Exosomes as bio-inspired nanocarriers for RNA delivery: preparation and applications

Exosomes are extracellular membranous vesicles (EMVs) possessing the special compositions of DNA, coding RNAs, non-coding RNAs, lipids, and proteins [1‐4]. These endocytic membranes-derived vesicles can deliver various signals to target cells, thereby mediating new cell-to-cell communication mechanisms [3]. In -molecular-scale studies, exosomes possess phospholipases and lipid-related proteins, proteins participating in the biosynthesis of multi-vesicular bodies (MVBs) (TSG101, Alix), proteins involved in transport and fusion of membranes (flotillin, GTPases, annexins), heat shock proteins (i.e., Hsp90, Hsc70), and tetraspanins (CD82, CD81, CD63, CD9) [5]. Until now, a total of 4500 various proteins have been detected with exosomes, often through mass spectrometry (MS) [6], likely serving as cargos for intercellular communications. In addition to the above-mentioned proteins, the membranous vesicles are enriched in special raft-related lipids, like phosphoglycerides with saturated fatty-acyl and long chains, sphingolipids, ceramide, and cholesterol (mostly B lymphocytes-derived exosomes) [7, 8].

The previous reports have proven that the exosomes derived from various fluids in the body [9, 10] and various cell lines [11] possess RNA molecules, especially mRNAs and miRNAs, which can be translated into proteins or regulate protein expression in the recipient cells, respectively [12‐15]. Current deep-sequencing studies have revealed that the membranous vesicles possess a variety of RNA cargoes comprising full-length RNAs with 25–250 nucleotides in lengths like tRNA and miRNA, as well as fragments of long RNA like rRNA, mRNA, even though mRNA molecules are presented in full-length types [16‐21].

Exosomes play multi-pronged roles in the tissues or cells of origin to facilitate movement of pathogens such as viruses [22] and prions from one cell to another cell [23], inducing tumorigenesis [24], coagulation [25], inflammation [26], angiogenesis [4], programmed cell death [27], antigen presentation [3, 28], improvement of the immune responses, and deletion of debris molecules [29]. Interestingly, exosomes have been proved to enable signaling and cell-to-cell communications and deliver macromolecular messages such as proteins and RNAs [1]. Interest towards these membranous vesicles ranging from in vivo roles to further uses like their application in therapeutics, biomarker development, and diagnostics (according to the assay of their protein and RNA extent) has substantially increased in the past decades [22, 26, 29].

Owing to their potential application in translational research, these vesicles have fascinated a lot of fieldwork attention on their functions in various diseases and health [8, 26, 30‐32]. These EMVs, especially mesenchymal stem cells (MSCs)-derived exosomes play an important role in kidney diseases, wound healing, liver diseases, neurodegenerative and autoimmune disorders, diabetes, spinal cord injury, and other diseases. Moreover, exosomes have attracted a great deal of attention as novel bio-carriers for gene and drug delivery because of the advantages such as less toxicity, the ability to cross biological barriers, and evade mononuclear phagocytic system (MPS) compared to synthetic nanoparticles [33]. Thus, highly efficient procedures for loading exosomes with drugs or biomolecules are prerequisites to achieving breakthroughs in the future [17, 22]. Although several studies have elucidated the detection, isolation, and characterization of exosomes along with their uses in drug delivery [1, 2, 4, 25, 26], the preparation and application of exosomes as the bio-inspired nanocarriers in RNA-based therapeutics have not been explored. In this study, the cutting-edge advances in the latest studies for the isolation and production of exosomes along with their clinical application in the delivery of RNA molecules, have been discussed. We revealed that exosomes with their unique properties and safety could open a new avenue for researchers in the development of efficient mRNA delivery system. Therefore, the establishment of large-scale RNA-loaded exosomes for clinical applications should be given further attention.

Exosome biogenesis starts with the generation of early endosome through inward budding of the cellular membrane followed by second budding of the endosomal membrane. The second budding leads to the production of late endosome. These endosomes including intraluminal vesicles (ILV), are known as multivesicular bodies. These bodies either follow the endocytic pathway for the exosome generation or fuse with lysosome to degradation [25]. During inward budding of the endosomal membrane, proteins, miRNA, mRNA, and DNA fragments are incorporated into the forming vesicles by very specific protein complex. Finally, by fusion of the ILV with plasma membrane the exosomes are released into extracellular space [26].

The term "exosome" was first presented in the 1980s for vesicles released by a cell line having ectoenzyme activities [34]. In fact, it refers to the vesicles secreted during the differentiation of reticulocytes [35], whereas, the EMVs are reported to be secreted through dendritic cells (DCs) and B lymphocytes via a similar path [24]. In the latter years, several cell kinds of nonhematopoietic/hematopoietic origin, like oligodendrocytes, intestinal epithelial cells, Schwann cells, neurons, platelets, cytotoxic T cells, and mast cells have been documented to release the EMVs [29, 36]. Moreover, the vesicles have been found in the various fluids of the body, such as bile [37], cerebrospinal fluid [28], amniotic fluid [38], ascites fluid [39], breast milk [40], urine [41], semen [42], saliva [43], and blood [44].

With the rapid progress in technology and science, many methods have been developed for exosome isolation in good purity and quantity. Each method exerts a specific characteristic of exosomes, like their surface proteins, size, shape, and density, to facilitate their isolation [22]. Some of the isolation procedures along with their mechanism of isolation and drawbacks and profits of the method, are summarized in Table 1 and Fig. 1. There are no efficient methods for exosome isolation, and the existing methods either encounter the problem of contamination with proteins and non-exosomal vesicles or are very expensive due to requiring special equipment [52]. The affinity-based methods are very pure; however, their high costs and low yields have prevented its extensive use in exosome purification. Researchers believe that a combination of two methods can boost the efficiency and purity of the isolated exosomes [53]. The field flow fractionation methods have received special attention due to their scalability and isolation of specific subpopulations of exosomes [54]. However, they require further improvement for use in clinical applications.

Procedures for the purification and preparation of extracellular vesicles in large amounts include exosome precipitation [55], ultrafiltration [56], sucrose density gradient ultracentrifugation, and differential ultracentrifugation [30]. In a study in which large-scale mRNA-loaded exosomes were prepared, two differential ultracentrifugation and density gradient ultracentrifugation methods were used to purify the exosomes. It was observed that the rate of mRNA recovery is similar in both procedures. At the same time, the chemicals used in the density gradient filtration method may not be removed entirely. Furthermore, more RNAs were concentrated in the exosome fractions when differential ultracentrifugation was used. Hence, differential ultracentrifugation was chosen as a preferable method to purify the exosomes [57].

Exosomes are natural intercellular transporters of RNAs that are responsible for different roles in vivo. Because of their intrinsic features, these EMVs can present a more effective procedure for transfecting RNAs in gene therapy than classic delivery vehicles of nucleic acids [63]. Despite many benefits, the exosome-based RNA delivery is restricted since generating adequate amounts of RNA-loaded exosomes for in vivo application is technically challenging [45, 64]. First, only a few cell sources have been observed to release an adequate quantity of exosomes that are needed for clinical translation [27, 65, 66]. Second, to produce a clinical dose of the EMVs, a great deal of cell culture should be incubated for multiple days, followed by RNA isolating and loading before the ultimate gene-enriched vesicles could be achieved. Although post-insertion of siRNA and shRNA plasmid into exosomes through electroporation technique has shown more remedial efficacies than synthetic nanocarriers in repressing oncogenic targets in the pancreatic cancer preclinical models [66], loading of large RNAs like mRNAs into exosomes remains challenging technically [67]. Herein, our group discussed several approaches to incorporate RNA molecules into exosomes for therapeutic applications and transcriptional manipulations. These approaches include guidance of signature sequences, electroporation, transfecting donor cell, transfection with specific reagents, producing hybrid exosome-liposome and cellular nanoporation [4, 63, 68]. In some loading methods such as electroporation, liposome-exosome hybrids, and transfection with specific reagents, exosomes are first purified from the appropriate sources, and RNA molecules are then loaded into its structure. However, in other techniques such as the guidance of signature sequences and transfecting donor cells, the cells are employed to load nucleic acids by engineering the exosome-producing cells. Therefore, the exosomes secreted from these cells will contain the desired RNA molecules.

Exosomes, a subset of extracellular vesicles, offer several benefits as drugs/biomolecules carriers over other drug nanocarriers, including the capability of loading different cargos and crossing impermeable biological barriers, facilitation of the cellular internalization of the cargos via endocytosis or membrane fusion, long half-life and circulation time, low immunogenicity, and small size [4]. These bio-inspired nanocarriers can deliver many cargos like proteins, small molecules, and nucleic acids such as DNA, siRNA, miRNA, and mRNA [22]. mRNAs play an essential role in the disease treatment and vaccine production and exosomes, owing to unique properties, can open up a new way for researchers in the development of a safe and efficient mRNA delivery system [148]. Although abundant evidence shows that lipid nanoparticles (LNPs) are highly efficient for mRNA transfer, some studies show that exosomes are more stable and less immunogenic than LNPs [52]. Recent in vitro studies have shown that transferring the CRISPR/Cas9-based RNA reporter system by exosomes has been much more satisfactory than the LNP [125]. However, due to the various challenges of using exosomes in the clinic, this extremely stable and efficient nano-carrier has not been employed in treatments yet [144]. One of the major problems is the lack of a standard and reliable method for exosome purification [149]. Although several strategies have been performed to load short nucleic acids like siRNA, shRNA, and DNA into exosomes [66, 68, 71, 72, 74], the efficient encapsulation of large mRNAs into exosomes remains a challenge. Therefore, the establishment of large-scale RNA-loaded exosomes for clinical applications should be given further attention.