The innate ability of exosomes to target tissues of interest besides their unique characteristics make them as an intriguing delivery agent in nanomedicine. Naïve exosomes accumulate within tumors due to the enhanced permeability retention (EPR) effect on leaky vasculature of tumors . The goal of this study is whether exosomes have preferential association and uptake by parental cells. Since exosomes are shuttling information between cells, the components and biological functions are considerably dependent on the donor cells. As tumor-derived exosome contents mimic parental cell constituents, thus may be cancer-derived exosomes effectively migrate to the same source they are released. Here, we hypothesized that the natural tropism of cancer-derived exosomes could be beneficial to design an exosome-based delivery system. Our results clearly showed that exosomes derived from TUBO cells exhibited higher binding to TUBO cells than HEK-derived exosomes. This is consistent with Kim et al. study in which delivery potency of epithelial-derived exosomes (HEK293) was compared with tumor-derived exosomes in SKOV3 xenograft. SKOV3 exosomes demonstrated better targeting efficiency due to the self-targeting of cancer-derived exosomes . On contrary, Smyth et al. reported that exosomes released by MCF-7, MDA-MB-231, and PC3 cancer cell lines did not preferentially interact with their “parent” cell lines. Most notably, their results further suggested that the exosomal lipid components are more responsible for increased cell association . Additionally by cleavage the surface proteins of exosomes they confirmed the importance of proteins in incorporation of exosomes. The involved mechanism for exosomes uptake after association with target cells are both endocytotic  and phagocytic pathways . Al-Nedawi et al. demonstrated the transfer of the mutated epidermal growth factor receptor (EGFRvIII) to cancerous cells by exosomes derived from highly aggressive brain gliomas . The short circulation half-life of exosomes have been reported in numerous studies presumably due to recognition by immune cells. To enhance the circulation time of exosomes and thereby increase the affinity of exosomes towards tissue of interest, previously we introduced targeting ligand on the surface of exosomes by fusion proteins. Indeed, targeting ligand resulted in an effective internalization by tumor cells, it appears that this elevated affinity is mediated by ligand and receptor reaction which in our targeted exosomes is conferred by DARPin ligand to HER2 receptors. Moreover, fusion with the cell surface lipid is another mechanism for adherence and internalization of exosomes . Yuki Toda et al. hypostatized that parental cell tropism is a critical factor for efficient exosome intercellular communication. They reported the incorporation of intact glioblastoma-derived exosomes into the parental and some other cancer cells. Furthermore, they demonstrated the underlying mechanism for the selective tropism and preferential uptake of exosomes can largely be attributable to the lipid component of U251-MG cell-derived extracellular vesicles . Due to the difference of plasma membrane lipid components of various cells, the extracted exosomes show variation . They attributed the higher uptake of U251exosomes to the higher polyethylenimine (PE) and lower percentages of sphingomyelin (SM) in U251 exosomes, and this difference is due to the lipid composition of the parental cell membranes they have derived. the cone-shape structure of PE promotes fusion by increasing negative membrane curvature . The abundance of PE in the plasma membrane of recipient cell and exosomes may be a key factor in self tropism. Smyth et al. investigated that apart from lipid composition, the distinct protein structures of exosomes also facilitate the internalization of exosomes in cancer cells . They confirmed the role of lipids in preferential association by interaction of liposome composed of exosomal lipids. The enrichment of exosomes by cholesterol, sphingomyelin (SM), and anionic phospholipids, most notably phosphatidylserine (PS) make their surface lipid components unique for better association. The high stability and rigidity of exosomes is owing to the presence of high cholesterol and SM concentrations. From the same group studies revealed that the cholesterol domains promote delivery both in vitro [29, 30] and in vivo  implicating the effect of lipids in exosome trafficking and likely extend exosomes circulation half-life . The enrichment of exosomes by trans membrane proteins such adhesion proteins most notably integrins [33, 34], tetraspanins , and the ICAM family of proteins  augment exosome adherence/internalization. Morelli et al discussed the presence of milk fat globule (MFG)–E8/lactadherin, CD11a, CD54, phosphatidylserine, and the tetraspanins CD9 and CD81 on the targeting of exosomes to DCs . They demonstrated that exosomes interaction with their parental cells significantly decreased when their surface protein has been cleaved. Therefore, the higher uptake of TUBO exosomes compared to HEK exosomes may be due to particular components of exosomal surface reportedly lipids and proteins involved in their uptake and association. Li Qiao et al. proved that isolated exosomes from Hela and HT1080 cancerous cell lines had preferential homing to their parent cancer cells. Furthermore, they found that exosomal integrin expression patterns might be responsible for the exhibited homing process . According to our results, fluorimetry analysis revealed the unspecific binding of blank exosomes in higher concentrations. Importantly, we observed that with increasing the dosage of exosomes on HER-2 positive cells, the prominent factor in the attachment would be nonspecific physical absorption . Here we could demonstrate that in lower concentrations of exosome, the uptake and affinity are selectively related to the exosome surface ligands and components, not to physical adsorption, which took part in the higher concentration of exosomes, thus decreasing the variation between two groups.
Here, again we reaffirmed the importance of incorporation of ligands to have better targeting efficiency. The most widely used method to produce targeted exosomes is the engineering of parental cells and expressing the desired ligand on the surface of exosomes beside a fusion protein [16, 38]. Although effective, some concerns about the engineering of exosome producing cells to express targeting ligand have been addressed. One of the main challenges in using fusion proteins, especially Lamp-2B, is the possible degradation of peptides fused to the N-terminal of lamp-2B protein. Hung et al. enhanced the half-life of Lamb-2B fusion protein by introducing a glycosylation motif . Moreover, the improper expression of the transgene and the difficulties in the transfection of some cell lines are the other limitations in exosome redecoration. Nevertheless, despite efficient genetic manipulation of some cells like HEK293, the low yield of produced exosomes hinder their translation in clinical therapeutic applications.
Here in this study, our aim was to employ the natural and intrinsic targeting ability of exosomes to overcome the drawbacks of exogenously added ligands for therapeutic purposes. As aforementioned, cancer-derived exosomes resemble the signature of the cells that they are released. Although cancer-derived exosomes might have the possibility to stimulate immune responses by packaging some cancer antigens, they can play major role in tumor progression too. Therefore, owing to these controversial roles of cancer-derived exosomes, care should be taken in designating an exosome-based delivery system . However, proteome array analysis are under progress in our team to unravel the involved mechanisms in preferential uptake by donor cells. We believe that understanding the contents of tumor derived exosomes is a promising avenue to overcome the unwanted side effects in cancer therapy. Several hurdles such as the clinical safety of cancer-derived exosomes required to be addressed prior to clinical translation.
As the physical property such as the size of exosomes might have a significant effect on cells ability to internalize them , the enhanced adherence of unmodified cancer-derived exosomes could be due to the greater size and density of cancer exosomes resulting in increased contact on the cell surface. Federica Caponnetto et al. determined the effect of physical properties of exosomes on their uptake and addressed the question whether the method of preparation of enriched exosomal fractions can affect their uptake by cells and their ability to trigger a response . They compared the polymer-based precipitation purification method of exosomes with ultracentrifugation extracted exosoms and confirmed that the smaller size of exosomes resulted by polymer-based method and led to higher and faster uptake rather than exosomes obtained by ultracentrifugation. This is corroborated with our previous study which we have isolated exosomes by differential ultracentrifugation procedure, and it yielded the lower amount of exosomes and bigger particle size distributions (172 nm) compared to the commercial kits precipitation method (120 nm). Here we confirmed the previous findings that NTA failed to recognize the smaller particles . Having efficient clinical translation of exosomes requires a reliable isolation method, rendering in highly pure exosomes without contamination. The gold standard method for exosomes isolation and purification is differential ultracentrifugation . But due to the high cost of ultracentrifugation, and protein aggregation we tried to purify exosomes using a commercial kit which precipitates exosomes by size exclusive chromatography. We assessed the size of various preparations of exosome by zeta sizer, and we could confirm that they had a similar size. Additionally, we homogenized the size of exosomes by filtrating the supernatants. In fact, engineered exosomes may show different properties such as uptake efficiency, kinetics and the internalization mechanism due to increase in size after introducing the transgene . But in case of our study, as the DARPin is a small ligand, the effect of this protein on the size of engineered exosome is negligible. The yield of exosomes depends on the isolation method and donor cells. It is believed that cancer cells generate more exosomes rather than healthy cells, which may be due to an enhanced growth rate or as a result of stimulation in response to stressful conditions. Here, TUBO cells secreted much higher exosomes than HEK cells according to the obtained results by Bradford and CD9 ELIZA assay. It has been already reported that exosome canonical markers are not equally expressed by different exosome subpopulations . But here TUBO and HEK293 exosomes relatively expressed CD9 in the same amount. We have used CD9 measurement due to the overestimation of exosome density by co-precipitation of proteins along with exosomes which makes the colorometric assays like Bradford impractical. By this alternative assay we can measure directly exosome population and avoid the risk of overestimation in future clinical studies. However, as different populations do not express the markers equally it would be ideally to measure the exosome density of the same source because exosomal secretion likely differs among cells. Taken together the innate tumor tropism of cancer-derived exosomes along with the high production rate, suggest that exosomes derived from cancer cells may represent a target for therapeutic applications. Here we compared the tropism and uptake of tumor-derived exosomes with engineered exosomes considering different factors such concentration and incubation time. To our knowledge our work is the first study comparing specific binding of tumor and engineered cells. Further studies are under investigation to unravel the mechanism of entry to establish whether the targeting of tumor derived exosomes would boost the efficiency of uptake in tumor models. Tumor cells regulate the movement of their exosomes via the modulation of exosomes components. The molecular components involving in specific uptake of exosomes are not fully elucidated and depends to the donor and recipient cells. The initial step on the recognition of exosomes by recipient cells is ligand receptor interaction which was the primary key element of targeted exosomes recognition by TUBO cells in the first 4 hr but with increasing the incubation time, TUBO cells had more time to recognize TUBO exosomes and facilitate their effective internalization. The increased absorbance and internalization of TUBO exsomes inside SKBR3 cells reveals that cancer cells have a mechanism to recognize cancer-derived exosomes.
In the pilot in vivo study we administered TUBO-derived exosomes into TUBO xenograft mice model to assess whether they are effective at traveling back to the parent cell line that produced them. To address this question, we studied the systemic biodistribution of tumor-derived exosomes, remarkably, we observed that the tropism exhibited by tumor-derived exosomes can be utilized in the future to shuttle the cancer therapeutic agents with no specific targeting capacity, to tumor tissues.