两种人脐带间充质干细胞源外泌体分离方法的比较

目的探索人脐带间充质干细胞(hUMSCs)来源的外泌体的最佳分离条件并对其进行生物学鉴定。方法无血清培养法培养hUMSCs,培养上清中收集外泌体,超高速离心法(ult-exo)和沉淀法分离外泌体(pri-exo),Western Blot检测外泌体标志性蛋白CD63、HSP70、HSP90和阴性对照蛋白TAPA1,透射电镜观察分离所得外泌体生物学形态,高清晰质谱分析外泌体包裹蛋白种类。结果无血清培养法能够在不改变细胞生物学形态的条件下获取hUMSCs源外泌体,透射电镜下可见超高速离心可以获得具有典型双层膜结构的外泌体,大小在100 nm左右,而沉淀法分离所得产物不具有典型的外泌体形态表征,大小在30 nm左右;Western Blot结果表明超高速离心法所得外泌体CD63、HSP70、HSP90均呈现阳性,阴性对照蛋白TAPA1呈现阴性,沉淀法只能检测到HSP90;高清晰质谱检测出超高速离心法分离所得外泌体有169种蛋白,沉淀法有102种,共有蛋白79种。结论超高速离心法和沉淀法所得外泌体在蛋白标志物、生物学形态和内含蛋白种类均有差别,本实验结果提示分离hUMSCs源外泌体超高速离心法优于沉淀法。     

高效包载酪氨酸羟化酶mRNA外泌体的制备

目的：酪氨酸羟化酶(TH)是多巴胺合成的限速酶,其对于帕金森病发生有着重要意义。外泌体是由细胞分泌的直径30～200 nm的微囊泡,被认为是可通过血脑屏障的潜在药物载体。拟利用古菌核糖体蛋白L7Ae和Kt环的结合特性,获得高效包载酪氨酸羟化酶mRNA的外泌体(TH-Kt-Exo),以实现mRNA药物穿越血脑屏障的递送。方法：通过构建带有Kt环的TH mRNA重组质粒,以及外泌体膜蛋白CD63和L7Ae融合表达的重组质粒pCMV-CD63-L7Ae-His,共转染HEK293F细胞,通过超速离心方法收取细胞分泌的外泌体,通过qPCR检测外泌体中TH mRNA的含量并转染受体细胞。结果：与单一酪氨酸羟化酶质粒转染得到的外泌体TH-Exo相比,利用该方法获得的外泌体TH-Kt-Exo在TH mRNA的装载水平上有显著提高。此外,该外泌体能够将其装载的mRNA转运至受体细胞中。结论：通过L7Ae和Kt环的特异性结合,可以有效提高目的mRNA在外泌体中的包载。     

人脐血血浆外泌体提取方法的比较北大核心CSCD

为探寻高效且稳定的提取人脐血血浆外泌体的方法,利用超高速离心法、蔗糖垫密度梯度离心法、改良超速离心法和聚乙二醇(polyethylene glycol, PEG)沉淀法提取人脐血血浆外泌体,并比较4种方法的优劣。利用透射电镜、动态光散射技术观察外泌体的形态、结构及大小;聚氰基丙烯酸正丁酯(bicinchoninic acid, BCA)法测定外泌体蛋白总量;Western blotting检测外泌体表面标志蛋白CD63、HSP70以及外泌体阴性蛋白GM130 (高尔基标志蛋白)的表达。结果表明,与提取外泌体的“金标准”,即超高速离心法相比,蔗糖垫密度梯度离心法稳定性好,获取的外泌体粒径较均一,但操作较复杂,耗时长;改良超速离心法操作较简单,纯度较高;PEG沉淀法提取的外泌体蛋白量最高,操作时间最短,但杂质较多。结果表明,4种方法均能从人脐血血浆中获取外泌体,但在操作时间、纯度、提取量等方面存在一定差异。因此,应根据实验目的和具体要求选择合适的提取人脐血血浆外泌体的方法。     

Muted蛋白介导CD63在嗜铬细胞大致密核粒的定位

大致密核心颗粒(Large dense-core vesicles,LDCVs)是一种溶酶体相关细胞器(Lysosome-related organelles,LROs),在细胞受到刺激时快速释放其内含物,从而调节机体生长发育、物质代谢和能量代谢等,维持机体的稳态。Muted蛋白是溶酶体相关细胞器生物发生复合体-1(Biogenesis of lysosomal organelles complex-1,BLOC-1)的一个亚基,参与调控溶酶体和多种细胞特异性LROs的生物学发生。四联体跨膜蛋白CD63最初被定位在内体-溶酶体系统,后来发现它也参与部分LROs膜的组成。CD63是否存在于LDCVs尚不清楚,其靶向运输过程是否依赖Muted蛋白也不明确。本研究以肾上腺嗜铬细胞为细胞模型,采用荧光共定位、活细胞追踪和密度梯度离心等实验鉴定CD63蛋白为LDCVs的膜组分,并探讨了其生物学功能。活细胞实验显示CD63-YFP特异性定位在NPY-dsRed标记的LDCVs上,并动态参与LDCVs膜的组成;密度梯度离心实验表明高密度区的CD63与LDCVs的标记蛋白VMAT1共同出峰;Muted蛋白缺乏的小鼠(Bloc1s5基因突变)是一种理想的Hermansky-Pudlak综合征(HPS)小鼠模型, 免疫印迹实验显示该突变体小鼠肾上腺组织中CD63蛋白含量明显减少,暗示Muted蛋白可能参与CD63的分选。以上结果表明CD63是LDCVs的膜成分,CD63在胞内的稳态水平依赖于Muted蛋白,为HPS的病理发生机制提供一定的理论依据。     

Muted蛋白介导CD63在嗜铬细胞大致密核心颗粒的定位

大致密核心颗粒(Large dense-core vesicles,LDCVs)是一种溶酶体相关细胞器(Lysosome-related organelles,LROs),在细胞受到刺激时快速释放其内含物,从而调节机体生长发育、物质代谢和能量代谢等,维持机体的稳态。Muted蛋白是溶酶体相关细胞器生物发生复合体-1(Biogenesis of lysosomal organelles complex-1,BLOC-1)的一个亚基,参与调控溶酶体和多种细胞特异性LROs的生物学发生。四联体跨膜蛋白CD63最初被定位在内体-溶酶体系统,后来发现它也参与部分LROs膜的组成。CD63是否存在于LDCVs尚不清楚,其靶向运输过程是否依赖Muted蛋白也不明确。本研究以肾上腺嗜铬细胞为细胞模型,采用荧光共定位、活细胞追踪和密度梯度离心等实验鉴定CD63蛋白为LDCVs的膜组分,并探讨了其生物学功能。活细胞实验显示CD63-YFP特异性定位在NPY-ds Red标记的LDCVs上,并动态参与LDCVs膜的组成;密度梯度离心实验表明高密度区的CD63与LDCVs的标记蛋白VMAT1共同出峰;Muted蛋白缺乏的小鼠(Bloc1s5基因突变)是一种理想的Hermansky-Pudlak综合征(HPS)小鼠模型,免疫印迹实验显示该突变体小鼠肾上腺组织中CD63蛋白含量明显减少,暗示Muted蛋白可能参与CD63的分选。以上结果表明CD63是LDCVs的膜成分,CD63在胞内的稳态水平依赖于Muted蛋白,为HPS的病理发生机制提供一定的理论依据。     

骨髓间充质干细胞外泌体miR-190a-5p通过靶向KLF15抑制肺癌细胞迁移和侵袭

目的:探讨骨髓间充质干细胞(BMSC)来源的外泌体miR-190a-5p对肺癌细胞的影响。方法:通过超速离心获得BMSCs外泌体,透射电镜观察外泌体形态,采用纳米颗粒示踪分析(NTA)检测外泌体粒径,利用Western印迹检测外泌体上的标志蛋白CD63、CD9及HSP70;选取肺癌细胞系A549、LK79、H1975和HCC827,以及人正常上皮细胞BEAS-2B检测对比miR-190a-5p在这些细胞中和BMSCs衍生的外泌体(BMSC-exosome)中的表达量;双萤光素酶报告基因检测验证Krüppel样因子15(KLF15)是否为miR-190a-5p的靶基因;定量PCR(qRT-PCR)和Western印迹检测miR-190a-5p对KLF15的表达调控;Transwell法检测外泌体对肺癌细胞迁移和侵袭的影响。结果:BMSCs外泌体呈圆形,粒径集中在150～200 nm,标志蛋白CD63、CD9及HSP70阳性表达;BMSCs外泌体中miR-190a-5p的相对表达量均高于在4种肺癌细胞及正常肺细胞BEAS-2B中的表达;双萤光素酶报告基因检测KLF15是miR-190a-5p的靶基因;BMSCs外泌体与miR-190a-5p mimics均能使肺癌细胞中的miR-190a-5p含量升高,并抑制KLF15的mRNA和蛋白表达,从而抑制肺癌细胞迁移和侵袭。结论:BMSCs外泌体miR-190a-5p通过下调KLF15抑制肺癌细胞迁移和侵袭,为肺癌的诊断和治疗提供了新的思路。     

近红外荧光蛋白标记乳腺癌细胞外泌体的构建及鉴定

外泌体(Exosomes)是一种大小为30~100 nm的细胞外膜囊泡,与细胞的生物学功能及细胞间的信号传递有着密切的关系,尤其在癌症的诊断及治疗等领域发挥重要作用。为将外泌体更好地应用于乳腺癌肿瘤传递机制的研究,本文首先通过分子克隆手段将近红外荧光蛋白iRFP682基因和外泌体标记蛋白CD63基因克隆到含腺相关病毒(Adeno-associated virus,AAV)末端倒置重复序列(Inverted repeat terminal,ITR)的质粒载体上,构建融合表达近红外荧光蛋白和CD63蛋白的重组真核表达载体。然后再与辅助质粒共转染AAV-293细胞,包装重组腺相关病毒、纯化测量滴度后用于感染乳腺癌细胞,最后通过荧光筛选出稳定表达近红外荧光蛋白的乳腺癌细胞株。通过对乳腺癌稳定株的分离、纯化及鉴定,最终得到一个新型生物标记物：iRFP682标记的乳腺癌细胞来源的外泌体,为后续研究外泌体在乳腺癌肿瘤微环境中的分布及信号传递提供保障。     

不同血清量标本外泌体提取及微小RNA的检测结果比较

本文旨在探讨Qiagen exoRNeasy Serum/Plasma试剂盒提取血清标本中外泌体所需的最适血清量。采用Qiagen exoRNeasy Serum/Plasma 试剂盒分别对250、500、1 000 μL血清中的外泌体进行抽提,使用透射电子显微镜检测分离的外泌体大小和形态,蛋白质免疫印迹法检测外泌体蛋白标记CD63和TSG101的表达,实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测外泌体中微小RNA-122(microRNA-122,miR-122)的表达。结果显示,透射电子显微镜下可见血清外泌体呈圆形或椭圆形,直径30～150 nm,有完整的膜结构。蛋白免疫印迹法检测外泌体CD63和TSG101阳性。实时荧光定量PCR检测慢性乙型肝炎患者250、500、1 000 μL血清外泌体中miR-122表达量,与正常人相比,分别上调22.44、21.48、20.69倍(P＝0.42)。结果提示,在临床血清样本体积有限的情况下,采用 Qiagen exoRNeasy Serum/Plasma 试剂盒提取血清中外泌体,减少血清量至250 μL也可达到所需实验目的。     

外泌体作为药物载体应用及其靶向给药策略

外泌体(exosomes)是一种由活细胞分泌并释放到胞外环境中、大小在60~100 nm的运输膜泡。外泌体以其天然的物质转运特性、相对较小的分子结构和优良的生物相容性,可递送化学药物、蛋白质及肽配基和基因药物等多种药物,在药物载体的领域具有巨大的潜力。该文重点介绍了外泌体运载多种药物的不同应用及靶向给药中三种主要策略和应用以及其他靶向给药的方向及应用,阐述了外泌体作为药物载体的优势和挑战。     

聚乙二醇沉淀剂有效分离尿外泌体

尿外泌体是病毒大小的胞外囊泡,是非侵入性获得肾及泌尿生殖道细胞生理病理信息的重要靶标。聚乙二醇沉淀 剂可经济高效地分离富集血清等外泌体,但未见用于尿外泌体富集的详细报道。本研究采用聚乙二醇沉淀剂分离鉴定尿外泌体,并对其RNA组分进行检测,以期建立一个经济、高效、简便的尿外泌体分离富集方法。采集10例健康志愿者晨尿20 mL,聚乙二醇沉淀剂分离尿外泌体。透射电镜观察到直径30～100 nm双层膜包绕的囊性小泡,中央有直径5～15 nm高电子密度区。Western印迹检测到外泌体标记蛋白CD63、CD9、TSG101、ADAM10和内标蛋白β-肌动蛋白的表达。纳米粒径仪测定粒子直径介于30～130 nm,并可见25.37 nm和95.07 nm二个粒子峰。qRT-PCR扩增得到β-肌动蛋白和RNU6 RNA产物带。上述结果表明,聚乙二醇沉淀剂可分离富集尿外泌体,该法简单、高效,不需要超速离心机等昂贵设备,且采用该法富集到的外泌体可用于后续蛋白质与核酸分析。该方法可望加速液体活检应用,尤其是肾及泌尿生殖道病变的无创检测。     

Heparanase activates the syndecan-syntenin-ALIX exosome pathway

Exosomes are secreted vesicles of endosomal origin involved in signaling processes. We recently showed that the syndecan heparan sulfate proteoglycans control the biogenesis of exosomes through their interaction with syntenin-1 and the endosomal-sorting complex required for transport accessory component ALIX. Here we investigated the role of heparanase, the only mammalian enzyme able to cleave heparan sulfate internally, in the syndecan-syntenin-ALIX exosome biogenesis pathway. We show that heparanase stimulates the exosomal secretion of syntenin-1, syndecan and certain other exosomal cargo, such as CD63, in a concentration-dependent manner. In contrast, exosomal CD9, CD81 and flotillin-1 are not affected. Conversely, reduction of endogenous heparanase reduces the secretion of syntenin-1-containing exosomes. The ability of heparanase to stimulate exosome production depends on syntenin-1 and ALIX. Syndecans, but not glypicans, support exosome biogenesis in heparanase-exposed cells. Finally, heparanase stimulates intraluminal budding of syndecan and syntenin-1 in endosomes, depending on the syntenin-ALIX interaction. Taken together, our findings identify heparanase as a modulator of the syndecan-syntenin-ALIX pathway, fostering endosomal membrane budding and the biogenesis of exosomes by trimming the heparan sulfate chains on syndecans. In addition, our data suggest that this mechanism controls the selection of specific cargo to exosomes.     

Identification of an inhibitory budding signal that blocks the release of HIV particles and exosome/microvesicle proteins

Animal cells bud exosomes and microvesicles (EMVs) from endosome and plasma membranes. The combination of higher-order oligomerization and plasma membrane binding is a positive budding signal that targets diverse proteins into EMVs and retrovirus particles. Here we describe an inhibitory budding signal (IBS) from the human immunodeficiency virus (HIV) Gag protein. This IBS was identified in the spacer peptide 2 (SP2) domain of Gag, is activated by C-terminal exposure of SP2, and mediates the severe budding defect of p6-deficient and PTAP-deficient strains of HIV. This IBS also impairs the budding of CD63 and several other viral and nonviral EMV proteins. The IBS does not prevent cargo delivery to the plasma membrane, a major site of EMV and virus budding. However, the IBS does inhibit an interaction between EMV cargo proteins and VPS4B, a component of the endosomal sorting complexes required for transport (ESCRT) machinery. Taken together, these results demonstrate that inhibitory signals can block protein and virus budding, raise the possibility that the ESCRT machinery plays a role in EMV biogenesis, and shed new light on the role of the p6 domain and PTAP motif in the biogenesis of HIV particles.     

Dynamics of MAL2 during glycosylphosphatidylinositol-anchored protein transcytotic transport to the apical surface of hepatoma HepG2 cells

Delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface takes place by transcytosis in hepatocytes and also probably in epithelial Madin-Darby canine cells. The integral protein MAL2 was demonstrated to be essential for basolateral-to-apical transcytosis in hepatoma HepG2 cells. Reduction of endogenous MAL2 levels impedes cargo delivery to the apical membrane, but, paradoxically, cargo does not accumulate in the subapical compartment where MAL2 predominantly resides but in distant endosome elements. To understand how transcytosis can be apparently mediated at a distance, we have analyzed the dynamics of machinery and cargo by live-cell imaging of MAL2 and transcytosing CD59, a GPI-anchored protein, in HepG2 cells. MAL2 was revealed as being a highly dynamic protein. Soon after basolateral endocytosis of CD59, a fraction of MAL2 redistributed into peripheral vesicular clusters that concentrated CD59 and that were accessible to transferrin (Tf) receptor, a basolateral recycling protein. Following Tf receptor segregation, the clusters fused in a MAL2(+)globular structure and moved toward the apical surface for CD59 delivery. All these processes were impaired in cells with reduced MAL2 content. Other GPI-anchored proteins examined behave similarly. As MAL2 is expressed by many types of epithelia, the sorting events described herein are probably of quite general utility.     

LMP1 association with CD63 in endosomes and secretion via exosomes limits constitutive NF-κB activation

The ubiquitous Epstein Barr virus (EBV) exploits human B-cell development to establish a persistent infection in ～90% of the world population. Constitutive activation of NF-κB by the viral oncogene latent membrane protein 1 (LMP1) has an important role in persistence, but is a risk factor for EBV-associated lymphomas. Here, we demonstrate that endogenous LMP1 escapes degradation upon accumulation within intraluminal vesicles of multivesicular endosomes and secretion via exosomes. LMP1 associates and traffics with the intracellular tetraspanin CD63 into vesicles that lack MHC II and sustain low cholesterol levels, even in 'cholesterol-trapping' conditions. The lipid-raft anchoring sequence FWLY, nor ubiquitylation of the N-terminus, controls LMP1 sorting into exosomes. Rather, C-terminal modifications that retain LMP1 in Golgi compartments preclude assembly within CD63-enriched domains and/or exosomal discharge leading to NF-κB overstimulation. Interference through shRNAs further proved the antagonizing role of CD63 in LMP1-mediated signalling. Thus, LMP1 exploits CD63-enriched microdomains to restrain downstream NF-κB activation by promoting trafficking in the endosomal-exosomal pathway. CD63 is thus a critical mediator of LMP1 function in- and outside-infected (tumour) cells.     

Isolation and proteomic analysis of exosomes secreted by human cancer cells in vitro

Exosomes are 20-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro and in vivo. Since exosomes contain both RNA (mRNA and microRNA) and proteins, which can be transferred to another cell, and be functional in that new environment, these vesicles may be involved in the communication between cells. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo suggest their participation in pathological situations. Our purpose here is to describe methods for the production, purification, and proteomic characterization of exosomes derived from human cancer cells in vitro. Based on exosomes' unique lipidic composition, we have developed the new approach to increase production of exosomes by cells in vitro. Secondly, we have developed quality control by laser correlation spectroscopy for exosomal assays based on the amount of MHC class I and CD63 molecules on their surface. At last, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was used after 2D electrophoresis for the proteomic analysis of exosomes derived from cancer cell lines. This study describes the protein composition of brain tumor cell-derived exosomes in more detail.     

Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.     

Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery

Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting.     

Hydrogel microchip as a tool for studying exosomes in human serum

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.     

Stabilization of Exosome-targeting Peptides via Engineered Glycosylation

Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics.