Mehrnoosh Bahadorani, Mahboobeh Nasiri, Kristen Dellinger, Shyam Aravamudhan, Reza Zadegan

Department of Nanoengineering, Joint School of Nanoscience & Nanoengineering, North Carolina Agriculture and Technical State University, Greensboro, NC, USA

Correspondence: Reza Zadegan, Department of Nanoengineering, Joint School of Nanoscience & Nanoengineering, North Carolina Agriculture and Technical State University, Greensboro, NC, USA, Tel +1(336)285-2857, Email [email protected]

Abstract: Exosomes emerge from endosomal invagination and range in size from 30 to 200 nm. Exosomes contain diverse proteins, lipids, and nucleic acids, which can indicate the state of various physiological and pathological processes. Studies have revealed the remarkable clinical potential of exosomes in diagnosing and prognosing multiple diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions. Exosomes also have the potential to be engineered and deliver their cargo to a specific target. However, further advancements are imperative to optimize exosomes’ diagnostic and therapeutic capabilities for practical implementation in clinical settings. This review highlights exosomes’ diagnostic and therapeutic applications, emphasizing their engineering through simple incubation, biological, and click chemistry techniques. Additionally, the loading of therapeutic agents onto exosomes, utilizing passive and active strategies, and exploring hybrid and artificial exosomes are discussed.

Introduction

Exosomes have gained increasing interest in therapeutic and diagnostic applications. Studies have demonstrated the protection and recovery potential of exosomes in several diseases. For example, the anti-inflammatory and neurogenesis effects of stem cell-derived exosomes in mouse models of traumatic brain injury, stroke, Alzheimer’s disease, and Parkinson’s disease have shown the potential of exosomes as therapeutic nanovesicles (Table 1). The contents of exosomes can affect the host cells since they transport different nucleic acids (DNA, various RNA, miRNA, and siRNA), lipids, and proteins. In addition to using intact exosomes, researchers are attempting to modify them for precise targeting. Exosomes may transport siRNA, miRNA, and chemotherapeutic drugs such as doxorubicin and gene editing systems.1 Wood’s group delivered siRNA to the mouse’s brain through the systemic infusion of exosome-derived dendritic cells, significantly decreasing the BACE1 mRNA and protein.2

Table 1 Using Stem-Cell Derived Exosomes or Engineered Exosomes to Target Neurodegenerative Diseases and Cancers

Table 2 The Lipid Sources for Exosome Engineering

Researchers have made extensive efforts to bring exosomes into clinical settings. Meanwhile, artificial exosome engineering is rapidly advancing, faithfully replicating the functionality of natural exosomes. In this field, modifications can be made to either the surface12–18 or cargo of the exosome.19–24 This engineering can involve modifying the parental cell to produce exosomes tailored for specific cargo loading20–22,24,25 or surface modification,17 or it can be applied directly to the exosome.24–26 Another method for creating artificial exosomes is mixing synthetic liposomes with natural exosomes to produce hybrid exosomes.1,27,28

Numerous exosome-related patents exist, such as WO2015110957A3, in which Joel de Beer et al patented a method using electrostatic interactions to guide targeted interactions between exosomes and liposomes. In this patent, incorporating the pH-sensitive lipids in liposomes prevents the hybridization of the exosome-exosome or liposome-liposome.29 Marban et al (US11660355B2) demonstrated that exosomes from various cell sources can be engineered with reporters, aptamers, or different segments of antibodies for effective tissue targeting. They modified the exosomes with a pegylated phospholipid linked to streptavidin, allowing biotinylated molecules to be added to the surface for improved tissue targeting and signal tracking.30

Noyes et al in Codiak BioSciences company (WO2020191369A1) demonstrated a significant advancement in extracellular vesicle (EV) purification with a large-scale process involving multiple chromatography steps using cation and anion exchange resin chromatography. This method yields highly purified EVs, characterized by reduced protein impurities such as perlecan, agrin, and/or total proteins, resulting in enhanced potency, uniformity, or a combination of both.31

In another patent (US20240075100A1), Choi et al introduced an effective technique for loading proteins into exosomes using an innovative optical reversible protein reaction. Their patent is based on the natural photoreaction of the photoreceptor cryptochrome 2 (CRY2) and CRY-interacting basic-helix-loop-helix 1 (CIB1) protein module, originally found in Arabidopsis thaliana under blue light. By altering CD9 with a shortened version of CIB1 and engineering the protein cargo to contain CRY2, they enabled the interaction to be triggered by blue light, facilitating cargo loading inside the exosome. Switching off the light tended to release the protein cargo.32

In less than a decade, pharmaceutical companies have increasingly invested in exosome research as nucleic acid carriers, potentially replacing the almost four-decade-old viral gene therapy approach.33 In this paper, we provide a comprehensive review of the biomedical applications of exosomes in diagnostics and therapeutics. We also explore different methods for engineering exosomes to enhance cargo loading and targeting capabilities.

Exosome Biogenesis

The process of exosome release from a cell involves three key steps: (i) the generation of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), (ii) the translocation of MVBs to the plasma membrane, and (iii) the fusion of MVBs with the plasma membrane. In the fusion step, cholesterol levels may impact the release of exosomes, as only high-cholesterol MVBs release exosomes (Figure 1). Factors other than cholesterol affecting exosome release include proteins, lipids, and cell types. The endosomal sorting complex required for transport (ESCRT) machinery is vital for exosome biogenesis and cargo loading. ESCRT comprises protein complexes of ESCRT (0, I, II, III) and the AAA ATPase Vps4 complex (AAA stands for ATPases associated with various cellular activities).34 Exosome secretion may be affected when levels of these proteins are reduced or depleted. For example, depletion of Hrs and Tsg101 (tumor susceptibility gene 101) of ESCRT-0 and STAM1 of ESCRT-1 reduces exosome secretion; nevertheless, knockdown of VPS4B (related protein) enhances exosome secretion. The depletion of ALIX, another related protein, reduces exosome secretion and alters its protein loading.34 ALIX is also involved in the transferrin receptor sorting and loading of miRNAs in rat and human liver cells, respectively.35

Figure 1 The biogenesis, contents, and cell uptake methods of the exosome. Exosomes, identified by markers CD63, CD9, and CD81, form when multivesicular bodies (MVBs) fuse with the plasma membrane, encapsulating nucleic acids, proteins, lipids, metabolites, and small molecules. MVBs can either be digested by lysosomes or release exosomes. Once released by donor cells, exosomes are absorbed by recipient cells through various pathways: pinocytosis, clathrin- and caveolin-mediated endocytosis, lipid raft endocytosis, macropinocytosis, or membrane fusion. Within recipient cells, exosome contents are sorted into various compartments or induce specific cellular responses. The figure was drawn using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution (https://creativecommons.org/licenses/by/4.0/).

The ESCRT-independent pathways are also crucial in exosome sorting and loading.35 The ESCRT-independent machinery includes tetraspanins, sphingomyelin, and ceramide,36 as well as lipid rafts.37 The tetraspanins (eg, CD9, CD63, CD81) have four transmembrane domains essential for exosome production and budding. The C-terminal of the CD9 interacts with the cytoskeletal protein (actin) and results in exosome fission from the cell.36 CD63 is involved in exosome packing and releasing since its knockdown decreases exosome release.38 In certain cell lines like Oli-neu cells (oligodendroglial precursor cell line), ceramide has a role in exosome release and promotes membrane budding. However, in bone metastasis cells (PC-3 cells), blocking neutral sphingomyelinase 2 (which produces ceramide from sphingomyelin) did not affect exosome release.34 Ceramides, a type of sphingomyelin, have a cone shape and help with blebbing and multivesicular evagination in the inner leaflet of the plasma membrane. The significance of ceramides in exosome secretion is supported by D609, an inhibitor of sphingomyelin converter that upregulates ceramide by inhibiting its conversion to sphingomyelin.39

Methyl-β-cyclodextrin, a lipid raft inhibitor, shows the role of lipid rafts in the exosome sorting process. Lipid rafts, specialized membrane domains enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol (GPI)-anchored proteins are pivotal in lipid raft-mediated cargo loading into exosomes. Proteins associated with these lipid rafts are effectively sorted into ILVs within MVBs. Shifting membrane proteins from non-lipid raft domains to lipid rafts could potentially enhance their incorporation into ILVs within MVBs. Upon fusion of MVBs with the plasma membrane, exosomes are released into the extracellular space, carrying the sorted cargo molecules. However, disruption of lipid rafts using methyl-β-cyclodextrin impedes the secretion of these proteins through exosomes. In summary, the involvement of lipid rafts in the exosome sorting process is demonstrated by their crucial role in facilitating cargo loading into ILVs, ultimately shaping the composition and function of exosomes.37

Exosome Contents

Exosomes contain a diverse range of biomolecules, which include lipids, proteins, nucleic acids, and other small molecules (Figure 1). The precise content of exosomes can vary depending on the cell type of origin and the physiological state of the parent cell. In the next section, we have outlined the diverse contents of exosomes. We have divided these into three main categories: lipids, proteins, and nucleic acids.

Lipids

Exosomes are composed of and transport different lipids, such as cholesterol, fatty acids, and eicosanoids, along with their related metabolic enzymes. Exosomal compounds may influence lipid transporters, nuclear transcription factors, programmed cell death, inflammatory and immunological responses, and other lipid metabolism-related activities.40 The imbalance in lipid metabolism causes atherosclerosis, cancer, non-alcoholic fatty liver disease, obesity, and Alzheimer’s disease.41 Following ceramide accumulation in neural cells, astrocyte-derived exosomes may promote brain cell death and Alzheimer’s disease. The exosomal miRNA regulates the lipid-metabolism genes like peroxisome proliferators-activated receptor-γ, -α (PPAR-γ and -α). Exosomal miR-155 and miR-27 inhibit the expression of PPAR-γ. Meanwhile, PPAR-γ controls the intake, storage, and metabolism of lipids in the liver.41 Inactivation of PPAR-α by exosomal miRNAs has been proposed to lead to obesity, where systemic injection of the obese mice’s exosome drives lean animals to acquire obesity-like characteristics. Mice with adipose-specific RNA interference develop hypertriglyceridemia, glucose intolerance, and dyslipidemia.42 Lipid metabolism also influences the biological processes of exosomes, such as biosynthesis and interactions with recipient cells. For example, researchers discovered that cholesterol efflux by different transporters affects exosome trafficking. ABCA1 (ATP-binding cassette transporter A1) increases exosome release, whereas SR-B1 (scavenger receptor class B member 1) may restrict exosome absorption in recipient cells.41

Proteins

Exosomal proteins are suitable biomarkers for the diagnosis of various diseases.43 Compared to other components, they are stable, and detecting a small amount of exosomal surface proteins is possible. Exosome tumor markers include epidermal growth factor (EGFR),44 epithelial cell adhesion molecule (EpCAM),45 EphA2 (ephrin type-A receptor 2),46 E cadherin (epithelial cadherin), and PD-1 (Program-death-1).47 Exosome proteins affect cancer angiogenesis, growth and metastasis, and therapeutic resistance. The protein profiles of exosomes can potentially be used as a noninvasive source of biomarkers for cancer diagnosis and therapy.43 However, exosomes can serve as a platform for generating aggregated abnormal proteins like α-synuclein and tau in Parkinson’s and Alzheimer’s, respectively. As reviewed by Howitt and Hill, studies revealed that these proteins can be disseminated to normal brain cells, contributing to the onset and progression of neurodegenerative disorders.48

Enhancing our understanding of protein trafficking within exosomes holds potential for refining diagnostic and therapeutic strategies. Notably, proteomic analyses have demonstrated shifts in the protein composition of exosomes derived from cancer cells. An illustrative case is evident in glioblastoma cells, where proteomics revealed the presence of oncogenic modifications such as the constitutively active expression of EGFR vIII (epidermal growth factor receptor variant III). This phenomenon leads to an exosomal cargo enriched with numerous pro-invasive molecules, underlining the dynamic nature of exosome-mediated protein transport.49

Researchers have demonstrated that Lamp2A plays a pivotal role in sorting proteins into exosomes. Depletion of this protein led to a substantial reduction in exosomal protein content, all of which shared a specific amino acid motif—KFERQ. This finding aligns with a mechanism wherein proteins containing a pentapeptide sequence, which exhibits biochemical similarity to the KFERQ motif, are selectively loaded into a distinct subpopulation of exosomes, contingent upon the presence of the membrane protein LAMP2A.50

Nucleic Acids

Exosomes can contain a variety of nucleic acids, including DNA and RNA. Exosomes can carry viral,51 bacterial,52 and parasite DNA and RNA during infections.53 For example, the DNA of Plasmodium falciparum, the parasite responsible for causing malaria, has been detected in the exosomes of infected red blood cells.54 This DNA facilitates the spread of the parasite to other red blood cells, contributing to the progression of the disease.55 Furthermore, Jeppesen et al showed that exosomes lack double-stranded DNA and incorporated histones.56 So, exosomes cannot transport genomic DNA from the cell’s nucleus, which has crucial implications for understanding exosome activities in many biological processes, such as disease progression and immunological response.56

The RNA content of exosomes comprises various RNAs: rRNA, circular RNA, miRNA, and long non-coding RNA. The miRNA can affect the gene expression of recipient cells, and they can be sorted. The target cells can affect the type of miRNA in the exosome since the knockout of the specific gene in target cells can decrease the miRNA sorting and packaging in the donor cells. Researchers showed that the miRNAs inside the exosome have specific motifs called EXO-motifs. This motif and its associated proteins regulate the loading of miRNA. RNA binding proteins, including Y-box protein 1, annexin A2, hnRNPA2B1, and SYNCRIP, are implicated in the loading of specific miRNAs—miR-144 and miR-233, viral RNA, miR-198, and Exo-motif RNA of GGCU, respectively.37 In addition, a short motif regulates the loading of microRNAs into exosomes, and this short motif is recognized by hnRNPA2B1 (heterogeneous nuclear ribonucleoprotein A2B1).57 Manka et al designed aptamers with lipid raft and exosome binding motifs, termed RAFT and Exo motifs. The researchers meticulously assessed these aptamers’ binding affinities within a liposome-based exosomal model, shedding light on their interactions. They assessed their affinity using a liposome exosomal model. MiRNAs containing RAFT (UUGU, UCCC, CUCC, CCCU) and Exo (GGAG, UGAG, CCCU, UCCU) motifs were encapsulated, suggesting potential for targeted therapeutic RNA transport. These motifs also imply specific lipid raft interactions and functional implications.

Exosome Uptake

Internalization of exosomes may occur via several processes, including membrane fusion, lipid-raft mediated endocytosis, phagocytosis, macropinocytosis, caveolin- mediated and clathrin-mediated endocytosis (Figure 1). Interactions between cells and exosomal proteins and glycoproteins may affect internalization. The uptake of exosomes by recipient cells can influence the function of the cell under both physiological and pathological conditions. Understanding the mechanisms behind this process can help study the various potential applications of this vesicle in cell functioning tests and theragnostic platforms, both diagnostic and therapeutic fields.58

Biomedical Applications of Exosomes

Subsequent investigations into exosomes hold the key to unveiling their substantial potential in translational medicine. This endeavor promises to usher in innovative pathways for advancing robust clinical diagnostics and sets the stage for developing highly effective therapeutic strategies. Coined as “exosome theragnostic”, the utilization of exosomes in these dual capacities underscores their pivotal role in bridging the gap between diagnostics and therapeutics and shapes the future landscape of medical research and application.

Diagnostics

Exosome profiling monitors donor exosomes to prevent fatal organ damage and compensable diagnosis.59 For example, Hu et al clinically detected antibody-mediated heart rejection. The donor exosome’s human leukocyte antibodies identify antibody-mediated rejection.60 Furthermore, exosome biomarkers can potentially detect and monitor pregnancy complications.59 Salomon et al discovered an increase of miR-486-1-5p and miR-486-2-5p in preeclampsia, which is helpful for early detection.61 The miRNAs of the exosome have a critical role in the theragnostic field, both diagnostic biomarkers and treatment strategy.59 For instance, overexpression of miR-9 and miR-124 are the biomarkers of acute ischemic stroke detected in serum exosomes.62 Exosomal DNA with more biological information than circulating DNA is another source of accurate prognosis prediction.59

Neurodegenerative Disorders and Exosome

Detecting neurodegenerative diseases at an early stage can help mitigate their most severe consequences. Hence, finding promising peripheral indicators is crucial. Aside from the results of NMR and mass spectrometric biometabolite research, exosomes may be a feasible option for identifying and treating neurodegenerative disorders.63 Some exosome biomarkers of neurodegenerative diseases include the formation of amyloid-beta, amyloid precursor proteins,64 and lactoferrin65 in Alzheimer’s diseases, aggregation of misfolded alpha-synuclein,66 muted protein deglycase (DJ-1)67 in Parkinson’s disease, lower activity of acetylcholinesterase68 and phosphorylated tau69 in Parkinson diseases, superoxide dismutase 1 (SOD1)70 and tar-DNA binding protein-43 (TDP-43) in Amyotrophic lateral sclerosis71 and mutant huntingtin protein (mHtt) in Huntington disease,72 as well as miRNA changing in all diseases.73 In Alzheimer’s disease, released amyloid-beta binds glycoprotein and glycolipid on the exosome. Lim et al demonstrated a distinct preference for aggregated amyloid-beta (Aβ42) to bind with exosomes. This finding suggests that exosomes are a stronger predictor of disease progression than free amyloid-beta. They innovated a sensitive plasmon nanostructure to detect Aβ42 and CD63 simultaneously in the limited number of 200 exosomes in Alzheimer’s plasma samples.74 The various RNAs in the exosome also have the potential application in monitoring neurodegenerative diseases. Sue et al settled a ceRNA network of mRNA, miRNA, lncRNA, and circRNA, which shows Alzheimer’s progress. They indicated the rising and falling of miR-27a-5p and miR-148a-5p in the exosome of the Alzheimer’s mice model, respectively.75

Cancer Diagnosis and Exosomes

Exosomes are also important in cancer diagnosis. Exosome-mediated interactions between tumor, immunological, and stromal cells in the tumor microenvironment may aid cancer diagnosis. Exosome contents and their signaling abilities impact cancer development by transferring factors that influence growth, transformation, and survival, facilitating communication among cancer cells. Screening for exosomal proteins is a standard method for detecting cancer biomarkers in various malignancies. For instance, caveolin and CD63 of blood exosomes rise in oral cancer, and exosomal CD81 and prostate antigen increase in prostate cancer.76 A particularly significant cancer marker, Glypican 1+ (GPC1+), is prominently present on exosomes released by cancer cells. Despite high costs and potential for false positives in magnetic resonance imaging (MRI), detecting circulating GPC1+ exosomes with mutated KRAS has proven effective in identifying early-stage pancreatic cancer in both mice and humans. This finding underscores the potency of exosomal analysis in achieving accurate cancer diagnosis.77

Moreover, the screening of exosome DNA in pancreatic cancer has shown a higher detection rate of KRAS mutation in the early stages, and it may be a supplemental diagnostic technique.78 Non-coding RNA may be another cancer marker, as the exosomal miRNA-21 and lncRNA-ATB significantly increase in hepatic cancer (hepatocellular carcinoma).79 Malignant forms of ovarian, esophageal, colorectal, liver, and breast cancers express miRNA-21 in blood circulation exosomes.76 In addition, the low miRNA let7 and the large quantity of RNA-binding protein Lin28B released by breast cancer stem cells may cause immunosuppression and lung metastasis.80 According to Yang et al circPSMA1, a circular non-coding RNA in the exosome may be a marker of triple-negative breast cancer (TNBC); circPSMA absorbs and suppresses miR-637, followed by metastasis and immunosuppression.81 Nevertheless, given the limited expression of miRNA, enhancing the sensitivity of detection methods at these minute quantities is crucial to ensure the smooth incorporation of these discoveries into clinical practices.76

Additionally, encapsulating carbon dots inside exosomes represents a promising approach for imaging tumors with high sensitivity, specificity, and biocompatibility. Recent research showed macrophage exosomes coated boron carbon nanodots after a two-hour coincubation at 37°C. The encapsulated boron carbon nanodots in the mouse model demonstrated a remarkable ability to accumulate in glioblastoma tumors and cross the blood-brain barrier. However, it is worth noting that this research also detected nanoparticle signals in other organs, such as the kidney, lung, and liver.82 In this study, the carbon dots lacked specific modifications on the surface for entrapment inside the exosomes. They could be released due to gradient differences following loading and subsequent washing steps. Furthermore, measuring loading and encapsulation efficiency percentages using UV-vis did not guarantee that the signal was confined within the exosome, as it could also be present in the solution due to potential release from the exosome-based on gradient differences.

Exosomes are crucial in transporting disease-specific biomolecules, enabling noninvasive sampling for early diagnostics. Beyond their diagnostic applications, the therapeutic potential of exosomes will be discussed in the following section.

Therapeutic Applications

Studies have investigated exosomes as a drug delivery system for treating neurodegenerative and cardiovascular diseases and cancer. According to Pinnell and Tieu’s review, studies have indicated that assessing α-synuclein levels in both plasma and saliva is a potential marker for Parkinson’s disease diagnosis.83 Targeting α-synuclein mRNA by exosome-loaded antisense oligonucleotide enhanced the movement in the Parkinson’s mice model (α-synuclein A53T).84 Furthermore, catalase-loaded exosomes demonstrated remarkable brain delivery through nasal administration in Parkinson’s mouse models.5 Kojima et al transferred the catalase mRNA in the brain of Parkinson’s mice model by subcutaneous implantation of engineered cell lines, which frequently released exosomes; these exosomes include the small reporter at 3ʹ terminal of CD63, RVG-Lamp2, and catalase mRNA for exosome tracing, brain targeting and anti-inflammatory effects respectively. This study addressed the challenge of rapid exosome removal through intravenous injection.85 In Parkinson’s model mice, dopamine-loaded blood cell-derived exosomes showed more than 15 times greater brain distribution, effectiveness, and less toxicity than free dopamine.86 In neurological applications, intranasal delivery of this nanocarrier is more effective than intravenous, which is cleared in the kidney and liver.6 Exosomes can be the carriers of siRNA to control gene expression. The siRNA delivery and gene silencing to the brain of the Huntington mouse model were significantly improved through engineered exosomes. Dar et al ameliorated siRNA exosome loading by creating an RNA binding system. They incorporated GAPDH (glyceraldehyde-3-phosphate dehydrogenase) with the phosphatidylserine binding domain and the RNA binding protein for attaching to the exosome surface and siRNA binding, respectively. The siRNA loading onto the exosome’s surface indicated a significant decrease in the activity of the Huntington’s gene following injection into mice. This study demonstrated that attaching siRNA to GAPDH protected it from degradation by nucleases in serum.18

Exosomes have several potential benefits over current cancer immunotherapy strategies, including carrying various antigens, activating various immunological pathways, and eliciting long-lasting immune responses. They can deliver tumor-associated antigens (TAAs) to antigen-presenting cells (APCs) like dendritic cells (DCs), which may activate T cells and generate an immune response against cancer cells. Exosomes are isolated from the patient’s tumor cells and then incubated with DCs in vitro. This incubation allows the loading of tumor exosomes onto the DCs, which may result in the presence of TAAs on the DC surface. T lymphocytes are then stimulated to detect and react to TAAs due to this presentation. Besse et al showed the boosting effect of this immunotherapy on the natural killer cells in patients with advanced non-small cell lung cancer (NSCLC). Exosomes are a viable treatment option for cancer patients since they are well-tolerated and have little toxicity.87 Using modified exosomes to transport drugs shows promising results in controlling cancer tumor development. Suppressing CD47 and its linking to SIRP-α on the mast cells helps cancer immunotherapy. Anti-CD47 and anti-SIRP designed exosomes were activated in mice’s acidic tumor microenvironment and reduced tumor growth.88

Exosomes have recently attracted interest as a possible transporter for therapeutic compounds, particularly those in gene editing. Researchers have harnessed these carriers to transport CRISPR Cas9 in various forms, including DNA, RNA, and ribonucleoprotein complexes (RNPs). Kim et al utilized cancer cell-derived exosomes to deliver CRISPR Cas9 plasmids, effectively inhibiting poly ADP-ribose polymerase-1 and inducing apoptosis in ovarian cancer.89 In another study, exosomes from red blood cells served as carriers for the mRNA form of CRISPR Cas9, demonstrating greater efficiency compared to plasmid form in breast cancer cells and leukemia, both in vivo and in vitro.90 Moreover, Wang et al successfully loaded the RNP form of CRISPR Cas9 into exosomes derived from HEK293T cells.91

To enhance the ability of exosomes to deliver large nucleic acids, Lin et al created a hybrid carrier by merging exosomes and liposomes to form a large vector of the gene editing system. To generate sgRNA-loaded exosomes, the researchers first transfected HEK293FT cells with the mRunx2 sgRNA plasmid. Then, the hybrid carrier was formed by incubating the sgRNA-loaded exosomes with liposomes and the dCas9 plasmid with an enhanced green fluorescent protein (EGFP) for 12 hours at 37°C. The generated hybrid exosomes successfully transferred the gene editing system to MSCs while dramatically reducing the targeted gene RNA (mRunx2 gene). This method circumvented the exosome delivery restrictions and increased the efficacy of gene editing in MSCs. These results imply that exosomes might be a potential tool for gene therapy applications when paired with other delivery vehicles.1 In another study, the exosome’s delivery of the gene editing system eliminated the mutant KrasG12D oncogenic allele in pancreatic cancer cells in vitro and in vivo.92

It should be noted that step-by-step exosome journey evaluation improves cargo delivery. The engineering of the natural exosomes helps with targeting and clearance avoidance. For example, polyethylene glycol (PEG), a hydrophilic agent, can reduce circulation clearance and increase cell targeting. Despite its positive role, PEG can decrease cell membrane fusion and endosomal escape. However, sheddable PEGylation may improve cell fusion, cargo delivery, and endosomal escape. In experiments using liposomes, pH-sensitive linkers such as diorthoester, vinyl ether, phosphoramidate, hydrazone, and β-thiopropionate caused detachment of PEG in low pH environments such as tumor microenvironments, endosomes, and lysosomes.93 Along with PEG, overexpression of CD47 on exosomes was shown to reduce macrophage phagocytosis and lengthen the exosome’s circulation time. The incorporation of CD47 on the modified exosome demonstrated the considerable cargo impact (siRNA) on KRAS G12D mRNA and decreased pancreatic tumor growth in the mouse model.14

Furthermore, exosomes showed the ability to deliver miRNA to target cells. Co-delivery of miRNA-21 inhibitor and 5-fluorouracil through engineered exosomes indicated colorectal anticancer efficiency in mice model.94 Since exosomal co-delivery of Everolimus and miR-7-5p improved the anticancer effects of Everolimus in lung cancer (non-small cell).95 In another study, exosomes were decorated with a peptide (KSLSRHDHIHHHC) to target mesenchymal epithelial transition factor (c-MET). Overexpressed c-MET on TNBC intended smart exosomes to deliver doxorubicin with remarkable effectiveness in animal model.96 Nanotechnology and site-specific targeted drug delivery may overcome tumor cells’ immunity, apoptosis, and multidrug resistance.97 Optimizing cell targeting and intracellular location are crucial components of optimal medication delivery in addition to biodistribution.98 Exosomes may become actively targeted carriers due to the surface engineering and cargo loading of exosomes using engineering technologies.

Engineering Exosomes

Exosome engineering involves the manipulation of exosomes to enhance their functionality by loading specific cargo or modifying their surface. Various methods, including biological, chemical, and physical techniques, are employed for creating modified exosomes.99 Biological techniques involve genetic engineering of the exosome-releasing cells. In this approach, the cells are genetically modified to produce exosomes with desired characteristics. Chemical methods rely on the conjugation of various chemical moieties to the surface of exosomes (Figure 2). This surface modification allows for targeted interactions with specific molecules or cells. Physical techniques, such as sonication, electroporation, extrusion, freeze and thaw, cell membrane permeabilization, and hypotonic dialysis, are utilized to load cargo (Figure 2). Researchers are exploring combinations of these engineering strategies to achieve remarkable results. By combining surface modification techniques with the transport of therapeutic compounds like nucleic acids, proteins, and other particles, enhanced therapeutic potential can be achieved.100

Figure 2 Schematic diagram of exosome engineering for cargo loading and surface modification. Techniques include gene editing, passive incubation, surfactant treatment, electroporation, heat shock, sonication, freeze-thaw, extrusion, and dialysis. Liposome hybridization enables surface and cargo loading. Cell extrusion produces biomimetic exosomes. Surface engineering employs non-covalent agents or click chemistry. One part of the figure was drawn using an image from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution (https://creativecommons.org/licenses/by/4.0/).

Engineering Exosome Through Gene Engineering

Utilizing cell synthesis machinery to express the viral or plasmid vectors can result in surface engineering and loading of therapeutic agents in the exosomes. In this method, the structural proteins and lipids of the exosomes can be exploited as a scaffold to fuse site-specific features. Exosome structural proteins, including CD9, CD63, CD81, and Lamp2, assist in fusing surface modifiers and cargo like miRNA and proteins.100

The genetic modification of parent cells might result in the removal or strong expression of CD47 on the surface of exosomes. This modification can have implications for the circulation and lifespan of the engineered exosomes. CD47 plays a crucial role in immune evasion and clearance of exosomes, so altering its expression can affect how long the exosomes remain in circulation.14 In addition, the hampering topology of exosomal structural molecules can be changed. The sequential splicing of the CD63 gene revealed the crucial function of the transmembrane 3 (TM3) segment in exosome membrane integration. This engineered CD63 can be a suitable platform for introducing the site targeted and cargo portions outside and inside exosomes, respectively.17 Exosomal membrane proteins can attach to cargo like reporter proteins,101 DNA vaccination,101 and cytosine phosphate guanine (CpG) motif.102

The surface of exosomes can be engineered by incorporating diverse peptide-based targeting moieties, improving delivery to specific cellular destinations. The fusion peptides can guide exosomes to precise locations within the body. For instance, RVG (rabies viral glycoprotein) allows for brain targeting, RGB for acetylcholine receptor targeting, and M12 for muscle tissues. These fusion strategies can be applied to critical exosomal proteins like CD63, CD81, and Lamp2b, as well as lipid components, ensuring a versatile and practical approach to engineered exosome-based delivery systems.

Exosome Membrane Engineering on Protein Platform

Researchers have used different proteins on the exosome as the platform for engineering their surface. For instance, CD63, CD9, and Lamp2B have been used for exosome engineering.

CD9 as Platform for Exosome Engineering

Yim et al engineered the photo-activated exosomal delivery system. Various proteins, such as the photoreceptor protein CRY2 and its binding protein from the plant Arabidopsis thaliana, were attached to CD9 using a linker of light-controlled protein-protein interaction. The linker induced the binding and releasing of the cargo proteins to the CD9-conjugated exosome in the presence and absence of blue light, respectively. These exosomes efficiently deliver their cargo inside the recipient’s cell cytoplasm in vivo and in vitro. In this study, they transfected HEK293T cells with two vectors: one containing luciferase-mCherry-photoreceptor (CRY2: Cytochrome 2) and the other with CD9-EGFP-photoreceptor interacting protein (CRY interacting protein (CIB1)). The blue light activated the cytochrome receptor and its binding protein, allowing the interested protein to be loaded into the exosome. The researchers demonstrated the efficient transport of various proteins, including mCherry, Bax, a super repressor of the IkB protein, and Cre recombinase13 (Figure 3A).

Figure 3 Engineering of the exosome using its protein platform. (A) The exosomal CD9 protein was modified with Enhanced Green Fluorescent Protein (EGFP) and Cry Interacting Protein (CIBN). This enables attachment to the photoreceptor protein CRY2 under blue light, and release of the photoreceptor and cargo protein in its absence. Reproduced with permission from Yim N, Ryu SW, Choi K, et al. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module. Nature Commun. 2016:9.13 (B) Using the bacteriophage library, the peptide CP05 with the best affinity for CD63 was selected. CP05 was conjugated with the muscle-targeting peptide (M12) and phosphorodiamidate morpholino oligomer (PMO). CP05-M12 and CP05-PMO were attached to exosomes, enabling targeting of dystrophy mice models (mdx). “i.v”. stands for intravenous. From Gao X, Ran N, Dong X, et al. Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy. Sci Transl Med. 2018;10(444). Reprintedwith permission from AAAS.103

CD63 as Platform for Exosome Engineering

Using muscle targeting peptide (M12) on the exosome’s surface showed its cargo’s significant effect (dystrophin correcting oligomer). In this study, Gao et al found the best exosome-capturing peptide CP05 by the bacteriophage library. CP05, with the sequence of CRHSQMTVTSRL, showed high affinity to CD63 on the surface of the exosome. They conjugated CP05 to muscle targeting peptide (CP05-M12). They also conjugated an exon-skipping oligo (phosphorodiamidate morpholino oligomer (PMO)) to CP05 using amid linker (CP05-PMO). The oligo antisense was used to mask exon 23 of the dystrophin protein. The CP05-M12 and CP05-PMO were incubated with exosomes and attached to CD63 on their surface. Following this engineering, dystrophin expression increased 18-fold in dystrophin-deficient mice103 (Figure 3B).

In another study, CD63 was attached to the gene-editing system CRISPR Cas9. Li’s group fused exosomal CD63 to GFP in one plasmid and gene editing components anti-GFP in another. The affinity of GFP and its antibody facilitated the loading of gene editing cargo in the exosome and efficiently edited the red fluorescent reporter gene. In this study, transfected cells produced exosomes in that CD63 linked to Cas9 protein by the GFP and anti-GFP bridge, respectively.104

In a distinct endeavor aiming to overcome challenges in attaching peptides to nucleic acids, Han et al introduced the exosomal anchor aptamer (EAA). EAA efficiently binds to proteins on exosomes from various sources, facilitating the effective loading of different nucleic acid drugs onto them. EAA’s strong affinity for exosomes and its capability to load nucleic acid drugs, such as the thrombin DNA aptamer inhibitor NU172 and Duchenne Muscular Dystrophy (DMD) phosphorodiamidate morpholino oligomers (PMOs), led to enhanced serum stability and therapeutic efficacy in dystrophic mice following systemic administration at low doses. However, this approach needs more deep research and optimizations.105

Lamp2B as Platform for Exosome Engineering

CD107b or Lamp2B (lysosomal associated membrane protein 2b) is another protein for exosome engineering. The ankyrin repeat protein (DARPin) was fused to Lamp2 and used to target HER2+ breast malignancy.12 Another approach to Lamp2B-based targeting of breast cancer cells involves fusing the iRGD peptide (CRGDKGPDC) to Lamp2B. This engineering on immature dendritic cells’ exosome efficiently targeted αv-integrin in MDA-MB 231 cells and delivered Doxorubicin in vitro and in vivo.106

Xing et al also employed Lamp2b to attach a HER2-specific single-chain antibody (P1h3) to exosomes, creating a delivery system for cancer immunotherapy. This system encapsulates GSDMD-N mRNA and incorporates Chlorin e6 (Ce6) and HER2 antibodies. By using puromycin in donor cells, they prevented premature mRNA translation, ensuring the stability of the therapeutic payload until it reached the target HER2-positive breast cancer cells. Upon delivery, sonodynamic therapy activates Ce6, neutralizes puromycin, and initiates mRNA translation. This triggers pyroptosis—an inflammatory form of apoptosis that stimulates a potent immune response, significantly suppressing tumor growth in breast cancer mouse models.24 Notably, Ce6 can induce reactive oxygen species (ROS), inhibit cell proliferation, and cause apoptosis through photodynamic therapy.107

Mentkowski designed cardiosphere-derived cells (CDCs) to secrete exosomes containing Lamp2b coupled to the cardiomyocyte-specific peptide (CMP). However, the highest distribution of these engineered exosomes in the lung, spleen, and liver without heart preference in the mice model indicated the low selectivity and specificity of the designed peptide.108

Fusing an RNA-binding protein (HuR) to Lamp2b is an alternative for a multi-targeted RNA interference approach. Li et al used HuR to target miRNA 155 and showed its reduction both in vitro and in vivo. The engineered exosome may drive RNA into the lysosome, deactivate it, and affect gene expression. The intravenous administration of these exosomes significantly reduced miR-155 in the liver and spleen and relieved liver fibrosis in the mice model (CCl4-induced). In this study, they mitigated the RNA content of the engineered exosome in an acidified medium to provide more free space for the interested RNA targeting.109 In another study, Lamp2b was fused to T7 peptide to target glioblastoma. T7 peptide on this engineered exosome targeted the transferrin receptor-rich-glioblastoma cells in the mice model. Furthermore, it efficiently delivered its cargo, miR21 antisense, which caused miR21 mitigation and reduced tumor size.110

Lamp2b fusion to RVG (rabies viral glycoprotein) peptide is neurotrophic and targets neural cells in vitro and in vivo.2 As RVG-engineered exosomes can also target the acetylcholine receptor, the intramuscular administration of these exosomes is used to deliver miR-29 and relieve muscle atrophy and renal fibrosis in the unilateral ureteral obstruction mice model.111 In another study, the BACE1 siRNA in this RVG-engineered exosome could knock down the BACE1 gene in the Neuro2A cell line and the brain of wild mice. However, it could not target muscle cells through muscle-specific peptide.2 The engineering of the exosomes and cargo loading is challenging since the fusion of different peptides on Lamp2b showed various outcomes; despite the success of the RVG exosomes, Alvarez-Erviti et al reported the weak abilities of the muscle targeting exosomes.2 To address this issue and sustain the surface-engineered peptides from degradation, Hung et al showed that the glycosylation group of targeting peptides plays a protective role against lysosomal degradation and higher delivery efficiency.112 However, the glycosylation motif on the protein platform in another study did not support endosomal escape,112 a substantial barrier to exosome therapy. Aside from using the protein platform for surface engineering, the lipid part of the exosome membrane is an anchor for the fusion of targeting agents.

Exosome Membrane Engineering on Lipid Platforms

The surface phospholipid of exosomes provides an additional platform for engineering exosomes. This anchor can be conjugated to biotinylated polyethylene glycol for various streptavidin probes or targeting agents.15 High concentrations of phosphatidylserine on the exosome membrane and its high affinity for C1C2 domain of the lactadherin has been used to fuse the targeting moieties like EGFR113 and HER2 antibodies.114 Kooijmans et al showed the selective affinity of the engineered exosome toward EGFR + cancer cells (A431 cells) in co-cultured cells (Neuro2A).113 Wang et al also demonstrated the significant result of selective binding of the engineered exosome to HER2+ cells in vitro and in mice models. The engineered HEK293FT cells released exosomes with an enzyme mRNA (HChRr6, an Escherichia coli enzyme). Expression of the enzyme in combination with its substrate (6-chloro-9-nitro-5-oxo-5H-benzo-(a)-phenoxazine (CNOB)) killed the targeted HER2+ cells and retarded the transplanted tumor in mice model.114

Several studies showed the role of GPI in boosting the antigens binding to exosomes.115 For example, stabilizing HER-2 attachment on exosomes through the GPI anchor demonstrated a robust immune response against breast cancer in vaccinated mice.116 In addition, the GPI fusion to IL-12 and anti-EGFR particles and on exosome boosted in vitro T cell proliferation and binding to EGFR-overexpressed tumor cells, respectively.115 In general, the genetic engineering of the parental cells may result in homogeneous vesicles with desired payloads.117 However, despite its effectiveness, the safety of viral and plasmid vectors99 and chemical transfection agents, as well as the lengthy procedures of this approach, pose challenges.100 The numerous challenges include safety concerns, inconsistent transfection effectiveness, potential genetic mutations, and limited targeting precision. Furthermore, this bioengineering process is time-demanding and must be repeated whenever a new molecular target is chosen. Therefore, there is a pressing need for accessible, quicker, more efficient, and expandable techniques to shift these nanovesicles from the lab to clinical settings.

Additionally, researchers have employed “click chemistry” to modify the surfaces of exosomes, using chemical groups like alkyne groups118 and copper-free azide-alkyne cycloaddition.16 Click chemistry offers benefits like a quick process, hydrophilic moieties, and more specific targeting with minimal changes in exosome size and properties.119 This approach allows attaching targeting agents to the exosome surface through chemical modifications involving antibodies, aptamers, and small molecules.

Engineering Exosome Using Covalent Binding (Click Chemistry)

The chemical means comprise covalent and noncovalent adjustments. Targeting specific tissues and cell types may be accomplished by “painting” the surface of exosomal lipids and proteins with various linkers. Exosome functionalization chemistry includes thiol-maleimide, EDC/NHS, azide-alkyne cycloaddition, and amidation. Despite the simplicity and speed of chemical methods, the immuno-compatibility of additional material has limited this application.99 Finding non-toxic reagents without affecting the function and robustness of the vesicles is pivotal.99

The researchers utilized a pH-sensitive linker, azide-dibenzocyclooctyne, to coat macrophage exosomes with antibodies that target CD47 and SIRP. These proteins enable cancer cells to evade the immune system, so targeting them offers considerable potential for cancer therapy. Dibenzocyclooctyne-conjugated antibodies and azido-modified exosomes detached from each other in the tumor microenvironment. The released antibodies effectively suppress CD47 on tumor cells and SIRP on macrophages in vitro and in vivo. As a result, this technique enables active targeting and has great potential for therapeutic benefits.88 Targeting CD47 can cause anemia and blood-related side effects by prompting the body to destroy red blood cells. Hence, it’s important to carefully determine the right timing, dosage, and consider individual health conditions. Additionally, administering erythropoietin alongside the treatment can be beneficial in managing these effects in clinical settings.120

In another study, incubating MDA-MB-231 cells with mannose-azide resulted in the release of azido-functionalized exosomes. These exosomes displayed a reaction propensity toward dibenzocyclooctyne-coupled fluorescent dyes. This potential ability enabled them to follow the exosomes in vitro and in vivo, offering possible uses in various medicinal sectors.121

Surface functionalizing using ligand as a cell-targeted component can be an easy method of exosome engineering. Sequential incubation without challenging steps of gene engineering can modify the exosome. The peptide of c(RGDyK) (cyclo(Arg-Gly-Asp-D-Tyr-Lys)) with high affinity to specific integrin (alpha v beta 3), targeted tumor cells in albumin-based nano-drug,122 then Tian et al utilized this peptide for surface functionalizing of exosomes to aim the cerebral endothelial cell. The azide-functionalized c(RGDyK) interacted with dibenzocyclooctyne on the exosomal amino group (proteins or phosphatidylethanolamine). This curcumin-loaded carrier efficiently arrested the apoptosis and inflammation in the mice model of the ischemic brain and demonstrated successful targeting and therapeutic benefits.16

Xing et al proposed a strategy to sequester engineered exosomes away from the fast bloodstream elimination. They modified MSCs- derived exosomes by attaching azide groups using metabolic glycan engineering. Additionally, they modified a collagen hydrogel with dibenzocyclooctyne (DBCO) molecules. The azide-labeled exosomes were then anchored to the DBCO-modified collagen using click chemistry. Subcutaneous implantation of the modified complex of exosomes and collagen hydrogel improved angiogenesis following the release of MSC exosome contents.123 However, further research and development are necessary before clinical application. Concerns about potential immune reactions to azide-exosomes or modified collagen also need to be addressed through thorough animal studies.

Engineered Exosomes Using Noncovalent Methods

Noncovalent methods of exosome engineering include hydrophobic and electrostatic integration, aptamer and peptide anchoring, and receptor and ligand interaction.124

Engineered Exosomes Using Hydrophobic Integration

Hydrophobic integration of the cholesterol-conjugated components is another surface modifier for signal amplification of colorimetric sensor125 and a targeted delivery system.126 He et al used cholesterol to anchor a DNA probe and improved the detection of exosomes by biotinylated oligonucleotides and streptavidin-labeled horseradish peroxidase (SA-HRP).125 The cholesterol-conjugated oligonucleotide is a useful anchor-spacer for attaching agents like small dyes, aptamers, and antibodies (Figure 2). DNA is a promising spacer for exosome modification due to its negative charge and capability for base pairing, as well as its configurable size and controlled release qualities. Yerneni et al induced in vitro and in vivo apoptosis in tumor cells through surface engineering of the exosome and conjugating cholesterol-biotinylated oligonucleotide-streptavidin Fas ligand.126

Engineered Exosomes Using Electrostatic Interaction

Nakase et al utilized the electrostatic interaction between cationic lipids and the exosomal membrane, along with the amphipathic GALA peptide, to enhance the uptake of exosomal cargo (Figure 2). GALA peptide consists of repeating sequences of glutamic acid-alanine-leucine-alanine. Incorporating the GALA peptide and cationic lipids increased cargo internalization. The GALA peptide transforms a helical structure at acidic pH levels, mimicking a viral protein. This helical conformation plays a crucial role in facilitating the escape of substances from endosomes. The GALA peptide exposes a hydrophobic leucine residue by adopting this helical shape. This exposure enables the fusion of exosomal and endosomal membranes, disrupting endosomal stability and the subsequent release of cargo into the cytosol. The negatively charged glutamic acid residues of the GALA peptide interacted with cationic lipids to accumulate on the exosome membrane and increased cellular uptake.127 However, studies showed that the harmful effects of cationic lipids following membrane thinning and erosion resulted in cell toxicity. So, the amount of the cationic lipid should be regulated to avoid making holes in the membrane.128

Engineered Exosomes Using Antibodies

An alternative technique for targeted drug delivery is antibody decoration for specific antigens. For example, since the overexpression of CD47 in cancer cells and interaction with SIRPα (signal regulatory protein α) helps cancer cells evade the immune system, inhibiting this interaction can control various cancers.129 The exosome-conjugated to anti-CD47 or anti-SIRP showed tumoricidal effects in vitro and in vivo.130 Although Xie’s group optimized releasing this exosome in acidic pH, intravenous treatment targeted all cells and dyed animals in less than a month.88 Considering that CD47 has an extreme distribution in the body, masking therapy of this target