**4. EVs as drug delivery vehicles**

Today's medicine is increasingly focused on personalized treatment methods, on targeted therapies that act at the molecular level. One of the concepts aimed at these aspects is that of theranostics, which aims at diagnosis, treatment, and concomitant follow-up of the response by using very specific drug delivery systems [76]. In this sense, EVs are an extremely useful tool for passive diagnosis (especially in neoplastic pathologies, through the ability to identify the tumor type based on the miRNA, mRNA, and mitochondrial RNA profile of EVs) and active (by associating EVs with advanced imaging methods). Thus, numerous platforms based on EVs technology have been developed for theranostic purposes, namely, transition metal-labeled exosomes, nanoparticle-loaded exosomes, bioluminescently labeled exosomes, nanocluster loaded exosomes, metabolically labeled exosomes. The main applications at present are those in the oncology field, but the lack of uniformity of clear classifications, increased immunogenicity of EVs, and the limited number of drugs that can be loaded at EVs highlight the need for future studies for widespread application of these diagnostic and therapeutic tools [77].

EVs are used as transporters for a variety of substances, ranging from small molecules, small interfering RNA, mRNA, and microRNAs to drugs with suboptimal pharmaceutical effects, carrying active constituents through biological barriers [4, 11].

Exosomal transporters present advantages, as they can travel efficiently between cells, smoothly passing their cargo along the cell membrane, keeping it biologically active, and crossing hard-to-penetrate barriers, such as the blood-brain barrier. Important issues regarding exosome-based drug delivery vehicles are the precise method of exosome loading, without altering the biological characteristics, and the scalable repeatable production of exosome categories [18, 78].

Exosomes tend to have special homing targets, influenced by their cell of origin [18]. Their membrane can be modified, to amplify the targeting of specific cells [18, 79].

The content of EVs can be loaded exogenously (integration of small proteins, RNA, or other molecules) or endogenously (assuring that cells possess the ways to integrate small molecules, proteins or RNA into EVs during their formation) [4, 80]. Exogenous changes of EVs can be done after their collection, incorporating the cargo into EVs through different methods: coincubation (with no modification of vesicle size distribution or integrity, electroporation, and sonication) [79, 80]. The endogenous loading can be obtained through artificial adjustment of the parental cell to overexpress certain proteins or RNA, that can be integrated into secreted EVs afterward [81].

Human MSCs, multipotent adult progenitors, could be an adequate source of exosomes for drug delivery. Their transplantation has been investigated in numerous trials and proved to be safe, MSCs also produce immunologically inert exosomes [18]. As a delivery system, studies have shown that EV derived from MSCs can transfer therapeutic drugs to diseased cells [19]. Their efficiency is based on the adhesion proteins on their surface (like integrins, extracellular matrix proteins, tetraspanins), which facilitate the penetration of the cellular membrane and the accumulation of EVs in the diseased cells [82]. Other characteristics that make EVs an ideal candidate for a drug delivery system are their decreased toxicity and immunogenicity, as well as their potential to cross the blood-brain barrier [83, 84]. A study conducted by S. Kamerkar *et al*. has shown that EVs produced by MSCs can transfer small interfering RNA targeting the oncogenic KRas (G12D) mutants to pancreatic cancer cells, increasing cells apoptosis and decreasing the risk of metastatic disease [85].

Exosomes have significant immune properties, modulating immunological responses and facilitating antigen presentation [11, 86]. Exosomes derived from dendritic cells can conduct MHC class I/peptide complexes to other dendritic cells for *in vivo* activation of cytotoxic T lymphocytes and promote T cell-dependent antitumor responses *in vivo* [86, 87]. Dendritic cell exosomes have been previously loaded with antigenic peptides, to activate T cell proliferation, with possible use as vaccines against infectious or neoplastic diseases. Due to the immunogenic nature of dendritic cell exosomes, their use as drug delivery vehicles is not ideal. A more suitable choice would be human ESCs-derived mesenchymal cells [86, 88, 89].

Clinical trials with therapies based on EVs are studied in malignancies, such as melanoma, non-small cell lung cancer, colon cancer, metastatic pancreatic cancer, bronchopulmonary dysplasia, malignant ascites, and pleural effusion, but also chronic kidney disease, type 1 diabetes, insulin resistance and chronic inflammation polycystic ovary syndrome, ulcers, and acute ischemic stroke [4]. Exosomes were shown to transport curcumin and chemotherapeutics, such as doxorubicin and paclitaxel [19, 90].

#### *Extracellular Vesicles as Intercellular Communication Vehicles in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.101530*

The implication of MSCs was also evaluated in patients with anthracyclineinduced cardiomyopathy. Mitochondrial transfer, mediated by large EVs, diminished injury determined by doxorubicin, in patient-specific induced pluripotent SC-derived cardiomyocytes. MSCs could ameliorate cardiac function in anthracycline-induced cardiomyopathy, regardless of regeneration effects [91].

Liposomes possess many favorable characteristics as drug delivery vehicles, being used in the transportation of anti-cancer drugs, anti-fungal medication, and analgesics [92–98]. Liposomes have a phospholipid membrane that helps with the incorporation of hydrophilic or hydrophobic drugs, and they can also deliver the carried drugs to the targeted points through plasma membrane breaching. To diminish the recognition by opsonins and their clearance, liposomes can be covered with polymers (PEG). Their membranes can be adapted, to present ligands or antibody elements, which can interact with specific cells and amplify the targeted drug delivery. Liposomes, with easy-to-control properties, can be loaded with drugs, DNA, diagnostic instruments, enzymes, or peptides. Drugs included in liposomes have attenuated toxicity and do not provoke unwanted toxic reactions [99]. Liposomal drugs have various routes of administration, such as parenteral, oral, topical, and even through aerosols [99].

Synthetic liposomes, although very useful, are overcome by EVs (naturally derived liposomes), which have lower toxicity [19]. Exosomes are considered superior drug delivery vehicles, as an alternative to liposomes. In contrast to the latter, exosomes are usually adequately tolerated by the human body and do not present intrinsic toxicity. They can deliver their content through the plasmatic membrane and protect against its early transformation and elimination [19]. Since exosomes can be found in a variety of biological fluids, such as blood, urine, breast milk the delivered drugs will be well tolerated, less toxic, and with a longer circulating half-life [19, 100].
