Exosomes as Carriers for Anti-tumor Therapy | ACS Biomaterials

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Review

Exosomes as Carriers for Anti-tumor Therapy Yanyan Li, Yongtai Zhang, Zhe Li, Kuan Zhou, and Nianping Feng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00417 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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ACS Biomaterials Science & Engineering

Exosomes as Carriers for Anti-tumor Therapy Yanyan Li1, Yongtai Zhang1, Zhe Li, Kuan Zhou, Nianping Feng*

Department of Pharmaceutical Sciences, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

* Corresponding author E-mail addresses: [email protected]; [email protected] 1

Authors contributed equally to this work

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Abstract Exosomes are bilayer vesicles with particle sizes of between 50 and 150 nm. Owing to their bilayer membrane structure, cell-to-cell communication, and good absorbability ， exosomes are increasingly used as carriers for drug delivery through the phospholipid membrane structure to the lesion site with enhanced targeting. Exosome sources and drug-loading methods are important factors affecting their use as drug carriers. There are various ways to pack species in exosomes, and researchers are constantly seeking new and improved approaches. In both in vivo and in vitro evaluations, exosomal vectors have achieved good results for anti-tumor therapy. Despite the importance of exosomes as drug delivery systems with accurate targeting ability and biocompatibility, improvements are needed to facilitate their wide clinical use. This review focuses on the preparation of exosomes as carriers and their utilization in anti-tumor research. Keywords: tumor targeting, biomimetic nanocarriers, nanomedicine


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1. Introduction Exosomes belong to family of membrane vesicles called extracellular vesicles, which typically include microvesicles, apoptotic bodies, and exosomes (Figure 1). As a nanoscale membranous vesicle, exosomes were first discovered during sheep reticulocyte culture and differentiation in the late 1980s1. However, they received little attention until Caby first detected exosomes in the body (blood) of healthy people in 2005, suggesting that they act as a molecular vehicle to facilitate the exchange of information between cells or organs2. Since 2005, the literature on exosomes has increased exponentially3.

Figure 1. Differences in size, markers, and origin among exosomes, microvesicles, and apoptotic bodies.

Exosomes are typically extracted from conditioned cell culture medium4 and extracellular fluids5-6 (e.g., urine, saliva, semen, breast milk, and cerebrospinal fluid). As small membrane vesicles, exosomes contain many functional biomolecules, such as proteins, lipids, nucleic acids (mRNA, microRNA [miRNA], ribosomal RNA, and long noncoding RNA [lncRNA])7-8. Regardless of the



source of exosomes, they share some characteristics, including the membrane that is rich in cholesterol, ceramide and sphingomyelin9. Exosomes also contain different specific sets of proteins, such as integrin and tetraspanin family proteins (CD9, CD63, and CD81), heat shock proteins (HSP60, HSP70, and HSP90), MVB synthesis proteins (Alix and Tsg101), membrane transporters, fusion proteins (Annexins, Rab GTPases, and flotillins), and cytoskeletal proteins (Figure 2)10.

Figure 2. Exosome composition and extraction sources. Exosomes are typically extracted from conditioned cell culture medium (cancer cells, dendritic cells, macrophages) and extracellular fluids (dairy products, blood, urine). The extracted exosomes carry important information such as lipids, proteins, DNA and RNA, while the exosome membrane is rich in a variety of specific proteins, such as the four transmembrane protein family and the heat shock protein family.

Exosomes have been used in recent studies focused on a wide range of diseases, such as cancer, neural diseases, liver disease, ischemic diseases, and immune diseases; in particular, anti-tumor research using exosomes is extensive. Cancer been a major focus of research worldwide, and


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numerous studies have evaluated the anti-tumor roles of exosomes. Exosome-mediated intercellular communication is a key link between cancer and the immune system11. Parolini et al. showed that the fusion of exosomes to target cells is effective under acidic conditions, indicating that exosomes could be preferentially ingested by tumors (which have an acidic microenvironment), rather than surrounding healthy tissues12. Recently, it has also been shown that exosomes induce immune responses and therefore can be used for cancer therapy13. In addition, exosomes can be used as biomarkers for the rapid detection of cancer at all stages14. It is beneficial to isolate circulating tumor exosomes to obtain more sensitive and specific biomarkers. For example, survivin present in exosomes released from breast cancer cells is a suitable diagnostic biomarker for patients with earlystage breast cancer15. Recent research has demonstrated that exosomes can be used for drug delivery systems and as carriers to deliver drugs to the desired site owing to their ability to move from cell to cell, which is highly advantageous for drug carriers16-17. Notably, exosomes can act as carriers to facilitate drug delivery through the blood–brain barrier (BBB)18-19. There are many adhesive proteins on surface of the exosomes, which provide a unique approach for the delivery of various therapeutic agents to specific target cells through cell–cell communications20. Furthermore, using exosomes as carriers, great achievements have been made with respect to the drug package that is delivered for anti-tumor treatment. As reported, exosomes are used by tumors for local and long-range cell communication as they are released in the extracellular environment; they are capable of transferring their cargo into recipient cells in an autocrine and paracrine manner21. Owing to the variety of functional substances, exosomes as drug carriers have ability to overcome natural barriers, intrinsic cell targeting properties, and stability in the circulation22. Therefore, it is



likely that using exosomes as carriers for drug delivery to tumor lesions can have a multiplier effect against tumors. In this review, we elaborate on exosome extraction methods, sources, loading methods, and empirical analyses of their targeting ability and anti-tumor effects, with an emphasis on the benefits of exosomes for cancer research. 2. Exosome sources Exosomes are generated by almost all kinds of cells through inward budding of the inner endosomal membrane followed by plasma membrane fusion23-24. Exosomes can be extracted from human body fluids, blood, cell culture media, milk, etc. For anti-tumor applications, the source of exosomes is important for the elimination rate and blood circulation time in vitro and in vivo. For example, when exosomes are derived from cancer cells, they effectively accumulate in the mononuclear phagocyte system and are rapidly removed from the blood25. In order to improve their pharmacokinetics, surface modifications to enhance their circulation time are necessary. Additionally, exosomes released from cancer cells affect the development of cancer in different ways, such as tumor growth, metastasis, drug resistance, angiogenesis, and immune system function, and modify the intrinsic motility and invasiveness of tumor cells26. Exosomes extracted from immune cells can function in tumor immunotherapy. Exosomes released from the primary fibroblast-like interstitial cells can bypass mononuclear cells and macrophages in immune clearance, leading to prolonged blood circulation. This may be related to the expression of CD47 molecules on the surfaces of exosomes and their binding partners thrombospondin-1 (TSP1) and signal-regulated protein alpha (SIRPα), which produce “don't eat me” signals in macrophages27. Differences in phospholipids and proteins on the surfaces of exosomes from different sources are associated with different mechanisms of action and therefore differences in their applications to specific diseases.


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3. Exosome Extraction and Identification The classic method for the extraction of exosomes is conventional differential ultracentrifugation, which is described in detail by Théry and colleagues28. Some conventional methods, including differential ultracentrifugation, density gradient centrifugation, size exclusion chromatography and immunoaffinity chromatography, have been reported for extraction of exosomes, but may undergo various drawbacks such as equipment requirements, expensive reagents, productivity, purity and operating procedures during the extraction process. This led to some novel extraction methods, such as ultrafiltration separation technology, integrated double filtration microfluidic device, nanoplasmon-enhanced scattering and membrane-mediated exosome separation29. However, these new methods are limited to theoretical stages, lack of preclinical research for their unknown scalability and reliability30. At present, the use of plasmonic heating to capture exosomes in a temperature gradient is another technical route31. The extracted exosomes are usually stored at 80°C32 or lyophilized33 for subsequent experiments. The production of exosomes is often accompanied by the synthesis of other vesicles, such as microvesicles and apoptotic bodies. In general, exosomes are between 50 and 150 nm in diameter, so they can be identified from other extracellular vesicles by determination of the particle size with the methods including nanoparticle tracking analysis, dynamic light scattering, resistive pulse sensing, flow cytometry, and observation of the size and morphology with electron microscopy34. Notably, the exosomes derived from the exocytosis of multivesicular bodies can be detected by endosomal markers, such as CD63, CD9, and CD8135. These protein markers carried by exosomes are very important for distinguishing them from microvesicles. Western blotting is widely used to



evaluate the levels of protein markers, including tetraspanins (CD63, CD81, CD9), cell adhesion molecules (EpCAM), and heterotrimeric G proteins. 4. Exosomes as carriers Exosomes have been evaluated as carriers for drug delivery to the lesion site with enhanced targeting20, 36. It has been reported that the parental cell membrane of exosomes carried cell-type specific proteins with specific cellular orientation and can be used to target diseased tissues37. With low toxicity and immunogenicity, the exosomes combined with therapeutic agents, or nanocomposites, is very valuable for cancer therapy. Exosomes can be used as a vector for biotherapeutic agents to achieve efficient delivery to target cells, increasing evidence shows that nucleic acids are protected by encapsulating into the exosome nanovesicles for the transport and intracellular delivery38. Methods for encapsulating substances by exosomes can be summarized into three categories: postloading method, pre-loading method and fusion method (Figure 3).

Figure 3. Three methods for encapsulating substances by exosomes: (A) post-loading method, (B) pre-loading method, and (C) fusion method.


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4.1 Post-loading method The post-loading method involves the extraction of exosomes and subsequent drug loading in exosomes by different approaches. Current methods for incorporating drugs or therapeutic agents into exosomes are incubation, electroporation, sonication, and extrusion. 4.1.1 Incubation Incubation methods are relatively common and simple. The paclitaxel stock solution was incubated with exosomes for 1 hour at 22 °C to form an excipient preparation with a loading efficiency of 9.2%. This approach is based on the high lipophilicity and poor water solubility of paclitaxel, and uses the passive diffusion of drugs loaded in the exosomes39. In addition, miRNAs can also be loaded into exosomes by co-incubation at 37 °C40. The incubation temperature is mostly 22℃ or 37℃, and the incubation time ranges from half an hour to twelve hours40-43. However, the loading efficiency is relatively low, especially for some large miRNAs, thus the incubation methods are often combined with other approaches for improving drug loading. 4.1.2 Electroporation Electroporation involves a high-intensity electric field, instantaneous increases in cell membrane permeability, and drug loading. Voltage settings typically vary from 150 V–700 V, depending on distinct types of donor cells, such as Hela cells, monocytes, and immature dendritic cells44. Exosomes and doxorubicin are mixed after electroporation with 350 V and 150 mF using a 0.4 cm electroporation method to ensure the complete recovery of the plasma membrane of exosomes. Transmission electron microscope and nanoparticle tracking analyses have shown that



electroporation does not significantly alter the physical properties. This maintenance of the original drug characteristics greatly helps the subsequent administration45. Despite the feasibility and success of electroporation, it has some limitations, e.g., discharges through macromolecules containing solutions, such as DNA, RNA, and proteins, can cause significant molecular aggregation46. 4.1.3 Sonication The principle of ultrasonic drug loading is that the microviscosity of the membrane is significantly reduced (typically by at least two-fold) by ultrasonic waves, and the hydrophobic drug enters through the membrane. Kim et al. isolated various concentrations of exosomes from macrophageconditioned medium and loaded paclitaxel into the exosomes by several methods. They found that sonication had a much better loading capacity compared to those of electroporation and incubation. Western blotting demonstrated that sonication does not affect the protein content of exosomes47. 4.1.4 Summary of the post-loading method The post-loading method is relatively intuitive and simple. Drugs are loaded by various methods (sonication, electroporation, or incubation). Less common methods include saponin-mediated permeabilization, freeze-thaw cycles, and extrusion48. As an entrapment method, the post-loading method shows little effect on the physical and chemical properties of the drug and exosomes. However, reports have shown that the actual encapsulation rate is far below the estimated value. Kooijmans found that the electroporation of extracellular vesicles using siRNAs is accompanied by substantial siRNA aggregates, which may result in the overestimation of the amount of siRNA actually loaded into the extracellular vesicle49. The encapsulation efficiency of post-drug loading methods is generally very low (generally only 20%), so improvements are needed.


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4.2 Pre-loading method Another successful loading method (pre-loading method) involves feeding desired compounds to cultured cells or genetically engineering cells to produce RNA or proteins, which then become incorporated into exosomes. Various reagents are commonly used for transfection to load a wide range of nucleic acids (siRNA, miRNA, mRNA, and plasmid DNA) into exosomes, including HighPerFect (Qiagen, Valencia, CA, USA), ExoFectin® (101 Bio, Palo Alto, CA, USA), and ExoFect ™ (System Biosciences, Mountain View, CA, USA)50. The drug or genetic material is usually cultured with cells, followed by extraction to obtain the exosomal substance. Paclitaxel was incubated with SR4987 cells (used as an mesenchymal stromal cell model) for 24 hours, paclitaxel-loaded exosomes were obtained by differential centrifugation, and the presence of paclitaxel in exosomes was confirmed by qualitative HPLC analysis51. Significant miRNA accumulation was detected from exosomes purified from primary melanoma cell lines expressing miR-222, and exosomal miR-222 was successfully prepared52. The J774 cell line was stimulated with the WEHI-164 cell lysate and HSP70-enriched WEHI-164 cell lysate to acquire exosomes that contain tumor antigens enriched with HSP7053. These results illustrate the feasibility of the pre-loading method, and the encapsulation of genetic substances has been greatly improved compared to the post-loading method. In a previous study, a hydrophobic photosensitizer was encapsulated by synthetic liposomes and treated with parental cells. Exosome separation allowed hydrophobic photosensitizers to penetrate both spheroids and in vivo tumors, thereby enhancing the therapeutic efficacy54. It is expected that future research will exploit new types of cargo to be delivered via exosomes, and may map the conditions suitable for a particular type of exosome-encapsulated cargo.



The pre-loading method is mainly implemented using cell culture medium, rather than extracellular fluid. The shortcomings of this approach include the lack of an intuitive, accurate, and convincing method to detect the amount of the substance(s) contained in exosomes. Accordingly, experiments using early purification may also require a large number of cells and drugs for treatment and incubation. Additionally, the extraction yield is not very high and the purity is not sufficient, which may affect its targeting ability and other biological characteristics. 4.3 Fusion method The encapsulation efficiency and drug loading of the exosomes were generally low. Although in vivo preparation and in vitro evaluation are effective, methods to improve the entrapment efficiency and other factors are needed to maximize the efficacy of drugs. The combination of exosomes and nanocomposites with a membrane structure by membrane fusion is a major recent innovation. The combination not only enables the sustained release of nanopreparations and improved absorption and effectiveness, but can also play an exocrine role involving the immune response, antigen presentation, cell migration, cell differentiation, and tumor invasion. Exosomes and nanoliposomes both have a phospholipid membrane structure, with similar particle sizes and structures. However, exosomes have a special surface composition and their origin is endogenous, so the half-life of exosomes in the circulation was longer than that of liposomes55. Therefore, researchers have developed exosome-liposome hybrids. Sato et al. combined liposomes with exosomes to form hybrid exosomes by freezing and thawing56. The hybrids were verified by fluorescence resonance energy transfer, which could detect changes in nanoscale distances of biological macromolecules in vivo. A recent study showed that liposome-exosomal hybrids


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efficiently encapsulate CRISPR-Cas9 large plasmids, and the resulting hybrid nanoparticles can be endocytosed by mesenchymal stem cells and can express the wrapped genes57. This hybrid effectively improves the defect that exosomes cannot encapsulate large nucleic acids due to their small size. Thus, this hybrid is a promising carrier candidate for targeted delivery of exogenous DNA. The combination of exosomes and nanocomposites can effectively increase drug encapsulation, and facilitate modification to improving targeting efficiency and controlling drug release, thus have unique advantages and non-negligible prospects as targeted delivery vehicles. 4.4 Summary of drug loading methods These three methods with their advantages are generally dependent on the characteristics of the materials used. The post-loading method is simple and easy to operate, but the general low drug encapsulation efficiency needs to be improved; the pre-loading method usually increase drug encapsulation ability and does not change the physical and chemical properties, but the premedication preparation dosage is large. The fusion method is the most innovative. It can be used to improve drug encapsulation and drug delivery. However, few studies have evaluated the combination of exosomes and nanocomposites, and there are less data on pharmacological efficacy in vivo. In order to co-loading different substances, the above various drug-loading methods can be used in combination. B16BL6 cells were transfected with a plasmid vector encoding a streptavidin (SAV)– lactadherin (LA) fusion protein to obtain a SAV-LA-exosome and then incubated with GALA to obtain a GALA-SAV-LA-exosome for the efficient cytosolic delivery of exosomal tumor antigens58. In this case, the pre-loading method and the post-loading method are respectively used to



encapsulate the substances to be entrapped in the exosomes. Li et al. coated surface-carboxyl Fe3O4 superparamagnetic nanoparticles (US) with high-density A33 antibodies (A33Ab-US) by coupling reaction, expecting A33 antibodies on the surface of the nanoparticles could bind to A33-positive exosomes to form A33Ab-US-Exo / Dox complex59. The preparation method of such a complex is also relatively novel and innovative. Multiple drug loading methods greatly expand the application space for nano drug delivery systems with exosomes as carriers. 5. Exosome vector for anti-tumor research In the field of anti-tumor research, experimental studies mostly use exosomes extracted from macrophages, mesenchymal stem cells, and tumor cells as vectors, and drugs with anti-tumor effects are loaded. Due to the bilayer vesicle structure of exosomes, the drug or gene substance is not expected to undergo any changes during administration and should maintain its original structural features. Pascucci et al., who showed that loaded paclitaxel in exosomes has a significant anti-tumor effect, indicating that the pharmacological activity of paclitaxel is unaffected by the physiological biology of exosomes51. Furthermore, recent studies have reported that exosomes reduce the immunogenicity of exosomes and inhibit lymphocyte activation by inhibiting the expression of CD3-ζ and down-regulating the JAK3-STAT5 signaling pathway60. In conclusion, exosomes as carriers for tumor immunotherapy may be very promising. Tumor targeting has always been a focus of research. Relatively mature formulations in tumor targeting studies include polyethylene glycol-modified nanocarrier composite drugs as well as nanoparticle-linked targeting moieties, such as antibodies, fragment antigen-binding, and small peptides61. Exosomes have proteins, nucleic acids, and other substances, enabling them to transport large quantities of biologically active substances between cells. Moreover, its properties enable it to


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bind to the surface of target cell membranes to target tumor cells and release drugs for tumor suppression (Figure 4). The application of therapeutic molecules to endosomes through the endocytic orbital is another mechanism by which exosomes target tumors62. Therefore, the exosomes as carriers for drug loading can play a supporting role in tumor targeting. A detailed overview of antitumor studies using exosomes from different sources as carriers was provided as follow.

Figure 4. Principle of exosomal drug tumor targeting. The exosomes are able to recognize specific receptors on the cell surface, then binding and targeting to the tumor lesions, releasing the drug for maximum efficacy.

5.1 Tumor cells Many studies have demonstrated that exosomes extracted from tumor cells successfully target tumor cells and are efficiently taken up by parental cells. Tumor-derived exosomes consist of cytoplasmic and membranous tumor antigens associated with antigen-presenting molecules63. Tumor cells are known to produce greater yield of exosomes compared to the normal cells64. The role of exosomes



in cell tropism and cell–cell communication might be important for applications to tumor biology. Therefore, exosome-based bionic nano-preparation and the selection of tumor cell-derived exosomes are often used to obtain anti-tumor effects. HepG2 cells were incubated with paclitaxel, etoposide, carboplatin, irinotecan, epirubicin, and mitoxantrone, and exosomes extracted from drugtreated cells have strong antiproliferative activity in the human pancreatic cell line CFPAC-165. Exosomes extracted from human melanoma cell lines transfected with exosomal miR-222 are abundant and result in the repression of the target gene p27Kip1, induction of the PI3K/AKT pathway, and inhibition of malignant melanoma, thus confirming the functional implications in cancer52. Tumor-derived exosomes are used as carriers to deliver drugs and have shown significant anti-tumor effects. Tumor cell-derived exosomes containing endogenous tumor antigens and antitumor immunity are induced by the transfer of tumor antigens to antigen-presenting cells66-67. The appropriate modification can enhance antigen presentation by endogenous autoantigens. For example, GALA (a pH-sensitive fusogenic peptide)-modified exosomes developed by the streptavidin-biotin interaction may be used to control tumor cell-derived exosomes to promote release and intracellular trafficking in an acidic tumor environment to enhance the tumor antigen presentation capacity by major histocompatibility complex (MHC) class I molecules58. Exosomes can also act mediators of intercellular communication between local or distant tumor microenvironments when exosomes are stimulated by hypoxia, which causes an increase in exocrine release in cancer cells. Exosomes boost tumor angiogenesis by maintaining endothelial cells in the local microenvironment or long-range vascular niches. This mode of transport protects the entrapped material from reaching the target cell without degradation and increases the activation of the corresponding signaling pathway. For instance, exosomes derived from the hypoxic


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glioblastoma cell line U87 induce the permeability of the blood–brain barrier by VEGF-A by reducing the expression of claudin-5 and occludin68. In addition to the tumor targeting and strong tumor inhibitory effects, bionic nano-preparations that use exosomes extracted from tumor cells have certain immunotherapeutic effects. Exosomes derived from tumor cells carry MHC-I molecules, tetraspanins, HSP70-80, LAMP1, and tumor rejection antigens, which cause MHC-I-restricted T cell responses and anti-tumor immunity69. Exosomal glycolipid-anchored-IL-12 (GPI-IL-12) significantly enhances the proliferation of T cells and subsequently increases the release of IFN-γ, and exosomal IL-12 could effectively induce antigen-specific cytotoxic T lymphocytes, demonstrating both enhanced immunogenicity and antitumor effects. However, recent studies have indicated that some tumor-derived exosomes may have immunosuppressive properties, promoting tumorigenesis and metastasis70. This property may limit its practical application; accordingly, researchers have focused on exosomes from other sources. 5.2 Cells of the immune system Exosomes extracted from immune cells are not only able to deliver drugs to a specific tumor site with a high rate of tumor inhibition, but also have certain tumor immunotherapeutic effects. The presence of MHC class I, MHC class II, and T cell costimulatory molecules on these exosomes is an important mechanism underlying antigen presentation71. Moreover, exosomes from these sources has not been reported to show the potential to induce tumor proliferation. Therefore, its ability to act as a carrier has received increasingly attractive. 5.2.1 Dendritic cells (DCs)



The surfaces of DC-derived exosomes being used as carriers express the tetraspanin CD9, which may result in membrane binding to target cells and thereby improve the cellular delivery of cargo. The lack of immunostimulatory markers on the surface of DC-derived exosomes, such as CD40, CD86, MHC-I, and MHC-II, can greatly reduce their immunogenicity and can result in anti-tumor immune responses, which is very helpful for the treatment of immunogenic tumors, such as lung cancer, stomach cancer, kidney cancer, and melanoma72. Tian et al. introduced a small αv integrin ligand, iRGD peptide, onto the DC-derived exosome surfaces, which was activated by an exogenous body membrane protein (Lamp2b) to obtain tumor targeting. Then, doxorubicin was loaded into the modified exosomes by electroporation, resulting in good tumor targeting and inhibition of breast cancer without overt toxicity in vivo and in vitro45. Exosomes loaded with αGC and model antigen ovalbumin (OVA) induce potent NK and γδ T-cell innate immune responses and synergistically amplify T- and B-cell responses that are OVA-specific, resulting in decreased melanoma tumor growth and increased antigen-specific CD8þ T-cell tumor infiltration73. Dendritic cells are powerful professional antigen-presenting cells in the body. Notably, the extracted exosomes have been demonstrated significantly enhanced the anti-tumor immune response and achieve good tumortargeting effects, thereby present great application prospects as a carrier for anti-tumor drugs. 5.2.2 Macrophages It has been reported that macrophage-derived exosomes can be loaded with low-molecular-weight chemotherapeutic agents, therapeutic proteins, or brain-derived neurotrophic factor74. Exosomes extracted from RAW 264.7 macrophages serve as carriers of paclitaxel increase drug solubility and overcome Pgp-mediated drug efflux, and paclitaxel-loaded exosomes accumulates in multidrugresistant cancer cells and shows excellent lung metastasis growth inhibition in the lewis lung


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carcinoma mouse model47. This experiment shows the unique achievements of macrophage exosomes in anti-multidrug resistance. The combination of the targeting ability and biocompatibility of an exosome-based drug formulation provides an efficient and novel delivery platform for anticancer therapy. Aminoethyl anisamide is a ligand with high affinity for sigma receptors, modification of polyethylene glycol can reduce the recognition of mononuclear phagocytic system (MPS) and significantly increases the circulation time in vivo. Based on the original paclitaxelloaded exosomes, a paclitaxel-loaded exosome preparation with an aminoethyl anisamidepolyethylene glycol (AA-PEG) carrier moiety has been developed and optimized to target the overexpression of lung cancer cell receptors75. It exhibits a higher load capacity and greater accumulation in cancer cells, resulting in a superior antineoplastic effect compared to that of paclitaxel-loaded exosomes. Behzadi et al. stimulated the J774 cell line with an HSP70-rich WEHI164 cell lysate to obtain Exo-HSP70, and found enhanced cancer immunotherapy by the generation of tumor-specific T cells able to identify and remove malignant cells and successfully inhibit fibrosarcoma52. The exosomes with multiple functions can be easily acquired from macrophages. No evidence has been reported that macrophage-derived exosomes have the risk of stimulating tumor growth and metastasis. 5.2.3 Mesenchymal stem cells Mesenchymal stem cells (MSCs) are generally extracted from the bone marrow, adipose tissue, umbilical cord blood, interdental papilla and other sources, and have increasing attention as potential therapeutic agents with regenerative properties76-78. MSCs may migrate towards inflammatory microenvironments and accumulate in the tumor sites79. Owing to their ability to home in on the tumor microenvironment and to deliver drugs without genetic manipulation, MSCs are considered



to be effective anticancer agent vectors. MSCs can communicate with cancer cells by gap junctional intercellular communication and through exosomes80. MSCs from different tissue sources have the ability to ingest and release drugs without any genetic cell manipulation. BM-MSCs were modified to express the exosomal siGRP78 combined with Sorafenib which could target GRP78 in hepatocellular carcinoma cells to inhibit the growth and invasion of the cancer cells and reverse the drug resistance81. MSCs can release large numbers of exosomes containing functional miRNAs82. An miR-146b expression plasmid was used to transfect MSCs, and exosomal miR-146b significantly reduced glioma xenograft growth in a rat model of primary brain tumor by decreasing EGFR, SMAD4, and NF-κB83.The loaded miR-146b and the miRNA on the surface of the exosomes achieved a multiplier effect. The mechanism by which MSC-extracted exosomes transport molecules into the tumor microenvironment contributes to its use as a carrier. MSCs-derived exosomes have the ability to penetrate the tumor sites based on enhanced permeability and retention (EPR) effect84. Therefore, packaging drugs in exosomes extracted from MSCs and delivery to the tumor site for tumor suppression and targeting is a promising approach. 5.3 Other sources Biomimetic nanostructures based on exosomes from other sources, such as epithelial cells, milk and plant, can also be used as vectors to achieve certain advances in anti-tumorresearch85-86. Epidermal growth factor receptor (EGFR), which is elevated in many epithelial-derived tumors, can serve as a receptor target in cancer drug delivery systems. HEK293 cells have been transfected with miRNA to express the transmembrane domain of the platelet-derived growth factor receptor fused to the GE11 peptide, and the modified let-7a miRNA was delivered to xenografted breast cancer tissues expressing EGFR with good inhibitory effect38. NK cell-derived exosomes carrying the tumor


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suppressor microRNA-186 as a potential therapeutic option alongside NK cell-based immunotherapy lead to inhibition of the tumorigenic potential of neuroblastoma and prevent TGFb1-dependent inhibition of NK cells87. Additionally, recent studies have reported that exosomes extracted from milk have similar nanostructures to those extracted from other sources, with good stability and biocompatibility. They are more stable in an acidic environment and are ideal carriers for drug delivery to tumor sites88. Milk-derived exosomes loaded with celastrol demonstrated enhanced antitumor efficacy as compared to free celastrol against lung cancer cell xenograft and improved poor bioavailability and off-site toxicity issues of celastrol89. 5.4 Summary of exosomes as carriers for anti-tumor research Exosomes extracted from various sources have been useful for tumor treatment, and each has its own characteristics and advantages. If the study involves tumor immunity, immune cells are more recommended. Exogenous DC cell-derived exosomes have weaker and less toxic effects on the body's immune response, but the disadvantage is that the amount of exosomes collected is relatively small, while MSCs and macrophages are secreted in greater quantities. Some experiments have compared the use of exosomes from different sources as carriers to load the same drug. It is found that the amount of exosomes extracted from different sources, drug loading efficiency, cytotoxicity experiments may vary, but there is no significant difference in cell uptake90. Exosomes from different sources may contain different biomolecules to support or antagonize the killing effect of drugs on tumor cells91-92. Its deeper anti-tumor mechanism is worthy of further research. Efforts have been made to modify original exosomes for improved tumor targeting (Figure 5). The results of these studies are quite exciting and this is a promising area of research on exosome carriers. There



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are many additional examples of exosome carriers used in tumor targeting studies, as summarized in Table 1.

Figure 5. Summary of modifications of exosomes and their targeting effects. Surface modification of exosomal vectors is grouped into four categories: targeting cell adhesion molecules, targeting cellular receptors, enhancing antigen expression, and targeting T cells. Abbreviations: iRGD (CRGDKGPDC), LFA-1 (Lymphocyte functionassociated antigen 1), AA-PEG (aminoethylanisamide-polyethylene glycol), GE11 (amino-acid sequence YHWYGYTPQNVI), RVG (rabies viral glycoprotein), GALA (a pH-sensitive fusogenic peptide), MHC (major histocompatibility complex).

Table 1. Tumor-related studies involving exosomes as gene/drug delivery vehicles.

Exosome

Cell source

Cargo

sources Cancer cells

B16BL6 cells

GALA peptide

Incorporation

Tumor-related

method

treatment

Transfection and

Tumor

incubation

presentation


antigen

References

58

Page 23 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LNCaP,

paclitaxel

PC-3 PCa cell

Incubation and

Prostate cancer, cell

centrifugation

cytotoxicity

Transfection

Melanoma

39

lines Human

miR‑222

melanoma cell

52

malignancy inhibition

lines HeLa,

RAD51 and RAD52

Lipofection and

HT1080 cells

siRNA

electroporation

HeLa,

hydrophobic

Lipofection

B16F10,

photosensitizers

93

Tumor targeting

solid

tumor

54

penetration

CT26 Neuro2A cells

EGFR

Transfection

Tumor cell targeting

94

Human renal

GPI-IL-12

Transfection

Enhances cytotoxicity

95

cancer

cell

of renal carcinoma in

line RC-2 Dendritic cells

mouse

vitro Doxorubicin

Electroporation

immature

Inhibition of tumor

45

growth

dendritic cells BMDCs

iNKT,

Incubation

αGC-OVA Macrophages

RAW264.7

Paclitaxel

of

73

pulmonary

75

Inhibition melanoma growth

Sonication

cells

Tumor

metastases and drug-



resistant

Page 24 of 45

cancer

treatment J774 cell

HSP70

Activation

of

cells

Fibrosarcoma cancer

53

immunotherapy and tumor regression

macrophages

Mesenchymal

MSCs

Acridine Orange

miR146b

stem cells SR4987

Paclitaxel

tumor

96

glioma

83

Incubation and

Melanoma

centrifugation

targeting

Transfection and

Reduce

electroporation

xenograft growth

Incubation and

Strong

transfection

antiproliferative

51

activity on CFPAC-1 MSC,

PLK-1 siRNA

Electroporation

HEK293 Others

HEK293 cells

Treatment of bladder

97

cancer let-7a miRNA

Transfection

38

Hepatocellular carcinoma and breast cancer inhibition

milk

Berry

Solution

and

anthocyanidins

ultracentrifugati

Ovarian

cancer

98

inhibition

on Abbreviations: GALA peptide: a pH-sensitive fusogenic peptide; EGFR: Epidermal growth factor receptor; GPI-IL12: glycolipid-anchored-IL-12; iNKT: Invariant NKT; αGC-OVA: immune cell ligand α-galactosylceramide-


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ovalbumin; HSP70: heat shock protein; SR4987: bone marrow mesenchymal stromal cell line; MSCs: Mesenchymal stem cells; BMDCs: Bone marrow–derived dendritic cell cultures.

6. Clinical Trials A number of clinical studies have examined the use of exosomes in treatment or prognosis, and some preliminary studies of the use of exosomes for drug delivery have been initiated. For example, tumor cell-derived exosomes from cancer patients have been studied in clinical trials of cancer immunotherapy, and exosomes with adjuvants are effective in generating a tumor-specific antitumor cytotoxic CD8+ T cell response99. In another clinical study at the James Graham Brown Cancer Center (NCT01294072), plant-derived exosomes were loaded with low-molecular-weight antiinflammatory curcumin and given orally to patients to treat colorectal cancer. So far, there have been many clinical trials evaluating the applications of exosomes as a therapeutic drug delivery vehicle based on experimental results suggesting potential clinical value99-101. Despite these clinical studies and applications of exosomes for drug delivery, relatively little clinical research has been performed, and this is expected to be a focus of future research. However, clinical applications still face many challenges. For example, exosome extraction methods are time-consuming and labor-intensive. Commercially available exosome isolation kits are now used to separate exosomes by precipitation with a polymer solution. These kits can be used to obtain a large quantity of exosomes, but the purity still needs to be strengthened, and the kits are expensive. Therefore, achieving large-scale production of exosomes for clinical use is challenging102. Plant-derived exosomes sound like a good choice, because plants show the advantage of large-scale production of exosome-like vesicles and have a certain role in anti-tumor



aspect103-104. And they are easier to extract and do not have the disadvantages which produced in animal cells105. Nanoparticle preparations with exosomes as carriers are still expected, but need to fully assess the possible risks of their clinical application. Currently, there are many techniques for direct, effective functionalization and capture of exosomes, such as the use of anchor peptide106, opto-thermophoretic technology31, however, their reliability and feasibility of industrial applications still need further evaluation. In addition, the risks associated with the use of exosomes, such as immunosuppression and reversal of tumorigenesis, are also worth considering and pondering107-108. 7. Discussion Drug delivery systems are becoming more and more sophisticated, but many factors, such as targeting and stability, are still major problems. The use of exosomes as carriers of nanopreparations is a relatively new field of research. Due to their double-membrane vesicle-like structure, exosomes can coat various drugs, genetic substances, and protein substances and can form new hybrid nano-preparations by membrane fusion. In summary, the exosome membrane structure composed of phospholipids, cholesterol, and protein substances, promotes cell exchange and avoids ingestion and early release of the cargo. This feature helps exosomes deliver therapeutic agents to precise sites via specific pathways. Various drug-loading methods have been introduced in this review. The post-loading method and pre-loading method have been used extensively to obtain good experimental results. However, the membrane fusion method of liposome and exosomes, has rarely been reported in the literature. Although membrane fusion is not common in exosomes, membrane fusion has some successful studies in other cell vesicles, so it has a good application prospect as a new drug carrier. Recently,


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fused red blood cell (RBC) membranes and platelet membranes have been used to develop dualmembrane-coated nanoparticles, i.e., nanoparticles coated with RBC-platelet hybrid membranes, which carry the properties of both source cells109. Goh et al. first proposed the fusion of cell-derived nanovesicles with liposomes to form EXOPLEXs with high loading efficiencies of greater than 65% with doxorubicin hydrochloride; better results have been obtained using these fusions than using simple liposomes in vitro110. The high entrapment efficiency and innovation of the nanoparticleexosome hybrids method makes it promising for future research. Tumor-derived exosomes play an important role in tumor progression and metastasis, but at present, there seems to be no clinical treatment based on these exosomes because of studies showing that tumor-derived exosomes have both the effect of inhibiting tumors but also the disadvantage of inducing tumor growth. Cancer cell proliferation, progression and malignant transformation are attributed to tumor-derived exosomes encapsulated microRNA post transcriptionally regulating gene expression in niche cells111. Liang et al. developed a siSphk2-loaded DC/CS nanocarrier that could effectively inhibit the tumorigenic potency of tumor-derived exosomes by effective ablation of exosomal miRNA112. The study proposes a new treatment for cancer by eliminating oncogenic miRNAs in malignant exosomes, which opens up a new path for tumor-derived exosomes. Exosome vectors have many advantages, especially their antineoplastic properties. Exosomes are robust and potent drug/gene delivery vectors that show potential in cancer therapy. Exosomes extracted from different sources have different characteristics, and these differences have important implications for in vitro and in vivo tumor studies. For example, Kim et al. found that cancer-derived exosomes have better cancer cell uptake than that of epithelial cell-derived exosomes, which may be affected by cell-type specific tropism113. According to the different characteristics of the sources,



extracted exosomes can be used as carriers for subsequent experiments. Revealing the sorting and loading mechanisms of exosomes may guide the way for purer exosome cargo, thus avoiding any physiological side effects mediated by the endogenous cargo of particular type of exosomes. These biomimetic nano-formulations have achieved great success with respect to anti- tumor activity, such as tumor targeting, tumor immunotherapy, and inhibition of tumor metastasis. In particular, exosomes can be effectively targeted to tumor lesions due to the cell communication ability and the interactions with receptors to exert the maximal efficacy. 8. Conclusion＆Future Perspective Exosomes are considered promising carriers owing to their cross-talk and transport abilities. Exosomes extracted from various sources have been demonstrated as drug carriers with many advantages, such as good biocompatibility, tumor-targeting, tumor immunotherapy, and inhibition of tumor metastasis. Future studies should focus on the clinical use of exosomes-based antitumor preparations, including reducing the manufacturing costs and in-depth evaluation of security.

Conflicts of interest There are no conflicts of interest to declare. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (81573619, 81673612).


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For Table of Contents Use Only Exosomes as Carriers for Anti-tumor Therapy Yanyan Li, Yongtai Zhang, Zhe Li, Kuan Zhou, Nianping Feng


Exosomes as Carriers for Anti-tumor Therapy | ACS Biomaterials

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