts the alveoli, impairing gas exchange, reducing lung compliance, and ultimately resulting in respiratory failure. Research has reported that EVs derived from dermal fibroblasts loaded with anti-inflammatory cytokines interleukin-4 and -10 (IL-4 and IL-10) genes, mRNA, and proteins can be targeted to mouse lung tissue, significantly reducing the secretion of pro-inflammatory cytokines, infiltration of neutrophils, and tissue damage. EVs loaded with miR-671-sp can also alleviate lung inflammation and injury through the NF-κB axis [36].

In 2019, COVID-19 emerged as a highly infectious respiratory illness with severe sequelae. Evidence from plasma lipidomics and metabolomics analysis suggests that EVs enriched in monosialoganglioside GM3 are associated with the pathogenesis of COVID-19, and GM3 levels increase with disease severity. Some severe COVID-19 patients develop a severe cytokine storm syndrome (CSS), a severe inflammatory immune response that threatens multiple organs. Recent clinical trials have shown that MSC-derived EVs are safe and effective for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related pneumonia [37]. MSC-derived EV therapy can significantly improve patient oxygenation and immune reconstitution within 72 hours without any side effects. mRNA lipid nanoparticle (LNP) vaccines for COVID-19 have been successfully developed [38] but their administration via intramuscular injection limits their pulmonary bioavailability. Inhalation therapy, on the other hand, can achieve effective drug concentrations in the lungs and has greater patient compliance, making it a favorable route for local administration. Pulmonary EVs are excellent candidates for inhaled therapy with nanoparticles, showing superior mRNA and protein distribution compared to commercial standards of bioengineered EVs and LNPs, thereby enhancing pulmonary bioavailability and therapeutic efficacy. Additionally, an inhalable COVID-19 vaccine has been designed that remains stable at room temperature for 3 months.

#### **4.6 Treatment of other diseases**

In addition to the diseases mentioned above, EV therapy also holds great potential in many other common diseases. For example, in patients with immune thrombocytopenia (ITP), mesenchymal stem cells (MSCs) in the bone marrow are damaged to varying degrees, resulting in abnormal immune regulation and immune tolerance and imbalance. MSC-derived EVs have been shown to regulate the secretion of antiplatelet antibodies by splenic cells, promote megakaryocyte generation of platelets, and alleviate bleeding symptoms in an ITP mouse model [39]. In stroke patients, ischemia/reperfusion injury further induces brain cell death in ischemic stroke patients. Ferroptosis, a novel form of cell death, is also present in ischemic stroke patients. Wang et al. found that intranasal administration of ADSC-derived EVs in mice can deliver miR-760-3p to cells undergoing ferroptosis, inhibit ferroptosis, improve motor and coordination abilities, and promote neural functional recovery [40].

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the targeted destruction of β-cells by self-reactive T cells. Recent studies have found that MSC-derived EVs can promote β-cell proliferation and have anti-apoptotic effects, improving T1D, possibly due to the high expression of PD-L1 in MSC-derived EVs. In the high glucose microenvironment of diabetic wounds, neutrophils are abnormally increased. Research has found that these abnormal neutrophils can induce apoptosis of human skin fibroblasts, leading to delayed wound healing. Conductive hydrogels loaded with EVs and metformin can promote angiogenesis and wound

healing in chronic diabetic wounds [41]. Additionally, researchers have discovered that miR-17-5p can target the MAPK pathway to prevent the formation of abnormal neutrophils. Under hypoxic conditions, the expression of miR-17-5p in EVs secreted by mesenchymal stem cells increases, as does EV production. Therefore, EVs from hypoxic mesenchymal stem cells can inhibit the progression of diabetic wounds, improve patients' quality of life, and provide a better understanding of diseases associated with abnormal neutrophils. The hypothalamus is the source of secretion for various hormones, and small extracellular vesicles (sEVs) derived from the hypothalamus can mediate hypothalamic AMP-activated protein kinase (AMPK) and target the central nervous system to treat obesity. Currently, research on many refractory diseases is also progressing, and with continued exploration of EV mechanisms, we believe that there will be more treatment options and better therapeutic outcomes in the future.

#### **5. Conclusion**

As drug delivery vehicles, EVs have shown tremendous potential, and clinical trials evaluating the efficacy and safety of EV-based therapies are gradually progressing. EV-based clinical drugs may be approved and enter clinical practice in the future. We believe that the production scale of EVs should be determined based on dosage, demand, and shelf life. Therefore, the production scale should be evaluated early to optimize the production process and avoid costly production expenses. Basic research on EVs should also gradually align with industrialization to lay the foundation for future applications based on EVs. We believe that by scientifically and reasonably combining these methods, the yield of EVs can be significantly improved, while also exploring and discovering new ways to increase production.

This chapter provides a brief overview of the generation, functions, and therapeutic applications of EVs in diseases. Due to their ability to achieve targeted delivery, high drug loading capacity, and controlled release, EVs are a promising drug delivery system in the field of disease treatment. However, the clinical application of EVs still faces many challenges, and the engineering and clinical translations of EVs remain major issues that need to be addressed. While increasingly complex therapeutic approaches expand our treatment options, they may also introduce additional regulatory barriers. Therefore, a careful balance between benefits and obstacles is required for the successful clinical translation of EV therapy.

In the future, what we can do is ① strive to solve the difficulties encountered in the engineering of EVs and complete the transformation of EVs research into clinical applications. ② Standardize EVs therapy and develop relevant standards. In addition, utilize the advantages of multiple disciplines to achieve mass production of EVs.

*Extracellular Vesicles for Therapeutic Applications DOI: http://dx.doi.org/10.5772/intechopen.113969*

### **Author details**

Jianbin Xu1,2,3,4\*, Liwei Wang1,2,3,4, Di Wang1,2,3,4, Kaicheng Xu1,2,3,4, Liang Chen1,2,3,4, Minjun Yao1,2,3,4 and Zhaoming Ye1,2,3,4

1 Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou City, Zhejiang Province, P.R. China

2 Orthopedics Research Institute of Zhejiang University, Hangzhou City, Zhejiang Province, P.R. China

3 Key Laboratory of Motor System Disease Research and Precision Therapy of Zhejiang Province, Hangzhou City, Zhejiang Province, P.R. China

4 Clinical Research Center of Motor System Disease of Zhejiang Province, P.R. China

\*Address all correspondence to: xu9709426@zju.edu.cn

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 2**

## Applications and Future Trends of Extracellular Vesicles in Biomaterials Science and Engineering

*Esra Cansever Mutlu, Georgios V. Gkoutos, Besim Ben-Nissan and Artemis Stamboulis*

#### **Abstract**

Extracellular vesicles (EVs) derived from natural resources and human cells are innovative biomaterials with vast potential for a wide range of applications. The applications of EVs are expanding rapidly, particularly in emerging fields such as biomaterialomics, information transfer, data storage, and 3D bioprinting, where principles of synthetic biology also come into play. These versatile structures exhibit diverse morphologies and compositions, depending on their cellular origin. As a result, they have been incorporated as key components in both medical and engineering fields. Their integration into these materials has facilitated research in various areas, including DNA and RNA storage, 3D printing, and mitochondrial transfer. Whilst the sustainable production of EVs using validated and standardized methods remains a significant challenge, it is crucial to acknowledge their tremendous potential and prepare for future scientific breakthroughs facilitated by EVs.

**Keywords:** biocomputer, DNA storage, exosome, data science, mitochondria

#### **1. Introduction**

Extracellular vesicles (EVs) play a crucial role in facilitating communication between different cellular compartments within the body by serving as carriers for the transfer of lipids, proteins, and nucleic acids [1, 2]. These vesicles are released by various types of cells and can be found in bodily fluids. Despite their sophisticated functions, only a limited number of these functions have been explored thus far.

The term "platelet-dust" was coined by Wolf in 1967 during his research at the University of Birmingham, UK, building upon previous studies that investigated the effects of platelet extraction protocols on coagulation [3–6]. Wolf's report focused on the indistinguishable properties observed amongst different platelet fractions obtained through the ultracentrifugation of plasma and serum, with regard to their coagulation activity [6].

**Figure 1.** *Schematic representation of EVs subtypes: (a) microvesicles; (b) exosomes; (c) apoptotic bodies.*

Researchers often categorize EVs (**Figure 1**) based on size, and exosomes (20–200 nm) (**Figure 2**) are particularly noteworthy due to their unique DNA and RNA content, which can be modified through cellular uptake. This ability enables them to regulate cells and tissues. Additionally, smaller vesicles than exosomes may intersect or co-release with microvesicles during the multivesicular endosomal pathway (0.1–1 μm). However, despite efforts to explore the diversity of EV subtypes, there is a lack of substantial biomaterialomics data regarding real-time release profiles [7]. Furthermore, no clear correlations have been established between the functions of EV contents. For example, recent research revealed that healthy-shaped mitochondria were found encapsulated in mitochondria-rich EVs derived from autologous stem cells [8], whereas other EV subtypes can carry mitochondria components such as mitochondrial DNA [9], or can transport mitochondrial proteins [10]. These subgroups, depending on the isolation techniques and protocols employed, can be purified as a combination of two or three subtypes. Although the relationship between motion tendency and cargo size has yet to be explored, utilizing specific biological vesicle types may provide the most effective means of targeted delivery.

**Figure 2.** *Schematic representation of an exosome structure.*

*Applications and Future Trends of Extracellular Vesicles in Biomaterials Science and Engineering DOI: http://dx.doi.org/10.5772/intechopen.113117*

#### **2. EVs in biocomputers: from synthetic biology to materials science**

Biocomputers are emerging computing devices that utilize biological macromolecules, such as DNA, RNA, and proteins, to perform computational tasks [11–14]. The field of biocomputing originated with a ground-breaking study by Adleman in 1994, which demonstrated that computational tasks could be carried out using DNA [12]. Since then, research has expanded to explore various approaches and applications of biomaterial science and synthetic biology, including DNA computing, RNA computing, and protein-based computing [15]. Notably, the CELLO algorithm has led to the development of genetic Boolean gates (**Figure 3**) and three-input Boolean circuits [16], highlighting the natural biochemical properties of these biological molecules for data storage, processing, and output.

Biocomputers rely on four main components: information storage [11, 17], information processing [18], protein-based computation [19, 20], and output-and-readout [21]. DNA consists of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), whilst RNA includes uracil (U) instead of thymine. By rearranging these bases in different-length fragments, information can be encoded and stored. Manipulating the conformation of DNA or RNA strands allows for data storage in biocomputing [22, 23]. Furthermore, these strands can be designed to selectively bind to specific target molecules or sequences, enabling logical operations such as AND, OR, and NOT gates. By combining multiple DNA or RNA strands, complex computations can be performed [24, 25].

Proteins play a crucial role in biocomputing by acting as switches or logic gates (**Figure 3**), facilitating signal processing and decision-making within a biocomputer system. Specifically designed proteins can perform computational operations, and enzymes can catalyze specific reactions [26]. Output in a biocomputer system can be achieved by detecting changes in DNA, RNA, or proteins. Alternatively, output signals can be analyzed through enzymatic reactions and translated into readable data allowing for effective communication of computational results [27].

Recently, the research on the use of exosomes in biocomputing has gained momentum, initially focusing on cell membranes and their potential as gates and

#### **Figure 3.**

*A typical example of an AND gate in digital circuits, where the two A and B inputs are exosomes. Proteins can act as switches or logic gates.*

signal processors. Fan et al. reported on the cost-effective and efficient utilization of exosomes for "Boolean response" in biocomputing. They proposed that the cell surface, with its complex array of surface molecules, could be harnessed for DNA computing, allowing for the design of intricate logic gates beyond traditional approaches [28, 29]. This concept involves using DNA aptamers to target biomarkers present on exosomes, triggering subsequent output signals through Boolean computation [30].

Huang et al. explored the use of nanovesicle surfaces as DNA-based logic gates, enhancing the targeting efficiency of encapsulated graphene carbon dots. They demonstrated the programmable elementary functions of nanovesicles derived from HCT116, a human colon cancer cell line, opening up possibilities for advanced biomedical applications [31]. Furthermore, exosome content derived from blood or cells can be detected from a biocomputing perspective. Oishi and Saito conducted research on hybridized gold nanoparticles (GNPs) that functioned as intra-particle DNA circuits, referred to as "DNA-walkers." Their study focused on the detection of miR-21 within or without fetal bovine serum (FBS)-derived exosomes, demonstrating the potential of this biosensor application for profiling endogenous miRNA in clinical samples from serum or cell lysates [32]. Recent studies have also shown that surface proteins of exosomes can be detected, or DNA/RNA computing devices can be employed for exosome detection [26]. Yu et al. designed a novel logic gate utilizing two types of hairpin DNAs that target specific surface proteins on different cancer cell lines. They successfully detected the presence of tyrosine kinase-like 7 (PTK7) and prostate-specific membrane antigen (PSMA) on CCRF-CEMsEV membranes, highlighting the potential of this approach [33].

More recent studies have revealed that natural vesicles, including EVs and exosomes, function as nano-machines on cell surfaces, capable of participating in biocomputation through active targeting strategies, in addition to passive accumulation [26]. In an earlier study by Yoshina et al., egg phosphatidylcholine vesicles ranging from 30 to 200 nm were investigated. The researchers utilized oligonucleotidetethered arrays of mobile vesicles, allowing for the separation of vesicle mixtures based on their sequence-specific binding to head-labeled antisense oligonucleotides. This approach enabled the sorting of vesicles based on their specific surface binding properties [34].

Building upon this concept, a technique was developed to create nano-biocomputing lipid nanotablets (LNTs), where DNA was used as surface ligands on small unilamellar vesicles. These DNA ligands served as input "molecular barcodes" that triggered biotin-streptavidin interactions through a supported lipid bilayer (SLB). The nano-bio-computing LNTs demonstrated sophisticated performance in capturing vesicle interactions, allowing for high-resolution spatiotemporal imaging and computational analysis [35]. Furthermore, Hao et al. provided a comprehensive review of promising studies utilizing DNA technology to amplify signals upon binding to tumor-derived exosomes, highlighting its potential for various applications [30].

In a study by Meng et al., a ratiometric electrochemical OR gate assay was developed for non-small cell lung cancer (NSCLC)-derived exosomes. DNA tetrahedrons were designed as aptamers and immobilized onto gold nanoparticles to detect NSCLC-derived exosomes. These exosomes were obtained from clinical samples, and the study demonstrated that the signal input and DNA OR logic gate could be harnessed for precise nanomedicine applications [36].

DNA storage offers remarkable advantages for long-term data storage (**Figure 4**), such as dense-durable-enormous capacity and low power consumption. However, it does have some limitations, including slow read/write speeds and the requirement

*Applications and Future Trends of Extracellular Vesicles in Biomaterials Science and Engineering DOI: http://dx.doi.org/10.5772/intechopen.113117*

#### **Figure 4.**

*Schematic representation of exosome usage in biocomputing applications.*

for specialized equipment or steganography abilities [37]. Notably, EVs and exosomes have been commonly utilized as a traditional and straightforward method for DNA storage, particularly for long-term storage at temperatures between −20°C and −80°C [38]. Furthermore, modern DNA data storage approaches involve dividing and encapsulating DNA within small vesicles called Data Blocks (DBs) to mitigate error rates by decreasing the oligos size [39, 40]. Recent studies have specifically focused on utilizing EV subtypes, particularly exosomes, for DNA or RNA storage [41, 42].

A study by Madisen et al. demonstrated that dry DNA could be stored for extended periods by using cell pellets derived from plasma, with intact DNA being preserved for 7–13 years in a solution at −20° [43]. Currently, biological membranes represent a growing field of interest for DNA storage, inspiring material scientists in applications related to the "Internet of Things" to devise a so-called "DNA-of-things" (DoT) storage architecture incorporating DNA storage into 3D-printed everyday materials [18, 44, 45]. In conclusion, synthetic biology is expected to increasingly focus on utilizing EVs and exosomes as versatile tools for data storage [46–48].

#### **3. EVs in 3D bioprinting, and mitochondrial transfer**

The largest portion of the body consists of soft tissues, including the skin, organ surfaces, and the eyes. Despite the advancements in versatile reconstruction materials, there are still significant challenges to overcome for true cell-based regeneration, necessitating the development of new biomaterials [49, 50]. One of the major obstacles in 3D tissue regeneration is the weak adhesion, immunologically non-inert nature, and long-term corrosion of materials, which hinders effective tissue repair [49]. Moreover, static or semi-static nature of these materials often results in insufficient information and energy for damaged tissues during the regeneration process, leading to inadequate tissue repair and unsatisfactory clinical outcomes [51].

Whilst some personalized cell-based trials have been conducted, the survival of cells on gliding and hydrophobic materials for long-term regeneration remains a challenge [49, 51]. However, crude EVs and exosomes show promise as self-dynamic regeneration guides and self-energy centres within biomaterials [51–53]. Motile exosome structures have the potential to deliver non-coding RNAs for cell-based regeneration, whilst motile mitochondria encapsulated in EVs can serve as energy centres during tissue repair [54, 55]. Zhang et al. in their review paper stated that secretion

of EVs increases in cells under hypoxia, resulting in indirect changes of the mitochondrial function of the receptor cells via the uptake of EVs content by the receptor cells facilitating tumor progression and ischemic damage. On the other hand, EVs derived from healthy cells can have a protective effect on the mitochondria of the recipient cells. Although it has been shown that EVs can have an effect on mitochondria regulation, it is still unclear whether the EVs content enters directly into the mitochondria of the recipient cells and whether exosomes and microvesicles play a different role in mitochondria regulation [56]. In an interesting review paper, Liu et al. [57] stated that intact mitochondria could be also present within exosomes, for example, the exosomes derived from airway myeloid-derived regulatory cells. Although, the size of mitochondria is larger than exosomes and, therefore, the case of encapsulated mitochondria in exosomes seems to be an unlike event, under certain conditions, mitochondria could be present in exosomes because the morphology of mitochondria is adapted to the demands of mitochondrial fusion, fission, and transport [58].

3D bioprinting is a promising technique that can standardize the production of tissue regeneration using crude EVs. This cell-free bioink production strategy can enhance tissue regeneration by providing self-elastic and self-bioenergetic sustainable biomaterials for 3D soft tissue repair [9, 59–61].

Mesenchymal Stem Cell-derived exosomes have gained significant attention in various regeneration products, thanks to their versatility in clinical applications similar to stem cells themselves [62]. However, a systematic review by Tan et al. emphasized the need for improved validation of animal studies with MSC-derived EVs before conducting human clinical trials, highlighting the importance of generating EV subtypes that are readily accepted and compatible for regenerating damaged cells [50, 63].

Therefore, there has been a growing focus on using EV subtypes, particularly MSC-derived exosomes, in 3D printing applications. Holkar et al. demonstrated that incorporating MSC-derived EVs within 3D hydrogel scaffolds can enhance their osteochondral healing potential [64]. Similarly, Huang et al. reported that incorporating exosomes into 3D printing scaffolds can effectively promote osteogenesis, angiogenesis, and cartilage repair [65–67].

In another study, Born et al. utilized methacrylated gelatin as a bioink along with EVs derived from mesenchymal stem/stromal cells (MSCs). The researchers investigated the in vitro bioactivity and release of EVs from the photo-cross-linked gel after 3D printing. The results demonstrated that EV bioinks retained the bioactivity of the gel and facilitated sustained release of EVs [68].

Bar et al. conducted a study on 3D-printed cardiac patches for the delivery of miR-199a-3p. They activated THP-1-derived macrophages (MΦ) and isolated their EVs. The EVs were then evaluated for bioavailability and incorporated into a RGDmodified alginate solution as a bioink after electroporation of miRNA into the EVs. The researchers used a FRESH-hydrogel solution for printing and found that the EV-based patches exhibited increased bioactivity for up to 5 days, although there is a need to improve their mechanical properties [69].

#### **4. Concluding remarks**

The field of Biomaterials Science and Engineering has evolved beyond the notion that "simplest is the best way." The Chengdu Declaration has introduced new definitions and concepts such as biomaterialomics, tissue-inducing materials, and

#### *Applications and Future Trends of Extracellular Vesicles in Biomaterials Science and Engineering DOI: http://dx.doi.org/10.5772/intechopen.113117*

bioink. Additionally, computational tools have enabled the synthesis of sophisticated biomaterials, necessitating the incorporation of extensive data and the utilization of machine learning and deep learning techniques by biomaterials scientists. Within this context, exosomes have emerged as an important tool in biomedical science, spanning a wide range of applications from 3D bioprinting to biocomputing [60, 70]. In the near future, biomaterialomics will pave the way for new studies, and exosomes will play a significant role within this field. Feng et al. introduced the term BioHEAs, highlighting the potential replacement of biological high-entropy alloys with highentropy alloys incorporated with exosomes [71]. Considering these advancements, it is clear that EVs and their subtypes will serve as fundamental components in the field of Biomaterials Science and Engineering. Whilst the sustainable production of EVs using validated and standardized methods and range of engineering and biomaterials remains a significant challenge, it is crucial to acknowledge their tremendous potential and prepare for future scientific breakthroughs facilitated by EVs.

#### **Author details**

Esra Cansever Mutlu1 , Georgios V. Gkoutos2 , Besim Ben-Nissan3 and Artemis Stamboulis1 \*

1 Biomaterials Research Group, School of Metallurgy and Materials, College of Engineering and Physical Sciences, University of Birmingham, Edgbaston, Birmingham, UK

2 Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK

3 School of Life Sciences, Innovative Biomaterials and Biomimetics Group, University of Technology Sydney, Broadway, NSW, Australia

\*Address all correspondence to: a.stamboulis@bham.ac.uk

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 3**

## Role of Extracellular Vesicles in Cardiac Regeneration

*Ceylan Verda Bitirim*

#### **Abstract**

Heart failure remains a leading cause of morbidity and mortality worldwide. Despite advances in medical management and device-based therapies, there is no cure for the damaged heart. The traditional therapeutic options for patients with heart failure, such as drugs, surgeries, and transplantation, have limitations and risks, leading to the need for innovative novel therapies. Clinical and preclinical studies have shown that extracellular vesicles (EVs) secreted by transplanted cells are more effective than direct stem cell transfer in the mechanisms involved in cardiac regeneration following ischemia. EVs have gained increasing attention as potential mediators of cardiac repair and regeneration. Preclinical studies have demonstrated the regenerative effect of EVs from a variety of cardiac cell types, including cardiac progenitor cells, mesenchymal stem cells, and iPS cells. Upon EV administration, the functional capacity of the heart improved, myocardial hypertrophy reduced, and necrosis resulted in a lesser degree. This indicates that EVs' ability to transport proteins, lipids, non-coding RNAs, and other biologically active factors plays a vital role in promoting cardiac restoration. At present, several clinical trials are exploring the therapeutic potential of EVs in heart regeneration approaches.

**Keywords:** stem cell, miRNA, cardiac regeneration, heart failure, mesenchymal stem cell, cardiac progenitor cell

#### **1. Introduction**

Through the pioneering pediatric cardiac surgery in 1938 and the introduction of open-heart surgery on cardiopulmonary bypass in the 1950s mortality rates caused by congenital heart disease significantly decreased [1]. Worldwide, ischemia injury-related cardiovascular disease continues to be the primary cause of death. Many of the more complicated types of congenital heart disease, however, still cannot be physiologically repaired; instead, patients need surgical relief, which allows for survival but leaves behind considerable residual volume and pressure loads in the heart [2]. These patients are susceptible to heart failure over time, and some will eventually need a heart transplantation. Especially, Myocardial infarction (MI) is considered the most severe clinical sign of coronary artery disease (CAD) and one of the life-threatening coronary events associated causes the loss of 1 million cardiomyocytes. Myofibroblasts that produce collagen are activated by infarction (MI), which quickly sets off an innate immune response to remove dead or dying cells and restructure the extracellular environment [3].

The limited capacity of mature mammalian cardiomyocytes to multiply is a significant biological issue that restricts therapy options for patients with congenital heart disease as well as people with acquired heart disease. Since mature, mammalian cardiomyocytes do not re-enter the cell cycle to replace ischemic tissue, cardiac muscle injury is irreversible [4, 5]. Mammalian cardiomyocytes exit the cell cycle shortly after birth, even though cardiomyocyte proliferation is responsible for fetal heart growth. There is demonstrable, if modest, cardiomyocyte turnover in adult cardiomyocyte turnover rate is 1% per year at age 20 and rapidly declines with aging [6].

There are few viable therapeutic options that effectively reverse cardiac damage and restore heart function, even though pharmacological medicines, such as statins and anti-hypertensive medications, have improved the management of coronary vascular disease and its associated symptoms. The need for more efficient methods to enhance heart function grows as the population continues to age.

The use of stem cells in therapy holds promise for the treatment of vascular disorders, particularly due to our understanding of the mechanisms underlying stem cell activation, homing, and differentiation during vascular remodeling and repair.

Adult stem cells (ASC) have been suggested as a treatment for the infarcted heart for more than 20 years and ASC therapy for the heart has attracted a lot of attention in the clinical and basic scientific fields over the past decade [1]. The bulk of animal research and early human investigations using ASCs including Hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), BM-derived mononuclear cells (BM-MNCs), and Cardiac stem/progenitor cells (CPC) therapy after Acute MI have shown an improvement in heart function.

Among these, bone marrow-derived MSCs (BM-MSCs) and cardiac progenitor cells (CPCs), have received attention, due to their unique properties. MSCs are CPCs allow both autologous and allogeneic transplantation. They can differentiate into endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and cardiomyocytes. However, injection of BM-MSCs [2] and CPCs [3] into the infarcted murine hearts has improved left ventricular ejection fraction (LVEF), contractility, angiogenesis, and decreased infarct size in pre-clinical investigations [4, 5]. These cells have shown the ability to differentiate into vascular endothelial cells and cardiomyocytes in vitro. However, according to pre-clinical or clinical studies, there is no exact conclusion about the differentiation of MSCs and CPCs into cardiac cell types in transplanted hearts [6]. However, further research failed to replicate these results in bigger animal experiments, revealing poor engraftment of the injected cells and a danger of tumor growth when employing pluripotent stem cells. Despite earlier findings that promised promising results. Additionally, there has been a wave of pessimism in the clinical community due to the potential immunogenic reaction linked to allogeneic and xenogeneic stem cell transplantation. Additionally, research has consistently demonstrated that BM-MSCs [7] and CPCs [8] transplanted in patients engraft successfully or do not survive past 3 weeks post-injection. These results suggested that differentiation following transplantation cannot be the key mechanism that causes the marked improvements in cardiac outcomes. Therefore, there is a therapeutic need to find a novel medication that gets around these constraints while still significantly enhancing heart function.

The "paracrine hypothesis" postulates an alternate process involving the secretion of soluble paracrine substances [4]. Extracellular vesicles (EVs) have attracted much attention as major players of paracrine systems.

Cell-to-cell communication can be carried out through direct contact or through secreted signaling molecules. The mechanism of intercellular communication is based

#### *Role of Extracellular Vesicles in Cardiac Regeneration DOI: http://dx.doi.org/10.5772/intechopen.113256*

on the release and uptake of membrane-bound vesicles, termed EVs. EVs are ranging in size from 30 nm to 10 μM. EVs are heterogeneous populations and can be broadly categorized based on size: apoptotic vesicles which are bigger than 1000 nm in diameter; microvesicles ranging from 100 to 1000 nm; and exosomes ranging between 30 and 100 nm. Exosomes are the subtypes of EVs and they are considered as smallest EVs. However, generally in a few studies it is suggested that the therapeutic EVs are exosomes. Due to this reason, we will use EVs as an "umbrella term" throughout this chapter. EVs are released from the endosomal compartments of living cells and carry cargo such as lipids, proteins, messenger RNA (mRNA), micro-RNA (miRNA), and long non-coding RNAs (lncRNAs) that participate in intercellular communication. They are taken into the target cell by endocytosis, fusion with the plasma membrane, or binding to receptors on the cell membrane [9]. EVs are released from many cell types and can be found in blood, urea, and serum in the body and the culture medium (media) in cell culture studies. The EV membrane-specific proteins such as CD63, CD81, Alix, and Tsg101 are used for characterization and experimental verification.

An increasing amount of empirical data substantiates the proposition that paracrine mechanisms, facilitated by substances produced by ASCs, are integral to the observed reparative process following stem cell mobilization or injection into hearts afflicted with infarction. Research has demonstrated that ADSs specifically MSCs and CPCs, have the capability to generate and release a diverse array of cytokines, chemokines, and growth factors. These bioactive molecules hold promise for their potential involvement in the process of heart repair.

#### **2. Paracrine hypothesis**

At the beginning of the story, the main idea was that transplanted stem cells would engraft, differentiate into cardiac cell types, and replace damaged cardiac tissues. However, no therapeutically important degree of transplanted ASC engraftment or differentiation has yet been shown through experimental or clinical studies. Instead, they show that transplanted cells release substances that lessen tissue damage and/or improve tissue repair.

In general, stem cell transplantation has demonstrated a modest though consistent enhancement in cardiac function in preclinical models of myocardial infarction [10]. The initiation of clinical cell treatment trials in patients following acute myocardial infarction (MI) occurred expeditiously. The utilization of unfractionated autologous bone marrow mononuclear cells was largely observed in randomized clinical trials. The trials demonstrated a limited enhancement in heart function among individuals who underwent cell treatment [11, 12]. It is noteworthy to add that a phase II clinical experiment using the intracoronary infusion of cardiac-derived stem cells exhibited a notable enhancement in cardiac function among patients with single ventricle physiology [13].

Previously, it was thought that the injected cells had the power to generate new cardiac tissue. However, there is increasing evidence indicating that these cells really exert their effects indirectly by releasing paracrine substances that stimulate internal systems of therapeutic value [1, 14]. Recent studies have indicated a connection between EVs and their role as a primary element in paracrine signaling, which regulates the beneficial effects of stem cells in a manner that does not involve the transplantation of complete cells, thus avoiding associated challenges and drawbacks. In light of numerous pre-clinical and clinical research, it is demonstrated that adult

stem cell therapy has beneficial benefits because of anti-apoptotic, immunomodulatory, and proangiogenic paracrine molecules that are released from cells.

EVs are considered as the major and active component of the paracrine secretion. Several recent studies have demonstrated the efficacy of utilizing EVs obtained from several types of stem cells, as a potential therapeutic approach to enhance heart function [15–17]. Therefore, it is believed that stem cells do not persist for an extended duration, but instead secrete paracrine factors that promote intrinsic myocardial

#### **Figure 1.**

*The diagram represents the local and distal connections of the heart mediated by EVs. Various cardiac cell types release EVs that have the potential to exert autocrine effects on the originating cell. EVs utilize intercellular communication through the transfer of proteins, lipids, and nucleic acids between cells. This transfer occurs through mechanisms such as endocytosis, membrane fusion, or gap junction-mediated transfer. The abbreviation CM refers to cardiomyocyte, CSC stands for cardiac stem cell, and EV represents EVs. (adapted from Sahoo et al. [18]).*

repair mechanisms, including heightened cardiomyocyte cell cycle activity, neovascularization, and augmented involvement of stem cells in myocardial repair (**Figure 1**).

The paracrine idea was supported by many clinical and pre-clinical studies showing how human CPC-derived exosomes can recapitulate in large part the advantages of stem-cell therapy and improve heart function following myocardial infarction.

This chapter elucidates the therapeutic potential of EVs formed from stem cells, in conjunction with therapies based on stem cells.

#### **3. Therapeutic potential of stem cell-derived EVs**

#### **3.1 Cardiac stem/progenitor cell-derived EVs**

The replacement of cardiomyocytes that have undergone cell death is not yet achievable by existing therapeutic interventions, leading to predominantly irreversible impairment of cardiac function. The use of stem cells has brought a new potential in the treatment of heart failure. To promote the regeneration of myocardial cells and vascular cells, hence enhancing cardiac function, potential strategies may involve the transplantation of pluripotent/adult stem cells into the injured area after area myocardial infarction. Before a decade ago, it was thought that the adult mammalian heart was a post-mitotic organ which has not the self-renewal ability.

The discovery of cardiac stem and progenitor cells, which are found in the heart and in extra-cardiac regions, has ushered in a new field of study centered on the application of endogenous cellular regeneration mechanisms to repair an injured heart. Nowadays it is widely accepted that the adult heart can still undergo cardiomyocyte turnover, possibly from resident cardiac stem/progenitor cells e(CPCs), according to current research. Islet 1+, Sca-1+, c-kit+, and cardiospheres are a few resident CPC populations that have all been found to support cardiac repair to varied degrees [19]. The identification of resident cardiac stem cells with the potential to differentiate into cardiac cell lineages was described [20, 21]. Following this discovery, it has been demonstrated through genetic destiny mapping studies that CPCs have a role in adult mammalian cardiomyocyte replacement after injury [22, 23]. In the hearts of numerous animals, including rats, dogs, pigs, and humans, endogenous CPC populations have also been discovered and isolated in a large number of additional studies. According to this research, CPCs can differentiate into a variety of cell types, including cardiomyocytes, endothelial cells, and vascular smooth muscle cells [24, 25].

CPCs are a variety of cells located in the atria, ventricles, epicardium, and pericardium of the heart. According to existing theories, CPCs are considered to remain quiescent and have minimal effects on cardiomyocyte renewal under physiologically normal circumstances. CPCs, on the other hand, can be triggered following injury and may differentiate into cardiomyocytes or vascular cells. Resident CPCs are a heterogeneous population. In the embryonic and adult heart, CPC populations have also been characterized by membrane markers such as c-Kit, Sca-1, Abcg-2, CD90, and transcription factors such as Isl-1, Nkx2.5, MEF2C, and GATA4 [20, 26].

The Cardiospheres/cardiosphere-derived cell (CDC) population is another CPC group that has been well characterized. CDCs are produced from atrial/ventricular biopsy tissue cultures and are a mixture of stromal, mesenchymal, and progenitor cells [27]. Due to the clonogenic and multilineage capability of CDCs in vitro, pre-clinical investigations have also shown that CDC transplantation is safe and

effective. Patients diagnosed with acute MI were randomly selected to receive CDCs via intracoronary infusion autologously. CDCs as part of the Cardiosphere-derived Autologous Stem Cells to Reverse Ventricular Dysfunction (CADUCEUS)- phase I research. After six months, there was no difference in the LV ejection fraction in this clinical trial. However, the group that was injected CDC considerable reduction in infarct size was observed. Patients receiving CDCs demonstrated decreased scar mass, increased mass of the viable heart, and enhanced local contractility [28].

Through both direct and indirect processes, CPC implantation in a damaged heart may result in myocardial healing. The cardiac stem/progenitor cells have been shown to possess transdifferentiation capability in the heart, as evidenced by data obtained from animal experiments. However, researchers have seen a limited presence of the transplanted cells in the heart after the initial few days of transplantation. Recent evidence suggests that indirect mechanisms may be responsible for the beneficial effects of CPC transplantation on a damaged heart. It was demonstrated that rather than direct differentiation into cardiomyocytes and vascular cells, releasing paracrine factors that cause existing cardiomyocytes to proliferate and hyperplasia, causing local endogenous CPCs to differentiate, and fusing transplanted cells with cardiomyocytes may cause an improved neovascularization and favorable alterations in the cardiac scar [4, 29].

It was published that EVs derived from human CPCs reduced apoptosis triggered by serum deprivation in neonatal mouse HL-1 cardiomyocytic cell lines [30]. Using human umbilical vein endothelial cells (HUVECs) is a very common method to sign an angiogenic activity. The angiogenic activity of CPCs was also demonstrated while promoting tube formation in HUVECs.

In the same studies, the efficacy of EV transfer was also demonstrated in vivo. The administration of human EVs released by CPCs into the region around the infarcted area of acute myocardial infarcted (MI) rats resulted in a decrease in cardiomyocyte apoptosis and scar formation while increasing the density of blood vessels in the infarcted area. This outcome also causes an improvement in ventricular function [31].

MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression by exerting post-transcriptional repression. The interchange of genetic material between cells through EVs has been recognized as a significant route for the transfer of mRNAs and miRNAs for the therapeutic benefit of EVs.

CPC-derived EVs are enriched with certain miRNAs, including miR-132,miR-210, and miR-146a-3p [31]. miR-210 and miR-132 involve invascular remodeling and antiapoptosis and the expression of miR-132 mediates the endothelial tube development. The activation of miR-146 leads to the induction of myocardial inflammation and dysfunction in cardiomyocytes through intercellular communication [32].

Two completed phase I clinical trials have shown that CDCs can improve cardiac function in patients with heart failure by reducing scarring and attenuating ventricular remodeling [33]. At 1-year follow-up, 4 patients receiving CDCs showed continued improvement in cardiac function despite only getting one injection at the beginning of the research. As a result of this discovery, it was postulated that transplanted CDCs largely extend their beneficial effects by secreting paracrine substances such as exosomes at the site of injury, resulting in short-term cardioprotection and longterm activation of endogenous cardiac healing. The study by Gallet et al. described the efficacy and safety of CDC-exosomes in treating acute and chronic myocardial damage in big animals [34]. The authors set out to find out whether human CDC exosomes display comparable characteristics when tested in swine with myocardial

#### *Role of Extracellular Vesicles in Cardiac Regeneration DOI: http://dx.doi.org/10.5772/intechopen.113256*

damage because CDCs have previously been found to be slightly immunogenic with no symptoms of systemic immune response or harm [33].

The application of hypoxia preconditioning, namely subjecting stem cells to low oxygen levels, has been demonstrated to boost the viability of transplanted stem cells and augment their therapeutic efficacy in cardiac repair [31]. Hypoxia preconditioning has the ability to promote the survival, migration, and angiogenesis of bone marrowderived cells, resulting in an improvement in their therapeutic effectiveness [35].

The last studies demonstrated with strong evidence that the release of antiapoptotic exosomes from explant-derived cardiac progenitor cells (CPCs) can be augmented by microenvironmental stimuli, such as hypoxia [31, 33].

Nevertheless, the investigation into the regulatory influence of traditional cardiovascular medications on the release of exosomes produced from human cardiac progenitor cells (CPCs) was elusive. Given the previous findings that the enrichment of plasma exosomes derived from transplanted cardiac progenitor cells provided protection to the ischemic myocardium, it was postulated that comparable outcomes could potentially be achieved through pharmacological stimulation of cardiacresident progenitor cells.

Importantly microarray analysis demonstrated that 11 miRNAs were significantly upregulated in exosomes derived from CPCs cultured under hypoxic circumstances compared to exosomes derived from cells cultured under normoxic settings. These microRNAs exhibit a covariation with angiogenic and antifibrotic responses. The transplantation of the exosomes produced from CPCs exposed to a 12-hour period of low oxygen levels (hypoxic) resulted in enhanced immediate and long-term performance, while also suppressing the development of fibrosis in ischemic heart [36].

The administration of conventional drugs such as ticagrelor to human cardiacderived mesenchymal progenitor cells also enhances the level of exosome release. These exosomes are enriched with anti-apoptotic HSP70 [37]. These pre-clinical outcomes bring the potential to contribute to the advancement of new pharmacological interventions that are clinically significant for exosome/EV-based therapies.

#### **3.2 Mesenchymal stem cell-derived EVs**

Smooth muscle cells, osteoblasts, chondrocytes, and adipocytes are just a few of the different cell types that mesenchymal stem cells can differentiate into. Mesenchymal stem cells (MSCs) are subsets of stromal cells and were first identified by Friedenstein in 1970 [38]. Beyond bone marrow, MSCs are found in a variety of tissues. Due to their unique characteristics, such as their capacity to develop into cardiovascular cells, immunomodulatory properties, and antifibrotic activities, MSCs have an important role in the treatment of cardiovascular disease (CVD).

The release of angiogenesis and arteriogenesis factors, according to some research groups, is the primary mechanism by which transplanted bone marrow-MSCs (BMSCs). In rat models of MI stimulate heart healing and angiogenesis in models of cardiac damage significantly increasing vascular density (80%) and decreasing collagen content (33%). The transplanted cells had the biggest inhibitory effect on the loss of heart function and enhanced vascular healing [39–41]. The efficacy of BMSC was also reported in big animals such as pigs. Amado et al. demonstrated that BMCS cells that were transplanted had been pre prepared from an allogeneic donor, and their successful integration without rejection represents a significant practical advancement in facilitating the widespread use of this therapeutic approach [42].

At the moment, bone marrow, adipose tissue, and cord blood are the primary sources of MSCs employed in clinical trials [43]. The most often employed cells in the therapy of CVD are adult allogeneic MSCs produced from bone marrow. The first clinical trial to employ intravenous allogeneic hMSC in MI was carried out in the United States in 2005 by Joshua Hare and associates (NCT00114452) [44]. In this study, MSCs were used in a clinical trial that was randomized and parallel-assigned to treat acute MI (heart attack). Phase I/II clinical study of the intramyocardial delivery of allogeneic human umbilical cord mesenchymal stem cells (HUC-MSCs) to patients with chronic ischemic cardiomyopathy was also reported by Ankara University in 2015 [45].

According to the research mentioned above, MSC therapy is safe and can enhance myocardial perfusion following MI. The clinical trials demonstrate the MSC transplantation's good safety profile and the absence of tumor formation that has been documented. However, there are certain possible dangers associated with the systematic administration of MSCs, including embolism and inflammation. Although researchers in CVDs have carried out several clinical trials, this form of trial is still in its very early stages.

Although researchers have documented the differentiation of MSCs into cardiomyocytes, it is commonly acknowledged that the main impact of MSCs in the therapy of CVDs depends on the paracrine action.

Numerous further investigations have demonstrated that MSCs take part in immunological modulation via paracrine mechanisms. Transplantation dramatically improved the cardiac function of MI mice by lowering the production of IL-1, IL-6, and TNF- alpha as well as the death of myocardial cells [46].

According to recent studies [4, 47], paracrine substances released by MSCs may be a mediator of some of these reparative effects. Numerous studies have found that MSCs release cytokines, chemokines, and growth factors that may be used to potentially heal damaged cardiac tissue, mostly through the formation and regeneration of cardiac and vascular tissue. This is evidence in support of the paracrine concept. According to Pittenger and Martin [48], this paracrine concept may offer an alternative to employing MSCs that are not cell-based for the treatment of cardiovascular disease. Contrary to cell-based therapies, non-cell therapies are considered as simpler and safer.

Using MSCs to treat a rat model of MI was found to improve cardiac function by lowering the number of CD68-positive inflammatory cells and monocyte chemotactic protein-1 (MCP-1) in the myocardium [49]. Studies demonstrate that MSCs that are transplanted into the vicinity of MI increase left ventricular remodeling and function by inhibiting miR-155-mediated profibrotic signaling and releasing HGF through direct cell contact [50]. Experimental underpinnings of the extensive clinical bone marrow-derived progenitor/stem cell transplantation trials that are currently being conducted in patients with myocardial infarction [2, 51] as these trials were largely founded on the presumption that bone marrow-derived cells produce significant amounts of cardiomyocytes through transdifferentiation.

MSCs are involved in paracrine pathways that promote angiogenesis. Ju et al. demonstrated that MSC-secreted exosomes enhanced capillary density and cardiomyocyte proliferation [52]. It was also shown that exosomes from hypoxia induced-MSCs induce angiogenesis both in *in vitro* and *in vivo* models through regulating HIF-1 [53, 54].

MSC-derived exosomes have also a cardioprotective impact on CPCs. The researchers observed that the proliferation, migration, and development of angiotubes in CSCs were enhanced after the treatment of CPCs with MSC-derived exosomes. In an experimental model of myocardial infarction in rats, the administration

#### *Role of Extracellular Vesicles in Cardiac Regeneration DOI: http://dx.doi.org/10.5772/intechopen.113256*

of mesenchymal stem cell-derived exosomes that were preconditioned with cardiac stem cells (CSCs) resulted in notable improvements in capillary density, reduction in cardiac fibrosis, and restoration of cardiac function.

The examination of miRNA profiling showed that a specific group of miRNAs exhibited significant alterations in CSCs following treatment with MSC-Exo. In an experimental model of myocardial infarction in rats, it was observed that CPCs that were preconditioned with exosomes derived from MSCs exhibited notable improvements in terms of engraftment, survival rates, capillary density, reduction in cardiac fibrosis, and restoration of long-term heart function [55].

As we mentioned above research conducted in pre-clinical settings has demonstrated that the application of hypoxia preconditioning could augment the therapeutic capacity of stem cells for the purpose of cardiac repair. Simultaneously, the administration of MSC exosomes has the capacity to modify the expression levels of microRNAs (miRNAs). It was shown that miR-210 and miR-744 exhibited an elevation in exosomal levels in response to hypoxia. miR-210 was selected as the principal factor in our investigation of myocardial protection providing protection to cells from harm through paracrine signaling. Moreover, some investigations have shown a correlation between exosomal-miR-210 and its role in safeguarding against ischemia injury. MSCs release exosomes containing a high concentration of miRNA-210 under hypoxic conditions [56].

Under conditions resembling peripheral arterial disease, MSCs lead to an upregulation of various angiogenic proteins which are encapsulated within exosomes and secreted by the MSCs. The released exosomes then stimulate angiogenesis in endothelial cells. The proteome of MSCs saw notable alterations when exposed to an environment resembling peripheral arterial disease. The examination of the angiogenesis interactome of proteins found in MSCs demonstrated that the strongest clustering of interactions with signaling proteins occurred with platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and NFkB nodes, suggesting proangiogenic capabilities of MSCs [57].

#### **3.3 The significance of EVs released by intracardiac cells**

The heart has not been traditionally considered as a secretory organ for a long time. Recent research indicated that myocardial tissue, including cardiomyocytes, endothelial cells (ECs), fibroblasts (Fbs), and cardiac progenitor cells (CPCs), releases EVs that play a significant role in facilitating intercellular communication within the cardiac system.

Communication between cardiac muscle cells and ECs includes EV-mediated pathways. The overexpression of HIF-1α in cardiomyocytes from newborn piglets exposed to hypoxia was found to result in the induction of Hsp20 which exhibits angiogenic properties by promoting the upregulation of vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells (ECs). Furthermore, it has been demonstrated that exosomes enriched with miR-126 and miR-210, derived from endothelial cells exposed to hypoxia, enhance the resistance of cardiac progenitor cells (CPCs) to hypoxic stress by activating the PI3K/Akt pathway and other pathways associated with cell survival [58, 59].

Cardiometabolic drugs also exert their effect on EVs secreted by ventricular cardiomyocytes under hyperglycemia. Ticagrelor treatment modulates the EVs released from H9c2 cells via reducing enhanced ROS production, ER stress, and autophagy

[60]. The investigation of the intricate signaling pathways involved in the activities of cardiometabolic drugs can offer a potentially advantageous pharmacological approach for safeguarding the heart from pathogenic stimuli.

#### **4. Engineered EV strategies in cardiac therapy**

On the contrary to the therapeutic efficacy of EVs, there are also limitations in EV-based therapies such as targeting, tracking, bioactivity, and internalization of transferred EVs.

In the past decade, engineered EV-based techniques have been developed to deliver specialized cargos to specific targeted tissues and promote the stability of EVs. The new strategies in the modulation of EVs including surface engineering and cargo loading provide promising therapies. There are two types of strategies; endogenous and exogenous methods in engineered EV-based therapies [61]. In the endogenous method, modulation processes are completed in paternal cells such as overexpression of nucleic acid and protein in the cells before isolation. In the exogenous method, EVs are directly engineered with nucleic acids and targeting ligands using physical or chemical methods following isolation from cells.

EV-biodistribution is a very important factor during the evaluation of the parameters such as efficiency of targeted delivery, administration route, concentration of EVs delivered, dispersion of EVs in body fluids, and the source of the EV-secreting cell in therapy [62]. For imaging purposes, EVs can be labeled after the isolation (exogenous method) or paternal cells can be genetically modified to EVs express a reporter protein (endogenous method). Fluorescence, nuclear (single photon emission computed tomography (SPECT) or positron emission tomography (PET)), and luminescence imaging techniques are used in studies to monitor EVs in vivo.

Cell source is a very effective parameter in the biodistribution of EVs. It is also suggested that EVs from different cell sources demonstrate a high tendency to accumulate in to originated- tissue [63]. On the other hand, a variety of studies reported that intravenously injected EVs show a higher preference to accumulate in mononuclear phagocyte system (MPS) organs like spleen, lung, and liver. Regardless of the delivery route, EVs are absorbed and destroyed by macrophages very quickly in blood flow. These kinds of limitations led the researchers to develop the methodologies for targeted delivery of EVs for treating heart diseases. Studies also demonstrated that intramyocardial delivery is more effective compared to intracoronary and intraperitoneally administration indicating that the delivery route of injection influences the distribution of infused EVs.

One method to increase the stability of EVs in circulation is modifying the EV membrane with polyethylene glycol (PEG). Following PEG covering, nanobodies specific to the targeted tissue are conjugated to prepare nanobody-PEG-micelles. This method increases the circulation time while reducing their uptake by non-specific cells at the same time [64].

Modification of the EV membrane with specific proteins or peptides that can interact with specifically cellular receptors or extracellular matrix components expressed in the cardiovascular system is considered as very reliable strategy to improve cardiac tropism.

Exosomes derived from cardiosphere-derived cells (CDCs) were engineered with a Lentivirus to express exosomal membrane protein Lamp2b that is fused to a cardiomyocyte-specific peptide (CMP), WLSEAGPVVTVRALRGTGSW targeting ischemic myocardium. This strategy caused an increase in the uptake of exosomes by cardiomyocytes and enhanced retention of EVs compared to un-modified exosomes [65].

To improve the bioactivity of EVs, secreting cells can be modulated by different exogenous strategies. Through these strategies, especially miRNA profiling of EVs could be changed in a cardioprotective manner. For instance, under hypoxia CPCs release proregenerative exosomes as a response. Differentially regulated miRNAs were indicated compared to normoxic conditions including miRNA-15b, miRNA-103, miRNA-20a, miRNA-210, miRNA-199a, and miRNA-292. Exosomes from hypoxic CPCs were delivered to the heart following ischemia–reperfusion injury. It was validated that exosomes from hypoxic CPCs improved cardiac function and reduced fibrosis compared to those cultured under normoxic conditions [36].

EV miRNA profiling also can be modulated by changing the culture medium or transfecting the paternal cells to upregulate/downregulate one (or more than one) specified miRNA expression level of released EVs to enhance and lead the cardioprotective effect of delivered EVs [66, 67].

Following intramyocardial injection of EVs which are isolated from miRNA-181a transfected-MSCs into mouse hearts improves heart functions compared to nonmodulated EVs [67].

Another important engineering strategy is a post-isolation modification of EVs. This method provides EV loading to modulate the enrichment of EVs. Through changing protein, lipid, and especially miRNA material of EV after isolation, we could lead the targeting and delivery regardless of their cell of origin. There are different loading strategies that are approaching such as electro-poration, heat–shock/ freeze–thaw procedures, sonication, or passive loading methods like incubating the EVs with interested drugs, or molecules [63].

For instance, EVs isolated CPCs enriched with miRNA-322 by electroporation and transferred to a mouse MI heart. This strategy causes an increase in angiogenesis and reduction of fibrotic area compared to the use of non-modified EVs [68].

All these strategies aim to enhance the efficiency of EV-based treatment for cardiovascular diseases. Hence, many studies are still required to use the advantage of EV engineering to improve the targeting, retention, and bioactivity abilities.

#### **5. EVs as biomarkers in heart diseases**

EVs carry information from cell-to-cell referring that they also respond to pathophysiological changes. It is well known that EVs are directly associated with physiological processes, such as thrombosis, apoptosis, inflammation, cell survival, endothelial dysfunction, and angiogenesis [69]. Especially, there is an increasing attention on plasma EVs as potential diagnostic/prognostic biomarkers for cardiovascular diseases.

Plasma EV proteins cystatin C, serpin C1, CD14, and serpin F2 levels are associated with an increased long-term major cardiovascular event risk after carotid endarterectomy suggesting cardiovascular risk markers [70].

Numerous studies reported that exosomal miRNAs such as miR320b, miR133a, miR143, miR150, miR155, miR214, miR223, and miR320b in circulation are diagnostic biomarkers for vascular inflammation and atherosclerosis [71].

In addition, exosomal miR208a, miR1, miR499-5p, and miR30a were identified as the early diagnostic markers of acute myocardial infarction [72].

#### **6. Conclusions**

To date, there has been significant attention focused on EVs due to their important properties, in relation to their potential for diagnostic and therapeutic applications in the treatment of cardiac diseases. According to preclinical findings, it is proposed that therapies utilizing EVs hold the potential to replace cell therapy in clinical settings.

The results indicate that the utilization of cardiac-derived stem cell therapy in patients is generally regarded as a safe approach. Nevertheless, the observed enhancements in cardiac function exhibit a somewhat modest and gradual pace of advancement. Furthermore, there is an ongoing debate surrounding the precise mechanism underlying the advantageous outcomes of stem cell therapy.

EVs demonstrate comparable advantages in stem cell transfer. Nevertheless, there remains a need to translate the results obtained from studies conducted on small animal models to a larger animal model that is clinically significant for investigating cardiac damage.

The therapeutic benefits of EVs in cardiac failures were very well reported in preclinical studies. However, due to the concerns about the off-target effects of EVs, the safety for use in human trials is required more pre-clinical studies.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Ceylan Verda Bitirim Ankara University Stem Cell Institute, Ankara, Turkey

\*Address all correspondence to: bitirim@ankara.edu.tr

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Role of Extracellular Vesicles in Cardiac Regeneration DOI: http://dx.doi.org/10.5772/intechopen.113256*

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#### **Chapter 4**

## Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical Review

*Manasa Varra, Girish Kumar Venkataswamy, B. Marinaik Chandranaik, Malkanna Topan Sanjeev Kumar and Nagalingam Ravi Sundaresan*

#### **Abstract**

Timely estrus detection is one of the critical factors for increasing reproductive efficiency in animals. Estrus physiology is under the influence of the endocrine signals that include a network of miRNAs. EV miRNAs are more stable than the other cell free miRNAs as they are doubly protected from endogenous RNase activity by means of cellular packing within the membrane-enclosed structures. Review of literature indicated the differential expression of miRNA at the estrus stage and other stages of the estrous cycle in various biological fluids, the role of miRNAs in oviductal function as well as their relation to the dynamics of preovulatory sex-steroid concentration or *vice-versa* by influencing the genes of miRNA biogenesis pathway. Interestingly, overlapping expression of miRNAs between tissues and EVs released from tissue fluids, as well as unique and differential expression of miRNA between bodily fluids and EV fractions of biological fluids has been identified. Studies focusing on the miRNA secreted in easily accessible urinary extracellular vesicles during the estrus stage in relation to the endocrine profile may pay the way for the identification of biomarkers for detecting estrus.

**Keywords:** estrus, extracellular vesicles, miRNA, biomarker, physiology

#### **1. Introduction**

Estrus is the stage of the estrous cycle when the animal shows sexual receptivity to the male, which is termed standing estrus [1]. Identifying females in estrus aids in either natural service by males [2] or artificial insemination (AI) [3]. During the estrus stage, the animals can facilitate the transport of spermatozoa through the uterus. The reproductive system of estrus animals will be in optimal environmental conditions, close to ovulation which follows the estrus stage, such that the animal can become pregnant. Detection of estrus in animals is also considered as an important prerequisite in the process of adoption of several protocols of estrus and ovulation synchronization, use of sexed semen, which are attempted to improve reproductive efficiency in the recent years [4, 5].

Estrus is dynamic, and the estrus cycle is regulated by interplay of the different organs and hormones inclusive of complex biological systems [6]. The onset of estrous cycles associated with the cyclic ovarian activity in animals occurs at the time of puberty, during which period, *via* the hypothalamic-pituitary-gonadal (HPG) axis, gonadotrophin releasing hormone (GnRH) stimulates the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) to induce steroid production and oogenesis in ovary. Multidirectional interactions between the oocyte, granulosa cells (GCs), theca cells and the HPG axis are crucial for all the complex developmental transitions of folliculogenesis [7, 8]. Ovarian cyclic activity/estrous cycle is a dynamic period between two consecutive ovulations and consists of a luteal phase (metestrus and diestrus) and a follicular phase (proestrus and estrus). Additionally, there are differences across species in the length of the heat and the estrous cycle [9].

Estrus physiology is influenced by the endocrine signals that also include a network of miRNAs [10–12]. Studies have indicated that, the miRNA present in biological fluids can indicate physio-pathological states. Extracellular vesicles (EVs) are found to be associated with cellular and molecular processes of ovarian cyclic activity [13]. Additional protection of miRNAs or proteins of EVs from degradation by endogenous RNase and protease activity [14–18] make them a better basis for identifying physio-pathological biomarkers, when compared to the cell free miRNA and/proteins. Further, a deeper comprehension of the patterns of follicle development in various species is necessary to create more efficient methods to manage domestic animal fertility. Hence, this chapter attempts to cover in detail the physiology of estrus, role of miRNA in estrus associated events, and the potential of extracellular vesicular miRNA as biomarkers to identify animals in estrus stage.

#### **2. Physiology of estrus**

Ovaries are critical organs of the female reproductive system associated with follicular development, ovulation, formation, function and subsequent regression of corpus luteum (CL). An adult ovary contains follicles at various developmental stages [19] with intensely coordinated cellular processes controlled by timely and spatially expressed genes and gene products [10, 12]. These cellular processes not only drive the folliculogenesis [7, 8, 20–23], but also the synthesis of ovarian steroid hormones which are important for estrus behavior [24–26].

#### **2.1 Cellular processes associated with ovarian cyclic activity and endocrinology of estrous cycle**

Studies on estrus cyclicity [27], cellular interactions during folliculogenesis [7, 8], follicular development in a wave-like fashion [28, 29], cellular and molecular processes occurring during folliculogenesis [30] have been put forth. FSH (Follicle stimulating hormone) and LH (Leutinizing hormone) signaling in the follicular cells are not only crucial for steroidogenesis [26, 31] and cellular signaling mechanisms for the ovulation to occur [21, 23, 24, 32–37], but also for driving estrus behavior in animals [38, 39] by guiding the biosynthesis of E2 and P4 at the estrus stage [27, 40, 41].

*Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical… DOI: http://dx.doi.org/10.5772/intechopen.113166*

#### *2.1.1 Ovarian follicular development and estrous cycle*

Follicular development occurs in a wave-like fashion with two waves [28] or three waves [29] as the most common pattern [32, 33]. GCs of small growing follicles secrete inhibin and acquire receptors for FSH (FSHR)*.* Further, it has been put forth that, each follicular wave begins with a transient FSH surge in the circulation, and the loss of dominance and the end of the follicular wave that does not result in ovulation are followed by a decrease in LH secretion [34, 35]. FSH controls the development of GCs by stimulating their proliferation and differentiation, and promoting the formation of the follicular antrum [21]. Subsequently, the dominant follicle acquires luteinizing hormone/choriogonadotropin receptor (LH/CGR) in its GCs, secrete more E2 than the subordinate follicles [24], triggering the LH surge that allows them to develop into the preovulatory follicle [23].

Various cellular processes like angiogenesis, steroidogenesis, basement membrane turnover, oocyte growth and maturation and follicular atresia [30] occur during folliculogenesis. Also, antioxidant system/reactive oxygen species (ROS) is found to modulate the events of folliculogenesis, ovulation, formation and activity of the CL and luteolysis [36]. Further, within the preovulatory GCs, initiation of the transcriptional upregulation and downregulation of genes, including cytokines, transcription factors (TFs) and matrix-remodeling proteins (MMPs) is caused by the LH surge [37]. However, the exact molecular mechanisms involved in the initiation of the growth of the primordial follicle and the further development of primary follicles up to the pre-ovulatory stage remains unknown [42, 43].

#### *2.1.2 Biosynthesis of ovarian steroid hormones*

The ovarian steroid hormones, progesterone (P4) and estradiol 17β (E2) are synthesized from cholesterol through the cooperative interactions of theca and GCs [26, 31]. LH binds to LH/CGR on the theca cell surface and stimulates the expression of the steroidogenic enzymes [steroidogenic acute regulatory protein (STAR), cholesterol side chain cleavage enzyme (CYP11A1), 3β-hydroxysteroid dehydrogenase (3BHSD), 17α-hydroxylase/17,20desmolase (CYP17A1)] necessary for androgen production. Pregnenolone synthesized from cholesterol gets converted to P4 by the action of 3BHSD. P4 then gets converted to androstenedione by the action of CYP17A1. Androstenedione produced by theca cells diffuses into GCs and gets converted to E2 *via* FSH signaling through FSHR and stimulates the expression of enzymes [17βhydroxysteroid dehydrogenase (HSD17B), aromatase (CYP19A1)] in GCs.

Though the gonadal hormones (P4 and E2) are important for estrus behavior, their circulatory levels are actually under the control of GnRH, FSH, LH and inhibins which have an important role in determining the expression of heat signs. Further, circulatory concentrations of FSH and LH were highest on the day of estrus compared to the other days of the estrous cycle [27, 40, 41].

#### **2.2 Differential expression of mRNA and proteins during various stages of estrous cycle**

Studies on differential expression of mRNA transcripts [44–46], heat shock proteins [47–50] and other proteins [44, 51–54] in various biological fluids and/or tissues collected at different stages of estrous cycle have been reported and the use of the same as biomarkers for the identification of estrus has been opined.

#### *2.2.1 Differential expression of mRNA transcripts at estrus stage*

The transcripts of lactoferrin (LF) and glutamate receptor-interacting protein 1 (GRIP1) were highly expressed in the uterine tissue of cattle during the estrus when compared to other stages of the estrous cycle [44]. Likewise, higher expression of mRNA transcripts of HSD17B1 and heat shock 70-kDa protein 1A (HSPA1A) in saliva during the estrus stage in both cyclic heifers and pluriparous buffaloes was reported [45]. In yet another study, it was reported that the mRNA transcript of tissue inhibitors of metalloproteinase 1 (TIMP1) was significantly over-expressed in cell free saliva at the estrus stage when compared to the diestrus stage in buffaloes [46].

#### *2.2.2 Heat shock proteins (HSPs) as estrus indicators*

Higher expression of heat shock protein-27 (HSP-27) in porcine endometrium during the estrus stage compared to other estrous cycle stages was demonstrated [47]. Likewise, higher expression of heat shock protein-70 (HSP-70) in cervico-vaginal fluid (CVF) was identified during the estrus stage when compared to the diestrus stage in buffaloes [48]. In yet another study conducted in sheep, higher expression of several HSP family proteins was identified in the luminal fluid samples collected from inner cervix, uterus, and oviduct during estrus stage when compared to other stages of estrous cycle [49], among which, heat shock protein-105 (HSP-105), heat shock protein-90-β (HSP-90-β) were abundant in cervical mucus, while heat shock protein-90-α (HSP-90-α) was expressed abundantly in both the uterine fluid as well as the cervical mucus. Further, the heat shock protein (HSP) family proteins (HSPH1, HSPAA1, HSP90AB1, HSPB1, HSPA4, and HSPA8) were found to be highly expressed during the estrus stage [50].

#### *2.2.3 Differential expression of other proteins at estrus stage*

Specific expression of β-enolase (ENO3) and TLR 4 (Toll like receptor 4) proteins in the saliva of buffaloes at the estrus stage was identified [51]. So also, some of the proteins that were discovered to be overexpressed in the saliva of buffaloes during the estrus stage included heat shock 70-kDa protein 1A (HSPA1A), lipocalin 1 (LCN1), odorant-binding protein (OBP), leukocyte elastase inhibitor (SERPINB1), vitelline membrane outer layer 1 (VMO1), 45-kDa calcium-binding protein (SDF4), and ENO3 proteins [54]. Likewise, LF and GRIP1 proteins were highly expressed in bovine cervical mucus during the estrus stage compared to other stages of the estrous cycle in cattle [44].

Further, the proteins, cullin-associated NEDD8-dissociated protein 1, HSP701A, HSD17B type 1, inhibin beta A chain, and testin were identified to be estrus specific by proteomic analysis of buffalo saliva using in-solution digestion and nano-Liquid Chromatography-Mass Spectrometry (LC-MS/MS) [52]. In yet another study, higher expression of LH in saliva was reported during the estrus stage compared to other estrous cycle stages in buffaloes [53].

The above studies on differential expression of mRNA transcripts, HSPs and other proteins during estrus, when compared to other stages of estrous cycle when compared to other different stages of the estrous cycle as well as unraveling their

relationship to the cellular processes associated with ovarian cyclic activity and/or the regulatory mechanisms involved in the differential expression of the same, may pave the way for the identification of a biomarker for estrus in animals.

#### **2.3 Role of microRNA (miRNA) in estrus associated events**

Studies on the role of non-coding RNA, especially the miRNA [55–59] associated with ovarian function as well as female reproduction [11, 60–64], regulatory mechanisms of miRNA on expression of genes associated with ovarian function [65–69] have been postulated.

MiRNA, circular RNA (circRNA), long non-coding RNA (lncRNA), small interfering RNA (siRNA) and PIWI-interacting RNAs (piRNA) constitute the non-coding RNAs [11, 70]. Among the non-coding RNAs, the role of miRNAs in female reproduction, primarily associated with the function of the ovary has been explored widely in the last decade [11, 62–64]. The role of miRNA on ovarian functions, the regulatory role of miRNA in maintaining the ovarian function, cyclicity and oocyte maturation has been postulated [11].

Earlier, miRNA was termed small temporal RNAs (stRNAs) for their sequential expression at specific times and regulation of various developmental events in *Caenorhabditis elegans* [71, 72]. In 2001, these small non-coding RNAs were given the term miRNAs [73, 74]. MiRNAs are found to be strongly conserved between vertebrates, invertebrates, and plants [65]. The miRNAs are 20–22 nucleotide long single stranded non-coding RNA molecules which have an important role in regulating gene expression by promoting mRNA degradation (via direct cleavage or by mRNA deadenylation) or preventing translation [66]. Further, the miRNAs are transcribed from individual genes, sometimes clustered and located intergenic or in introns or exons of protein-coding genes [67].

Various *in vitro* and/or *in vivo* studies have been conducted to identify the miRNA associated with follicular/luteal development in the mammalian ovary. Further, the analysis of whole ovaries was very useful for comprehensively identifying miRNA sequences. Study of small RNA population in the ovaries of humans and other species using molecular techniques like microarrays, high-throughput quantitative polymerase chain reaction (PCR) and next-generation sequencing (NGS) [55–58] has revealed that miRNAs constitute the most abundant class of small RNAs in the ovary.

Regardless of the species, it was discovered that the let-7 family miRNAs miR-21, miR-99a, miR-125b, miR-126, miR-143, miR-145, and miR-199b were the most prevalent miRNA populations in the ovary [61]. Likewise, the study of the expression profiling of miRNA in pigs [58], mice [57], cows [75] and sheep [76] has confirmed the role of miRNAs in the different functions of ovary. In yet another study, miR-21, miR-143, let-7 family, miR-26a and miR-125b were identified as highly abundant miRNAs in mammalian ovaries as revealed by cloning based technologies or NGS, while the miR-21 has been identified to promote follicular cell survival during ovulation and the miR-17-5p and let-7b were identified as pro-angiogenic and therefore opined to be essential for the development of CL [60].

Above all, specific sets of miRNAs were found to be expressed within the ovary and are found to strictly regulate the gene expression patterns in ovary spatiotemporally [59]. Further, various genes involved in follicular development, oocyte maturation and implantation have been reported to be regulated post-transcriptionally by miRNAs [68, 69]. In yet another study, it was reported that gene ontology (GO)

analysis and bioinformatic screening revealed that the targets genes of predominantly expressed miRNA in the mammalian ovaries were associated with a number of biological pathways or molecular networks, including cellular growth, development, and proliferation, cell to cell signaling, cell cycle regulation, cell death, endocrine system disorder, and various pathways underlying ovarian functions [56].

Therefore, miRNA appears to be involved in regulating estrus-associated events. Hence, the literature on the miRNA associated with ovarian cyclic activity, biogenesis and nomenclature of miRNA is being reviewed below.

#### *2.3.1 Biogenesis of miRNA*

MiRNAs are generated from its precursors, *i.e.,* long strands of RNA called primary RNA (pri-miRNA). Pri-miRNA upon cleavage by the enzyme Drosha, a nuclear RNAse III endonuclease and simultaneous binding of RNA-binding cofactor, DGCR8 (DiGeorge syndrome critical region gene 8) leads to the formation of hairpin shaped precursor miRNA (pre-miRNA) within the nucleus which is of 70–100-nucleotide (nt) in length [77].

This Pre-miRNAs get transported out of the nucleus into the cytoplasm by exportin 5 [78]. The RNAse III endonuclease Dicer breaks down the pre-miRNAs in the cytoplasm, decreasing the hairpin loop and creating a duplex miRNA. The final active miRNA is created when the miRNA-induced silencing complex (miRISC), which is composed of proteins from the Argonaute (AGO) family among others, attaches to one of the strands of this duplex miRNA. This complex then binds to the complementary 3′ or 5′-untranslated region (UTR) of the target mRNA [79, 80], open reading frames (ORF) or promoter regions [81] and exert their effects. This miRNA biogenesis pathway involving Drosha/DGCR8 is universal to all mammalian miRNAs and is known as the "canonical" pathway of miRNA biogenesis which is depicted in **Figure 1**.

#### **Figure 1.**

*The canonical pathway of miRNA biogenesis (adapted from [82]).*

*Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical… DOI: http://dx.doi.org/10.5772/intechopen.113166*

#### *2.3.2 Nomenclature of miRNA*

Based on the discovery sequence, the initial nomenclature was followed for miRNA [83]. For example, miR-3 was the third miRNA to be discovered using the guidelines. The let-7 and lin-4 are exceptions to the numerical naming rule, whose original names were given based on their historical significance (www.mirbase.org). Lin-4 and let-7 were the first two miRNAs originally discovered in the nematode, *Caenorhabditis elegans* and control the timing of stem-cell division and differentiation [71, 84]. With the increasing research on the role of miRNAs in disease and health, the list of miRNAs is becoming large and many other publicly available miRNA databases are now available [85].

The prefix added to the specific miR signifies the species (hsa, human; mmu, murine; bta, cow, etc.). For example, bta-miR-21 refers to cow miR-21. Suffixes have also evolved over time. A number placed after the miRNA name (i.e., miR-218-1 or miR-218-2) indicates the exact same mature miRNA sequence. Still, it suggests that it was derived from independent gene loci (in this case, human chromosomes 4 and 5, respectively).

The passenger strand of a miRNA duplex, which is less frequent and is thought to have no biological purpose, was previously mentioned and was denoted by an asterisk (\*). However, it has been established that the guide miRNA strand may not always be present or as physiologically active as the miRNA\* in some cells or tissues. The \* has been replaced by a -5p or -3p mark. The -5p/-3p suffix is a critical differentiator since these miRNA target very diverse groups of mRNAs based on their various seed sequences (i.e., bases 2–8 from the 5′ end of the mature miRNA).

The bases used by the majority of algorithms to find possible targets are assumed to be the main bases that direct the mi-RISC to certain mRNA targets. A note (e.g., miR-34a, miR-34b, etc.) can be included after the miRNA name to identify two miR-NAs with closely comparable sequences and 100% homology in the seed sequence.

#### *2.3.3 MicroRNA associated with ovarian cyclic activity*

The role of miRNA related to endocrine regulation of ovarian cyclic events has been postulated [12, 26, 60, 86–92].

The miRNA regulating aromatase expression (encoded by CYP19A1 gene) during follicle development include miR-224, miR-378 and miR-383 [60]. So also, miR-378 was found to suppress the E2 release in swine GCs by targeting the CYP19A1 gene [87]. In yet another study, it was revealed that, *in vivo* injection of miR-320 in mouse was associated with decreased E2 levels by targeting the transcription factor (TFs) genes, elongation factor 1 (E2F1) and steroidogenic factor 1(SF-1) [90]. While, miR-764-3p was found to target the gene SF-1, thereby suppressing E2 release in mouse [92]. In contrary to the above findings, various studies conducted in mouse have revealed that miR-224, miR-383, miR-133b and miR-132 can suppress the E2 release in cultured GCs by targeting the genes SMAD4, RBMS1, FOXL2 and NURR1 respectively [86, 88, 89, 91].

Therefore, the biosynthesis of the two main ovarian steroid hormones, E2 and P4 is regulated in the ovary by miRNA at all levels that includes (1) transcription of genes (STAR, CYP11A1, 3B-HSD-II, SR-BI, 17B-HSD-I, CYP19A1, RBMS1, FOXL2, RUNX2) coding for essential enzymes (2) direct post-transcriptional regulation of the enzymes involved in hormone biosynthesis and (3) expression of enzymes that catalyze the conversion of these hormones to inactive metabolites [26].

Further, the role of miRNA in follicular atresia [93] and the miRNA transcriptome dynamics of different stages of CL during an estrous cycle [94] has been postulated. Notable miRNA families and clusters that have been functional during the process of follicular atresia, which is mainly indicated by GC apoptosis include the let-7 family, miR-23-27-24 cluster, miR-183-96-182 cluster and miR-17-92 cluster [93]. Many researchers have applied the *in vitro* gain- and loss-of-function studies to explore the effective functions of miRNAs during atresia. The use of small RNA sequencing technology for studying the miRNA transcriptome dynamics at different timely defined CL classes using ovaries collected from cycling German Fleckvieh cows covering the entire physiological estrous cycle revealed that, bta-miR-143 expression reached its peak in the regressed CL (rCL, days >18 of the estrous cycle), whose expression was significantly downregulated in the early CL (eCL, day 5–7 of the estrous cycle). In addition, regardless of CL developmental or functional status, bta-miR-21-5p and bta-miR-143 were found to be abundantly expressed [94].

Above all, differential expression of miRNA in GCs of preovulatory dominant and subordinate follicles [95], the housekeeping role of miRNA in the ovary during anestrus stage [56, 95–100] was documented. When compared to the subordinate follicles, the preovulatory dominant follicles significantly downregulated the miR-17-92 cluster, btamiR-409a, and bta-miR-378, according to a study of the expression pattern of miRNAs in GCs of bovine preovulatory dominant and subordinate follicles during the late follicular phase of bovine estrous cycle [95]. This suggests that miRNAs may play a role in the post-transcriptional control of genes involved in bovine follicular development during the late follicular phase of the estrous cycle. This is supported by the unique sets of miRNAs found in the GCs of preovulatory dominant and subordinate follicles.

Further, among the highly abundant miRNAs expressed in the ovary across the estrous cycle in various species [56, 96, 98, 99], the miRNAs, *viz.*, bta-miR-10b, bta-miR-26a, bta-miR-27b, bta-miR-30d, bta-miR-30a-5p bta-miR-92a, bta-miR-99b, bta-miR-125a, bta-miR-143, bta-miR-148a, bta-miR-186, bta-miR-191, bta-let-7a-5p, bta-let-7f and bta-let-7i, were found to be expressed irrespective of the stage of follicular development [95, 97]. Therefore, the housekeeping role of the afore mentioned miRNAs in maintaining the normal physiological processes in mammalian female reproduction was opined [100].

In yet another study, the acquisition and combinatorial analysis of the databases of miRNA, circRNA, lncRNA, and mRNA obtained from the ovary of estrus synchronized Xinong Sannen goats in estrus and diestrus stages of the estrous cycle revealed the following [98]: (a) Differential expression of miRNA, circRNA, lncRNA (noncoding RNAs) and mRNA (coding RNA) at estrus and diestrus stages was evident. (b) Screening of differentially expressed (DE) non-coding RNAs and coding RNAs illustrated their regulatory role in ovary to maintain the homeostasis. (c) As taken from the network, differentially expressed miRNAs and mRNAs that are important in controlling the estrous cycle include miR-21-3p, miR-202-3p, and miR-223-3p. (d) Tissue inhibitors of metalloproteinases (TIMP1), matrix metallopeptidase 9 (MMP9), 3-hydroxysteroid dehydrogenase (3BHSD), and prostaglandin I2 synthase (PTGIS) were chosen from the differentially expressed mRNAs in order to screen the differentially expressed miRNAs and circRNAs that may have regulated their expressions by creating circRNA-miRNA-mRNA networks. (e) Differentially expressed miRNAs were found to target the mRNA of TIMP1, 3BHSD and PTGIS, but no miRNA that can target the mRNA of MMP9 was identified.

It is important to note that, most of the research has been focused on studying the regulatory role of miRNA in ovary by various *in vitro* studies. As a result of the *Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical… DOI: http://dx.doi.org/10.5772/intechopen.113166*

complexity of the physiological mechanisms underlying inter- and intracellular signaling, *in vitro* investigations are still crucial for unraveling the secrets of cellular communication [101]. However, *in vivo* studies have reported the role of miRNAs, let-7, let-7b, 17-5p, 21, 125b, 181a, 224 and 430a in specific cells of the ovary.

#### *2.3.4 Physiological stage specific miRNAs in biological fluids*

It is apparent from the above that miRNA is involved regulates the molecular events associated with the ovarian cyclic activity. Accordingly, the literature indicating the differential expression of miRNA in biological fluids for identifying physiological states is being reviewed below.

Differential expression of miRNA at estrus stage and other stages of estrous cycle in various biological fluids [46, 102–104], the role of miRNAs in oviductal function as well as their relation to the dynamics of preovulatory sex-steroid concentration or *vice-versa* by influencing the genes of miRNA biogenesis pathway [105, 106], overlapping expression of miRNAs between tissues and EVs released from tissue fluids [102, 105–107] as well as unique and differential expression of miRNA between biological fluids and EV fractions of biological fluids [102, 107] and differential expression of miRNA in urine in accordance with the season of breeding [108] has been postulated.

Study on plasma miRNA profiles during the estrous cycle in estrus synchronized Holstein-Friesian heifers using NGS and PCR-based platforms (PCR array analysis and RT-qPCR analysis) [103] revealed the differential expression (up to 2.2-fold increase, P < 0.05) of let-7f, miR-125b, miR-145 and miR-99a-5p in the plasma on the day of estrus (Day 0) when compared to the days 8 and 16 of the estrous cycle and therefore the feasibility of using circulating miRNAs as biomarkers of reproductive function were opined. Likewise, differential expression of bta-miR-99a-5p in buffalo urine between estrus and diestrus stages of estrous cycle was reported [104]. In yet another study, higher expression of miR-141 was found in cell free saliva at the estrus stage compared to the diestrus stage in buffaloes [46].

Further, the circulatory steroidal concentration of E2 and P4 was found to influence on the miRNA expression in the oviduct of estrus synchronized Nellore cow animal models [106]. Interestingly, the periovulatory sex-steroid milieu was also found to affect the miRNA processing machinery (by influencing the expression of genes, DROSHA, DICER1 and AGO4 involved in miRNA processing pathwaycomponents) and the expression of specific miRNA levels (miR-125b, miR-200b, miR-30d, miR-375, miR-92a) in bovine oviductal tissues. The fact that the previously mentioned differentially expressed oviductal miRNAs were also discovered in the EVs of the bovine oviductal fluid suggests that the miRNAs discovered in the oviduct may be charged into EVs and released into the oviductal fluid and have an impact on embryonic survival and development [105]. It was apparent from the findings of [106] that the miRNAs have a role in the oviductal function and their role on the post-transcriptional control is subjected to the dynamics of periovulatory sex-steroid concentration which can vary widely between individual animals.

Likewise, controlled ovarian hyperstimulation (COH) of estrus synchronized Simmental heifers intended to stimulate the growth of multiple dominant follicles with ovulating capability [107] by maintaining an elevated level of circulating FSH gonadotrophin was found to induce the differential expression of circulatory miRNA in bovine follicular fluid (FF) and blood plasma when compared to the unstimulated heifers. Further, bioinformatics analysis of the differentially expressed circulating miRNAs indicated that their potential target genes are associated with various

pathways including transforming growth factor-beta (TGF-β) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, pathways in cancer and oocyte meiosis. Above all, it was discovered that the majority of these miRNAs could be located in the FF and blood plasma's exosomal and Ago2 protein complex fractions. According to the results of qRT-PCR, there are variations in the expression patterns of miR-103, miR-127-3p, miR-134, miR-147, miR-221 and let-7 g depending on the stage of the estrous cycle. The exosomal component of the miRNAs, which were found in both blood plasma and FF, had a higher abundance. However, it was discovered that only the exosomal fraction could detect miR-182 from FF and miR-221, miR-103, and miR-127-3p from blood plasma [102].

Interestingly, obtaining the urinary miRNA profile by NGS in male goats has revealed differential expression of 40 miRNAs in urine during the breeding season compared to the non-breeding season. Among the differentially expressed miRNAs, miR-1246 was the most downregulated microRNA during the breeding season characterized by elevated testosterone levels. Further, testosterone through androgen receptors (AR) was found to be involved in the regulation of miR-1246 expression and other miRNA genes, whose expression differed between breeding and non-breeding season, indicating that miRNAs could serve as intermediaries of testosterone preparation of the male urogenital tract for high metabolic demands of the breeding season [108].

The preceding studies on differential expression of miRNA in biological fluids at various stages of the estrous cycle, the role of miRNAs in oviductal function as well as their relation to the dynamics of preovulatory sex-steroid concentration or *viceversa* by influencing the genes of miRNA biogenesis pathway, overlapping expression of miRNAs between tissues and EVs released from tissue fluids as well as unique and differential expression of miRNA between biological fluids and EV fractions of biological fluids, differential expression of miRNA in urine in accordance with the season of breeding speculate the presence of EVs in biological fluids like urine as well as the differential expression of EV miRNA, if any in animals at the estrus stage and other stages of estrus cycle.

#### *2.3.5 Extracellular vesicular miRNAs as indicators of physio-pathological states*

EVs are cell-derived vesicles of 30–1000 nm in diameter, includes both exosomes and microvesicles and carry protein, lipid, RNA and miRNA [109]. EVs can be detected in all biological fluids as they are being shed by any cell type in the organism [110]. The recipient cells can take up EVs released into the extracellular space and modulate their biology [111].

Studies on various means of extracellular release of miRNA, reasons for the use of extracellular miRNA especially, EV miRNA &/protein as an alternative source for the search of biomarkers [15, 16, 18, 112–115] and differential expression of miRNA in EVs of biological fluids and/or reproductive cells associated with various reproductive diseases has been postulated [113, 116, 117].

The tissue/organ specific miRNAs find their way into serum or plasma by several cellular release mechanisms [112, 115]. For instance, mature miRNA synthesized in the cell can either bind to RNA-binding proteins or lipoproteins, or get loaded inside microvesicles or exosomes when they are to be released. Since the miRNAs secreted in the plasma can mediate the uptake of miRNA at distant sites in the body, miRNA in body fluids has been regarded as hormones by some authors [113]. Various means of extracellular release of miRNA from a cell is depicted in **Figure 2**. Additionally, it

*Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical… DOI: http://dx.doi.org/10.5772/intechopen.113166*

**Figure 2.** *Different forms of extracellular miRNA (adapted from [118]).*

was discovered that extracellular miRNAs serve as novel biological tools for intercellular cross-talks across cells in several organs, including the female reproductive system [114].

Above all, extracellular miRNAs could be used as the novel potential biomarkers of various physio-pathological conditions for the following reasons: (a) Stability of the miRNA in the extracellular environment against the activity of RNases, extremes of pH and/or high temperature. (b) Distinct expression profiles of extracellular miRNA in different body fluids such as blood, urine and follicular fluid and even in cell culture media. (c) Protection of extracellular miRNAs from degradation either by packaging in the lipid vesicles as EVs/exosomes or forming complexes with the RNA-binding protein, Argonaute 2 protein (Ago2) [14, 17, 18]. (d) Circulating miRNA with in the microvesicles and the miRNAs associated with Ago2, high density lipoprotein (HDL) and nucleophosmin 1 (NPM1) [119, 120] are protected from endogenous RNase activity [15, 16].

Therefore, cell free miRNA (Extracellular miRNA) found in various biological fluids like serum, plasma, urine and saliva can be broadly of two types, miRNA found in EVs and miRNA associated with proteins. The existence of primarily exosomal or vesicle-free miRNA can depend on the miRNA itself, the cell type from which they arise, and/or other factors impacting the secretion of miRNA in a particular individual.

Exosomes from biological fluids and/or exosomes from particular reproductive cells have also been proposed as biomarkers for a variety of reproductive diseases, including uterine fibroids, preeclampsia, polycystic ovary syndrome (PCOS), endometriosis, ovarian cancer, and Asherman's syndrome. The need for research to specify the functions and mechanisms of exosomes has been highlighted by many researchers [113, 116, 117].

#### **2.4 Extracellular vesicular miRNA associated with physiological states**

Proteins and miRNAs carried within the EVs are found to be the major regulatory components and are associated with the alteration of the cell biology of the recipient cells up taking the EVs [121, 122]. MiRNAs of exosomes can be delivered to distant target cells, which is recognized as an important mode of cell-cell communication [123]. Further, EV miRNAs are more stable than the other cell free urinary miRNAs as they are doubly protected from endogenous RNase activity by means of cellular packing within the membrane-enclosed structures [124].

Exosomes, microvesicles and apoptotic bodies are the three main subtypes of EVs. The EV subtypes are distinguished according to their biogenesis, size, release pathways, cargo and functions [125, 126]. It is evident that EVs present in various biological fluids can be used to identify physiological stage specific biomarkers. Therefore, the literature pertaining to the studies on EV miRNA and/or proteins related to physio-pathological states along with their biogenesis is being reviewed below.

#### *2.4.1 Biogenesis of EVs*

All types of cells secrete exosomes, which are EVs. Exosomes are created as intraluminal vesicles (ILVs) and range in size from 30 to 150 nm. They originate from the endosomal compartment. ILVs are created by the early endosomal membrane budding inward, encasing proteins, lipids, and cytosol for later degradation, recycling, or exocytosis. After early endosomes develop into late endosomes, multivesicular bodies (MVBs) are created as a result of the buildup of several ILVs. These MVBs may either fuse with lysosomes and get degraded or fuse with the plasma membrane (PM) to release their sequester ILVs as exosomes in the extracellular space [127].

Microvesicles are larger EVs than exosomes and are 100 nm to 1 μm in diameter and bud directly from the plasma membrane (PM). The EVs involved in cellular communication are characterized by protein markers such as tetraspanins (CD9, CD63, and CD81) and adhesion integrins. Both exosomes and microvesicles form a source of cellular clearance and are involved in cell–cell communication both between local and distant cells [128].

The size of apoptotic bodies varies from 50 to 5000 nm in diameter and are released into the extracellular space as blebs of cells undergoing apoptosis [125] and therefore contain nuclear fragments and cell debris. Apoptotic bodies do not play a part in cellular communication or exhibit surface markers characteristic for exosomes or microvesicles [129].

#### *2.4.2 Studies on physiological stage-specific EV miRNA in biological fluids*

Studies on the presence of EVs in biological fluids of female reproductive tract [130–135], role of EV miRNA of biological fluids [136], differential expression of uterosomal EV miRNA during pregnancy [137], the impact of estrous cycle on the miRNA content of FF EVs [138] and the influence of lactation and energy status on miRNA content of follicular fluid [139] has been postulated.

Oviductal luminal fluid (OLF) is one of the reproductive secretions which is rich in EVs (Oviductal EVs or Oviductosomes, OVs) containing both exosomes (50–100 nm) and microvesicles (100–1000 nm) which are known to play a role in inter cellular communication [130]. Likewise, EVs isolated from human uterine flushing's and *in vitro* cultured human endometrial epithelial cells contained the

#### *Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical… DOI: http://dx.doi.org/10.5772/intechopen.113166*

miRNA [131]. EVs found in the uterine fluid are termed uterosomes and EVs present in the uterine lumen flush (ULF) of pregnant animals contain EVs emanated from both the endometrial epithelia of the uterus and trophectoderm of the conceptus [135]. In particular, OVs have been found in media of cultured bovine epithelial cells [132, 133], bovine oviductal secretions (*in vivo*), human oviductal fluid [134] and in mouse [134].

The following was discovered as a result of the research done to determine whether OVs contain miRNAs during the estrous cycle and whether particular miR-NAs in the estrus oviductosomal cargo can be transferred to sperm during their communication inside the oviduct in mice: (a) MiRNAs in OVs of the mouse were found to have similar expression levels for the majority throughout the murine estrous cycle (b) OVs can transfer miRNAs to sperm, notably miR-34c-5p, which is only produced from sperm in the zygote and is necessary for the initial cleavage. (c) Particularly the miR-34c-5p, which is strongly concentrated at the centrosome where it is known to function, miRNAs transported by OVs to sperm are mostly localized in specific head compartments [136].

Further, the uterosomal miRNA cargoes of pregnant and non-pregnant sheep were found to be significantly different [137]. Likewise, EVs present in the uterine lumen flush (ULF) of early pregnant sheep were found to modulate trophectoderm cell growth by decreasing the proliferation of ovine trophectoderm cells and promoting the secretion of interferon tau (IFNT) without affecting gene expression*.* EVs have been reported to decrease early in pregnancy in the ovine uterus, to carry lipid and protein cargo, and to control trophectoderm cell proliferation. Hence, EVs' protein and/or lipid cargo along the nucleic acid cargo in the lumen of the pregnant ovine uterus in conceptus development has been highlighted [135].

In yet another *in vitro* study conducted with the ovaries obtained from crossbred Nellore cows to know the impact of the estrous cycle on the microRNA content in EVs of FF that modulate bovine cumulus cell transcripts that in turn influence the oocyte maturation revealed the following: (a) 161 miRNAs were significantly upregulated in the small EVs of FF with low P4 levels when compared to the ones with high P4 levels, (b) the uptake of small EVs by cumulus cells started after 1 h of *in vitro* maturation (IVM) as evident from live cell imaging, (c) within just 9 hours of IVM, small EVs derived from low and high P4 FF can induce a specific RNA profile in the cumulus cells and (d) cumulus cells supplemented with EVs obtained from FF of low P4 group presented a large number of genes that are upregulated that modulate biological processes involved in reproduction and immune responses [138].

Further, a study conducted to know the association between postpartum negative energy balance (NEB) and EV-coupled miRNA signatures in follicular fluid (FF) of cows revealed that NEB in postpartum cows is mainly associated with downregulation of EV-coupled miRNAs in FF and *vice-versa*. Moreover, regardless of the energy status, lactation induced changes in FF EV-coupled miRNA profiles in dairy cows compared to the heifers [139].

#### **3. Conclusion**

MiRNA are involved in regulating molecular events at the level of mRNA and proteins related to the biogenesis of E2 and P4. MiRNA are also identified to be involved in follicular atresia and have a housekeeping role in the function of the ovary across the estrous cycle. Differential expression of mRNA transcripts, HSPs and other proteins

connected to the cellular processes of ovarian cyclic activity has also been identified in various species of animals. Differential expression of miRNA in GCs of preovulatory dominant and subordinate follicles have been detected. Differential expression of miRNA in biological fluids at various stages of estrous cycle, overlapping expression of miRNAs between tissues and EVs identified in tissue fluids as well as biological fluids, unique and differential expression of miRNA between biological fluids and EV fractions of biological fluids was demonstrated. Importantly, EV miRNAs are more stable than the cell free miRNAs making them promising biomarkers for the identification of physiological states. Estrus being dynamic and EVs identified as associated with cellular and molecular processes of ovarian cyclic activity around the estrus stage, disclose the potential of EV miRNAs in easily accessible biological fluids to act as biomarkers.

### **Author details**

Manasa Varra1 \*, Girish Kumar Venkataswamy2 , B. Marinaik Chandranaik3 , Malkanna Topan Sanjeev Kumar4 and Nagalingam Ravi Sundaresan5

1 Department of Veterinary Biochemistry, College of Veterinary Science, Sri Venkateswara Veterinary University, Proddatur, India

2 Department of Veterinary Physiology and Biochemistry, Centurion University of Technology and Management, Orissa, India

3 Institute of Animal Health and Veterinary Biologicals, Hebbal, Karnataka Veterinary, Animal and Fisheries Sciences University, Bangalore, India

4 Department of Veterinary Microbiology, Veterinary College, Gadag, Karnataka Veterinary, Animal and Fisheries Sciences University, Bangalore, India

5 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

\*Address all correspondence to: dr.manasa88@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical… DOI: http://dx.doi.org/10.5772/intechopen.113166*

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#### **Chapter 5**

## Extracellular Vesicles in Kidney Disease

*Chunyan Lv*

#### **Abstract**

The kidney is the mainly apparatus in the human body, with a complex organizational structure and diverse pathological changes closely related to other organs. Extracellular vesicles are vesicles with diameters ranging from tens of nanometers to several micrometers, originating from multiple intracellular vesicles or local cell membranes. They carry various information from the source cells and operate between various cells in the kidney and extrarenal organs, conveying substances between cells. They play a large part in signal transmission within the kidney and between the kidney and other organs. Detecting changes in extracellular vesicles and their cargo can monitor both renal neoplastic and nonneoplastic diseases. Extracellular vesicles derived from various stem cells, loaded with bioactive substances, can be applied to some extent to treat kidney diseases. Bioengineering drugs using extracellular vesicles as carriers are also playing an increasingly big role in treating kidney diseases. Research on extracellular vesicles has achieved certain results and has some preclinical applications, but there is still a process for large-scale and widespread application.

**Keywords:** kidney disease, extracellular vesicles, cell-to-cell communication, biomarker, therapeutic potential

#### **1. Introduction**

Extracellular vesicles (EVs) are a heterogeneous population of bilayered lipid vesicles [1–3] regarded as a vital interactive courier between cells and secreted by most types of cells. EVs can be differentiated into three main categories according to their origin and size: exosomes, local microdomains assembled in endocytic membranes with the size of 30–100 nm; microvesicles, released from the plasma membrane with a size of 100 nm-1 μm; apoptotic bodies, released by cells undergoing apoptosis with a size of 1 μm–5 μm [4, 5]. The giant tumor-derived vesicles (oncosomes) and mitochondrial-originating vesicles (mitovesicles) are also considered as EVs [6, 7].

Despite the heterogeneous and diverse nature of EVs, they are released by normal cells and move in endless cycles in humor, with a major role in numerous physiological and pathological conditions. EVs are abundant in body fluids and are easy to separate and enrich, with complicated cargo reflecting the physiological and pathological condition of the source cells. EVs can be efficient transport through the cell membrane

of target cells, thus enabling communication between cells and modulating the gene expression and function of target cells [8, 9].

The kidney is one of the important organs to remain steady in the internal milieu. EVs feature in renal physiology and are widely involved in the occurrence and progression of various kidney diseases. It can be used for the diagnosis of kidney diseases and be instrumental in the treatment of renal diseases [10, 11].

#### **2. Role of extracellular vesicles in cell-cell communication in kidney**

As the characteristics mentioned above, EVs act major affection in nephron intercellular communication [12]. Proteomics results show that the exosomes in nephron mainly come from glomerular podocyte and tubular cells located in the proximal tubules, the thick segments of ascending medullary branches, the distal tubules, and collecting ducts. Some studies have confirmed that EVs transmit information in the nephron between the glomerulus and renal tubules. The upper tubule cells can release EVs, which are internalized by the cells in the lower segment of the tubule, transmitting activated cell molecules. The earliest research found that EVs could be expelled and internalized by murine renal collecting duct cells in cultivation and transfer the functional aquaporin 2 [13]. Further research has found that the EVs from proximal renal tubules can be absorbed by the distal tubules and collecting ducts [14]. A study on podocyte RNA labeling found that this RNA was subsequently detected in renal tubular cells, thus confirming the material transfer between glomerulus and renal tubular cells [15].

Not only that, the maintenance of electrolyte and acid-base equipoise is one of the crux functions of the kidney, which is fulfilled by tubular transport. A variety of proteins and transporters rich in urinary exosomes come from cells in the nephron, for example, Na+-Cl symporter (distal tubule), aquaporin (AQP)-2 protein (concentrated pipe), Na+-K+-2Cl symporter (thick ascending limb), AQP-1 (proximal tubule), podoglycocalyx protein (podocyte). The urinary extracellular vesicles (uEVs) produced at the top membrane of kidney tubular cells, cargo water, electrolyte, and acid-base transporters. When the proximal tubular cell line is affected by inflammation, they excrete more EVs from apical and basal membrane, both of which have different molecular and functional characteristics [16]. It has proved that miRNAs acted as a major role in EV-mediated intercellular communication. Jella et al. recorded by unipath patch clamp that the probability of ENaC opening Xenopus cells and isolated splitopen tubules could be reduced by proximal EVs, and this could be weakened by EVs transfected with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitors [17]. Another research confirmed that ENaC activity could be regulated by EVs through purinergic signaling [18].

The distant tissues can release EVs into circulation and mediate inter-organ crosstalk. EVs released from the placenta of preeclampsia can carry anti-angiogenic factors and lead to maternal glomerular endothelial dysfunction and proteinuria [19]. EVs released from autoimmune diseases, such as antiphospholipid syndrome, thrombotic microangiopathy, systemic lupus erythematosus, and ANCA-vasculitis, can promote coagulation, thrombosis, and immune-mediated renal pathology [20].

Increasing evidence shows that circulating cells (monocytes, neutrophils, and red blood cells) and platelets attacked by toxins release the EVs, and the latter are the major elements of pathogenesis in hemolytic uremia syndrome [21]. The ability of EVs derived from mesenchymal stem cells (MSC) to alleviate myocardial

#### *Extracellular Vesicles in Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.113200*

injury in experimental metabolic renovascular hypertension is partly mediated by IL-10-containing EVs [22]. The bioactive cargo of EVs in kidney transplantation (KT) includes implant antigens, cytokines, growth factors, costimulation/inhibitory molecules, and functional microRNAs (miRNAs) that may regulate gene expression in receptor cells, which act as the immune modulators and play a crucial role in maintaining complex crosstalk between graft tissue and innate/adaptive immune system. EVs are of great importance in allogeneic recognition, ischemia–reperfusion injury (IRI), autoimmunity, and allogeneic immunity and are expected to become biomarkers and therapeutic tools for KT [23].

The kidneys can also release EVs, affecting the function of other tissues and organs. A study on the proteomics of EVs in murine hearts found that some proteins come from other organs, including the kidneys [24]. Cardiovascular dysfunction is caused by a high level of circulating endothelial cell-derived particles in chronic kidney disease (CKD).

These particles may mediate inflammation, vascular wall damage, and remodeling and act as an incremental risk of Vascular calcification (VC) [25–30]. VC is a pathological manifestation with high mortality, mainly manifested as abnormal calcium deposition in the vascular wall. During the process of VC, vascular smooth muscle cells (VSMCs) suffer osteogenic transformation and secrete EVs with various sources and compositions. The secreted EVs may obtain calcium-promoting properties, thus acting as the nucleation focus of hydroxyapatite crystallization and calcium transmission [31]. The serum calpain particles (CPPs) and EVs in uremia are meaningful participants in the extensive calcification mechanism of CKD, and cGRP (a protein rich in Gla) plays an inhibitory role in preventing calcification at the system and tissue levels [32].

#### **Figure 1.** *Extracellular vesicles as delivery vans in cell-cell communication between renal cells and extrarenal organs (cells).*

The deterioration of renal damage during liver or heart disease may be caused by EVs, while the advancement of hepatorenal or cardiorenal syndrome may involve EVs [33, 34]. Nonvalvular atrial fibrillation is associated with kidney disease because the increase in thromboembolism is mediated by a higher level of EVs from prethrombotic endothelial platelets rather than by thrombotic status and other markers of cell activation, even in anticoagulant patients [35]. In patients with hypertension, the presence of EVs indicating podocyte injury and peritubular capillary injury is detected [36]. The endothelial cells derived EVs released from peritubular capillaries were checked in the primary and renovascular hypertension patients' urine, with the density of EVs directly associated with clinical parameters and the scarcity of capillaries, but was inversely proportional to renal perfusion [37]. Therefore, the change of urinary EVs in hypertensive patients can be regarded as an early marker of renal injury caused by peritubular capillary injury, and it will change with the improvement of renal function after drug treatment in patients with primary and renovascular hypertension (**Figure 1**).

#### **3. Extracellular vesicles as biomarkers of renal diseases**

EVs are a type of disc-shape vesicles with single concave, secreted by active cell and enveloped by phospholipid bilayers [38], with sizes ranging from nanometers to micrometers [39–42]. EVs grow out from the straight sprout of small vesicles wrapped in membranes or through blend on the superficial multivesicular bodies, engage in the intercellular commutation of materials and message, with cargo proteins, lipids, and nucleic acids (microRNA, messenger RNA, circ RNA, and lncRNA, etc.) [43–47]. With the pivotal roles in an exchange between renal cells and target cells, EVs are expected to serve as new molecular markers for the detection of kidney diseases, carrying specific molecular substances in source renal cells with the lipid structure to protect the contents from being degraded [48, 49]. At present, the study of EVs, especially exosomes, has been involved in the field of liquid biopsy [50, 51].

#### **3.1 Extracellular vesicles as liquid biopsy markers for renal cell carcinoma**

Renal cell carcinoma (RCC) is a kind of malignant tumor in the urinary system originating from the renal tubular epithelium, accounting for about 3% of adult malignant tumors, ranking second in the urinary system. The partial or total nephrectomy are the current therapeutic strategy, following or not following with immune-checkpoint-inhibitors-based targeted therapies to which patients are often drug-resistant. It lacks specific symptoms at the early stage [52]. Diagnosis mostly relys on ultrasound and other examinations. At the initial diagnosis, about 17% of RCCs have distant metastasis, with a 5-year survival rate close to 12% [53]. Therefore, early diagnosis and prediction of metastasis risk are crucial to the treatment and prognosis of patients. Data has shown that EVs are the proficiency biomarkers for tumor liquid biopsy due to their specific content, species conservation, stability, and high abundance under different sources and physiological and pathological conditions [54].

#### *3.1.1 EVs as biomarkers for the diagnosis and staging of RCC*

Some researchers conducted multi-omics analysis on tumor-related proteins and mRNA in EVs from different RCC cell lines, identified multiple candidate molecular markers, and confirmed that EVs could not only distinguish between RCC and benign

#### *Extracellular Vesicles in Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.113200*

lesions, but also assisted in determining the subtypes of RCC, with great potential for clinical transformation [55]. Research has found that compared to the control HK-2 cell line, the expression level of miR-150 in EVs from the 786-O RCC cell line was upregulated by 5.2 times, while miR-205 was downregulated by 10,000 times, indicating that miR-205 and miR-150 in EVs were new prospective biomarkers for the diagnosis of RCC [56].

However, *in vitro* cell models cannot fully and in real-time reflect tumor conditions, and it is still essential to screen and validate in clinical specimens. There are reports that compared to healthy control, the expression of miR-149-3p and miR-424-3p in plasma EVs of RCC patients are upregulated, while miR-92a-1-5p is obviously downregulated, which is helpful for the diagnosis of RCC [57]. Zhang et al. observed that the levels of miR-210 and miR-1233 in serum EVs were markedly upregulated in patients with clear cell renal cell carcinoma (ccRCC) than those in the control group, and their expression were dramatically reduced after tumor resection, they has worthwhile value for RCC diagnosis and surgical effect monitoring [58]. Additionally, the expression of miR-210 is also related to their clinical prognosis in serum EVs of RCC patients, and the diagnostic performance for early patients is better than that of serum miR-155 [59]. Similarly, serum EVs miR-4525 can serve as a potential diagnostic marker for advanced RCC patients [60]. EVs from other sources of bodily fluids, besides blood also have the diagnostic potential for RCC. Urinary exo-miR-30c-5p may regulate the expression of a protein related to the progression of ccRCC (heat shock 70 kDa protein 5) and is regarded as a latent diagnostic biomarker for early ccRCC [61]. Some researchers have found that a combination detection of urine EVs miR-126-3p and miR-449a or miR-34b-5p can prominently differentiate between ccRCC and healthy individuals [62].

Recently, although most research on EVs as molecular markers of RCC has focused on miRNAs, it is also important to recognize that other specific molecules (proteins, mRNA, DNA, etc.) in EVs can also be used for auxiliary diagnosis of RCC [63]. Palma et al. found the obvious diverse in urinary exosomal shuttle RNA (esRNA) type of ccRCC patients compared to healthy population and non-ccRCC, and observed that this specific pattern increased to reach the normal level one month after partial or radical nephrectomy. They identified that the urinary esRNA levels of CEBPA, GSTA1, and PCBD1 were downregulated in patients than the healthy subjects, then suggested the RNA content in urinary EVs could be the prospective diagnostic applications for the ccRCC [64]. Some scholars describe CA9, CD147, and CD70 in EVs as tumor markers that are specific to ccRCC [65].

Some scholars characterized CA9, CD147, and CD70 as tumor-specific markers on EVs in CCRCC [65]. Vergori observed that CA9 in circulating large EVs plays a part in the diagnosis and prognosis of ccRCC [66]. Researchers regarded Polymerase I and transcript release factor (PTRF)/CAVIN1 in urinary exosomes as prospective biomarkers of ccRCC [67]. Li et al. believed that exosomal circRNAs may be the potential cancer diagnosis marker [68]. The above studies have confirmed that EVs and their contents are expected to become diagnostic and staging markers for RCC.

#### *3.1.2 EVs as markers for RCC metastasis monitoring*

The messages in EVs actively loading by cancer cells can support tumor spread; the double layer of EV protects the peculiarity. Thus EVs play a major role in metastasis by improving the content's half-life and stability, and then EVs act as markers for metastasis monitoring. Many scholars have confirmed this by animal and cell experiments.

EVs released from RCC cell line (786-O) can transfer long-chain noncoding RNA (LncRNA)\_human lung adenocarcinoma of the metastasis related transcription factor 1 (MALAT1) to adjacent nonmetastatic RCC cells and enhance their growth, invasion, and metastasis abilities [69]. Meanwhile, CD103+ EVs separated from cancer stem cell (CSC) of ccRCC patients targeted regulate the protein level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), which is tightly relevant to cell migration, by transporting miR-19b-3p to ccRCC cells; Cancer stem cell EVs also can be guided by CD103 to target cancer cells and organs, making ccRCC have higher lung metastasis capacity [70].

In addition, the expression of miR-1293 decreased, and the expression of miR-301a-3p increased in plasma EVs of RCC patients with metastasis, which are regarded as presumable biomarkers for the metastasis of RCC [71]. The downregulated miR-483-5p was observed in ccRCC, and Wang et al. believed that it contributed to cell proliferation, metastasis, and inflammation [72]. Evs-derived proteins also can be the marker of RCC metastasis monitoring. Some investigators observed that the serum EVs of prostate-specific membrane antigen (PMSA) levels in metastatic patients of RCC were sharply increased compared with nonmetastatic patients by establishing a sandwich ELISA method, and also believed that PMSA can reflect the angiogenesis of the primary tumor and metastasis, then monitoring the metastatic RCC in real time [73]. Furthermore, the surface or content of EVs can provide targeted recognition sites for tumor metastasis, providing a theoretical basis for developing the predicting RCC metastasis markers and targeted therapy strategies.

#### *3.1.3 EVs as biomarkers for drug resistance and prognosis in RCC*

The prognosis of RCC patients is closely related to tumor staging, grading, metastasis, and patient sensitivity to therapeutic drugs. It is deemed that partial nephrectomy is the preferred treatment strategy because some researchers observed a better ending and better postoperation renal function in the advanced stage RCC patients treated by partial nephrectomy than those that undergo radical nephrectomy [74]. The survival rate of early RCC is 90%, while that of locally advanced or metastatic cases is only 13%. Therefore, early diagnosis and treatment of RCC is extremely prominence for improving the 5-year survival rate of this disease. Liquid biopsy techniques provided for early RCC detection may bring about a superior outcome. EVs detection can guide clinical rationalization, individualization, and precision of medication by assessing the sensitivity of patients to drugs. Ras-related protein Rab-27B is one of the chief proteins involved in the secretion of EVs. Previous studies have confirmed that the high expression of this protein is related to the poor prognosis of hepatocellular carcinoma, ovarian cancer, and colorectal cancer. Tsuruda et al. identified that Rab-27B was significantly overexpressed in sunitinib-resistant renal cell carcinoma cell lines [75]. Dias et al. found that compared with patients with nonmetastatic in situ ccRCC, patients with metastatic disease had higher levels of matrix metalloproteinase inhibitor of metalloproteinase-1 (TIMP-1) mRNA in plasma EVs and lower overall survival rate. This means TIMP-1 mRNA derived from EVs is a potential prognostic marker for ccRCC [76].

Additionally, researchers also found that after 1 day of cryoablation treatment, the miR-17-5p, miR-126-3p, and miR-21-3p in mouse serum-derived EVs rapidly decreased, reflecting the number of active tumors, which can be used to evaluate tumor clearance efficacy and dynamically monitor tumor burden [77]. Du et al. applied Cox regression analysis and Kaplan Meier analysis to confirm the significant overall survival (OS) association of three EVs source miRNAs (miR-let-7i-5p, miR-615-3p, and miR-26a-1-3p. Then, these miRNAs in plasma exosomes

#### *Extracellular Vesicles in Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.113200*

were used as prognostic markers for metastatic kidney cancer [78]. Nakanori et al. found that intracellular miR-224 increases in ccRCC and is obviously relevant to cancer metastasis and invasion. Then, they explored the relevance between the level of exo-miR-224 and the prognosis of 108 ccRCC patients, and they observed that progression-free survival and overall survival are shorter in the group with high exo-miR-224 expression level. Thus, they deemed that extracellular miR-224 in ccRCC patient's exosomes is a prospective prognostic marker [79]. Sunitinib is a novel multi-targeted oral drug for treating tumors and is also used for treating advanced renal cell carcinoma. The drug resistance is the main challenge of current treatment. Sun et al. detected lncRNA Activated in RCC with sunitinib resistance (lncARSR), a lncRNA related to the clinically poor response to sunitinib. LncARSR may improve the level of AXL and c-MET in RCC cells by competitively binding to miR-34/miR-449 and recovery the drug resistance of sunitinib. In addition, this lncRNA also transmits its drug resistance mechanism to sensitive cells through exosomes. Therefore, it is suggested that lncARSR may predict sunitinib resistance and a potential therapeutic target (**Table 1**) [80].



#### **Table 1.**

*A list of candidate dysregulated EV contents as molecular markers for RCC.*

#### **3.2 Extracellular vesicles as diagnostic markers for renal tubulointerstitial injury**

Some biomolecules carried in urine EVs are closely related to the degree of renal tubulointerstitial inflammation and fibrosis; the detection of these markers in EVs can reflect the degree of renal inflammatory fibrosis progression. After genetic screening of EVs in urine of chronic kidney disease (CKD) patients, it was found that miR-29c and CD2AP mRNA were positively correlated with the degree of renal fibrosis and renal function, which may have meaningful diagnostic value for the progression of renal fibrosis [81, 82]. Based on previous findings that EVs CCL2 mRNA released by renal tubular epithelial cells (TECs) can directly transfer to macrophages and promote tubulointerstitial inflammation, some researchers found that the expression of CCL2 mRNA in urine EVs of IgA nephropathy (IgAN) patients is related to the level of eGFR and can predict the progress of renal function, which may become a new marker for IgAN prognosis monitoring [83]. Hypertension is a noteworthy cause of CKD, and the loss of peritubular capillaries (PTC) is one of the characteristics of hypertension; the level of endothelial microparticles (EMP) in the loop can report the endothelial injury systematically. Urinary EMPs levels were examined as possible markers of PTC and decreased fibrotic density. Ptc-emps were peculiar proteins that were identified as plasmalemmal vesicle-associated protein (PL-VAP) positive urinary exosomes, which were showed in PTC and Vas deferens endothelium but not in glomeruli and arteries and are supposed to be a novel biomarker of intrarenal capillary loss [84]. In obstructive kidney disease, uEVs may help assess the risk of developing renal dysfunction, and some studies have observed that the profibrotic factor TGF-β1 level in uEVs is related to glomerular filtration rate [85]. It was the upregulated of the urinary exosomal miR-181a (200-fold) in CKD patients [86] and the downregulated of the exosomal level of secreting transglutaminase-2 (a fibrosis-activating enzyme) in UUO mice [87]. CKD patients have a decrease in miR-200b of urinary exosomes, with

#### *Extracellular Vesicles in Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.113200*

the greatest reduction in urinary exosomes originating from cells outside the proximal renal tubules [88]. The upregulation of the urinary exosomal ceruloplasmin in CKD patients and animals with passive Heyman nephritis was observed [89]. Moreover, osteoprotegerin, an inflammatory marker, was shown to be increased in the uEVs of patients with CKD [90].

The kidney is a major organ that regulates the body's water and salt metabolism. Ions and aquaporin are distributed in different segments of TECs. During the injury of TECs, these ions and water transporters in urine EVs also changed correspondingly. For example, the decrease of aquaporin (AQP) expression in urine EVs was observed in the rat model of Acute kidney injury (AKI) induced by cisplatin and ischemia/reperfusion, suggesting its potential impact on the change of renal concentration function [91]. The level of EVs Na + transporters in urine may also indicate certain pathological conditions in the kidneys. The activation of renin angiotensin aldosterone system (RAAS) after sodium-restricted diet acute aldosterone infusion in patients with hypertension increases the content of epithelial sodium channel (ENaC) in urine EVs by nearly 20 times, suggesting that it may be a novel means to monitor RAAS activation [92]. Moreover, the significant reduction of furosemide-sensitive sodium potassium chloride cotransporter (NKCC2) and sodium chloride cotransporter (NCC) in urine EVs has been used to distinguish different phenotypes of hereditary desalinization renal tubular disease [93, 94]. These studies suggest that changes in ion and water transport proteins in urine EVs may reflect renal tubular damage.

#### **3.3 Extracellular vesicles as diagnostic markers for glomerular injury**

EVs derived from glomeruli are continuously released into urine under physiological conditions, so changes in urine EVs can reflect the degree of glomerular disease, including podocyte damage. In early studies, Wilms Tumor Protein 1 (WT1) has been identified as a key regulator of podocyte expression, and the WT1 target gene is crucial to maintain the glomerular filtration barrier [95]. WT1 in uEVs was confirmed to be detectable before obvious glomerular sclerosis and urinary EVs WT1 appeared prior to proteinuria and glomerular histological damage in focal segmental glomerulosclerosis (FSGS) animal models [96]; moreover, urinary exosomal WT-1 was significantly decreased in patients in remission for either FSGS or steroid-sensitive nephrotic syndrome (SSNS) or following steroid treatment of patients with SSNS [97]. Compared with patients with minimal change nephropathy (MCN), the WT1 mRNA expression was increased significantly in urine EVs of patients with diabetes nephropathy (DN) and was associated with decreased eGFR; thus the high expression of WT1 mRNA in urine EVs distinguishes patients with DN from patients with MCN [98]. In addition, hyperglycemia can stimulate the release of WT1 from podocyte to urine EVs, so the detection of WT1 in urine EVs can indicate early damage of podocyte in diabetes patients. WT-1 may be a biomarker for early diagnosis of podocyte injury, as suggested by these data. Recently, it has been found that the expression of the upper pitt-specific transcription factor ELF3 protein in urine EVs is in patients with DN, but not in patients with MCN. Urine EVs ELF3 can predict the decline of eGFR in patients with DN in the next few years [99]. It is a practical diagnostic tool that uEVs is applied to distinguish early IgA nephropathy (IgAN) and thin basement membrane nephropathy with microscopic hematuria in children and adults. Moon et al. discovered four various biomarkers that have different expressions in the uEV of these patients and the high levels of aminopeptidase N and vasoactive precursors

in the thin basement membrane nephropathy group, while α-1-Elevated levels of antitrypsin and ceruloplasmin were higher in the IgAN group [100].

#### **3.4 Extracellular vesicles as diagnostic markers for other kidney disease**

The biological processes involved in cytoskeleton-regulating and Ca(2+)-binding proteins are closely related to the pathogenic state of renal tubular epithelial cells in autosomal dominant polycystic kidney disease (ADPKD). Some study found by iTRAQ-based quantitative proteomics that this differential expression of proteins in urine EV of ADPKD demonstrates the possibility of using urine EV to monitor patient status [101, 102]. Fabry disease, also known as Anderson-Fabry disease, is the most common lysosome accumulation disease [103]. It is an X-linked congenital defect in the pathway of glycosphingolipid metabolism, which causes the accumulation of globotriaosylceramide (Gb3) in a variety of lysosome, leading to a series of clinical manifestations. Fabry nephropathy is kidney impairment, mainly manifested as hypertension, hematuria, mild proteinuria and fatty urine, and various renal tubular dysfunction, such as concentration and dilution function [104]. Some scholars observed the increased expression of miR-29a-3p and miR-200a-3p in uEVs of Fabry nephropathy patients may reveal an attempt by this organism to inhibit the progression of renal lesions leading to end-stage renal disease (ESRD).


#### *Extracellular Vesicles in Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.113200*


#### **Table 2.**

*Deregulated EVs and their contents as the molecular marker of nonneoplastic kidney disease.*

Meanwhile, the expression of miR-30b-5p increased within 10 years in uEVs of patients without renal dysfunction, which may play a defensive part in podocyte trauma and may play a vital role in Fabry nephropathy [105].

In summary, the "liquid biopsy" based on EVs currently shows good application prospects in the diagnosis of kidney disease. In future research, it is necessary to further explore the standardization of urine sample separation, storage, and processing [106], strengthen the large-scale cohort study of the diagnostic, prognostic and predictive value of EVs biomarkers in different kidney diseases, and promote the technical research on the traceability of urine EVs kidney cells (**Table 2**).

#### **4. Extracellular vesicles in the therapeutic role**

Renal pathophysiology is a multivariate procedure concerned with different kidney structures. Acute kidney injury (AKI) is a group of clinical syndromes, the manifestations include oliguria, anuria, edema, loss of appetite, etc., with the character of tubular necrosis and glomerular hyperfiltration. The maladaptive repair after AKI can easily lead to chronic kidney disease, also known as CKD. CKD is a growing and irretrievable damage of kidney function, the athological manifestation being fibrosis that could lead to ESRD. Currently, kidney disease therapies based on EVs can be divided into two categories: firstly, certain specific cells, such as stem cell-derived EVs, can be directly applied as therapeutic drugs due to their carrying bioactive molecules; Secondly, EVs can serve as delivery carriers for various types of drugs for the treatment of kidney diseases [119, 120]**.**

#### **4.1 Application of stem cell-derived EVs in the treatment of kidney disease**

Timmers et al. first confirmed that the conditioned medium of human mesenchymal stem cells had a protective effect on myocardium in 2007, they also confirmed that the main active substances involved this were at 100–220 nm by size analysis [121]. The team further isolated and identified the substance in 2010, identifying it as exosomes. Since then, more and more studies have confirmed that the EVs derived from mesenchymal stem cell (MSC-EVs) play a major part in tissue damage repair and immune regulation, and have developed their therapeutic potential in the matter of regenerative medicine. In recent years, stem cell-derived EVs have been studied in different animal kidney disease models in vivo, including AKI, diabetes nephropathy, hypertensive nephropathy, unilateral ureteral obstruction, and subtotal nephrectomy.

#### *4.1.1 Acute kidney injury (AKI)*

AKI is a general clinical condition, and there is no clear and effective treatment method. In recent years, stem cell EVs have been proven to play therapeutic roles in AKI, including anti-apoptotic, anti-inflammatory, and antioxidant stress. BRUNO et al. proved that in the glycerol-induced AKI mouse model, the bone marrow mesenchymal stem cells derived EVs accelerate the repair of damaged renal tubular cells, promote renal tubular cell proliferation, and protect cells from apoptosis [122]. Extracellular vesicles derived from bone marrow mesenchymal stem cells were also verified in the toxic AKI model induced by cisplatin and gentamicin [123, 124], which can improve renal function, reduce histological damage, and alleviate renal fibrosis. The bone marrow mesenchymal stem cells derived EVs have also achieved the same effect in renal ischemia–reperfusion (I/R) injury models [125, 126].

There are many mechanisms by which stem cell-derived EVs can improve AKI. Studies have found that the bone marrow mesenchymal stem cells derived EVs may carry specific mRNAs to activate the proliferation process of surviving renal tubular cells after injury, so that the impaired cells can re-enter the cell cycle.

Recently, CAO and his team sequenced human umbilical cord mesenchymal stem cells derived EVs and found that they are rich in miR-125b-5p and can inhibit G2/M cell cycle arrest and apoptosis of TECs by targeting p53, thereby promoting renal tubular repair and improving ischemic AKI [127]. Li et al. found that human urine-derived stem cells EVs can defend renal function of ischemia–reperfusion rats by carrying miR-146a-5p, which can target the 3′-untranslated coding region of

#### *Extracellular Vesicles in Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.113200*

IL-1 receptor-associated kinase (IRAK), thereby inhibiting NF-κ activation of the B signaling pathway and infiltration of inflammatory cells [128]. Lately, someone conducted EVs derived from human bone marrow combined with pulse-focused ultrasound therapy on a cisplatin-induced mouse model and found that the decreased expression of NLRP3 inflammasome and its downstream pro-inflammatory cytokine IL-1βand IL-18 decreased promoted renal repair after AKI [129]. These studies suggest that MSC-EVs exert the therapeutic effect of AKI through anti-inflammatory effects. The study also found that MSC-EVs can also inhibit mitochondrial damage in the IRI model through various pathways, thereby alleviating AKI. MSC-EVs derived from human placenta activate the Keap1-Nrf2 signaling pathway, stimulate mitochondrial antioxidant defense mechanisms to maintain the stability of TEC mitochondrial structure and regulate mitochondrial function, participate in TEC damage repair, and promote renal function recovery [130]. In addition, there are reports that MSCs-EVs can reduce mitochondrial damage and inflammation caused by AKI through the mitochondrial transcription factor A (TFAM) pathway [131]. EVs originating in umbilical stalk blood mesenchymal stem cells promote dedifferentiation and proliferation of renal tubular cells; extracellular vesicles derived from umbilical cord Wharton glue mesenchymal stem cells stimulate cell proliferation, reduce inflammation, and apoptosis through mitochondrial protection. 3D cultured placental mesenchymal stem cells derived EVs more effectively inhibit cell apoptosis, inflammation and improve renal function [132–134]; Human urinary mesenchymal stem cells derived EVa accelerate renal recovery, stimulate renal tubular cell proliferation, reduce the expression of inflammatory and injury markers, restore endogenous Klotho loss, and thus protect renal function [135, 136].

#### *4.1.2 Chronic kidney disease (CKD)*

It is a significant pathological manifestation in CKD progression of persistent kidney tubulointerstitial fibration. Scholars have found that hBMSC-EVs can inhibit TGF through their rich miR-294/miR-133-β1 mediates epithelial-mesenchymal transformation in CKD rats, thereby alleviating renal interstitial fibrosis (RIF) [137]. Moreover, particles derived from renal-derived MSCs can improve peritubular capillary sparsity in the kidney and delay the progression of renal injury by inhibiting tubulointerstitial fibrosis in mice UUO [138]. EVs produced by bone marrow mesenchymal stem cells derived EVs alleviate UUO renal fibrosis, partially by inhibiting the RhoA/ROCK pathway [139]. In a mouse CKD model induced by chronic cyclosporin, bone marrow derived EVs can improve the kidney function in the inflammatory microenvironment [140]. Extracellular vesicles rich in miR-196b-5p mediate crosstalk between proximal tubular epithelial cells and fibroblasts, which may be related to the STAT3/SOCO2 signaling pathway and mediate aldosterone-induced renal fibrosis with diabetes [141]. However, miR-221 in the EVs derived from podocyte reversed DN by inhibiting Wnt/β-Catenin signaling mediated proximal tubular cell damage [142]. EVs from human liver stem cell through miR29b reduce renal fibrosis by disturbing the β-Catenin pathway [143]. In a recent study, researchers synthesized a biological scaffold, which integrates MSC-EVs, extracellular matrix, poly (lactic acid Glycolic acid) copolymer, poly (Deoxyribonucleotide), etc. This biological scaffold achieved renal tissue remodeling in a partial nephrectomy mouse model by promoting cell proliferation, angiogenesis, and inhibiting fibrosis and inflammation [144]. Eirin et al. found that intrarenal injection of adipose-derived mesenchymal stem cells EVs improved pigs with metabolic syndrome and renal

artery stenosis disease by ameliorating renal inflammation and fibrosis. Further research confirmed that these renal protective effects were mediated by the carrier of anti-inflammatory cytokine IL-10 carried by MSC-EVs [145]. The secretion of EVs from mesenchymal stem cells stimulated by melatonin inhibits fibrosis in renal tissue by regulating cell apoptosis and proliferation of fibrosis related cells [146]. Stem cell-derived EVs also improve CKD related lesions. CHOI et al. found that renal mesenchymal stem cells expressing erythropoietin EVs improve renal anemia in mice with chronic kidney disease [147].

#### *4.1.3 Diabetic nephropathy (DKD)*

Diabetes nephropathy (DKD) is one of the elemental etiology of terminal-stage kidney disease [148]. For a long time, there is no specific drug for DKD, and the current treatment is limited to blood glucose control, blocking of renin-angiotensinaldosterone system, and changes in lifestyle [149]. MSC-EVs can protect cells from high glucose-induced damage by promoting regeneration through anti-apoptosis, anti-fibrosis, and autophagy. Jin et al. confirmed that miR-486 carried by EVs derived from fat MSCs can be transferred to podocyte, which leads to increased autophagy flux and decreased apoptosis by inhibiting the Smad1/mTOR signal pathway of podocyte in DKD mice [150], thereby improving podocyte injury in DKD mice. In addition, MSC-exos can reduce the overexpression of TGF-βto improve tubulointerstitial fibrosis in DN mice and regulate the expression of ICAM-1 for inhibiting inflammatory cell infiltration, thereby reducing diabetes kidney damage [151]. Xiang et al. found that umbilical cord mesenchymal stem cells derived EVs can reduce inflammatory factors (IL-6, TNF-α) in renal tubular cell expression, reduce inflammatory cell infiltration, interstitial fibrosis, and other pathological changes of diabetes nephropathy in renal tissue [152]. Mesenchymal stem cells derived EVs can regulate autophagy through the mTOR pathway, upregulate autophagy proteins such as LC3 and Beclin-1, and observe an increase in autophagic vesicles under electron microscopy, and the treatment of the MSC-derived EVs decreased the urine protein and serum creatinine in diabetes nephropathy mice. Renal biopsy showed that the renal pathological changes of diabetes nephropathy such as mesangium expansion and fibrosis were alleviated [153]. Jin et al. observed that the adipose stem cells derived exosomes could reverse the damage of renal function caused by high glucose environment and further found that miR-486 is a critical determiner in the reverse process, which can reduce the expression of Smad1, increase cell autophagy, and reduce podocyte apoptosis [150].

Recently, researchers injected two doses of MSC-EVs into patients with CKD phase III and IV in a single center, stochastic, placebo-contrast phase II/III clinical trial, and the results showed that the renal inflammation and function were effectively improved in the treatment group, providing worthwhile clinical evidence for the employ of MSC-EVs in the therapy of CKD. Unfortunately, this study did not investigate its mechanism and related renal pathological types [154]. Obviously, it has extremely clinical significance to further strengthening research.

#### **4.2 Application of EVs based drug targeted delivery in the treatment of kidney disease**

In 2010, Sun et al. wrapped curcumin with anti-inflammatory and antioxidant effects in EVs for the first time to treat sepsis in mice and achieved good therapeutic effects [155]. Since then, targeted drug development based on EV carriers has continued to heat up. Existing research suggests that EVs can successfully load various types of drugs such as nucleic acid drugs, protein drugs, and small molecule drugs for the treatment of kidney disease.

#### *4.2.1 EVs as nucleic acid drug carriers*

Various types of nucleic acids are the main ingredients of EVs; therefore, it has aroused widespread interest in using EVs as delivery carriers for nucleic acid drugs. Previous studies have shown that EVs can load and transport nucleic acids such as mRNA, miRNA, and small interfering RNA (siRNA) for disease treatment [156]. Among them, siRNA therapy has shown great potential in the treatment of human diseases. However, due to its instability and missing target effect, the clinical application of RNA interference technology has been limited. Recently, TANG et al. successfully developed a siRNA delivery system based on red blood cell-derived EVs, which utilizes renal injury molecule-1 targeted peptide A Kim-1-binding peptide (LTH) to modify red blood cell-derived extracellular vesicles (REVs) and successfully deliver siP65 and siSNAI1 to the injured renal tubules, effectively improving renal tubulointerstitial inflammation and fibrosis induced by ischemia-reperfusion and UUO, and blocking the chronic progression of AKI [157]. Meanwhile, they also applied muscle-targeted peptide-modified EVs with loaded miR-26a, significantly improving Sarcopenia in 5/6 nephrectomized CKD mice [158]. Combining EVs with a kidney-targeting peptide, Rabies Virus Glycoprotein (RVG), and loading miR-29a into it, can not only alleviate the myopenia in mice with UUO but also improve renal fibrosis through EVs-mediated communication between skeletal muscle and renal organs [159]. Therefore, the targeted therapy of nucleic acid drugs based on EVs can not only delay the progression of kidney disease but also improve its complications. A different strategy was applied and studies have shown that EVs from engineered MSCs overexpressing miR-let7c can shift miRNA to renal cells and inhibit RIF [160]. In addition, BM-MSC-derived EVs overexpressing miR-34a embellished by lentivirus inhibited TGF-β1 begotten epithelial-mesenchymal transition (EMT) in human renal tubular cells [161].

#### *4.2.2 EVs as protein drug carriers*

Protein deficiency and dysfunction are important causes of many diseases. Therefore, it is one of the methods for treating diseases by increasing the corresponding protein levels. However, protein drugs themselves have drawbacks, such as high molecular weight and poor stability, which limit their clinical application. The loading performance and modifiability of EVs have brought dawn to this field. Recently, Researchers constructed a cytokine IL-10 delivery system (IL-10 + EVs) using macrophage-derived EVs as a vector. The loading of EVs not only improved the stability of IL-10 but also demonstrated a unique ability to target kidney damage. Further mechanism research has found that IL-10 + EVs can promote mitochondrial autophagy in TECs by suppression the sensitization of the mTOR signaling pathway, significantly improving renal injury and chronic lesions induced by ischemia-reperfusion [162]. Kim et al. utilized a novel photogenetic engineering technique to load NF-κ inhibition protein srIκB into EVs, effectively improving the inflammatory response and cell apoptosis in the kidney after ischemia [163].

#### *4.2.3 EVs as small molecule drug carriers*

Research has found that the encapsulation and delivery of EVs can improve the targeting, cell uptake efficiency of small molecule drugs, also improve drug stability, and reduce toxic side effects [119, 164]. MSCs-EVs have been considered as another prospective acellular therapy for AKI. A Supermolecule hydrogel containing Arg-Gly-Asp (RGD) peptide has been manufactured to enhance the efficiency in the therapy of AKI. Data shows that RGD-EV hydrogel has a good rescue influence on renal function at the early stage of AKI, by dwindling tubular damage and facilitating cell proliferation through the combination of RGD and integrin [165]. Recently, researchers constructed M2 macrophage-derived EVs loaded with dexamethasone (DEX) and found that it not only targets damaged kidneys but also has effective anti-inflammatory and anti-fibrotic effects. Moreover, it significantly alleviates the adverse effects of DEX on blood glucose and the hypothalamic-pituitary renal gland axis in mice impact [166]. In the AKI model, EVs derived from MSCs ameliorative to over-express the octamer binding transcriptional factor 4 (OCT4) showed decreased expression of Snail, a trigger factor for epithelial-mesenchymal transition (EMT). Therefore, the administration of EV-ameliorated MSCs has achieved better renal tissue recovery, cell proliferation improvement, cell death elimination, and the initial fibrosis process block [132].

All in all, EVs have become a worthwhile carrier for the next generation of targeted drug delivery and have received widespread attention in recent years based on the advantages of good biocompatibility, low immunogenicity, and modifiability of EVs Cell therapy. Of course, there are still issues that need to be addressed: producing EVs that are repeatable, large-scale, high-throughput, and meeting the clinical application level; suitable parental cells selecting, culture systems such as cell factories, bioreactors, and hollowing fiber tubes establishing to achieve large-scale expansion production [167]; standardized EVs separation technology suitable for large-scale production building, a high-throughput detection platform developing, the quality of the EVs production process control and quality monitoring at the single particle level achieving, thereby further achieving standardized quality control and improving drug loading efficiency.

#### **5. Conclusion**

In the past 20 years, research on EVs has made rapid progress. More and more preclinical studies have shown that biomarkers and related treatment technologies based on EVs have great prospects in detecting and treating kidney disease, laying the foundation for their clinical application. Further strengthening the basic and clinical research on the role of EVs in making a diagnosis and giving treatment of kidney diseases, developing standardized, clinical-level EVs separation, purification, and quality control, and strengthening clinical queue research will offer technical support for the clinical transformation and application of electric vehicles.

*Extracellular Vesicles in Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.113200*

#### **Author details**

Chunyan Lv Basic Medical College, Chengdu University, Chengdu, China

\*Address all correspondence to: lllcccyyyy@126.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## BP-EVs: A Novel Source of EVs in the Nanocarrier Field

*Cristina Lorca, María Fernández-Rhodes, Jose Antonio Sánchez Milán, María Mulet, Julia Lisa, Xavier Gallart-Palau and Aida Serra*

#### **Abstract**

Extracellular vesicles (EVs) represent a complex mechanism of molecular exchange that has garnered significant attention in recent times. Nonetheless, identifying sustainable sources of biologically safe EVs remains challenging. This chapter delves into the utilization of fermented food industry by-products as a circular and secure reservoir of biocompatible EVs, dubbed as BP-EVs. BP-EVs demonstrate excellent oral bioavailability and biodistribution, with negligible cytotoxicity, and a preferential targeting capacity toward the central nervous system, liver, and skeletal tissues. The ease of editing BP-EVs is also depicted using the most common EV editing methods in this chapter. Globally, these groundbreaking findings are poised to unlock significant avenues for leveraging BP-EVs as an optimal source of biocompatible nanovesicles across a wide array of applications within the bioeconomy and biomedical fields. These applications primarily target molecule delivery into the central nervous system and skeletal tissue but are not limited to these two organism systems.

**Keywords:** extracellular vesicles, circular economy, nanocarriers, upcycling, compound delivery

#### **1. Introduction**

Extracellular vesicles (EVs) represent a sophisticated method of intercellular communication, characterized by resilient lipid membranes, specific molecular contents, and embedded signaling molecules. This mechanism has recently garnered tremendous interest [1]. These spherical vesicles are released and assimilated by almost all cell types across all domains of life [2]. Thanks to these unique attributes, EVs have been suggested to play crucial roles in both health and disease [3]. Furthermore, they have gained recognition as promising carriers for delivering drugs and bioactive compounds, sparking growing interest in their potential applications as nanocarriers [4].

While EVs possess favorable characteristics as nanocarriers—such as low immunogenicity, the capability to cross biological barriers, stable circulation, and organ targeting—there are significant challenges impeding their optimal use [4]. Issues related

to safety, scalability, and the identification of compatible physicochemical attributes present limitations [5]. These challenges arise partly because the primary sources of nanocarrier EVs in research are immortalized cell lines, which raise concerns about human safety and resource availability [6]. Similarly, the progress of EV mimetics, involving the laboratory-based creation of artificial EVs and liposomes as potential nanocarriers, is hindered by the limitations of current EV sources [7].

Here, we present food industry by-products derived EVs (BP-EVs) (European Patent PCT/EP2022/080507) [8], a novel nanocarriers platform based on the use of EVs obtained from fermented food industry by-products (FFBP). FFBP represents a safe, circular economy friendly, and inexpensive source of EVs. BP-EVs exhibit no cytotoxicity, as they highly resemble the daily consumed food-derived EVs, and display excellent oral and intravenous bioavailability as well as specific organ targeting capacity, with preferential targeting capacity toward the central nervous system (CNS), skeletal tissue, and liver. Additionally, BP-EVs can be easily edited by different methods. Collectively, we believe that BP-EVs will open substantial venues as an optimal source of biocompatible nanovesicles in manifold applications of the bioeconomy and biomedical fields.

#### **2. Extracellular vesicles**

Extracellular vesicles (EVs) are a heterogeneous group of spherical nanoparticles ranging in size from 30 to 5000 nm, naturally produced by cells, and delimited by a phospholipid (PL) bilayer membrane [9]. These rounded structures, which cannot replicate themselves, consist of lipids, proteins, and nucleic acids, and function as tiny vehicles for transporting, protecting, and delivering a wide diversity of cargoes [9]. Despite the crucial functions performed by these vesicles and their excellent properties as biomarkers for human diseases, EVs were long considered mere residues, often referred to as "cellular dust," and were overlooked for decades [10]. Nowadays, we know that EVs are produced by most, if not all, cell types and can be found in all biological fluids. According to the International Society for Extracellular Vesicles (ISEV), the term "EVs" should be reserved for those particles enclosed by a cellular membrane, naturally released by cells, and incapable of replication. Based on the ISEV's classification, EVs are further categorized into exosomes, microvesicles, and apoptotic bodies, depending on their biogenesis pathway [11] as illustrated in **Figure 1**.

EVs have been confirmed as a potent communication system between both same and different organisms, capable of serving as vehicles for signaling molecules between cells at close and distant locations, thus functioning as mediators at all four levels of communication (autocrine, direct, paracrine and endocrine) [9]. In contrast to other signaling particles, such as hormones, EVs not only have the ability to simultaneously package and protect a variety of messenger molecules, including hydrophilic and hydrophobic substances, but they also have complex compositions that provide direct information from the progenitor cell [12]. Additionally, it has been confirmed that some of these vesicles possess the ability to preferentially deliver their contents to specific cell types or tissues [13]. This groundbreaking discovery has opened up an entirely unexplored and multidisciplinary research field, offering promising potential for using these particles as tools to better understand and eventually intervene in cellular cross-talk. Furthermore, recent evidence points to EVs as the particles with the highest potential for use as editable and specifictarget nanocarriers in medicine in the near future [13].

#### **Figure 1.**

*EVs biogenesis pathways. 1. Potential EV components and cargoes are dispersed throughout the cytosol and membranes. 2. Specific elements and cargoes that will constitute future EVs are clustered and recruited. 3. The recruited elements contribute to membrane bending and invagination. 4. Membrane scission leads to the generation of microvesicles (A) or intraluminal vesicles (B). 5. Transport of the multivesicular body (MVB) to the plasma membrane. 6. Fusion of the MVB with the plasma membrane and release of the intraluminal vesicles (now exosomes) into the extracellular space.*

#### **2.1 EVs from different life kingdoms**

#### *2.1.1 Fungal EVs*

Although first observed in the early 1970s, fungal EVs (F-EVs) were not properly described until 2007 in the ubiquitous encapsulated yeast *Cryptococcus neoformans* [14]. Similar to bacterial and plant cells, fungal cells are protected by a thick cell wall composed of glycoproteins and complex carbohydrates, including the fungal-specific polysaccharide chitin [15]. Although the cell wall was initially thought to prevent vesicular transit due to its rigidity and absence of large pores, we now know that cell walls are flexible structures that can be easily rearranged for cellular division or during EV generation. In the last decades, it has been shown that F-EVs are fundamental for key biological functions, such as cell wall remodeling [16], biofilm matrix formation [17], and host-pathogen interactions [18]; and that these particles are clearly able to modify the behavior of recipient cells [19]. To date, F-EV production has been confirmed in several fungal species, including yeasts such as *Saccharomyces cerevisiae* [16], known for its role in beer production, or the opportunistic pathogen *Candida albicans* [20], which is also a common member of the human microbiota. F-EV production has also been proven in filamentous fungi such as *Aspergillus fumigatus* and *A. flavus* [21], which cause invasive aspergillosis.

F-EVs share a classification similar to that of mammalian EVs based on their biogenesis into exosome and microvesicle-like structures. While the full extent of EV production in fungi remains incompletely understood, there are similarities in pathways and genes with mammalian vesicles [22]. Notably, the production processes, such as the endosomal pathway, are highly evolutionarily conserved mechanisms. In addition to this, in fungi, periplasmic vesicles (PVs) have been observed between cells and their cell wall. However, it remains uncertain whether PVs are identical structures or fulfill similar roles to F-EVs found outside cells [23].

#### *2.1.2 Plant EVs*

While plant extracellular vesicles (P-EVs) were first observed in 1967, predating the discovery of mammalian EVs, they have received considerably less attention, and the pathways responsible for their generation remain largely unexplored. These pathways encompass various mechanisms, including fusion of multivesicular bodies (MVBs) with the plasma membrane, resulting in the production of exosomes, as well as budding from the plasma membrane, leading to the formation of microvesicles or apoptotic bodies. Additionally, there is the process of exocyst-positive organelle (EXPO)-mediated secretion, which generates EXPO-positive vesicles [24]. Furthermore, the mechanisms facilitating the passage of P-EVs through the cell wall to reach the extracellular space, along with the diverse secretion pathways, remain incompletely understood. Consequently, the classification of P-EVs is still in its early stages. P-EVs could potentially be categorized based on the presence of specific markers associated with the particular generation mechanism of P-EVs, giving rise to at least three subcategories [25]: TET-positive exosomes, characterized by the presence of TETRASPANIN (TET)-like proteins and originating from MVBs; EXPO-derived EVs, which originate from the EXPO organelle; and PEN1-positive EVs, composed of the penetration 1 protein, whose origin is as yet unknown but which appears to play a role in stress responses [25]. Similar to other biological kingdoms, P-EVs serve as pivotal regulators in essential processes, with their noteworthy contribution extending to cellular defense against pathogens [25].

#### *2.1.3 Bacterial EVs*

Bacterial extracellular vesicles (B-EVs) typically range from 20 to 400 nm in diameter and can be classified based on their structure, composition, and origin. The primary distinction lies in whether they are produced by Gram-negative or Gram-positive bacteria, as their differing structures are reflected in the vesicle's architecture [26].

#### *2.1.3.1 Gram-negative bacteria-derived EVs*

Gram-negative bacteria possess two double-layered membranes: the outer membrane, which is rich in lipopolysaccharide (LPS) on its outer leaflet, and the inner or cytoplasmic membrane, separated by a periplasmic space containing peptidoglycan [27]. Among the extracellular vesicles (EVs) produced by Gram-negative bacteria, outer membrane vesicles (OMVs) are predominant, typically ranging in size from 50 to 250 nm. As their name suggests, OMVs originate directly from the outer membrane, making them covered in LPS and enriched in outer membrane proteins. They also contain periplasmic components and exhibit specific lipid compositions [28]. Additionally, OMVs may contain cytoplasmic molecules, although the presence and sorting mechanisms for cargo selection have not been fully elucidated [27]. In contrast to conventional EVs, outer-inner membrane vesicles are encased by not one but two bilayer lipid membranes, corresponding to the outer and inner membranes of Gram-negative bacteria. Similar to OMVs, these particles feature a rich outer membrane with LPS and contain peptidoglycan in the periplasmic space [26].

#### *2.1.3.2 Gram-positive bacteria-derived EVs*

In contrast to Gram-negative bacteria, Gram-positive bacteria have a plasma membrane covered by a thick layer of peptidoglycan, which must be crossed by secreted

#### *BP-EVs: A Novel Source of EVs in the Nanocarrier Field DOI: http://dx.doi.org/10.5772/intechopen.113891*

vesicles to reach the extracellular space [29]. The term "cytoplasmic membrane vesicles" (CMVs) is used to describe B-EVs generated by Gram-positive bacteria. Since these B-EVs lack an outer membrane, B-EVs originating from dying cells are also considered CMVs. In both cases, the production of CMVs is triggered by endolysin, enabling these vesicles to bud into the extracellular space, crossing the peptidoglycan barrier [26]. Established functions for B-EVs include the transmission of virulence factors, nucleic acids, and defense factors to hosts, antibiotics, or bacteriophages [26]. Additionally, B-EVs play an intriguing role in ecosystems, such as their participation in the carbon cycle in marine environments [30].

#### **3. EVs as nanotransporters**

The efficacy of drug delivery faces constraints due to the instability of drugs within the body and their inability to reach the target tissue. This often necessitates the use of carriers for efficient drug delivery. Drug delivery systems encompass technologies that package and transport drugs within the body, overcoming pharmacokinetic challenges and natural bodily limitations to enhance their effectiveness [4]. In the quest for effective vectors for targeted drug delivery, various approaches have been explored in recent decades. A diverse range of synthetic vehicles, including liposomes, microspheres, and polymeric nanoparticles, has been employed to distribute drugs throughout the body. These systems can transport various types of drugs, including small molecules, proteins, nucleic acids, and antibodies [31]. However, a drawback of these synthetic carriers is their tendency to elicit a toxic immune response when recognized as foreign particles.

Extracellular vesicles (EVs) have emerged as promising nanotransporters in medicine due to their ability to deliver various bioactive molecules, such as proteins, lipids, and nucleic acids, to specific cells and tissues. EVs possess unique properties and offer appealing advantages over synthetic nanocarriers, including superior cellular uptake, high stability, biocompatibility, low immunogenicity, the capability to cross biological barriers, such as the BBB, cargo protection, and the potential for targeted delivery of bioactive molecules [32]. When used as drug nanocarriers, EVs can significantly enhance pharmacological efficacy while reducing drug toxicity. Other potential applications of EVs encompass gene therapy, immunotherapy, vaccine development, and tissue engineering [33]. For example, they can be utilized to deliver bioactive molecules that promote tissue repair [34]. Consequently, developing proper strategies for employing EVs as nanocarriers would enable their application in a wide range of medical scenarios [35].

#### **3.1 Endogenous EVs advantages over synthetic nanocarriers**

Synthetic nanoparticles have been extensively studied for their potential as carriers [36]. In general, synthetic nanoparticles are much simpler in structure compared to EVs, offering appealing characteristics such as ease of mass production in a costeffective and time-efficient manner, known composition, and simpler standardization protocols [37]. However, when applied to living organisms, these engineered particles present inherent challenges that have hindered their full realization as efficient nanocarriers [36]. One of the primary concerns lies in their lack of biocompatibility and potential toxicity. Engineered materials can trigger immune responses and cause cytotoxicity, limiting their application in biomedical settings [38]. Moreover,

synthetic nanoparticles often encounter accumulation issues, which can lead to potential long-term adverse effects [39]. Additionally, challenges arise regarding the cellular delivery capacity of synthetic carriers and their ability to traverse physiological barriers such as the blood-brain barrier (BBB), which restricts their access to vital target tissues, such as the brain [40].

In contrast, native EVs, which are naturally produced by cells, possess inherent biocompatibility, leading to reduced immunogenicity [41] and fewer toxicity concerns. As endogenous carriers, EVs are readily recognized by recipient cells and exhibit longer retention times in circulation compared to synthetic counterparts [42]. Their natural ability to interact with specific cell types facilitates targeted delivery [43], minimizing off-target effects and enhancing therapeutic efficacy. Furthermore, EVs have shown remarkable potential in crossing physiological barriers, including the BBB [44], which facilitates the delivery of cargo to the CNS for targeted brain therapies. Notably, this attribute makes them particularly attractive for treating neurological disorders and brain-related diseases. Nonetheless, EVs are not without challenges. Obtaining EVs in sufficient quantities remains one of the major obstacles [45]. Safety is another concern, as EVs derived from cancerous cells have been shown to be tumorigenic, raising concerns when choosing EVs derived from immortalized cell cultures, which currently represent one of the main sources [46]. Additionally, standardizing isolation protocols can be intricate due to the high heterogeneity of these particles [45]. Although naturally produced EVs emerge as a superior choice for biomedical nanocarriers, outperforming synthetic nanoparticles in terms of biocompatibility, targeted delivery, and overcoming physiological barriers, there are still critical challenges that need to be addressed to fully harness their biomedical potential [47].

#### **3.2 Challenges to implement EVs as nanocarriers**

Given the potential of EVs as nanocarriers, there is currently a significant and growing interest in researching EVs as pharmacological transporters for various substances, including chemotherapeutics [48]. However, numerous obstacles still exist that impede the clinical utilization of these particles [49]. The major challenges include obtaining a sufficient yield, isolating, storing, standardizing procedures, characterizing EVs, ensuring safety, loading them with cargo, and editing their targeting capabilities [50]. Currently, one of the most common sources for generating EVs for use as therapeutic nanocarriers is immortalized cell lines, including adherent stem or immune cells grown in 2D cultures [51]. However, this presents a significant limitation as cell cultures produce EVs in small quantities [52], and adapting these settings to grow in suspension can be nontrivial, hindering their transferability to the pharmaceutical and biotechnology industries. Another challenge arising from the use of cell lines as a source of EVs is safety concerns [53], especially when dealing with EVs derived from tumor cell lines that may have tumorigenic effects. To function as transporters, EVs need to be loaded with the desired cargo, and different strategies can be applied depending on the physicochemical properties of the cargo [53]. These various loading strategies will also be reviewed in this chapter. Finally, depending on the application, the EV surface may need to be modified to achieve the desired biodistribution and targeting properties while preserving other relevant traits, such as low immune recognition and stability [54]. Despite these challenges, the interest and potential of these vesicles are evident, reflected not only in the increasing academic research each year but also in the growing number of companies offering products for EV isolation, purification, characterization, and engineering, as well as conducting preclinical and clinical trials [55].

### **4. BP-EVs: a novel approach for safe and accessible nanocarriers**

BP-EVs, an asset protected under patent (PCT/EP2022/080507) since November 2021, are EVs enriched from FFBP derived from the production or processing of animal-based foods, such as kefir and other fermented dairy items, as well as plantbased foods like beer and wine, among others [8]. Generally, plant-based BP-derived BP-EVs are produced by yeast and bacteria, while dairy-based BP-derived BP-EVs are predominantly produced by mammalian EVs [8]. With diameters ranging from 30 to 950 nm, with 50% falling below 200 nm, signifying exosome enrichment, BP-EVs share similar lipidomic and proteomic attributes with food-derived EVs, demonstrating no cytotoxicity [8]. BP-EVs exhibit remarkable oral bioavailability, showing no disparity compared to intravenous administration, and exceptional biodistribution. Notably, these vesicles are rich in exosome markers and demonstrate distinctive *in vivo* targeting, particularly toward the CNS, liver, and skeletal tissues. The most efficient method for obtaining BP-EVs, as elaborated in the patent, involves an initial centrifugation phase to separate cells and insoluble debris from the BP solution, followed by a sequence of washing and filtration steps to eliminate soluble components from the source material and concentrate the vesicles [8].

When considering the challenges of utilizing EVs as nanocarriers, particularly regarding the acquisition of these particles in sufficient quantities, BP-EVs have successfully demonstrated that FFBPs provide a practical and cost-effective alternative for obtaining safe and biocompatible EVs. These EVs have the potential to be employed as targeted nanotransporters [8]. The industrially scalable strategy, coupled with the sister industrial technology of tangential filtration, for obtaining EVs from FFBPs offers an innovative and practical solution to overcome some of the major challenges in EV research and exploitation as nanocarriers [8].

#### **4.1 BP-EVs: transforming industrial waste into a valuable asset for human health**

Waste from food production is generated in large quantities, posing significant environmental problems and resulting in substantial handling expenses. During the development of BP-EVs, we embraced circular economy principles in line with the Sustainable Development Goals of the European Union. By harnessing FFBPs as a sustainable source for safe and biocompatible EVs, we created an innovative, upcycling, and environmentally conscious strategy. This strategy transforms otherwise discarded food industry waste into advanced nanocarriers, representing a valuable biomedical resource that addresses industrial waste challenges while unlocking research opportunities for EVs in biomedical applications. Altogether, BP-EVs are positioned as treasured assets for their potential as nanocarriers.

#### **4.2 Drug delivery and potential applications of BP-EVs**

BP-EVs possess a notable advantage due to their inherent low immunogenicity and cytotoxicity, which stems from their biological origin. Additionally, BP-EVs have demonstrated the capability to traverse biological barriers, potentially opening new avenues for treatments targeting organs that have historically been challenging to access, such as the brain [8]. The primary potential market for the application of BP-EVs lies in human health within the bioeconomy and biomedical fields. It is worth noting that the scope of potential applications of BP-EVs extends beyond the biomedical field. These versatile particles have the potential to be used in other industries,

impacting various sectors, including cosmetics and nutrition, among others. While researchers continue to unravel the intricate mechanisms underlying the physiology and behavior of these remarkable particles, these endeavors bring significant hope for the early application of these vesicles as targeted nanocarriers, thereby improving the effectiveness of multiple therapeutic interventions.

Given their natural capacity to reach the brain, BP-EVs could facilitate passage through the BBB and effectively transport drugs to target the CNS. It is worth noting that the market for CNS-focused treatments represents a global market estimated at \$612 million in 2022, and it is expected to grow at a compound annual growth rate (CAGR) of 8.9%, reaching \$938 million by 2027. An example of a potential application would be the treatment of glioblastoma multiforme, the most common and deadliest form of brain cancer in adults, which currently has a 5-year relative survival rate of only 5% [56]. However, the treatment of other CNS diseases, such as neurodegenerative diseases, psychiatric disorders, and various types of CNS cancers, could also be substantially improved through the use of these advanced nanocarriers.

Additionally, given their ability to preferentially accumulate in bone tissue, it is plausible to employ BP-EVs for delivering therapeutics specifically to the bone. This is particularly relevant because bone is a tissue that can be difficult to reach due to the avascular cartilage, often necessitating high doses for effective treatment, which can lead to elevated off-target toxicity [57]. An illustrative example of a potential application is in the treatment of osteoporosis, a metabolic disorder that compromises bone strength, resulting in an increased risk of fractures and contributing to morbidity and mortality for patients [58]. Notably, the market for osteoporosis treatment was valued at \$14 billion in 2022, with a CAGR of 3.8%. It is important to note that the liver targeting capacity of BP-EVs is not discussed in this section, as the presence of vesicles in this organ may be associated in part with detoxification and excretion [59]. Further research is needed to investigate the potential use of BP-EVs for targeting the liver in the treatment of liver diseases.

#### **5. Editing methods**

There are different editing methods that enable the engineering of BP-EVs and EVs in general to serve as nanocarriers, allowing the modification of their properties to achieve desired therapeutic effects. EVs can be tailored to carry molecules of interest either internally or externally, to reduce their clearance by natural systems (such as the immune, hepatic, and renal systems), to exhibit tropism to specific microenvironments (e.g., low pH), to target specific cell types or tissues, to enhance their intracellular cargo delivery capabilities, or to activate cells in specific ways, among other objectives. Editing methods can be categorized based on their objectives, which may involve modifying molecules transported by EVs or influencing EV targeting and interactions within their environment. This section provides a detailed overview of the most common methods for editing EVs.

#### **5.1 Cargo loading**

#### *5.1.1 Passive cargo loading*

The simplest and most convenient technique for cargo loading involves incubating desired cargoes with EVs. This method relies on a passive transport mechanism that takes advantage of the concentration gradient, allowing for passive diffusion.

#### **Figure 2.**

*Transmission electron microscopy images of a. unloaded BP-EVs and B. BP-EVs loaded by passive diffusion with the plant extract BacA from the nootropic plant Bacopa monieri. Ultrastructure of BP-EVs was not altered during the loading process.*

This facilitates the spontaneous incorporation of hydrophobic cargos into EVs or EV-secreting cells [60]. The loading efficiency of this strategy is generally low and influenced by the polarity of the cargo [61]. Other factors influencing the process include temperature, incubation time [35], pH, and cargo concentration [54]. Passive cargo loading typically does not affect the ultrastructural properties of EVs, as demonstrated in **Figure 2**.

Within passive cargo loading strategies, the pH gradient method is based on the fact that exosomes typically have an internal pH of 9. This method creates a pH gradient between the inside (at pH 9) and outside of the EVs (ideally at pH 4.5). The generated pH gradient can increase loading efficiency by up to three times [62].

Another variant of passive cargo loading is hypotonic dialysis, which involves mixing cargo and EVs in a dialysis membrane or tube to obtain cargo-loaded EVs as a result of the differential concentration gradient [63]. This method has been reported to increase loading efficiency by more than 11-fold compared to incubation [64].

#### *5.1.2 Active cargo loading*

#### *5.1.2.1 Physical methods*

In the freeze-thaw method, the mixture of EVs and cargoes is exposed to several cycles (typically from 5 to 10) of freezing temperatures below −70°C, followed by rapid thawing at room temperature [65]. This temperature shift disrupts membranes, allowing for the loading of small molecules. This simple technique can cause EVs to fuse and has also been successfully used to merge EVs with liposomes, creating EV-mimetic particles [66]. However, it has a moderate encapsulation efficiency, lower than that of other physical techniques such as electroporation or sonication [49].

Electroporation uses short and high-voltage electrical pulses to temporarily create small holes in membranes, increasing their permeability and allowing hydrophilic cargoes to diffuse into EVs [67]. This technique is one of the most commonly used for EV loading. The applied potential can vary significantly, ranging from 0.1 to 1000 kV, depending on the specific case. This method offers good loading efficiency and is easy to operate [55]. However, electroporation can potentially affect membrane integrity [67].

Sonication applies sound waves to generate transient pores or even break down and reconstitute EVs [68], allowing for the passive diffusion of cargo molecules within the membrane. This method offers high loading efficiency [69]; however, it may compromise the structural integrity of EVs and generate smaller particles compared to other techniques [70].

Extrusion involves forcing the vesicles through small pores, which can result in mechanical destruction and reformation of EVs. It employs an extruder device equipped with a heating block and polycarbonate filters with specific pore sizes, typically ranging from 100 to 400 nm. The cargo can enter the EVs by repeatedly pushing the mixture of vesicles and cargo through the filters [71]. This method provides relatively high packing efficiency and a uniform EV size distribution [72]. However, this process can damage the vesicles, including their membranes (which can alter their zeta potential) and proteins [64].

#### *5.1.2.2 Chemical methods*

Surfactant treatment involves the use of a reagent such as saponin or Triton, which creates pores in the membranes of EVs or cells, increasing their permeability. This allows cargo to enter more easily and significantly enhances the loading rate [73].

Transfection employs a specific vector to facilitate cargo loading into EVs. Among the agents used are reactives like calcium phosphate [74], diethylaminoethyl-dextran [75], polyethyleneimine [76], or cell-penetrating peptides [77]. Structures such as liposomes can also be employed to introduce larger cargoes, such as the CRISPR/Cas9 system, through merging with EVs [78]. It is important to note that some vectors may potentially damage EVs, cells, or cargoes [62].

#### **5.2 Surface editing**

#### *5.2.1 Chemical modification of EV surface*

There are different surface engineering strategies available to enhance the ability of EVs to successfully deliver their cargo to specific destinations. Chemical modification techniques use either covalent or non-covalent interactions to bind specific molecules to EV membranes without disrupting them [79]. Through covalent binding, various functional molecules such as small peptides, proteins, or polymers can be strongly attached to EV surfaces [49]. However, these techniques may require toxic chemicals and should be used with caution for editing therapeutic EVs [80]. Additionally, they often necessitate further purification steps [81]. Non-covalent binding can also be employed to modify EV membranes in a stable manner [82]. Another strategy for non-covalent binding is based on multivalent electrostatic interactions, which allow for the coating of EVs with a positive charge. This enhances their ability to target biological membranes, which typically have a negative charge, and facilitates their uptake by cells [83]. It is important to note that cationic nanomaterials can potentially cause cytotoxicity by disrupting membranes [84].

#### *5.2.2 Other methods for the modification of EV surface*

Other strategies for EV surface editing include hybridization with liposomes, resulting in the generation of larger, mimetic particles with mixed surface

properties. This technique has been used not only to incorporate larger cargos into mimetic-EVs [85], but also to enhance stability, prolong retention time, and improve cellular uptake [80].

#### **6. Conclusion**

The extensive details provided in this chapter position BP-EVs as ideal candidates for the next generation of nanocarriers in the field of biotechnology and biomedicine. Their potential applications are manifold, but primarily focused on the delivery of drugs into the CNS or poorly vascularized skeletal tissue. Thus, their impact on current and future developments in the field is poised to be transformative.

EVs have shown remarkable potential as advanced nanocarriers for delivering substances with a wide range of applications in the fields of biotechnology and biomedicine. BP-EVs exhibit a predominantly exosomal nature and possess exceptional biocompatibility. They also demonstrate the ability to traverse biological barriers and offer excellent oral bioavailability. BP-EVs can be easily edited by physical methods, although other strategies can also be applied to extensively modify their external and internal composition.

While BP-EVs offer numerous advantages as potential nanocarriers for drug delivery and other applications, they also have some drawbacks and challenges. Although BP-EVs are generally considered safe and biocompatible, safety concerns can arise if they are derived from specific food by-products that may contain allergens (i.e., BP-EVs derived from dairy FFBP). Thorough safety assessments are necessary to ensure their suitability for medical applications. Regarding particles diversity, like other types of EVs, BP-EVs can exhibit heterogeneity in terms of size, cargo content, and surface properties. This heterogeneity can complicate their characterization and standardization for specific applications. Finally, it is important to consider that like any novel therapeutic or drug delivery system, BP-EVs may face regulatory hurdles and require extensive testing and approvals before they can be used in clinical applications.

#### **Acknowledgements**

Support for this work was provided by the National Institute of Health/Instituto de Salud Carlos III-ISCIII, Spain (PI22/00443 to X.G.-P.), grant co-funded by the European Union; the Ministry of Science and Innovation-MCIN, Spain, and the National Research Council/Agencia Estatal de Investigación-AEI, Spain (PID2020- 114885RB-C21 to A.S.) funded by MCIN/AEI/ 10.13039/501100011033; the MCIN with funds from the European Union NextGenerationEU (PRTR-C17.I1) and from the Autonomous Community of Catalonia "Biotechnology Plan Applied to Health" [(EVBRAINTARGET-Y7340-ACPPCCOL007 to X.G.-P., A.S., & A.R.M.) coordinated by the Institute for Bioengineering of Catalonia (IBEC)]; the Diputació de Lleida, Spain (PIRS22/03 to X.G.-P.) and the Catalan Research Council-AGAUR (AGAUR 21SGR010065 to E.V.). X.G.-P. acknowledges a Miguel Servet program tenure track contract (CP21/00096) of the ISCIII, awarded on the 2021 call under the Health Strategy Action, co-funded by the European Union (FSE+). A.S. acknowledge Ramón y Cajal program tenure track contracts (RYC2021-030946-I) of the AEI, funded by

MCIN/AEI/10.13039/501100011033 and by "ESF Investing in your future"; M.F.-R.'s postdoctoral contract is funded by (PRTR-C17.I1) and (EVBRAINTARGET-Y7340- ACPPCCOL007). C.L.'s PhD is funded by the European Social Fund for the recruitment of predoctoral researchers (PEJD-2019-PRE/BIO- 16475); M.M.'s PhD is funded by the MCIN-AEI (PR2021-097934); J.A.S.M.'s PhD is funded by AGAUR (2023 FI-1 00054); J.A.S.M. contributions have also been supported by Diputació de Lleida, Spain "Ajuts al Talent en Investigació Biomèdica"; J.L.'s PhD is funded by AGAUR, Spain (2022 DI 100) and by the company Algèmica Barcelona S.L.; IRBLLEIDA, J.A.S.M., X.G.-P. and A.S. are co-funded by CERCA Program/Generalitat de Catalunya; X.G.-P. is member of the ExoPsyCog Consortium, funded by IKUR-Neurobiosciences – Basque Government.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Cristina Lorca1,2, María Fernández-Rhodes1,2, Jose Antonio Sánchez Milán1,2, María Mulet1,2, Julia Lisa1,2, Xavier Gallart-Palau1 \* and Aida Serra2 \*

1 +Pec Proteomics Research Group (+PPRG) - Neuroscience Area, Biomedical Research Institute of Lleida Dr. Pifarré Foundation (IRBLLEIDA), University Hospital Arnau de Vilanova (HUAV), Lleida, Catalonia, Spain

2 +Pec Proteomics Research Group (+PPRG), Department of Medical Basic Sciences, University of Lleida (UdL) - Biomedical Research Institute of Lleida Dr. Pifarré Foundation (IRBLLEIDA), Lleida, Catalonia, Spain

\*Address all correspondence to: xgallart@irblleida.cat and aida.serra@udl.cat

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Manash K. Paul*

This edited volume, *Extracellular Vesicles - Applications and Therapeutic Potential*, provides a comprehensive overview of the emerging data confirming the involvement of extracellular vesicles (EVs) in disease development. It also discusses the scientific advancements that have enabled the characterization and manipulation of EVs, leading to their utilization as tools in biomarker discovery, disease diagnosis, prognosis, and therapeutic interventions. The book examines the latest research efforts in the field by international authors and opens new possible research paths for further novel developments.

### *Tomasz Brzozowski, Physiology Series Editor*

Published in London, UK © 2024 IntechOpen © 123dartist / iStock

Extracellular Vesicles - Applications and Therapeutic Potential

IntechOpen Series

Physiology, Volume 24

Extracellular Vesicles

Applications and Therapeutic Potential

*Edited by Manash K. Paul*