Targeting Efficient Drug Delivery With Exosome Nanoparticles
Exosomes are small vesicles (typically 30-120 nm), formed through the inward budding of endocytic compartments and secreted through fusion of these vesicle-containing endosomes with the plasma membrane. Exosomes are secreted by most cell types and may be found in bodily fluids such as plasma, urine, saliva, serum, and cerebrospinal fluid.1 Increasing evidence suggests that exosomes play an important role in cell-to-cell communication through the transport and delivery of cellular components such as lipids, proteins, and nucleic acids.
Exosomes have gained a considerable interest recently as potential biomaterials for drug delivery. First, because exosomes contain endogenous cellular components, certain exosomes may be equipped to target particular cell types and tissues, enabling them to overcome biological barriers, like the blood brain barrier. Secondly, owing to their endogenous origin, exosomes are less likely to be immunogenic or cytotoxic than other synthetic delivery systems. Finally, the lipid bilayer of exosomes may protect the drug from rapid blood clearance, and may reduce cytotoxicity associated with off-target drug effects.
What Makes Exosomes Efficient Drug Delivery Tools?
The intrinsic characteristics and components of exosomes make them an ideal candidate for drug delivery.2 Exosomes are large enough to avoid rapid renal clearance, yet small enough to escape uptake by the reticuloendothelial system. Small nanoparticles like exosomes (typically 10-100 nm) preferably accumulate at solid tumor sites due to leaky vasculature and abnormal lymphatic drainage, making them an attractive candidate for drug delivery in cancer certain types of cancer.3
Like other vesicles, exosomes are composed of a lipid bilayer, which forms an aqueous inner compartment and lipophilic outer layer. This structure enables the loading of both hydrophobic and hydrophilic materials into exosomes. Although exosomes vary widely in the type and amount of cellular components they carry, certain lipids, proteins, and nucleic acids appear to be more common than others. Exosomes contain high amounts of cholesterol, sphingolipids, phosphoglycerides, ceramides, and saturated fatty acid chains,4 and the incorporation of these rigid molecules appears to contribute to the stability of exosomes.
A variety of both membrane-bound and intracellular proteins can be found in exosomes. The most common of these include membrane transport and fusion proteins, major histocompatibility complexes, heat shock proteins, tetraspanins, proteins of the endosomal sorting complex required for transport (ESCRT) complex, and lipid raft-associated proteins.2 Exosomes also contain an enrichment of proteins specific to the cell type from which they are secreted. For example, exosomes derived from dendritic cells are enriched with heat shock cognate protein (hsc73), a protein which may play a role in the anti-tumor effects observed with dendritic cell-derived exosomes.5
Exosomes also contain nucleic acids, including microRNA, non-coding RNA, and messenger RNA. Interestingly, these RNAs can either be found inside the aqueous compartment of the exosome or tethered to the outside of the exosome by the argonaute2 protein.6 Certain microRNAs may be targeted and packed into the exosome by a protein that recognizes short motifs in the RNA,7 suggesting that RNAs may be selectively packaged into exosomes.
Examples of Exosome-based Drug Delivery
Since exosomes are released by a variety of cells types, a number of options exist for the selection of a donor cell from which to isolate exosomes. Two important factors that play a role in the selection are biological properties of the exosomes and yield of exosomes from the specific cell type. For example, it has been shown that dendritic cell-derived exosomes stimulate stronger antitumor immune responses than EG7 tumor cell-derived exosomes, suggesting that dendritic cell-derived exosomes contain more molecular factors that can stimulate T cell proliferation and differentiation or interact with T cells more efficiently than tumor-derived exosomes.8 Another factor in exosome donor cell selection is the amount of exosome released by a particular cell type. Mesenchymal stem cells have been identified as a particularly promising cell type for many reasons among which are their high production of exosomes, ease of isolation, ability to expand to large scale cultures,9,10 and promote cell survival.11,12
Ensuring the purity and abundance of exosomes, whether from cell culture media or from bodily fluids, is critical for the development of exosome-based drug delivery. Typically, exosomes are purified using differential centrifugation and their yield measured through various methods of protein determination.13 Other alternative strategies include filtration, immunoaffinity isolation, and microfluidic techniques that rapidly isolate exosomes for structural and physical analysis, however, it is not clear whether exosomes purified using these new techniques would be viable for therapeutic delivery.
Therapeutic agents have been loaded into exosomes both using in vitro14 and ex vivo15 techniques. Several ex vivo techniques, including freeze thaw cycles, saponin permeabilization, sonication, and extrusion procedures, have been used to load drugs into exosomes, and the structure and activity of the drugs have been maintained after exosome loading. In addition, it appears that exosomes are stable when stored from -20⁰C to -80⁰C and when subjected to multiple freeze-thaw cycles.2
Delivery of Therapeutic Agents through the Blood Brain Barrier and Future Directions
One of the greatest challenges in chemotherapeutics has been delivery of drugs across the certain biological barriers, particularly the blood brain barrier. Exosomes released by glioblastoma have been detected in serum, suggesting that endogenous exosomes cross the blood brain barrier.16 Furthermore, preparations of exosomes loaded with curcumin, a polyphenol with anti-inflammatory properties, were delivered intranasally in mice and resulted in microglial cell apoptosis, indicating that vesicle preparations may pass through the blood brain barrier.17 Finally, exosomes derived from brain epithelial cells and loaded with anticancer drugs were shown to cross the blood brain barrier and induce cytotoxicity in tumor cells in zebrafish.18 Taken together, these findings suggest that exosomes may be particularly useful in drug-delivery in the brain.
While exosome-based drug delivery appears to be a promising direction for therapeutics, a few key issues need to be addressed before it can be safely and efficiently implemented. First, the exosome isolation and purification processes need to be standardized to eliminate contaminants such as protein aggregates and to improve reproducibility. Second, donor cells that provide a stable source of exosomes need to be identified, and exosomes from those donor cells need to be fully characterized. Finally, more efficient processes to load drugs into exosomes need to be developed in order to maximize the delivery of therapeutics.
1. El Andaloussi S, Mäger I, Breakefield XO, Wood MJA. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013;12:347–357. doi: 10.1038/nrd3978.
2. Ren J, He W, Zheng L, Duan H. From structures to functions: insights into exosomes as promising drug delivery vehicles. Biomater Sci 2016;4:910–921. doi: 10.1039/c5bm00583c.
3. Prabhakar U, Maeda H, Jain R, et al. Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology. Cancer Res 2013;73:2412–2417. doi: 10.1158/0008-5472.CAN-12-4561.
4. Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002;2:569–579. doi: 10.1038/nri855.
5. Théry C, Reganault A, Garin J, et al. Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73. J Cell Biol 1999;147:599–610.
6. Arroyo JD, Chevillet JR, Kroh EM, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U.S.A. 2011;108:5003–5008. doi: 10.1073/pnas.1019055108.
7. Villarroya-Beltri C, Gutiérrez-Vázquez C, Sánchez-Cabo F, et al. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat Commun 2013;4:2980. doi: 10.1038/ncomms3980.
8. Hao S, Bai O, Yuan J, et al. Dendritic cell-derived exosomes stimulate stronger CD8+ CTL responses and antitumor immunity than tumor cell-derived exosomes. Cell Mol Immunol 2006;3:205–211.
9. Lai RC, Yeo RWY, Tan KH, Lim S. K. Exosomes for drug delivery - a novel application for the mesenchymal stem cell. Biotechnol Adv 2013;31:543–551. doi: 10.1016/j.biotechadv.2012.08.008.
10. Yeo RWY, Lai RC, Zhang B, et al. Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery. Adv Drug Deliv Rev 2013;65:336–341. doi: 10.1016/j.addr.2012.07.001.
11. Zhang J, Guan J, Niu X, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med 2015;13:49. doi: 10.1186/s12967-015-0417-0.
12. Zhang Z, Yang J, Yan W, et al. Pretreatment of Cardiac Stem Cells With Exosomes Derived From Mesenchymal Stem Cells Enhances Myocardial Repair. J Am Heart Assoc 2016;5:1. doi: 10.1161/JAHA.115.002856.
13. Théry C, Amigorena S, Raposo G, Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Editor. Board Juan Bonifacino Al (2006) Chapter 3, Unit 3.22. doi: 10.1002/0471143030.cb0322s30.
14. Pascucci L, Coccè V, Bonomi A, et al. Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: a new approach for drug delivery. J Control Release Off J Control Release Soc 2014;192:262–270. doi: 10.1016/j.jconrel.2014.07.042.
15. Haney MJ, Klyachko NL, Zhao Y, et al. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. J Control Release Off J Control Release Soc 2015;207:18–30. doi: 10.1016/j.jconrel.2015.03.033.
16. Al-Nedawi K, Meehan B, Micallef J, et al. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 2008;10:619–624. doi: 10.1038/ncb1725.
17. Zhuang X, Xiang X, Grizzle W, et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain. Mol Ther J Am Soc Gene Ther 2011;19:1769–1779. doi: 10.1038/mt.2011.164.
18. Yang T, Martin P, Fogarty B, et al. Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. Pharm Res 2015;32:2003–2014. doi: 10.1007/s11095-014-1593-y.