The enteral route, including oral administration of drugs is the preferred delivery method to treat systemic diseases or local gastrointestinal (GI) pathologies due to its minimal invasiveness (pain-free), relatively low cost, and ability to self-administer [1]. However, these advantages are challenged by acidic conditions in the stomach and degrading conditions in the intestine, which affect the stability, absorption, and bioavailability of various therapeutic molecules, limiting the diversity of therapeutic compounds that can be prescribed orally [2]. Indeed, macromolecules such as proteins, peptides, or nucleic acids as free agents show only slight absorption when administered orally, as they are degraded by GI enzymes, have low stability at acidic pH and limited permeation through biological barriers [3]. Likewise, several hydrophilic and lipophilic drugs also have limitations for their oral intake since their absorption is greatly conditioned by their molecular weight, logP value, and gastrointestinal permeability, often requiring nanocarriers to generate a biological effect [4].

Nanocarriers are a colloidal transport system for drugs with a nanometric particle size (typically < 500 nm) [5]. In their oral administration, they allow the safe transport of active therapeutic molecules, improving pharmacokinetics, biodistribution, and stability, reducing toxicities, and offering a controlled release and delivery of drugs to specific sites [5]. Small extracellular vesicles (sEVs) are one of the most studied nanocarriers due to their biological origin that endows them with different and natural attributes that favor their biomedical use [6]. Defined as cell-derived nanostructures enclosed in a lipid membrane, they transport and protect an active molecular cargo composed of nucleic acids, proteins, and lipids ^{[7][8][9][10]}[7,8,9,10]. In the physiologic and pathologic processes, they play a role in the regulation of intercellular communication ^[11][12][11,12]. As nanocarriers, they have advantageous attributes related to their proven rapid internalization, low immunogenicity even at repeated doses, high stability in physiological conditions, and the capability of modifying their internal and superficial components to generate a controlled and specific release of endogenous or loaded therapeutic molecules [13]. Compared with synthetic nanocarriers, sEVs exhibit substantial benefits in targeting, safety, and pharmacokinetics, being considered the next-generation drug delivery platform [14].

Since its introduction as a drug delivery nanosystem, research has focused on understanding the pharmacokinetics and biodistribution of intravenously (i.v.) administered sEVs [15]. The valuable information collected on this subject contrasts with the need for more knowledge about the safety, stability, pharmacokinetics, and biodistribution of orally administered sEVs, the preferred route of administration for doctors and patients. Although there are few quality studies, the data to date show that, remarkably, sEVs can withstand the harsh environment of the GI tract and reach the intestine, where they accumulate heavily. It is from the intestinal lumen where sEVs penetrate the epithelium, which is the innermost layer lining the entire GI tract and selectively regulates transport from the lumen to the underlying tissue compartment [1]. Although most of these studies are limited to a single source of sEVs, cow’s milk, they remarkably demonstrate the efficacy of using sEVs as a nanocarrier system for a therapeutic payload to elicit a desired biological effect.

2. Challenges of Orally Administered sEVs

sEVs are non-replicative lipid-based vesicles classified by a hydrodynamic diameter inferior to 200 nanometers (nm) [16]. They are secreted by most known cells and can be found in every biological fluid (blood, urine, saliva, breast milk, among others) [13]. Besides, their biogenesis mechanism allows the transport and protection of bioactive molecules (nucleic acids, proteins, lipids, or metabolites) from a donor towards an acceptor cell, modifying their transcriptional profile, function, or phenotype [13]. Unique properties such as high relative stability, biocompatibility, permeability, low toxicity, and low immunogenicity determine its success as a novel cell-free therapeutic agent. Their versatile properties may be modified by different bioengineering allowing the insertion of targeting motifs for specific cellular lineages in their surface, a load of therapeutic drugs or macromolecules in their membrane or lumen, or even they can be modified to increase blood circulation time (Figure 1) [17]. Orally administered sEVs must survive the harsh degrading conditions of the digestive system as moisture, lubricants, mechanical forces, digestive enzymes, emulsifiers, pH neutralizers [2], commensal microbiota and their derivates [18] to successfully reach the intestine and deliver their therapeutic payload regionally or be absorbed intact for systemic distribution (Figure 2). The latter is the most challenging since once the sEVs penetrate the intestinal mucus layer, they must cross the intestinal epithelium to reach the lamina propria and cross the endothelium of blood vessels for systemic distribution [19]. When synthetic nanoparticles are orally ingested, most are degraded or eliminated, and a small fraction is effectively absorbed [20]. Nonetheless, it is currently unknown if the same proportion of orally administrated sEVs is degraded or eliminated since sEVs differs in their surface molecules, expressing receptors, peptides, saccharides, and lipids from a biological progenitor. To date, it has been described that different types of nanoparticles can cross the intestinal epithelium using different mechanisms, such as paracellular transport [1]. Paracellular transport consists of the diffusion of particles between cells through tight junctions that form the intestinal epithelial barrier. However, due to limited physical dimensions between cells in physiological conditions, only particles ranging between 0.5 and 20 nm should be considered for this mechanism in a relevant proportion [21]. Conversely, a pro-inflammatory context in the intestine disrupts the epithelial barrier, allowing the passing of larger particles, as demonstrated by Tulkens et al. [22]. Thus, based on the reported results, the paracellular transport of an oral administration of sEVs (>200 nm) should only be considered in inflammatory diseases of the intestine or treatments that disrupt the epithelial barrier as side effects. The second mechanism described by which nanoparticles cross the intestinal layers and reach systemic circulation is transcellular transport by epithelial cells (mainly enterocytes that constitute 90–95% of the cells in the GI tract) and M-cells (specialized phagocytic cells that represent 1% of the intestinal epithelium) [1]. Transcellular transport consists of the endocytosis of particles in the apical face, the intracellular transit, and posterior exocytosis in the basal face [20]. The main challenge for this route of absorption is avoiding the transport and fusion of the intracellular vesicles with lysosomes enriched in degradative enzymes. A unidirectional transport of bacterial-derived extracellular vesicles (bEVs) was demonstrated by epithelial cells in vitro, where a small proportion was “re-secreted” towards the basal face, partially supporting the proof of concept of orally ingested sEVs potential absorption and lysosome avoiding [23]. A better understanding of the molecular mechanism of uptake by these intestinal cells could contribute to an engineered increased interaction with sEVs. Macropinocytosis, caveolin, and clathrin-dependent endocytosis are the principal mechanisms described for particle uptake in enterocytes. In M-cells, the most studied are the uptake by phagocytosis and receptor-mediated endocytosis [20].

3. Biodistribution, Stability, and Safety of Oral Delivery of Native and Drug Loaded sEVs

Murine studies identifying the biodistribution pattern of orally administered sEVs are few and focus mainly on cow’s milk-derived sEVs, although some studies using plant-derived exosomes-like particles. These studies show that sEVs/exosomes-like particles manage to withstand the hostile environment of the gastrointestinal tract, associated with their transit through acidic conditions in the stomach and degradative conditions in the gut, in various murine models ^[24][25][26][24,25,26]. Cow’s milk-derived sEVs cross the upper gastrointestinal tract and reach the intestine in relatively short times (1–6 h) ^[27][28][27,28]. The absorption of sEVs seems to occur in the gut through mechanisms that are not well understood, but that facilitates the entry of sEVs into the systemic circulation and their distribution in other organs, essentially localized in the abdominal cavity ^{[27][28][29][30][31]}[27,28,29,30,31]. Unlike the “trapping” of sEVs in the organs of the mononuclear phagocytic system (liver, spleen, and lung) after systemic injection of sEVs [17], oral ingestion allows a considerable accumulation of sEVs in the intestine ^{[27][28][30][32][33][34]}[27,28,30,32,33,34]. In the other organs of the body, the accumulation of sEVs is less but notably shows a homogeneous distribution among them. Figure 3 shows the biodistribution pattern in mice after oral and systemic administration.