Engineered Extracellular Vesicles as a New Class of Nanomedicine.

Cells release many different types of EVs. In general, these EVs can be classified into two major categories, namely, ectosomes and exosomes. Ectosomes are vesicles generated by the direct outward budding of the plasma membrane. Ectosomes include microvesicles, microparticles, and large vesicles in the size range of ∼50 nm to 1 mm in diameter. On the other hand, exosomes originated from endosomes in the cell and in a size range of ∼40 to 160 nm in diameter. Exosomes are of particular interest because of their generation involves a distinct intracellular process, which is thus tightly regulated and likely determines their composition and functions. 2 , 5 , 6 This Review focuses on exosomes, unless stated otherwise. As mentioned above, the biogenesis of exosomes is linked to endosomal pathways. More specifically, the exosome biogenesis process involves the following steps: (1) Invagination of the plasma membrane forms a cup-shaped structure that includes cell-surface proteins and soluble proteins associated with the extracellular milieu. (2) This leads to de novo formation of an early sorting endosomes (ESE), which may merge with a preexisting ESE. The trans-Golgi network and endoplasmic reticulum can also contribute to the formation of the ESE. (3) ESEs mature into late-sorting endosomes (LSEs) and eventually generate multivesicular bodies (MVBs). This process gives rise to MVBs containing several intraluminal vesicles (ILVs), which are future exosomes. 4) The MVBs can either enter the degradation pathway (usually by fusing with lysosomes or autophagosomes), or fuse with the plasma membrane to release the ILVs as exosomes to the extracellular environment. 7 − 10 Interestingly, recent research has found that autophagy can also lead to exosome secretion, in a process termed secretary autophagy. 11 The exosome biogenesis pathway intersects with other molecular pathways associated with the trafficking of intracellular vesicles. Rab GTPases, a large family of small GTPases, play central roles in ensuring precise transport in these vesicles. 12 The ESCRT (endosomal sorting complexes required for transport) machinery plays a key role in the generation of ILVs in MVBs. 13 The fusion of MVBs with the plasma membrane to release exosomes is mainly mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). 14 Other important molecular players in the exosome biogenesis include Sytenin-1, TSG101 (tumor susceptibility gene 101), ALIX (apoptosis-linked gene 2-interacting protein X), syndecan-1, phospholipids, tetraspanins, ceramides, and sphingomyelinases. 7 − 10 Exosome surface proteins include tetraspanins (e.g., CD63, CD9, and CD81, commonly used as biomarkers of exosomes), integrins, immunomodulatory proteins, and others. Exosomes can contain different types of cell surface proteins, intracellular protein, RNA, DNA, amino acids, and metabolites. 15 , 16 Figure 1 shows a schematic illustration of the biogenesis process and molecular content of exosomes. Biogenesis and identification of exosomes. Reproduced with permission from ref ( 2 ). Copyright 2024 The American Association for the Advancement of Science. The fact that exosomes contain many molecular contents from the source cells gives rise to exosomes’ diverse biological functions. The molecular contents can be further altered by environmental or innate (e.g., oncogenic) changes. 17 , 18 The inherent properties of exosomes in regulating complex intracellular pathways offers their potential utility as therapeutics of a wide range of diseases, such as immunogenic diseases, tissue damage, neurodegenerative diseases, and cancers. 2 For therapeutic purposes, exosomes are sometimes called “cell-free cell therapeutics”, because they can impart the therapeutic functions of the source cells while bypassing the potential problems (e.g., tumorigenesis and mutagenesis risks) associated with using live cells. 19 Exosomes have been used to regulate immune responses. Exosomes from distinct source cells, e.g., immune cells, epithelial cells, and mesenchymal stem cells (MSCs), contain molecular cargos that can influence the activities of recipient cells of both the innate and adaptive immune system. These molecular cargos include but are not limited to miRNAs, antigenic peptides, DNAs inducing cGAS-STING (cyclic GMP-AMP synthase stimulator of interferon genes) signaling, and surface ligands inducing various signaling pathways. For example, exosomes from antigen-presenting cells (APCs) carry p-MHC-II (major histocompatibility complex II with antigenic peptide) and costimulatory signals, and directly present the peptide antigen to specific T cells to induce their activation. 20 MSCs-derived exosomes can suppress the pro-inflammatory M2 macrophage phenotype, suppress pro-inflammatory Th17 cells, and cause T-regulatory cell polarization. 21 , 22 Thus, these exosomes have been explored as immunomodulatory therapeutic for inflammatory diseases, autoimmune diseases and graft-versus-host diseases. 23 − 25 In an asthma mouse model, EVs generated from adipose-derived MSCs (AMSCs) were found to enhance the FoxO1 signaling pathway, by inhibiting miR-183–5p, thereby inhibiting the M1 macrophage marker iNOS (inducible nitric oxide synthase). 26 In a mouse model of bacterial pneumonia and COVID-19 infection, MSCs-derived EVs promoted the polarization of macrophages to an anti-inflammatory M2 phenotype when delivered via inhalation. 27 MSCs-derived EVs reduced the expression of pro-inflammatory cytokines, increased the expression of anti-inflammatory cytokines, and improved pathological scores in acute lung injury. Exosomes have also been used for tissue repair and regeneration of various organs. 19 Exosomes can reduce cell apoptosis and tissue inflammation while increasing cell proliferation, survival, angiogenesis, and lineage specific differentiation. The most popular option as the cell source of exosomes is so far MSCs. Other reported cell sources of exosomes for tissue regeneration include embryonic induced pluripotent and tissue-specific stem cells, primary precursor cells and mature cells from the tissue to be regenerated or from immune/endothelial origin. 28 − 30 For example, intravitreal administration of MSCs-derived EVs 24 h after the ischemic event resulted in improved functional recovery, reduced neuro-inflammation, and decreased apoptosis. 31 In another example, a phase II clinical trial of COVID-19-associated acute respiratory distress syndrome (ARDS) showed that intravenous administration of MSCs-derived EVs significantly reduced the 60 day mortality rate of critical patients. In this trial it was also reported that allogenic MSCs-derived EVs caused no significant increase in treatment-related serious adverse events compared to the placebo. 32 In addition to functioning as therapeutics themselves, EVs have also been explored as delivery carriers for pharmaceuticals. Potentially, EVs can circumvent several key problems associated with synthetic nanoparticles (e.g., liposomes, lipid nanoparticles, and polymer nanoparticles) as drug delivery carriers. Compared with these synthetic nanoparticles, EVs are less immunogenic, less toxic, and are more capable of crossing biological barriers such as the blood-brain barrier and extracellular matrix. 33 The advantages of EVs in low immunogenicity and low toxicity largely originate from the fact that they are derived from biological cells. 34 Interestingly, it has been reported that CD47 on exosomes derived from normal fibroblast-like mesenchymal cells results in a “don’t-eat-me” signal, protecting them from phagocytosis and limiting their clearance from circulation. 35 In another important mechanistic study, it has been shown that the reasons why EVs (from mouse mesenchymal stromal cells) can traverse dense extracellular matrix (with pore sizes smaller than those of EVs) are likely because (1) matrix stress relaxation allows EVs to overcome the confinement, and (2) water permeation through aquaporin-1 mediates the EV deformability. 36 There are many unanswered questions regarding the biology of EVs. For example, the exact roles of EVs in communications between cells within the same organ and between organs remains unclear. Also, it is unclear whether the biogenesis and molecular content of EVs change with age. Answers to these questions would help guide the design of EVs-based therapies.

Many of the beneficial features of EVs for therapeutic efficacy bring challenges to characterization and understanding of how EVs work in a therapeutic context. EVs are heterogeneous, multicomponent, and dynamic. These features render the following analytical techniques particularly useful: dynamic bioimaging (tracking), single-particle/single-molecule/single-cell/single-vesicle analysis, and multiplexed bioanalysis (omics and others). With these techniques, researchers attempt to answer the following fundamental questions: (1) What are the trajectories, modes, and rates of transport of EVs at organ, tissue, cell, and subcellular levels? (2) What are the molecular mechanisms behind these motions? (3) What are the molecular contents of EVs? (4) What are the molecular mechanisms of the therapeutic effects of EVs? (5) What are the molecular mechanisms of the undesired side effects of EVs? Answers to these questions are imperative for guiding the development of EVs (including engineered EVs) in the clinic. Animal imaging is typically used to understand the transport of EVs at the organ and tissue levels. In vivo animal imaging techniques of nanomedicines usually include fluorescence imaging, bioluminescence imaging, magnetic resonance imaging (MRI), X-ray computed tomography (X-ray CT), ultrasound imaging, positron emission tomography (PET), and single-photon emission computed tomography (SPECT). Virtually all of these imaging techniques have been adopted for imaging and tracking EVs in live animals. Each of these techniques has its unique benefits and limitations. For example, fluorescence and bioluminescence imaging are easy to perform and have good spatial resolution; but they do not offer high sensitivity and absolute quantification. On the other end of the spectrum is nuclear imaging techniques such as PET and SPECT. These techniques provide high sensitivity with minimal background noise and ability of absolute quantification; but they are very expensive and their availability is often limited by lack of access to instrument or/and lack of expertise. Bimodal/multimodal imaging strategies have been employed to combine the benefits and overcome the limitations. Below are several representative examples from the literature of applying imaging techniques to the study of EVs in live animals. Lai et al. designed a sensitive and versatile probe that enables bimodal EV imaging in live animals, and tracking EV biodistribution and clearance over time ( Figure 6 ). 92 The authors genetically engineered EVs to display a membrane reporter called EV-GlucB, consisting of Gluc (Gaussia luciferase) fused to a biotin acceptor domain, which is metabolically biotinylated when expressed in mammalian cells in the presence of biotin ligase. When incubated with the Gluc substrate coelentrazine (CTZ), these engineered EVs exhibit a strong bioluminescence signal. Furthermore, biotin on the surface allows EVs to be conjugated to any labeled streptavidin, which can then be imaged in vivo using different techniques, including but not limited to fluorescence-mediated tomography (FMT). Ex vivo analysis of tissue, blood, and urine with the Gluc assay permits evaluation of biodistribution and clearance of EVs. The bimodal imaging capacity originates from the combination of Gluc and biotin. Gluc emits flash bioluminescence (480 nm peak); meanwhile biotin allows another imaging modality such as MRI, SPECT/PET, and FMT. Schematic diagram of developing EV-GlucB reporter for in vivo multimodal imaging of EVs. (A) Membrane-bound Gluc (GlucB) or Gluc (control) and the secreted form of humanized bacterial biotin ligase (sshBirA) were delivered via lentivectors to HEK 293T cells for stable expression. (B) Upon expression and EV production by the cells, the sshBirA tags the BAP sequence of GlucB with a single biotin moiety at a specific lysine residue, which is then displayed on the cell surface as well as on the EV surface. EVs were isolated from conditioned medium of cells and injected intravenously (iv) via tail or retro-orbital veins into nude mice for bioluminescence and fluorescence-mediated tomography (FMT) imaging. For bioluminescence imaging, coelentrazine, a Gluc substrate, was iv-administered immediately prior to imaging. For FMT imaging, isolated EVs were conjugated with streptavidin-Alexa680 prior to administration into nude mice. (C) EVs derived from cells synthesizing naturally secreted Gluc were used as controls as Gluc is not present in the EVs. Abbreviations: BAP, biotin acceptor peptide; CMV, cytomegalovirus; GFP, green fluorescent protein; hBirA, humanized biotin ligase; hGluc, humanized Gaussia luciferase; IRES, internal ribosome entry site; SA, streptavidin; ss, signal peptide; TM, transmembrane domain of platelet-derived growth factor receptor. Reproduced with permission from ref ( 92 ). Copyright 2024 American Chemical Society. A PET/MRI platform was reported by Banerjee et al. to track EVs in vivo . 93 Cu 2+ was selected as the radiotracer because it can be produced on a large scale with high specific activity. Cu 2+ was conjugated to EVs in two steps. First, the EV surface was conjugated with the metal chelator 1,4,7,10-tetraazacyclodode-cane-1,4,7,10-tetraacetic acid (DOTA), using the free thiol groups in the EV membrane. Second, DOTA was complexed with Cu 2+ . The stability of the EV-DOTA-Cu complex was evaluated in serum, PBS, and animal blood to be stable for at least 24 h. The potential influence of Cu labeling on the bioactivity of EVs was also examined. It was found that the prosurvival activity of EVs with Cu labeling against ischemic endothelial cells was higher than those without Cu labeling. Nanoparticles have also been loaded to EVs for in vivo imaging, as shown in a study by Betzer et al. 94 For X-ray CT, exosomes were incubated with glucose-coated gold nanoparticles for 3 h at 37 °C, achieving labeling of exosomes with gold nanoparticles (contrast agent for X-ray imaging). In a mouse model of stroke, the gold nanoparticle-labeled exosomes were delivered into the brain by intranasal administration. The gold nanoparticles permitted X-ray CT imaging and tracking of the exosomes in the mouse brain. In vivo CT imaging revealed the selective accumulation of exosomes in the stroke region. A comprehensive comparative study was conducted by Lázaro-Ibáñez et al. to examine whether imaging probes would alter the biodistribution of EVs. 95 The following imaging probes (and the associated imaging modalities) were investigated: a noncovalently labeled fluorescent dye DiR, a covalently labeled nuclear imaging probe 111 Indium-DTPA, a genetically engineered fluorescent label mCherry, and genetically engineered bioluminescent label Firefly (Fluc) and NanoLuc (Nluc) luciferases. It was found that 111 Indium-DTPA and DiR provided the most sensitive in vivo imaging of the EVs. The biodistribution study revealed that NanoLuc fused to CD63 altered the EV biodistribution, resulting in high accumulation in the lungs. Visualizing the behaviors of EVs at the cellular and subcellular level is usually conducted by fluorescence/bioluminescence microscopy. The results in the literature so far appear to indicate that the cellular uptake mechanisms of EVs are usually endocytosis, and a large portion of the endocytosed EVs is colocalized in intracellular vesicles such as lysosomes. 96 − 100 In addition to endocytosis, membrane fusion was also found to be involved in some studies. 101 EVs have been found to cross the blood-brain barrier by transcytosis. 102 More detailed and systematic studies are clearly needed. It is challenging to image both the EVs and their molecular contents (e.g., mRNAs, miRNAs) simultaneously. It is also challenging to image the biological impacts of EVs at the molecular level (e.g., translation of the mRNA cargos of EVs in the recipient cells). Lai et al. developed a genetically engineered probes-based imaging platform technology to address these challenges. 103 To generate fluorescent reporters of EVs (including all the subpopulations), a palmitoylation signal was genetically fused in-frame to the N terminus of fluorescent proteins (EGFP or tdTomato), forming PalmGFP or PalmtdTomato, respectively. The palmitoylation enables association of proteins with cellular membranes and thus all EV membranes. These reporters permitted multiphoton intravital microscopy of the EV dynamics in tumor tissue. It was observed that the lowest EV densities were typically in the areas of the highest tumor cell density at the core of the tumor parenchyma; in contrast, more peripheral regions, where the tumor parenchyma interfaces with the tumor stroma, showed the highest densities of EVs. In order to simultaneously visualize EV-packaged mRNAs and EVs, a dual-function reporter was designed. In this reporter scheme, PalmtdTomato protein was used for EV visualization, while its transcript was tagged with a repeated MS2 RNA binding sequence in the 30 UTR (PalmtdTomato-MS24X) for EV-RNA detection by bacteriophage MS2 coat protein fused with EGFP (MS2CP-GFP) expressed in the same cells. In order to detect and monitor translation of EV-delivered mRNA in parallel with EV uptake, fluorescent (PalmtdTomato) and bioluminescent (EV-GlucB) reporters were multiplexed. If the recipient cells translate EV-delivered GlucB mRNA, an increase in the GlucB signal would be observed, whereas treatment with cycloheximide (a protein translation inhibitor) would prevent GlucB mRNA translation and reduce any increase in the signal. Notably, an increase in the intensity of the GluB signal was observed as early as 1 h post-EV exposure, indicating that translation of EV-delivered GlucB mRNA started shortly after EV uptake by the recipient cells. In an effort to address the challenge of distinguishing EVs-generating cells and EVs-receiving cells, Zomer et al. developed a Cre/LoxP-based imaging scheme, in which EV uptake led to Cre-induced red-to-green color conversion. 104 , 105 This color switch occurred specifically in reporter-expressing cells that take up EVs released from cells expressing Cre recombinase (Cre+ cells). With this EV imaging scheme, the authors were able to identify tumor cells that take up EVs in vitro and in vivo in a mouse model (using intravital fluorescence microscopy). It was found that EVs released by malignant tumor cells were taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carried mRNAs involved in migration and metastasis. It was further shown that the less malignant tumor cells that take up EVs displayed an enhanced migratory behavior and metastatic capacity. CD63 has often been used to label and track exosomes and MVBs. However, it is challenging to observe the fusion events of MVBs with the plasma membrane, because the fluorescence emitted by the endosomes and MVBs labeled by CD63-fluorescent proteins is extremely bright, leading to poor signal-to-noise ratio during the fusion events. To solve this challenge, Sung et al. employed a pH-sensitive GFP derivative, pHluorin, to image the vesicle fusion events. 106 pHluorin is virtually nonfluorescent under acidic conditions but fluoresces at neutral pH, making it an ideal reporter to observe the fusion of acidic late endosomal MVBs with the plasma membrane. The authors developed a pHluorin-tagged CD63 reporter and used it to show that MVB fusion precedes adhesion formation in spreading cells by 1–2 min. Later, the same research group further improved the stability and brightness of pHluorin-CD63 by incorporating a single amino acid mutation, M153R. 107 Using this construct, they were able to track multiple aspects of the exosome lifecycle including MVB motion within cells before fusion, endocytosis of extracellular exosome deposits, and acidification of exosome-containing endocytic compartments. Each EV contains numerous molecular species, and this is a main reason why EV therapy can mimic cell therapy as a conventional drug only contains one (or a very small number of) molecular species. Given this feature of EVs, omics-based analysis has become invaluable for understanding molecular compositions of EVs and molecular mechanisms of the physiological functions of EVs. 108 − 111 Transcriptomics has been widely used in studying EVs, offering comprehensive information about mRNAs, miRNAs, lncRNAs, and circRNAs. Meanwhile, proteomics permits the comprehensive identification and quantification of proteins. Primarily due to the heterogeneity of EVs and limited detection sensitivity of omics, reproducibility (biological reproducibility or technical reproducibility) is currently a major challenge in using omics for studying EVs. 112 , 113 Thus, multiomics analysis and validation by other analytical methods are often needed. 112 , 113 EVs are heterogeneous. Techniques that can analyze single vesicles are thus highly valuable in unraveling the heterogeneity of EVs. Some of these techniques are label-free methods, while others need labeling (e.g., fluorescence labeling) on the vesicles. Label-free methods can circumvent the potentially undesirable influence of labels on EVs, but these methods often produce weak signals. The most commonly used single-vesicle analysis techniques for EVs are nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). In NTA, individual vesicles are detected by scattering the light of a laser beam; the trajectories of diffusing vesicles are tracked and analyzed to yield the hydrodynamic sizes of individual vesicles. In TEM, individual vesicles are directly visualized, producing information about morphology and size. Raman tweezer microspectroscopy (RTM), also known as laser tweezer Raman spectroscopy (LTRS), can be employed to investigate the chemical content of single EVs. RTM utilizes a tightly focused laser beam for both optical trapping of single (or a very small number of) vesicles in water and excitation for subsequent Raman scattering, which offers a vibrational chemical fingerprint from the trapped constituent biomolecules. This technique can be used to analyze both the surface and the interior of single EVs, revealing specific molecular signatures of proteins, lipids, nucleic acids, and carotenoids. 114 , 115 Other less frequently used, label-free methods include atom force microscopy (AFM) and single-particle interferometric reflectance imaging sensor (SP-IRIS). 116 , 117 Label-based methods are dependent on the detection of signals from fluorescent molecules or signal-enhancing nanoparticles. High-resolution flow cytometry (hrFC) has been extensively used to analyze single EVs. This technique permits the quantification of the size distribution and diversity of EV populations by detecting multiparametric scattered light and fluorescence emitted by the labeled EVs. Using antibody-fluorophore conjugates, this technique can be used to profile the protein or nucleic acid content of a EV population at single vesicle level. 118 Fluorescence microscopy has been widely used for localizing fluorescently labeled targets. In particular, total internal reflection fluorescent microscopy (TIRF) is now commonly used for single vesicle analysis. This technique can be used in an aqueous environment to image fluorescent molecules located near a highly refractive solid substance. TIRF allows for fluorescence tracking of single EVs in live cells. It should be noted that in TIRF the fluorophores can be excited only within a few hundred nanometers from the solid substrate. 119 Another particularly useful fluorescence-based imaging technique is fluorescence (or Förster) resonance energy transfer (FRET) imaging. The phenomenon of FRET occurs via resonance energy transfer at distances <10 nm. FRET imaging is capable of producing single-vesicle fluorescence information very fast. This imaging technique offers unique abilities for assessing kinetic and structural dynamics of EVs (e.g., lipid-mixing process). 120 − 122 Super-resolution microscopy (SRM) is a group of advanced microscopy techniques that enables visualization of biological features smaller than the optical diffraction limit (typically ∼250 nm axial and ∼500 nm lateral resolution). This feature is especially useful for imaging single EVs and investigating their biological functions. 123 − 125 Finally, the applications of microfluidics have great potential in single EV analysis. 126 , 127

Modification of the content and structure of EVs, for enhanced or integrated or new functions, can be achieved by many different methods. Here, these methods are broadly categorized into three classes, namely, cell culture condition control, genetic engineering, and chemical engineering. Figure 2 is a schematic summarizing the various methods under the three broad classes. Schematic illustration of the various methods of modifying the content and structure of EVs, for enhanced and integrated and new functions. The content and secretion rate of EVs can be influenced by the external environment of the source cells. Culturing the source cells in stress-inducing conditions, e.g., hypoxia, serum starvation, or inflammation, has been widely used. 37 For example, a short-term hypoxia treatment on MSCs yielded EVs with increased levels of several therapeutically important miRNAs such as miR-103 (with apoptosis inhibition function), miR-210 (with pro-angiogenesis and cardio-protection functions), and miR-17 (with fibrosis inhibition, pro-proliferation and pro-angiogenesis functions). 38 Preconditioning adipose-derived stem cells with endothelial differential medium increased the secretion of microvesicles and enhanced the angiogenic effect of the secreted microvesicles in vitro . 39 Gorgun et al. conducted a study to dissect the effects of preconditioning with inflammatory cytokines and hypoxia on the two different portions of MSC secretome, namely soluble factors and EVs. It was found that the inflammation stimuli strongly inhibited the pro-angiogenic capacity of the soluble factors, but did not significantly affect that of the EV portion. 40 Liao et al. showed that treating MSCs with metformin, a widely used oral antihyperglycemic drug for diabetes, promoted the release of EVs from MSCs. 41 Further mechanistic studies found that the increased EV secretion occurred via an autophagy-related pathway. Proteomics analysis revealed that metformin increased the protein content of EVs involved in cell growth. Yang et al. reported that heat shock treatment on bone marrow MSCs (BMSCs) led to upregulation of the 70 kDa heat shock protein (HSP70) in BMSC-derived exosomes, and alleviation of cisplatin-induced ototoxicity in mice. 42 Multiple reports indicated that 3D culture of the source cells could significantly change the contents (including miRNAs and proteins) and amount of EVs. 43 − 45 The source cells can be genetically modified to produce EVs with a specific content enriched or added. For example, Tao et al. transduced human synovial MSCs with lentivector miR-140–5p. The EVs derived from the thus-formed miR-140–5p-overexpressing MSCs were found to improve cartilage tissue regeneration and prevented osteoarthritis of the knee in a rat model. 46 The content of therapeutic proteins in EVs can be modified in a similar way as for therapeutic miroRNAs. Yu et al. transduced bone marrow MSCs with the protein GATA-4 by a retroviral expression system. GATA-4 is a transcription factor important for the regulation of angiogenesis and cell survival. EVs derived from these GATA-4-overexpressing MSCs were shown to reduce apoptosis, restored cardiac contractile functions and reduced infarct size in a regional myocardial ischemia/infarction rodent model. 47 Yang et al. reported a modified electroporation treatment on the source cells (called “cellular nanoporation method” in the paper). 48 The cellular nanoporation was achieved with a custom-designed biochip, which permits a monolayer of source cells to be cultured above the chip surface containing an array of nanochannels (approximately 500 nm in diameter). These nanochannels enables the passage of focal and transient electric pulses to the source cells, to load nucleic acids (e.g., mRNA and DNA plasmids) into the cells. Remarkably, it was shown that, compared with conventional “bulk” electroporation treatment, the cellular nanoporation method yielded a large increase in production rate of exosomes containing the above-mentioned nucleic acids. Mechanistically, it was found that the increase in exosome biogenesis was the result of a stress response. More specifically, it was caused by thermal shock through increased production of heat-shock proteins via the p53–TSAP6 signaling pathway. The cellular nanoporation method was successfully applied for gliomas treatment 48 and for collagen replacement therapy in small animal models. 49 Fusion of the genetic sequence of a selected protein with that of a protein abundantly present on an EV membrane has become a successful strategy to load the selected protein on an EV membrane. This strategy has been employed to achieve targeted delivery of EVs to difficult-to-reach tissues. 50 − 52 A limitation of this strategy is that usually only a fraction of the EV population is labeled by the selected protein, thus, limiting the targeting ability to just a subset of EVs. To overcome this limitation, Gupta et al. conducted a systematic screening of EV-loading protein moieties, which resulted in optimization of the joint display of two different therapeutically relevant protein receptors on EVs namely cytokine-binding domains derived from tumor necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST). 53 The optimization of surface display of EVs led to enhanced targeting ability and improved therapeutic efficacy of anti-inflammation in three different inflammatory mouse models. In a sophisticated utilization of genetic engineering for EV production, Stranford et al. successfully integrated three complementary functional components into EVs, all by genetic engineering of the source cells of EVs. 54 This work addressed several problems in genome editing of T cells for immunotherapy, including lack of endocytosis in T cells, targeting to T cells, loading of Cas9-based genome editing tools, and release of molecular cargo to cytoplasm of T cells. The authors termed their platform technology GEMINI (genetically encoded multifunctional integrated nanovesicles). In this work, the source cells used for EV production were HEK293FT producer cells; multiple populations of EVs were collected and used for delivery of Cas9 ribonucleoproteins (RNPs) into T cells. GEMINI incorporates the following three main components. First, to enhance targeted binding of EVs with T cells, antibody single-chain variable fragments (scFvs) were genetically engineered on the surface of EVs to facilitate specific interactions with CD2, a costimulatory receptor that is highly expressed on T cells. The authors optimized the coding sequence of the scFv display construct for expression in human cells using a sliding window algorithm. This optimization led to a >100-fold increase in EV binding to CD2+ Jurkat T cells over nontargeted EVs. Second, to both enhance cargo protein loading and increase the likelihood that a given EV will incorporate both a cytosolic cargo protein and the membrane-bound scFv, the authors designed a small molecule dimerization-based loading system. This loading system is based on the plant hormone abscisic acid (ABA)-inducible interaction between truncated versions of the abscisic acid insensitive 1 (ABI) and pyrabactin resistance-like (PYL) proteins. This loading system has several benefits: the association is rapid; the dimerization is reversible, presumably allowing for cargo release in recipient cells; ABA is inexpensive and nontoxic; and small-molecule-regulated loading is readily applicable to biomanufacturing. Third, to enhance EV uptake and cargo release in T cells, fusion proteins were genetically engineered on the EV surface. Two fusion proteins, namely vesicular stomatitis virus glycoprotein (VSV-G) and T cell-specific measles virus glycoproteins hemeagglutinin (H) and fusion (F), showed positive outcome in enhancing EV fusion with T cells. By combining the above three genetic engineering methods, the authors achieved EV-mediated Cas9 editing of the CXCR4 locus (which encodes the HIV coreceptor CXCR4) in primary human T cells. In addition to treating the source cells, EV engineering can also be achieved by treating the EVs after they are generated from the source cells. These methods include loading therapeutic molecules into EVs, loading nanomaterials into EVs, surface modification of EVs, and the encapsulation of EVs into hydrogels. The first report of exogenous drug loading into EVs was published in 2011, in which therapeutic siRNA was loaded by electroporation of exosomes. 55 Delivered by exosomes, the siRNA exhibited enhanced ability of crossing the blood-brain barrier and led to significant downregulation of an Alzheimer-associated gene in a mouse model. Electroporation was later also used for exogenous loading of small molecule drugs into EVs. 56 Fuhrmann et al. performed a systematic study on several different methods of exogenous loading of small molecule drugs, using hydrophobic porphyrins as the model drugs. 57 The examined drug loading methods included electroporation, surfactant, extrusion, and dialysis. These “active loading” methods were found to be up to 11-fold more efficient in drug loading than the passive loading method, i.e., simply incubating the drug with the EVs. Dehghani et al. conducted another systematic study on different methods of exogenous loading of tofacitinib (TFC), a janus kinase (JAK) inhibitor for treating psoriasis (overactive immune system causing skin cells to overgrow). 58 The authors evaluated five different methods to load tofacitinib into exosomes of keratinocytes (the primary cell type found in the epidermis): (i) TFC incubation with donor cells of exosomes, (ii) TFC incubation with exosomes, (iii) freeze–thaw cycles of exosomes, (iv) probe sonication of exosomes, and (v) ultrasonic bath of exosomes. After particle size, zeta potential, drug loading efficiency, and release efficiency were compared, the probe sonication method was selected. The thus-prepared TFC-loaded exosomes showed a significant therapeutic effect in a mouse model of psoriasis. In an effort to treat lung cancer and promote systemic immunity, Liu et al. loaded IL-12 mRNA into human embryonic kidney cell-derived exosomes (HEK-Exo) through electroporation, yielding IL-12-Exo. 59 IL-12 (interleukin-12) stimulates interferon-γ (IFNγ) production, turning “cold” tumors to “hot” ones to augment the cytolytic potentials of immune cells. Liposomes loaded with IL-12 mRNA (IL-12-Lipo) were used as a control for comparison. The authors delivered the exosomes by inhalation into mice with lung tumors. Remarkably, compared with IL-12-Lipo, IL-12-Exo showed superior performance in tumor microenvironment (TME) biodistribution and minimized toxicity. The inhaled IL-12-Exo promoted IFNγ-mediated immune activation, systemic immunity, and immune memory, culminating in lung tumor suppression and heightened resistance against tumor rechallenges. Nanomaterials, such as quantum dots (QDs), magnetic nanoparticles, liposomes, and polymer nanoparticles, possess functional or/and structural properties that can enhance the utility of native EVs. For example, QDs, if incorporated in EVs, can emit bright and stable fluorescence for the long-term tracking of EVs. On the other hand, polyester nanoparticles, with hydrophobic internal structure, can be used to load a large amount of hydrophobic drugs into EVs. Peng et al. reported a multifunctional EVs, which incorporated curcumin-loaded superparamagnetic iron oxide nanoparticles (SPIONs) in MSC-derived exosomes, to treat Parkinson’s disease (PD) in a mouse model. 60 MSC-derived exosomes have inherent ability to treat PD, given their miRNA and protein contents, particularly miR-188–3p, miR-106b, and miR-133b. The magnetism of SPIONs offered two new functions to the exosomes: first, enhanced delivery into the brain guided by a magnet; second, tracking by magnetic resonance imaging (MRI). The curcumin loaded on SPIONs helped PD treatment by reducing α-synuclein aggregates. The curcumin-loaded SPIONs were incorporated into the exosomes by passing both of the SPIONs and exosomes in an extruder ( Figure 3 ). After treatment with these multifunctional EVs, PD model mice showed significant improvement in movement and coordination ability. Multifunctional EVs, composed of MSC-derived exosomes incorporating curcumin-loaded SPIONs, for PD treatment. Reproduced with permission from ref ( 60 ). Copyright 2024 American Chemical Society. Rheumatoid arthritis is an autoimmune disease that involves several factors, namely, immune tolerance breakdown, local synovial inflammation, and tissue destruction, gradually leading to systemic and chronic disability. These factors act cooperatively, creating a vicious cycle. The current clinical treatment, which is based on anti-inflammatory drugs, can relieve only symptoms. MSCs-derived EVs could help to restore normal immune functionality but lack the ability to offer immediate suppression of inflammatory symptoms. Koo et al. developed a hybrid structure to provide both immune function normalization and immediate inflammatory suppression. 61 In this hybrid structure, ceria nanoparticles were conjugated to MSCs-derived vesicles by the thiol-maleimide reaction. The ceria nanoparticles (Ce NPs) can provide immediate inflammatory suppression, due to their ability to remove reactive oxygen species (ROS) by cycling between two reversible ionic states, Ce 3+ and Ce 4+ , to scavenge overproduced ROS in RA-inflicted knee joints, and to induce M1-to-M2 macrophage polarization. In a mouse model of collagen-induced arthritis, this hybrid structure was shown to successfully treat and prevent rheumatoid arthritis by both relieving the main symptoms and restoring the immune system through the induction of regulatory T cells. Liposomes have been integrated with EVs to form hybrid structures in multiple reports. 62 − 64 Sato et al. used a freeze–thaw method to fuse liposomes with EV membrane, forming a hybrid nanoparticle. 63 The EVs were genetically engineered to overexpress HER2. By doing so, the authors showed that genetic modification can be combined with EV membrane engineering for rational design of engineered EVs. Ma et al. utilized dexamethasone-loaded liposomes (Dexlip) to tackle the limitations of MSCs in treating systemic lupus erythematosus (SLE). 64 Dexlip were coincubated with MSCs for 24 h. Dexlip was found to induce a shift in MSCs toward an anti-inflammatory phenotype by triggering the glucocorticoid receptor (GR) signaling pathway. It was further found that these MSCs released exosomes (Dex-MSC-EXOs) with elevated levels of expression of the anti-inflammatory protein CRISPLD2. In a mouse model of SLE, the combined method was shown to be more effective in treating SLE than Dexlip or MSC alone Wang et al. reported chemical conjugation of lung-derived exosomes to a recombinant SARS-CoV-2 receptor-binding domain (RBD), forming an inhalable COVID-19 vaccine. 65 The RBD antigen was first conjugated with (1,2-distearoyl- sn -glycero-3-phosphoethanolamine-poly(ethylene-glycol)- N -hydroxysuccinimide) (DSPE-PEG-NHS) to form RBD-PEG-DSPE. Then, RBD-PEG-DSPE was incubated with exosomes derived from lung spheroid cells (LSCs) for 24 h, resulting in RBD-decorated exosomes. In mice, the vaccine elicited RBD-specific IgG antibodies, mucosal IgA responses, and CD4 + and CD8 + T cells with a Th1-like cytokine expression profile in the animals’ lungs and cleared them of SARS-CoV-2 pseudovirus after a challenge. In hamsters, two doses of the vaccine attenuated severe pneumonia and reduced inflammatory infiltrates after a challenge with live SARS-CoV-2. Chemical conjugation to the functional groups of proteins in the EV membrane is a common strategy used for the surface modification of EVs. However, this strategy is limited by the low density of protein molecules in the EV membrane. Bhatta et al. developed a metabolic glycan tagging approach to address this issue ( Figure 4 ). 66 A common metabolic labeling agent, tetraacetyl N -azidoacetylmannosamine (Ac 4 ManAz), was synthesized and used for metabolic labeling of various types of cells. The cells were treated with Ac 4 ManAz for 3 days and further incubated with DBCO-Cy5 for 30 min. EVs from Ac 4 ManAz-treated cells showed significantly higher Cy5 fluorescence intensity, confirming the successful surface tagging of EVs (with Cy5 as the model here). The authors showed that this chemical tagging approach was applicable for many different cell types including cancer cells, MSCs, dendritic cells, and T cells. They also showed that, in the context of tumor vaccines, toll-like receptor 9 agonists could be conjugated onto EVs, thereby enabling timely activation of dendritic cells and generation of superior antitumor CD8 + T cell response. Metabolic tagging of EVs. Reproduced with permission from ref ( 66 ). Copyright 2024 Springer Nature. In the context of heart repair and regeneration post infarction, in order to improve the targeting ability of MSCs-derived EVs to ischemia-injured myocardium, Zhang et al. modified their surface by membrane fusion with monocyte mimics. 67 Taking advantage of the recruitment feature of monocytes after MI-RI (myocardial ischemia-reperfusion injury), the thus-formed modified EVs exhibited enhanced targeting efficiency to injured myocardium, and improved therapeutic outcome in a mouse MI/RI model An interesting surface engineering method for EVs was reported by Chen et al., who modified the EV surface with spherical nucleic acid (SNA). 68 The authors mixed natural EVs with cholesterol-modified oligonucleotides; driven by hydrophobic interaction, the cholesterol-modified oligonucleotides coassembled with the natural EVs, forming an oligonucleotide shell on the EVs. The thus-formed new nanostructure, called extracellular vesicle spherical nucleic acid (EV-SNA), showed programmability: EV-SNA can respond to AND logic gates to achieve vesicle assembly manipulation. EV-SNA also displayed superior cellular delivery ability compared with liposome SNA. The native environment of EVs is often an extracellular matrix (ECM), which has a hydrogel structure. Cells receive feedback from the ECM and use intracellular processes to regulate the biogenesis of EVs. After secretion, various biochemical and biophysical factors determine whether EVs are locally incorporated in the matrix or transported out of the matrix. In principle, understandings of how EVs and ECM interact could be used to develop engineered hydrogels in which EV production, retention and release can be precisely controlled to yield desirable therapeutic outcomes. 69 Lenzini et al. examined the transport of EVs, in comparison with synthetic nanoparticles, in ECM. It was found that EVs can readily transport through nanoporous ECM, in contrast to synthetic nanoparticles. The mechanisms of this transport phenomenon were revealed to have two aspects. 70 First, matrix stress relaxation allows EVs to overcome the confinement of pores in the ECM. Second, water permeation through aquaporin-1 (AQP1) mediates the deformability of EVs, which further supports EV transport in the ECM. Encapsulating EVs in a hydrogel, forming an EVs-In-Gel structure, can be beneficial for the applications of EVs in several different ways. First, after local injection, EVs are often washed out, resulting in a large loss of the therapeutic agents. Encapsulating EVs in a hydrogel can help to immobilize the EVs in a desired site. 71 − 73 Second, a main limiting factor of the current EV-mediated therapies is the need for the prolonged presence of EVs at a specific location. This translates to multiple injections over the course of several weeks, which not only is disadvantageous in terms of patient compliance but also causes varying concentration over time. Encapsulating EVs in a hydrogel can offer sustained release of EVs, thereby yielding desirable efficacy, minimized side effects and improved patient compliance. 74 − 76 Third, the stability of EVs is of concern; loss of EV functionality occurs fairly quickly upon storage, likely due to membrane instability or degradation of proteins and RNAs in EVs. Encapsulating EVs in hydrogel can enhance the EV stability, as indicated in several reports. 77 − 79 The polymers used for the hydrogel often include natural biopolymers, such as hyaluronic acid, alginate, chitosan, collagen, and amphiphilic peptides. 80 − 88 The considerations for the design of the polymer composition include the biomedical history, degradation, gelation kinetics, mechanical properties, and EV release kinetics. The pore size of a hydrogel is crucial to EV release. Because the diameter of exosomes is between 50 and 150 nm, the hydrogel needs to be nanoporous or microporous to allow efficient exosome release. On the other hand, if the applications of EVs are tissue regeneration, the pores of hydrogel need to be large enough to allow for ingrowth of cells. In general, there are two different ways to encapsulate EVs into a hydrogel, as illustrated in Figure 5 . 74 In the first approach, the polymers and EVs are mixed together; subsequently gelation is initiated by addition of a cross-linker or by activating an external trigger. In the second approach, polymers, cross-linkers and EVs are mixed together in solution, which is injected by a syringe (often a dual chamber syringe) at a specific site (e.g., the site of injury); subsequently in situ gelation occurs. Compared with the first approach, the second approach (injectable gel) is preferred in terms of the noninvasiveness of treatment. 89 The driving forces of cross-linking for injectable gels usually involve intermolecular forces (rather than covalent bonds), including guest–host interactions, ionic interactions, and hydrophobic interactions. Overview of two different general approaches for encapsulating EVs into a hydrogel. (A) EVs and polymers are mixed, after which a cross-linker and/or an external trigger (e.g., heat, UV light) starts the gelation process. (B) Polymers, cross-linker and EVs are added simultaneously in a dual chamber syringe to achieve in situ gelation at the target site. Reproduced with permission from ref ( 74 ). Copyright 2024 Elsevier. In addition to hydrogel encapsulation, other methods have been used to achieve a sustained release of EVs for improved therapeutic outcomes. In one example, Hu et al. coated stents with MSCs-derived exosomes, forming exosomes-eluting stents, to overcome limitations of drug-eluting stents as implants after ischemic injury. 90 Through a series of reactions, the surface of the stents was conjugated with 1,2-distearoyl- sn -glycero-3-phosphoethanolamine (DSPE). Further incubation with exosomes led to insertion of DSPE into the lipid membrane of exosomes via hydrophobic interaction, yielding exosomes-coated stents. The exosomes showed a sustained release profile over several days. The release was accelerated by ROS accumulated in the ischemic site due to a ROS-responsive linker used in the above-mentioned surface conjugation. In rats with renal ischemia-reperfusion injury, compared with drug-eluting stents, the exosome-eluting stents showed more rapid reendothelialization and reduced in-stent restenosis 28 days after implantation. In another example, Bao et al. encapsulated EVs with microcapsules made of the common biodegradable polymer poly(lactic- co -glycolic acid) (PLGA), for treatment of vitreoretinal diseases. 91 In a mouse model of retinal ischemia-reperfusion injury, intravitreal injection of MSCs-exosomes-encapsulated PLGA microcapsules gave rise to prolonged release of exosomes for over one month and restoration of retinal thickness to nearly that of the healthy retina.

Many difficult-to-treat diseases, such as neurological diseases and immunological diseases, lack single targets to effectively treat by conventional drugs. EVs, with a large number of molecular species, could potentially be an ideal solution to these diseases. Furthermore, EVs exhibit an excellent capacity to cross delivery barriers. To realize the full potential of EVs, many techniques have been developed to modify native EVs, forming engineered EVs. These engineered EVs have shown great efficacy and minimal toxicity in treating an array of major diseases, demonstrating potential as both a new class of nanomedicine and a new class of cell therapy. Looking forward, interdisciplinary and collaborative work is needed to tackle challenges in the following aspects to translate this potential to the clinic and industry. First, quality control and scale up of production are challenging for all nanomedicines and especially so for engineered EVs. In addition to the complexity and heterogeneity of engineered EVs, these challenges arise from the fact that the production process involves steps that are difficult to control. In the upstream of the production process, EV biogenesis is difficult to control. In the downstream of the production process, isolation of EVs based on ultracentrifugation (the dominant method used in the current practice) is difficult to control and scale up. Second, clinical translation requires systematic and deep understanding of the transport behaviors and molecular mechanisms underlying the efficacies and toxicities of engineered EVs. Gaining this understanding needs application-specific development of advanced analytical techniques.

As a new class of nanomedicine as well as a new class of cell therapy, EVs (native EVs and engineered EVs) face important challenges in translation to the clinic and industry. A major challenge comes from the manufacturing of EVs on a large scale with satisfactory quality control to meet the needs of clinical trials and industry production. This is due to the inherent complexity of EVs, size heterogeneity, and natural batch-to-batch variations in the production. In the source cell culture step, the methods can include multilayered culture flasks, bioreactors, and hollow fiber cartridges. Small-scale manufacturing can be performed in shake flasks, spinners, roller bottles, wave bags, and bioreactors. Large-scale cell culture can be conducted in stainless steel bioreactors (up to 20,000 L scale), platform-rocker wave bags (up to 500 L scale), or disposable bioreactors (up to 2000 L scale). Genetic drift and contamination need to be monitored closely. In the EV harvesting and isolation step, the commonly used method, namely, ultracentrifugation, poses difficulties for large-scale production. Many other methods are being examined such as tangential flow filtration, size exclusion chromatography, affinity chromatography, and magnetic isolation. In the drug loading step, sonication, surfactant, etc . have been used to facilitate the diffusion of drug molecules into EVs. The EV products are often stored at 4 °C or −80 °C. Lyophilization has been tested for long-term storage of EVs; but its impact on EV integrity needs to be carefully studied. Quality control by characterizing the critical quality attributes (CQAs) is needed. These include but are not limited to viability and surface marker expression of the source cells, quantity, size, and surface marker expression of the EVs, microbial contamination (for example, detection of endotoxin and mycoplasma), and functional activities specific to the applications. Further, for clinical translation of EVs as therapeutics, “biological unknowns” and “pharmacological unknowns” need to addressed. 185 Addressing the biological unknowns includes resolving the functional ambiguity of EV action and elucidating the subpopulations and payloads of EVs. Addressing the pharmacological unknowns includes mapping out the optimal dose, mode of administration, systemic distribution, and pharmacokinetic, pharmacodynamics, and other properties of EVs. To address these issues, developing advanced characterization techniques (e.g., multiomics analysis, single-cell analysis, dynamic imaging, and super-resolution microscopy) for EVs is essential. Finally, standardization efforts are highly important. An example of such efforts is the standardization of EV isolation and characterizations by the International Society for Extracellular Vesicles (ISEV), resulting in the publication of “MISEV” (Minimal information for studies of extracellular vesicles). So far three editions of MISEV have been published (publication year: 2014, 2018, 2023). 186

The therapeutic applications of engineered EVs are being pursued in a large number of diseases and the number is growing. Thus, this section is not meant to be exhaustive but rather aims to provide representative examples, in which key methods and main findings are highlighted. Tables 1 and 2 briefly summarize reported preclinical studies and clinical studies of engineered EVs, respectively. OM: olfactory mucosa; SBC: sodium bicarbonate; hUCBMSCs: human umbilical cord blood-mesenchymal stem cell; PDAC: pancreatic ductal adenocarcinoma; DoxApt: AS1411 aptamer and doxorubicin; AApt-Lips: AS1411 aptamer-conjugated liposomes; HUVECs: Human umbilical vein vascular endothelial cells; HAMA/PVA MNP: hyaluronic acid methacrylate/poly(vinyl alcohol) microneedle patch; HBMECs: human brain microvascular endothelial cells; MSC: mesenchymal stem cell; 6-OHDA: 6-hydroxydopamine; PTX: paclitaxel; DTX: docetaxel; ECM: extracellular matrix; HESC: human embryonic stem cell; ACE2: angiotensin converting enzyme; PM: plasma membrane; CTS: cyptotanshinone; aT: antitumor necrosis factor-α antibodies; CEC: corneal epithelium cell; SPION: superparamagnetic iron oxide nanoparticles; CHIP: carboxyl terminus of Hsc70-interacting protein. Data obtained from ClinicalTrials.gov using “engineered exosome”, “engineering exosome”, and “exosome” as search keyword. The categorization of diseases follows the system by Clinical Trials.gov. ARDS: acute respiratory distress syndrome; FH: familial hypercholesterolemia; IBS: inflammatory bowel disease; N/A: not applicable; LDLR: low density lipoprotein receptor. EVs have been reported to be involved in many physiological and pathological processes in cardiovascular system, such as angiogenesis regulation, blood pressure control, cardiomyocyte hypertrophy, apoptosis, survival, and cardiac fibrosis. 145 In preclinical research, therapeutic potential of EVs has been demonstrated in cardiovascular regeneration and protection. 145 The therapeutic functions of EVs have been enhanced by EV engineering in many studies in the literature. Hypoxia preconditioning of olfactory mucosa MSCs was reported to produce EVs with improved ability of angiogenesis promotion, via enrichment of miR-612. 146 Forced overexpression is another commonly used approach for improving therapeutic efficacy. In one study, forced Tcf21 overexpression suppressed transforming growth factor-β signaling and myofibroblast differentiation, in the context of myocardial fibrosis treatment. 147 In another study, miR-126, as a critical regulator of angiogenesis, was overexpressed to promote vascular endothelial cell repair. 148 Surface modification of EVs with targeting ligands has been used to enhance the targeting ability of EVs for improved efficacy in cardiovascular diseases. For example, Wang et al. engineered exosomes with ischemic myocardium-targeting (IMT) peptide on the surface, and showed that the IMT peptide significantly increased the accumulation of exosomes in ischemic heart area, and improved the therapeutic efficacy. 149 Embedding EVs in hydrogels has been used to enhance the efficacy of treating cardiovascular diseases. For example, Hu et al. found that incorporating MSC-derived exosomes in a class of angiogenin-1 hydrogel stents increased the efficacy, by prolonging the residence time of exosomes at the site of cardiac damage in the ischemic microenvironment. 150 Chen et al. showed that incorporating endothelial progenitor cell-derived EVs into shear-thinning hydrogels improved angiogenesis and promoted function after myocardial infarction, by sustained release of the EVs from the hydrogel. 151 Neuronal cells-derived EVs have been identified as vehicles for transferring misfolded proteins or coding materials between neurons. Transporting these disease-associated cargos may transform healthy cells into dysfunctional cells, contributing to the progression of neurological diseases such as Alzheimer’s dementia (AD), PD, Huntington’s disease, and prion diseases. 152 , 153 Because of the inherent ability of EVs to cross the blood-brain barrier, they have been employed as a delivery vehicle or/and a therapeutic agent to treat diseases in the brain. 50 , 154 For example, EVs isolated from human blood and loaded with dopamine have been shown to deliver dopamine to the central nervous system through the transferrin receptor. 155 EVs derived from murine dendritic cells containing siRNAs targeting α-synuclein have resulted in reduced levels of mRNA and protein of α-synuclein in the brains of mouse models of Huntington’s disease. 156 EVs also showed protective capability for the encapsulated biomolecules, such as enzymes. 157 Surface modification of EVs with the brain-tumor-targeting cyclic RGDyK peptide has been shown to enhance drug delivery across the blood-brain barrier. 158 Finally, MSCs-derived EVs have been shown to promote neuronal repair and regeneration; engineering these EVs (e.g., by loading a drug) could further enhance the functions. 60 A landmark paper of using engineered EVs for cancer treatment was published in 2017. 35 In this article, clinical-grade exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry siRNA or shRNA specific to oncogenic KRASG12D, a common mutation in pancreatic cancer. The engineered exosomes were found to target oncogenic Kras with an enhanced efficacy (compared with liposomes) that is dependent on CD47. In multiple mouse models of pancreatic cancer, the engineered exosome treatment suppressed cancer and significantly improved overall survival. In other studies, chemotherapeutic drugs, sonosensitizers, and photoacoustic imaging agents were loaded to EVs for enhanced anticancer therapeutic efficacy. 159 − 161 It is worth noting that tumor cells-generated exosomes can be a cause of cancer metastasis. 162 − 164 In a recent report, human dermal fibroblasts-derived EVs were loaded with mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1), and induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. 49 In another study, human adipose MSCs-derived EVs were loaded with miR-21–5p, and exhibited potent promotion effect on diabetic wound healing. 165 Similarly, MSCs-derived EVs loaded with pioglitazone promoted collagen deposition and ECM remodeling by regulating P13K/AKT/eNOS pathway and enhancing neovascularization, thereby accelerating wound healing in diabetes. 166 In employing EVs for treating diabetic wound healing, silk fibroin patches 167 or hydrogels 168 − 170 have been utilized for EV immobilization and prolonged release at the wound site. EVs can be functional in immune regulation, likely due to the transfer and presentation of antigenic peptides, delivery of DNA-inducing cGAS-STING (cyclic GMP-AMP synthase stimulator of interferon genes) signaling in recipient cells, gene-expression manipulation by exosomal miRNA, and induction of different signaling pathways by surface ligands present on the EVs. 2 , 171 Engineered EVs have been successfully used to elicit adaptive and innate immune reactions, suggesting their utility as therapeutics. In one example, a hybrid nanoparticle of ceria and vesicles was prepared to modulate both innate and adaptive immunity in a collagen-induced arthritis model. The individual components of the hybrid structure worked synergistically to alleviate inflammation and modulate the tissue environment into an immunotolerant-favorable state, by bridging innate and adaptive immunity. 61 Engineered EVs are being investigated to treat reproductive disorders including premature ovarian insufficiency (POI), polycystic ovarian syndrome (PCOS), recurrent spontaneous abortion (RSA), intrauterine adhesion (IUA), and endometriosis (EMS). 172 The molecular mechanisms of the therapeutic efficacy have been attributed to the molecular cargos of the EVs, including miRNAs, circRNAs, lncRNAs, proteins, and small molecules. 173 − 175 Combining EVs with a scaffold has been shown to enhance the therapeutic functions. In one study, a collagen scaffold was loaded with human umbilical cord-derived MSCs (CS/UC-MSCs) and was applied for treating IUA. CS/UC-MSCs showed EVs-mediated endometrial regeneration in a model of endometrial damage, by promoting endometrial stromal cell proliferation and apoptosis inhibition. 176 In another study, a collagen scaffold was laden with MSCs-derived exosomes for treating IUA. In a rat endometrium-damage model, the treatment was found to promote endometrium regeneration and restore fertility through macrophage immunomodulation. 177 Multiple studies have been reported on applying engineered EVs for treating respiratory diseases. In one study, Tu et al. constructed an engineered EV system by loading miR-511–3p into exosomes derived from HEK293T cells, and then decorating the exosome surface with the RNA nanoparticle PRNA-3WJ (three-way junction of the bacteriophage phi29 motor packaging RNA) with mannose surface modification. 178 The mannose decorated EVs were designed to target macrophages through the mannose receptor Mrc1. In a mouse model of asthma, intratracheal inhalation of these engineered EVs effectively penetrated the airway mucus barrier, delivered functional miR-511–3p to lung macrophages, and successfully reversed the increased airway inflammation. Complement C3 (C3) was identified as a major target of miR-511–3p. In another study, Xie et al. developed an engineered EV system as a COVID-19 therapy. 179 By fusing the S-palmitoylation-dependent plasma membrane targeting sequence with ACE2 (a key cell surface receptor interacting with the viral spike protein), we engineered EVs enriched with ACE2 on their surface were engineered. The engineered EVs showed neutralization potency against SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) in human ACE2 transgenic mice and efficiently blocked viral load of SARS-CoV-2, thereby protecting the host against SARS-CoV-2-induced lung inflammation. Engineered EVs have shown remarkable potential in treating ophthalmic diseases, including both anterior (front-of-the-eye) and posterior (back-of-the-eye) diseases. In order to treat corneal damage (a major anterior eye disease), Tang et al. used a thermosensitive chitosan-based hydrogel to encapsulate exosomes generated by induced pluripotent stem cell-derived MSCs (iPSC-MSCs). 180 The hydrogel permitted sustained release of exosomes, which effectively promoted repair of damaged corneal epithelium and stromal layer, downregulating expression of mRNAs coding for the three most enriched collagens in the corneal stroma and reducing scar formation in vivo. Mechanistically, it was found that the therapeutic effect was via miR-432–5p in the exosomes, which suppresses translocation-associated membrane protein 2 (TRAM2), a vital modulator of the collagen biosynthesis in the corneal stromal stem cells, to avert the deposition of extracellular matrix (ECM). In order to treat retinal ischemia-reperfusion injury (IRI) (a major posterior eye disease), Yu et al. studied the effects of exosomes derived from gingival MSCs. 181 Exosomes from gingival MSCs were isolated by ultracentrifugation and were injected into the vitreous of mice. It was found that TNF-α stimulation of the source cells enhanced the neuroprotective effects of exosomes in IRI. Mechanistic studies revealed that the enhancement effect was due to enrichment of miR-21–5p in the exosomes. Increasing evidence in preclinical models demonstrates the potential of using EVs, especially those derived from stem cells, to treat kidney diseases such as acute kidney injury (AKI) and chronic kidney diseases (CKD). 182 Engineered EVs can further augment therapeutic performance, as suggested by multiple reports. Tang et al. developed interleukin-10 (IL-10)-loaded EVs by engineering macrophages for treating ischemic AKI. 183 The utilization of EVs enhanced not only the stability of IL-10, but also the targeting to the kidney due to adhesive components on the EV surface. In a mouse model, the treatment with IL-10-loaded EVs ameliorated renal tubular injury and inflammation caused by ischemia/reperfusion injury, and prevented transition to CKD. In an effort to use hydrogel encapsulation to enhance therapeutic efficacy of EVs, Zhang et al. developed a hydrogel based on RGD (Arg-Gly-Asp) peptide. 184 RGD peptide binds strongly to integrins, which are present on the membrane surface of MSC-derived EVs. The EVs-encapsulated hydrogel increased the retention and stability of the EVs. After intrarenal injection into a mouse AKI model, the EVs-encapsulated hydrogel showed superior performance in rescuing renal function in early stages of AKI and in antifibrosis in chronic stages.

All biological cells release vesicles, namely, extracellular vesicles (EVs). These vesicles, which enclose their molecular contents within membranes, were initially thought to be merely a form of waste of the cells. In 2007, a groundbreaking paper from Jan Lötvall’s group at Göteborg University showed that exosomes, a type of EVs, can transport functional nucleic acids between different cells. 1 Since then, numerous papers have been published demonstrating that this is in fact a general phenomenon. 2 It appears that EVs can work as mediators of communication between cells. Among different types of EVs, exosomes are of particular interest because their cellular generation involves a distinct intracellular regulatory process which likely dictates their composition and functions, after being released into the extracellular environment. 2 , 3 As fundamental research into the biology of EVs continues, efforts to apply EVs as tools to diagnose or treat diseases have also flourished. For disease diagnosis, exosomes are attractive because they could be used as minimally invasive liquid biopsies with the potential for longitudinal sampling to follow disease progression. 2 , 4 For disease treatment, exosomes and other EVs are being investigated as drug delivery vehicles or/and therapeutics themselves. 2 , 5 , 6 This review article focuses on the disease treatment aspect. In the efforts to treat diseases with EVs, a new trend is emerging, i.e., modifying the structures of EVs to enhance certain functions or to integrate with additional functions. The resulting EVs, called engineered EVs here, are analogous to engineered organisms, e.g., genetically modified rice with vitamin enrichment or genetically modified corn with insect resistance. Yet, the sizes of engineered EVs are at nanometer-scale, which are much smaller than those of engineered organisms. Thus, engineered EVs can also be considered as a new class of nanomedicine (nanometer-scale matters as medicine). This article reviews the burgeoning field of engineered EVs as nanomedicine, discussing how to generate engineered EVs, their medical applications, probing their biological fates, and their clinical/industrial translation.