4.2.2. Exosomes

As it was described previously, exosomes are also considered NPs that are originate from cells and transmit information between tissue microenvironments and can influence the function and differentiation of target cells. They are secreted by all cell types, including immune cells (such as B and T cells, DC cells), cancer cells, stem cells and endothelial cells; in addition, exosomes and are present in the human proximal fluids such as blood, urine and breast milk. In general, exosomes are constitutively released by tumour cells or in a regulated manner by immune cells (i.e., B cells). Exosomes biogenesis is produced by internal germination of late endosomes and produce multivessel bodies that fuse with the plasma membrane and are released into the microenvironment [203]. Structurally exosomes are composed of a lipid bilayer expressing ligands and surface receptors, which contains a hydrophilic nucleus. In the nucleus, there is a high rich content from RNA, proteins and other components that come from the source cells. Thus, exosomes carry information in the form of mRNA and miRNA that will correspond to the normal or pathogenic processes of the cells from which they come [202], such as the elimination of unwanted proteins, the presentation of antigens, genetic exchange, immune responses, angiogenesis, inflammation, tumour metastasis and the spread of pathogens or oncogenes [204,205].

Regarding the content on membrane proteins, exosomes contain very interested ligands such as integrines, tetraspanines, and receptors in native conformations, including the co-receptors needed for in vivo signalling [206], among others. The adhesion molecules (i.e., integrines, selectins, etc.) contained in exosomes are known to be expressed on the cells from which they originate; for example, DC-derived exosomes express CD80 and CD86 [207], B-cell derivatives express CD19 [208]. From the proteome point of view, exosome proteomes have been analysed in several studies because, although they constitute only a small part of the total plasma proteome, they are enriched in altered proteins under different pathological conditions and might therefore be considered diagnostic markers [203,209]. Therefore, in lung cancer, colorectal cancer and diabetes, specific expression patterns of serum miRNA have been identified as biomarkers for the detection of these diseases in human physiological proximal fluids [210].

Bearing in mind all these inherent properties of exosomes, they seem ideal biological nano-carriers. Moreover, due to origin, exosomes present biocompatible, such as immune tolerance, which allows them to avoid elimination through adaptive response [206]. They also escape phagocytosis, because they could fuse with cell membranes and avoid lysosome envelopes, and are more stable in the blood [211]. These exosomes can be modified either endogenously at the cellular level or exogenously in cell cultures. Endogenous modification is based on modifying exosome components, such as proteins, at the level of production of the cell from which they originate [206]. Exogenous modifications are important in understanding the extent to which the contents and function of exosomes of different biological origins could be manipulated. These exogenous modifications provide information on how exosomes target and interact with tissue-specific microenvironments in vivo, which would allow for new applications in diagnosis and therapy. The structure of exosomes allows for three types of exogenous modifications: 1.-modifying exosome surface molecules to allow specific targeting and monitoring of exosomes, 2.-loading hydrophobic therapies onto the membrane, and 3.-loading hydrophilic drugs or therapeutic cargo into the nucleus. These modifications facilitate the use of exosomes as nanomedicine approaches in immune-onco-therapy [202].

One of the described applications, it is based on the pre-existing surface receptors themselves, which could be also adapted for use in therapeutic applications. In one recent study, it was shown that mesenchymal stem cell (MSC) exosomes can transmit membrane and ligand receptors to attenuate the function of self-reactive CD4 T cells isolated from mice with experimental autoimmune encephalitis. The ligand PD-L1, TGF-β and galectin-1 were transferred to the T cells and decreased secretion of IL-17 and IFN-γ by 50% by the T cells after treatment with the exosomes [212].

MSCs produce a greater number of exosomes than other cell types and this production is not compromised in terms of quantity or quality thanks to the immortalization of these cells to generate permanent cell lines that guarantee the reproducible and sustainable production of exosomes from MSCs [213]. These exosomes, in addition to surface markers CD9 and CD81, express adhesion molecules that are also expressed on the MSC membrane, such as CD29, CD44 and CD73. MSCs recruit and regulate T cells, either by cell to cell contact or paracrine. Cytokine secretion and ligand–receptor inhibitory interactions are believed to be an important function of MSCs [203]. Exosomes derived from these cells act as mediators that induce peripheral tolerance of self-reactive cells by carrying MSC-specific tolerance molecules such as PD-L1, Gal-1 and TGF- β. These exosomes have been shown to inhibit the proliferation of self-reactive lymphocytes and promote the secretion of anti-inflammatory cytokines such as IL-10 and TGF-β, among others [214]. Therefore, MSC-derived exosomes are mediators that induce peripheral tolerance and modulate immune responses, and could therefore be used in the treatment of auto-immune diseases [81]. These exosomes have also been used in graft-versus-host disease (GVDH), which has been shown to delay its appearance in mouse models and to increase Tregs cells [215].

Another relevant study, described by Bo Yu et al., was based on the idea that MSCs have different effects on tumour growth, as they may favor tumour initiation or inhibit the progression of established tumours. Thus, exosomes released by MSCs also have varied effects [203]. One effect is the increased incidence and growth of tumours induced by certain cell lines, which indicates that MSC-derived exosomes promote tumour progression as do MSCs in vivo [216]. Another study showed that MSC-derived exosomes suppress tumour progression and angiogenesis by negatively regulating VEGF expression in in vitro and in vivo tumours. The miRNA-16 is believed to be responsible for the anti-angiogenic effect, as MSC-derived exosomes are enriched in this miRNA which targets VEGF [217].

Another exosome modification strategy is using in RNA administration mediated by in exosome. Exosomes have the inherent ability to transmit mRNA and miRNA between cells [218]. Among other methods, electroporation facilitates the loading of exogenous siRNA into exosomes. The efficiency of electroporation depends on the concentration of exosomes and the applied voltage. Using this method, Wahlgren et al. found that plasma-derived exosomes loaded with MAPK-1 siRNAs suppressed the levels of MAPK-1 mRNAs in monocytes and lymphocytes [219].

Momen-Heravi et al. charged B-cell exosomes with the miRNA-155 inhibitor by electroporation. When cells were stimulated with LPS, miRNA-155 increased their production of TNF-α. Exosomes loaded with the miRNA-155 inhibitor were able to reduce the production of TNF-α by LPS-treated macrophages. This strategy allows reducing the negative inflammatory component in different disease processes. The importance of choosing the correct exosome subpopulations for the therapeutic application of interest is highlighted. In this case, the isolation of exosomes was performed using anti-CD36 immunomagnetic microspheres [220]. This type of isolation and enrichment is very useful to separate exosome subpopulations for biomarker studies [221].

Exosomes may also be used as immunotolerant nano-carriers for hydrophilic chemotherapeutic load, such as doxorubicin [202]. Tian et al. designed immunotolerant immature dendritic cell (iDC) exosomes that expressed a chimeric Lamp2b fusion protein and the integrin-specific iRGD peptide α-V. Electroporation was used to load doxorubicin into the exosomes and the encapsulation efficiency was 20%. The iDC exosomes were able to target and accumulate in breast tumours expressing α-V integrin in mice and inhibit their growth. In contrast, free doxorubicin or untargeted doxorubicin exosomes had no effect on tumour growth. In addition, tumour growth inhibition with iDC exosomes did not result in observable toxicity and therefore the use of iDC exosomes as biocompatible nanoporters was validated [222].

Another recent study, it has also been conducted with modified exosomes to treat NSCLC. Here, exosomes loaded with paclitaxel were modified with PEG and AA (ligand) to increase blood circulation time and attack lung metastases. In this way, the drug selectively targets the target cancer cells and increases the survival rate of patients with lung cancer [223].

B-lymphocyte-derived exosomes have also been shown to have immunomodulatory function, triggering specific CD4+ T-cell responses and thus performing a role as transporters of MHC class II peptide complexes between immune cells [224]. In the case of DC-derived exosomes, they have been

shown to express MHC class I, class II and co-stimulatory T-cell molecules and to suppress the growth of T-cell-dependent murine tumours [225]. For this reason, these exosomes may begin to be considered as cell-free "vaccines" in cancer immunotherapy [226].

Attempts are still being made to determine the endogenous function of the various exosome subtypes and subpopulations. This is even more important in the design of nano-carriers with tumour exosome subtypes that may have a pathogenic burden, which must be neutralized so that it does not impede the therapeutic efficacy of these exosomes [206]. In contrast, tumour antigen retention may also be beneficial for the development of tumour exosome-based immunotherapies [227]. The various therapeutic applications will require the selection of the optimal exosome subtype for conversion to nano-carriers and this requires an understanding of the normal function of exosomes and the ability to predict the function of modified exosomes.
