Desirable features of exosomes have made them a suitable manipulative platform for biomedical applications, including targeted drug delivery, gene therapy, cancer diagnosis and therapy, development of vaccines, and tissue regeneration. Although natural exosomes have various potentials, their clinical application is associated with some inherent limitations. Recently, these limitations inspired various attempts to engineer exosomes and develop designer exosomes. Mostly, designer exosomes are being developed to overcome the natural limitations of exosomes for targeted delivery of drugs and functional molecules to wounds, neurons, and the cardiovascular system for healing of damage. In this review, we summarize the possible improvements of natural exosomes by means of two main approaches: parental cell-based or pre-isolation exosome engineering and direct or post-isolation exosome engineering. Parental cell-based engineering methods use genetic engineering for loading of therapeutic molecules into the lumen or displaying them on the surface of exosomes. On the other hand, the post-isolation exosome engineering approach uses several chemical and mechanical methods including click chemistry, cloaking, bio-conjugation, sonication, extrusion, and electroporation. This review focuses on the latest research, mostly aimed at the development of designer exosomes using parental cell-based engineering and their application in cancer treatment and regenerative medicine. Graphic Abstract

Exosomes are the best options for gene targeting, because of their natural, nontoxic, non-immunogenic, biodegradable, and targetable properties. By engineering exosome-producing cells, ligands can be expressed fusing with exosomal surface proteins for targeting cancer cell receptors. In the present study, HER2-positive breast cancer cells were targeted with a modified exosome producing engineered HEK293T cell. For this purpose, the HEK293T cells were transduced by a lentiviral vector bearing-LAMP2b-DARPin G3 chimeric gene. Stable cells expressing the fusion protein were selected, and the exosomes produced by these cells were isolated from the culture medium, characterized, and then loaded with siRNA for subsequent delivery to the SKBR3 cells. Our results showed that stable HEK293T cells produced DARPin G3 on the surface of exosomes. These exosomes can bind specifically to HER2/Neu and are capable of delivering siRNA molecules against TPD52 gene into SKBR3 cell line down-regulating the gene expression up to 70%. Present approach is envisaged to facilitate genes and drugs transfer to HER2 cancer cells providing additional option for gene therapy and drug delivery.

PurposeExosomes are small 30–100 nm vesicles secreted by various cell types. They are released by most cell types, indicating their important role in physiological and pathological processes, including signaling pathways, cell-to-cell communication, tumor progression, and molecule transferring. As natural nanovesicles, exosomes can be a good candidate for drug delivery due to low immunogenicity and ability to enter tissues and even cross the blood–brain barrier. In an effort to improve the efficiency of exosomes for targeted drug delivery with minimal effect on normal cells, we expressed ligands against HER2+ cells.MethodsTo purify exosomes, transduced mesenchymal stromal cells were cultured to reach 80% confluency. Next, the cells were cultured in serum-free media for 48 hours and the supernatant was harvested to purify exosomes. These exosomes were then labeled with PKH67 and added to BT-474, SKBR3 (HER2+), and MDA-MB231 (HER2−), cell lines and their binding to HER2+ was evaluated by flow cytometry. Exosomes were loaded with doxorubicin and quantified using intrinsic fluorescence of doxorubicin at 594 nm.ResultsTargeted exosomes were preferably uptaken by HER2+ cells. Therefore, untargeted exosomes showed lower binding to HER2+ cells compared to their targeted counterparts. MTT assay was performed to analyze cytotoxic effect of exo-DOX (exosome encapsulated with doxorubicin). Efficiency of exo-DOX and free DOX (doxorubicin) delivery with different concentrations, to the BT-474 cell line, was compared, and no significant difference was observed.ConclusionOur results imply that targeted exosomes are preferentially uptaken by HER2+ cells relative to HER2− cells and have the potential to be used as an efficient drug delivery system.

Background Exosomes are natural nanovesicles with unique characteristics, such as long circulating half-life, the intrinsic ability to target tissues, biocompatibility, and minimal or no inherent systemic toxicity. Mesenchymal stem cells produce large amounts of exosomes with regenerative properties and more stability in human plasma. TUBO breast cancer cell lines overexpress rat HER2/neu protein. Methods Targeted exosomes were isolated from transduced bone marrow mesenchymal stem cells. Doxorubicin was encapsulated into exosomes by electroporation. Flow cytometry was used to assess the attachment of exosomes to the target cells. The in vitro cytotoxicity effect of targeted doxorubicin-loaded exosomes on TUBO cells was determined using MTT assay. Selective delivery of doxorubicin to tumor tissues was analyzed by measuring the auto-fluorescence of doxorubicin by in vivo imaging system. Moreover, tumor growth inhibition and body weight were monitored following injection of free doxorubicin, and targeted and untargeted doxorubicin-loaded exosomes in a TUBO breast cancer model. Finally, mouse tissues were examined for the presence of intrinsic fluorescence of doxorubicin. Results Flow cytometry results revealed significant differences in binding of targeted exosomes to HER2-positive (46.05%) and HER2-negative (13.9%) cells. The results of MTT assay showed that cytotoxicity of targeted doxorubicin-loaded exosomes was higher than free doxorubicin at 72 hours. Selective distribution of targeted doxorubicin-loaded exosomes in the target tissues of the murine breast cancer model suggested specific delivery of doxorubicin by targeted exosomes, rather than untargeted exosomes. Free doxorubicin and untargeted doxorubicin-loaded exosomes showed insignificant effects, whereas targeted doxorubicin-loaded exosomes reduced the tumor growth rate. Conclusion Herein, we report efficient delivery of targeted doxorubicin-loaded exosomes in vitro, corroborated with a significant reduction of murine breast cancer model tumor growth rate.

No abstract