Targeted Exosomes for Drug Delivery: Biomanufacturing,Surface Tagging,and Validation

Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers, such as low productivity and ineffective cancer targeting, need to be overcome before wide clinical applications. The present study aims at creating a new biomanufacturing platform of cancer‐targeted exosomes for drug delivery. Specifically, a scalable, robust, high‐yield, cell line based exosome production process is created in a stirred‐tank bioreactor, and an efficient surface tagging technique is developed to generate monoclonal antibody (mAb)‐exosomes. The in vitro characterization using transmission electron microscopy, NanoSight, and western blotting confirm the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy demonstrate that mAb‐exosomes have strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, romidepsin, a histone deacetylase inhibitor, is loaded into mAb‐exosomes. The in vitro anti‐cancer toxicity study shows high cytotoxicity of mAb‐exosome‐romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validates the cancer targeting specificity, anti‐cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, new techniques enabling targeted exosomes for drug delivery are developed to support large‐scale animal studies and to facilitate the translation from research to clinics.     

Exosomes are secreted at similar densities by M21 and PC3 human cancer cells and show paclitaxel solubility

Exosomes are cell-secreted vesicles less than ≈150 nm in size that contain gene-encoding and gene-silencing RNA and cytosolic proteins with roles in intercellular communication. Interest in the use of exosomes as targeted drug delivery vehicles has grown since it was shown that they can bind specific cells and deliver intact genetic material to the cytosol of target cells. We isolated extracellular vesicles (EVs), consisting of a mixture of exosomes and microvesicles, from prostate (PC3) and melanoma (M21) cancer cell lines using serial ultracentrifugation. Interrogation via western blot analysis confirmed enrichment of CD63, a widely recognized EV surface protein, in the EV pellet from both cell lines. Nanoparticle tracking analysis (NTA) of EV pellets revealed that the two cell lines produced distinct vesicle size profiles in the ≈30 nm to ≈400 nm range. NTA further showed that the fraction of exosomes to all EVs was constant, suggesting cellular mechanisms that control the fraction of secreted vesicles that are exosomes. Transmission electron microscopy (TEM) images of the unmodified PC3 EVs showed vesicles with cup-like (i.e., nanocapsule) and previously unreported prolate morphologies. The observed non-spherical morphologies for dehydrated exosomal vesicles (size ≈30–100 nm) are most likely related to the dense packing of proteins in exosome membranes. Solubility phase diagram data showed that EVs enhanced the solubility of paclitaxel (PTX) in aqueous solution compared to a water-only control. Combined with their inherent targeting and cytosol delivery properties, these findings highlight the potential advantages of using exosomes as chemotherapeutic drug carriers in vivo.     

Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro

Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. The in vivo targets of exosomes are poorly understood; however, they are believed to be important in cell-to-cell communication and may play a prominent role in cancer metastasis. We aimed to elucidate whether cancer derived exosomes can be used as drug delivery vehicles that innately target tumors over normal tissue. Our in vitro results suggest that while there is some specificity towards cancer cells over “immortalized” cells, it is unclear if the difference is sufficient to achieve precise in vivo targeting. Additionally, we found that exosomes associate with their cellular targets to a significantly greater extent (> 10-fold) than liposomes of a similar size. Studies on the association of liposomes mimicking the unique lipid content of exosomes revealed that the lipid composition contributes significantly to cellular adherence/internalization. Cleavage of exosome surface proteins yielded exosomes exhibiting reduced association with their cellular targets, demonstrating the importance of proteins in binding/internalization. Furthermore, although acidic conditions are known to augment the metastatic potential of tumors, we found that cells cultured at low pH released exosomes with significantly less potential for cellular association than cells cultured at physiological pH.     

Exosomal sorting of the cytoplasmic domain of bovine leukemia virus TM Env protein

Exosomes are small membrane vesicles that are released into the extracellular compartment as a consequence of fusion of multivesicular endosomes with the plasma membrane. To unravel the molecular basis of protein sorting into exosomes, we have made a chimeric protein containing the cytosolic domain of the transmembrane subunit of the viral Env protein of BLV and the ectodomain of CD8 (CDTM-BLV-CD8). When expressed in K562 cells known to constitutively secrete exosomes, the chimera was found to be very efficiently targeted to the released vesicles. Very interestingly, the cytosolic domain of the Env protein contains peptide motifs potentially recognized by components of the ESCRT machinery that could be related to chimera sorting into the vesicles. Then, quantifying the chimera secretion, we investigated the site of exosome biogenesis in K562 cells using a pharmacological approach. We present different arguments indicating that CDTM-BLV-CD8-containing exosomes are likely formed from a recycling endosomal/TGN compartment.     

外泌体介导的靶向基因运送及在心血管疾病中的应用

外泌体是体内几乎所有细胞分泌的具有双层脂质膜结构的纳米级小囊泡。外泌体大小均匀,平均直径为40～120 nm,存在于所有体液中。外泌体曾一度被认为是细胞成熟过程中清除废弃细胞器的‘垃圾袋’。但近年研究显示：外泌体含有丰富的来源于‘供体细胞’的信号分子,如蛋白质、DNA、mRNA、miRNA以及lncRNA等。当外泌体与‘受体细胞’融合时,这些信号分子便被运送到‘受体细胞’,从而实现细胞 细胞之间的通讯,影响‘受体细胞’的生理病理过程。虽然外泌体的研究目前主要集中在癌症等疾病的预防、诊断与治疗中,但是越来越多的研究显示,外泌体在心血管系统的生理及病理过程中同样发挥着重要作用。本文将对外泌体的起源、分离与纯化方法及外泌体介导的‘细胞 细胞’之间的通讯机制进行综述,并重点论述利用基因工程技术对外泌体进行靶向运输的方法及靶向外泌体运送在心血管疾病治疗中的应用。     

Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes

To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes-endogenous nano-vesicles that transport RNAs and proteins-can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.     

A novel peptide, THALWHT, for the targeting of human airway epithelia

Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr-His-Ala-Leu-Trp-His-Thr (THALWHT). Subsequent binding studies confirmed that THALWHT-displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non-airway-derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non-viral and viral gene therapy vectors.     

Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73.

Exosomes are membrane vesicles secreted by hematopoietic cells upon fusion of late multivesicular endosomes with the plasma membrane. Dendritic cell (DC)-derived exosomes induce potent antitumor immune responses in mice, resulting in the regression of established tumors (Zitvogel, L., A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, P. Ricciardi-Castagnoli, G. Raposo, and S. Amigorena. 1998. Nat. Med. 4:594-600). To unravel the molecular basis of exosome-induced immune stimulation, we now analyze the regulation of their production during DC maturation and characterize extensively their protein composition by peptide mass mapping. Exosomes contain several cytosolic proteins (including annexin II, heat shock cognate protein hsc73, and heteromeric G protein Gi2alpha), as well as different integral or peripherally associated membrane proteins (major histocompatibility complex class II, Mac-1 integrin, CD9, milk fat globule-EGF-factor VIII [MFG-E8]). MFG-E8, the major exosomal component, binds integrins expressed by DCs and macrophages, suggesting that it may be involved in exosome targeting to these professional antigen-presenting cells. Another exosome component is hsc73, a cytosolic heat shock protein (hsp) also present in DC endocytic compartments. hsc73 was shown to induce antitumor immune responses in vivo, and therefore could be involved in the exosome's potent antitumor effects. Finally, exosome production is downregulated upon DC maturation, indicating that in vivo, exosomes are produced by immature DCs in peripheral tissues. Thus, DC-derived exosomes accumulate a defined subset of cellular proteins reflecting their endosomal biogenesis and accounting for their biological function.     

MHC II in Dendritic Cells is Targeted to Lysosomes or T Cell-Induced Exosomes Via Distinct Multivesicular Body Pathways

Dendritic cells (DCs) express major histocompatibility complex class II (MHC II) to present peptide antigens to T cells. In immature DCs, which bear low cell surface levels of MHC II, peptide-loaded MHC II is ubiquitinated. Ubiquitination drives the endocytosis and sorting of MHC II to the luminal vesicles of multivesicular bodies (MVBs) for lysosomal degradation. Ubiquitination of MHC II is abrogated in activated DCs, resulting in an increased cell surface expression. We here provide evidence for an alternative MVB sorting mechanism for MHC II in antigen-loaded DCs, which is triggered by cognately interacting antigen-specific CD4+ T cells. At these conditions, DCs generate MVBs with MHC II and CD9 carrying luminal vesicles that are secreted as exosomes and transferred to the interacting T cells. Sorting of MHC II into exosomes was, in contrast to lysosomal targeting, independent of MHC II ubiquitination but rather correlated with its incorporation into CD9 containing detergent-resistant membranes. Together, these data indicate two distinct MVB pathways: one for lysosomal targeting and the other for exosome secretion.     

综述与专论: 适配体功能化外泌体在肿瘤靶向治疗中的应用

传统的肿瘤治疗方法因缺乏足够的靶向性而会产生严重的毒副作用。外泌体（exosome）是一种天然的纳米囊泡,参与细胞间的信息传递,并且作为药物递送载体具有出色的性能优势,包括低免疫原性、低毒性和能够穿越天然屏障等特点。然而以外泌体为载体的药物递送系统的靶向能力仍有不足。适配体（aptamer）是一类化学合成的单链核酸分子,具有分子质量小、易于修饰和免疫原性低等特点,可作为亲和性配体与靶向分子特异性结合。通过在外泌体表面修饰适配体,药物可以被精确递送到肿瘤细胞发生部位,从而实现对肿瘤的靶向治疗,提高肿瘤治疗效果,减少毒副作用。本篇综述将重点讨论适配体功能化外泌体药物靶向递送系统在各种肿瘤治疗方面的应用,并对其未来的挑战和机遇进行阐述。     

Cell to Cell Signalling via Exosomes Through esRNA

Exosomes are small vesicles of endosomal origin that can be released by many different cells to the microenvironment. Exosomes have been shown to participate in the immune system, by mediating antigen presentation. We have recently shown the presence of both mRNA and microRNA in exosomes, specifically in exosomes derived from mast cells. This RNA can be transferred between one mast cell to another, most likely through fusion of the exosome to the recipient cell membrane. The delivered RNA is functional, as the mRNA can lead to translation of new proteins in a recipient cell. The RNA shuttled between cells via exosomes is called esRNA. We propose that several types of exosomes may exist, and that an additional function of exosomes is to communicate to neighboring cells through delivery of RNA-signals.     

Towards analyzing the potential of exosomes to deliver microRNA therapeutics

Exosome selectivity mechanisms underlying exosome–target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigation towards establishing functional exosome‐based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC‐5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo‐transmission electron microscopy; (c) cellular uptake studies of rhodamine‐labeled exosomes in normal and cancer cells, providing to exosomes either “autologous” or “heterologous” cellular delivery environments; and (d) loading exogenous Alexa Fluor 488‐labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC‐5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising “bio‐shuttles,” being pharmacologically exploitable in the context of theranostic applications.     

Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles

Dendritic cells constitutively secrete a population of small (50-90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1alpha) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.     

Exosomes for drug delivery — a novel application for the mesenchymal stem cell

Exosomes are the most extensively characterized class of secreted membrane vesicles that carry proteins and RNAs for intercellular communication. They are increasingly seen as possible alternatives to liposomes as drug delivery vehicles. Like liposomes, they could deliver their cargo across the plasma membrane and provide a barrier against premature transformation and elimination. In addition, these naturally-occurring secreted membrane vesicles are less toxic and better tolerated in the body as evidenced by their ubiquitous presence in biological fluids, and have an intrinsic homing ability. They are also amenable to in vivo and in vitro loading of therapeutic agents, and membrane modifications to enhance tissue-specific homing. Here we propose human mesenchymal stem cells as the ideal cell source of exosomes for drug delivery. Mesenchymal stem cell transplantation for various disease indications has been extensively tested and shown to be safe in numerous clinical trials. These cells are also prolific producers of immunologically inert exosomes. Immortalization of these cells does not compromise the quantity or quality of exosome production, thus enabling infinite and reproducible exosome production from a single cell clone.     

Exosomes containing glycoprotein 350 released by EBV-transformed B cells selectively target B cells through CD21 and block EBV infection in vitro

Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes by inward budding of the endosomal limiting membrane. They have immune stimulatory-, inhibitory-, or tolerance-inducing effects, depending on their cellular origin, which is why they are investigated for use in vaccine and immune therapeutic strategies. In this study, we explored whether exosomes of different origins and functions can selectively target different immune cells in human peripheral blood. Flow cytometry, confocal laser scanning microscopy, and multispectral imaging flow cytometry (ImageStream) revealed that exosomes derived from human monocyte-derived dendritic cells and breast milk preferably associated with monocytes. In contrast, exosomes from an EBV-transformed B cell line (LCL1) preferentially targeted B cells. This was not observed for an EBV(-) B cell line (BJAB). Electron microscopy, size-distribution analysis (NanoSight), and a cord blood transformation assay excluded the presence of virions in our LCL1 exosome preparations. The interaction between LCL1-derived exosomes and peripheral blood B cells could be blocked efficiently by anti-CD21 or anti-gp350, indicating an interaction between CD21 on B cells and the EBV glycoprotein gp350 on exosomes. The targeting of LCL1-derived exosomes through gp350-CD21 interaction strongly inhibited EBV infection in B cells isolated from umbilical cord blood, suggesting a protective role for exosomes in regulating EBV infection. Our finding also suggests that exosome-based vaccines can be engineered for specific B cell targeting by inducing gp350 expression.     

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.     

Exosome biogenesis,bioactivities and functions as new delivery systems of natural compounds

A rapidly growing body of experimental evidence has begun to shed light on the wide ranging molecular mechanisms which modulate intra- and inter-cellular communications. A substantial quantity of the available knowledge has only been uncovered in recent years, and we are learning that donor cells release nanovesicles, known as exosomes, which regulate the cellular behavior of recipient cells following uptake. Based on the impressive capacity of exosomes in delivering their “payload”, different therapeutic agents, are currently being tested using this delivery method for more effective therapy. This review summarizes the most recent developments in exosome bioactivities and discusses the biochemical nature of exosomes and their biogenesis. It also summarizes the use of exosomes as delivery vehicles for drugs and natural compounds to the targeted site.     

Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides

The protein invasin expressed on the cell surface of the pathogenic bacteria Yersinia pseudotuberculosis mediates the entry of this bacterium into cultured mammalian cells. We have developed a system for expression of random peptides on the cell surface of Escherichia coli (E. coli) by creation of a fusion hybrid between a peptide and the invasin protein. The fusion protein constructs consist of part of the outer membrane domain of the invasin protein, six proline spacers, and a decamer of random peptides flanked by cysteine residues (CX(10)C). Peptides were constitutively expressed on the cell surface in the resulting random decamer peptide library, which we designated as ESPEL (E. coli Surface Peptide Expression Library). The ESPEL was systematically screened for its binding affinity toward human cultured cells. Several bacterial clones were identified whose binding to human cells was mediated by peptides expressed on the bacterial cell surface. Flow cytometric analysis showed that both the identified bacterial clones and these corresponding chemically synthesized peptides bound to human cells specifically. The techniques described provide a new method that uses E. coli random peptide library to select targeting peptides for mammalian cells without any knowledge of the human cellular receptors.     

Exosome nanotechnology: an emerging paradigm shift in drug delivery: exploitation of exosome nanovesicles for systemic in vivo delivery of RNAi heralds new horizons for drug delivery across biological barriers

The demonstration that dendritic cell (DC)-derived exosomes can be exploited for targeted RNAi delivery to the brain after systemic injection provides the first proof-of-concept for the potential of these naturally occurring vesicles as vehicles of drug delivery. As well as being amenable to existing in vivo targeting strategies already in use for viruses and liposomes, this novel approach offers the added advantages of in vivo safety and low immunogenicity. Fulfilment of the potential of exosome delivery methods warrants a better understanding of their biology, as well as the development of novel production, characterisation, targeting and cargo-loading nanotechnologies. Ultimately, exosome-mediated drug delivery promises to overcome important challenges in the field of therapeutics, such as delivery of drugs across otherwise impermeable biological barriers, such as the blood brain barrier, and using patient-derived tissue as a source of individualised and biocompatible therapeutic drug delivery vehicles.     

Targeting endothelial exosomes for the prevention of cardiovascular disease

Exosomes are small lipid bilayer-enclosed 30–140 nm diameter vesicles formed from endosomes. Exosomes are secreted by various cell types including endothelial cells, immune cells and other cardiovascular tissues, and they can be detected in plasma, urine, cerebrospinal fluid, as well as tissues. Exosomes were initially regarded as a disposal mechanism to discard unwanted materials from cells. Recent studies suggest that exosomes play an important role in mediating of intercellular communication through the delivery and transport of cellular components such as nucleic acids, lipids, and proteins and thus regulate cardiovascular disease. Further, the underlying mechanisms by which abnormally released exosomes promote cardiovascular disease are not well understood. This review highlights recent studies involving endothelial exosomes, gives a brief overview of exosome biogenesis and release, isolation and identification of exosomes, and provides a contemporary understanding of the endothelial exosome pathophysiology and potential therapeutic strategies.