Engineered extracellular vesicles: A new approach for targeted therapy of tumors and overcoming drug resistance

3.1 EVs

EVs were identified in reticular cells in 1983 and were initially assumed to be responsible for cellular waste removal [48]. However, in-depth investigations into EVs in recent years have revealed their unique characteristics. EVs are ubiquitously produced and released into the extracellular fluid by almost all cells. They are about 30-120 nm in size and are considered natural endogenous NPs [49, 50].

EV production starts with the invagination of the cell membrane, which enters the cytoplasm to become early endosomes. These early endosomes receive particulate matter from cells to form late endosomes, which eventually develop into multivesicular bodies (MVB) under endocytosis. An MVB then combines with certain portions of the cell membrane before being released into the extracellular matrix as vesicles (Figure 2) [51, 52]. Since EVs can be released by almost all cells, they possess excellent circulatory stability and have negligible cytotoxicity in vivo [53, 54]. At the same time, EVs can be extracted from patients, so they have low immunogenicity, which has more advantages than NPs [55-57]. EVs offer distinct benefits in treating brain diseases owing to their capacity to quickly penetrate the blood-brain barrier (BBB), according to recent research [58]. Compared with directly delivering live cells, EVs have shown encouraging results in the treatment of brain diseases [59, 60].

3.2 Characterization of EVs

As for how to identify EVs, the International Society for Extracellular Vesicles proposes that at least one of the following proteins needs to be evaluated (e.g., the plasma membrane-associated or endosome-associated transmembrane or glycosylphosphatidylinositol-anchored extracellular proteins and lipid- or membrane protein-binding cytoplasmic proteins) to determine whether they are EVs [61]. In most studies, electric vehicles are typically characterized using three methods: transmission electron microscopy, the particle diameter analysis system and western blotting [61-63]. EV transmission electron microscopy can identify the size and shape of EVs to determine the presence of an EV-like structure in the sample, which is usually in a saucer shape with a concave center or a hemispherical shape with a concave side [64]. The original state of EVs can be ensured by measuring and counting the diameter distribution of particles in the sample through the particle diameter analysis system. Western blotting identification of EV surface protein markers: usually the detection of protein markers, CD9, CD63, and tumor susceptibility 101 (TSG101), is used to determine the presence of EVs. In addition to the above three general methods, EV markers can also be detected by flow cytometry, which has the advantages of fast analysis and fewer samples required. However, this technology is time-consuming and labor-intensive, which limits its applications [65]. In addition, a time-resolved fluorescence immunoassay based on NPs has been developed, which can capture EVs directly from urine and cell supernatant and identify disease-specific markers on the surface of urine EVs from patients [66]. Therefore, researchers can actively select and develop a variety of markers to characterize EVs in many ways according to their own needs and conditions.

3.3 Isolation of EVs

There are many methods for isolating EVs, and different separation methods can be used according to different purposes and needs. Among them, ultracentrifugation, ultrafiltration, precipitation, immunoaffinity capture, and size exclusion chromatography are the five most commonly used techniques, and these techniques may be used in combinations in different ways.

Ultracentrifugation is the gold standard for the isolation of EVs and is the most commonly used isolation technique. This method separates components in a solution based on differences in size and density [67], and the operation is simple. In addition to the initial instrument, the price is low, and a high concentration of EVs can be separated [68]. However, its disadvantages are time-consuming, high equipment requirements, and damage to the structure [69].

Ultrafiltration is a selective separation based on the size of particles. This method is faster than ultracentrifugation, and the activity of EVs is not affected. However, there are problems, such as damage to EVs due to the applied shear stress and low purity of recovery [70].

The precipitation method uses PEG to harvest EVs by reducing the solubility of EVs [71]. This method is relatively simple to operate and is used to separate large-dose samples. However, the purity and recovery rate of EVs are low, which is not conducive to subsequent functional experimental analysis [72].

Immunoaffinity capture is based on the fact that EVs can be separated by recognizing their unique surface markers (such as CD9, CD63, CD81 and Alix) through antibodies immobilized on surfaces such as magnetic beads, chromatography column resins, multi-well plates and microfluidic device [73]. This method has the characteristics of strong specificity, high sensitivity and high purity [74]. However, there are also some problems such as high cost and unsuitability for large-scale separation [75].

Size exclusion chromatography, the mildest chromatographic technique, takes advantage of size differences, that is, the differential elution of particles of different sizes through gravity flow, thus effectively separating EVs from most proteins (such as albumin) in body fluids. This method can maintain the integrity and biological activity of EVs and has high yields, but it is time-consuming and has a low recovery rate [76].

In general, each method has advantages and disadvantages, and researchers need to integrate and innovate existing methods according to their own needs. For example, to improve the separation purity, some studies have tried to combine ultracentrifugation and ultrafiltration with size exclusion chromatography to isolate EVs [77, 78]. The results showed that ultrafiltration combined with size exclusion chromatography could reduce the level of impurity cytokine (interleukin-10) in isolated EVs.

3.4 Therapeutic potential of EVs

The secreted EVs can engage with corresponding target cells through a diverse range of mechanisms [79]. Upon receptor binding, it initiates fusion and endocytosis processes, subsequently triggering relevant biological responses [80, 81]. Nucleic acids, including microRNA (miRNA), long non-coding RNA (lncRNA) and DNA, may be transported via EVs to facilitate cell-to-cell communication. According to Liu et al. [82], EVs from activated T cells contain miRNAs that regulate immunity by targeting antigen-presenting cells (APCs). Similarly, EVs produced by donor dendritic cells (DCs) have been shown to include functional donor major histocompatibility complex molecules and APC activation signals, which may increase allograft rejection [82]. Moreover, EVs can convey proteins expressed by parent cells to facilitate intercellular communication. For example, EVs can transfer a variety of proteins to promote the development of illnesses, including Alzheimer's disease and prions [83, 84].

These observations highlight the natural EVs' effectiveness as a drug delivery platform in light of their biodistribution, biocompatibility, and minimal immunogenicity. EVs have a high permeability and are capable of traversing most biofilms, including the BBB (Figure 2) [57]. Moreover, as they come from the patients’ own body, the likelihood of immune response, allergic reaction, toxicity, and biodegradability issues is low [85]. Given these advantages, EVs hold promise as candidates for cancer therapeutic delivery.

4.1 Chemical modification

Chemical modification of EVs can improve their physical stability and increase the effectiveness of both their targeting and medication delivery [87]. One effective method for co-binding small and macromolecular organisms to the EVs’ surface is click chemistry, which has a quick reaction time, high specificity, and aqueous compatibility. In addition, click chemistry has minimal effects on the function of EVs [88]. To address the challenges of treating gliomas, in which the BBB often restricts the entry of drugs into the brain, thus affecting the treatment of tumors, Jia et al. [89] coupled the EVs’ membrane with the neuroderm-1 targeting peptide by click chemistry to deliver curcumin and NPs using EVs. The resulting EVs could pass through the BBB and treat gliomas simultaneously. Similarly, c(RGDyK) peptide, which demonstrated a strong affinity for integrin α(v)β3 in reactive cerebral vascular endothelial cells after ischemia [90, 91], was found attached to the EVs’ surface through a chemical reaction, and the curcumin-loaded c peptide was then intravenously delivered into a mouse model of transient middle cerebral artery blockage [59]. The c peptide EVs can effectively penetrate the BBB and inhibit inflammation and apoptosis in the lesion area [59].

Chemical modification can also be used to inhibit tumor immune escape. “Don't eat me” signals are activated when CD47 binds to the signal regulatory protein (SIRP) on innate immune cells, including macrophages and DCs, which allow the tumor to evade phagocytosis [92, 93]. By using click chemistry via a pH-sensitive junction, Nie et al. [94] coupled an M1 macrophage-modified EVs with dibenzocyclooctyne-modified antibody of CD47 and SIRP. After systemic administration, the modified EVs recognize active tumor targeting and effectively reprogram into an M1-dominated tumor microenvironment (TME).

Nucleic Apts are a promising tool for specific targeting owing to their strong affinities and selectivity for target molecules [95]. The EVs' surface can be modified with nucleic Apts to improve their ability to target cells and increase the effectiveness of medication delivery [96, 97]. For instance, Apts (AS1411) that target nucleolin, a protein that is overexpressed on the surface of BC cells, were added to EVs. These AS1411-modified EVs efficiently delivered small interference RNA (siRNA)/miRNA to BC cells in mice, resulting in effective tumor inhibition [98]. Another study used an Apt (sgc7) that recognizes protein tyrosine kinase 7 (PTK7) to modify the surface of EVs for targeted delivery of chemotherapeutic drugs to cancer cells expressing PTK7 [87]. Additionally, EVs have been modified with RNA Apts (such as PSMA-Apts and EGFR-Apts) to efficiently deliver siRNA/miRNA to corresponding tumor sites, leading to substantial tumor growth suppression. Overall, EV nucleic Apt modification has the potential to significantly increase the specificity and effectiveness of targeted medication delivery [99].

The EVs’ surface can also be modified by other chemical methods. Qi et al. [100] created a cluster of superparamagnetic NPs based on EVs with dual functions that connect transferrin-bound superparamagnetic NPs to the EVs derived from blood reticulocytes by transferrin-transferrin interaction, which not only enhances the specificity and efficiency of cancer targeting but also enables real-time imaging and monitoring of the drug delivery process. Nakase et al. [101] combined cationic lipid treatment with the negatively charged membrane of EVs and added lipodiamine to the EVs’ surface, which greatly improved the cell uptake efficiency. CP05 anchoring modification has been proven to retain the natural size and morphological characteristics of EVs without affecting their distribution in vivo and can change their cell targeting behavior, thus more accurately treating tumors [102].

However, the availability of certain functional groups on the surface of the EVs may restrict chemical modification, and it may not be possible to target certain molecules using this approach [103]. Furthermore, it is crucial to properly account for any possible safety concerns brought on by chemical alterations, especially when designing EV-based therapies for clinical use. The modified EVs must not be harmful and must not cause the patient to develop an immunological reaction. Additionally, regulatory hurdles may need to be addressed before modified EVs can be approved for clinical use.

4.2 Genetic engineering

Compared to chemical modification, genetically engineered EVs offer several advantages for the expression and stability of the targeted parts [104]. Moreover, the EV membrane's diversity of transmembrane proteins, including lysosomal-associated membrane protein (LAMP) and glycosylphosphatidylinositol, can be fused with ligands or homing peptides to enhance the transport of EVs to their intended destination [105].

By using genetic engineering, the gene sequences for the relevant surface membrane protein and the ligand or homing peptide may be combined. A common target for the genetic engineering of EVs for targeted distribution is LAMP-2B, a transmembrane protein that is abundant in EVs generated from DCs [106]. In this approach, the targeting protein or peptide's gene is fused with LAMP-2B's gene to generate a chimeric protein that is produced on the EVs’ surface. Despite this limitation, targeted medication delivery using genetically modified EVs has shown encouraging results, and the potential for therapeutic use is now being extensively investigated. Kumar et al. [107] used EVs derived from DCs to fuse the EVs’ membrane protein LAMP-2B into the central nervous system specific rabies virus glycoprotein (RVG) peptide. The RVG peptide can specifically bind to neuronal cells to express acetylcholine receptors. In mice, glyceraldehyde-3-phosphate dehydrogenase siRNA-loaded RVG-targeting EVs were injected intravenously. The results demonstrated that EVs for RVG targeting might selectively target brain neurons and result in specific gene silencing. This provides a promising vector for the treatment of glioblastoma and gene therapy of chronic neurodegenerative diseases [108]. Given that immature dendritic cells (imDCs) can generate EVs with low immunogenicity and toxicity [109, 110], Tian et al. [111] employed imDCs and fused the N-terminal of the mouse EV LAMP-2B protein with the rabies virus glycoprotein (iRGD) peptide, which is specific for integrin α(v)β3. Experiments revealed that iRGD EVs could specifically deliver adriamycin or doxorubicin to integrin-positive BC cells. Bai et al. [112] fused the targeted peptide tLyp-1, a ligand of selective targeting neuropilin 1 and neuropilin 2 [113, 114], to LAMP-2B and successfully constructed targeted tLyp-1 EVs, which have a high transfection efficiency for use in gene therapy. Given that endosomal proteases may destroy the peptides on the surface of EVs during their synthesis, Hung et al. [115] used genetic engineering methods to combine the N-terminus of the targeting peptide-LAMP-2B fusion with the glycosylation sequence to prevent peptide degradation.

LAMP-2B is not the only transmembrane protein that can be used for membrane display. Another option is to use platelet-derived growth factor receptor (PDGFR), a transmembrane protein. Cheng et al. [116] combined antibodies targeting T cell CD3 and cancer cell-associated EGFR with the transmembrane domain of human PDGFR to create EVs that showed powerful anti-cancer immunity against EGFR-positive triple-negative BC cells in vitro and in vivo.

In addition to PDGFR and LAMP-2B, tetraspanin superfamily proteins CD63, CD9, and CD81 can be designed to display targeting sequences or probes [117-119].

However, genetic engineering-based modification is limited by the encoded gene sequence, and its application is challenging. Ongoing researches are focusing on improving this method. In summary, surface modification of EVs significantly enhances their stability and targeting, but it is a challenging process that requires strict control of reaction conditions to prevent destruction and non-specific accumulation of EVs due to inappropriate temperature and pressure [120].

5.1 Delivery of nucleic acids

The objective of gene therapy is to cure diseases brought on by faults and aberrations of genes without causing harmful side effects by introducing foreign, normal genes into target cells [128]. Various diseases, including cancer, can be treated with gene therapy using non-coding RNA (ncRNA) [129, 130]. miRNA and siRNA can specifically inhibit the expression of any cancer-related genes/mRNA through RNA interference (RNAi) [131], such as Bcl-2 [132], c-Myc [133], KRAS [132, 134], etc. However, ncRNA is a macromolecule that is difficult to deliver within the body. Excitingly, a study demonstrated that the EVs secreted by THP-143 cells transfected with miR-1 mimics in vitro can successfully load miR-143 and can be targeted to tumor cells in mice [135]. Given the absence of delivery vectors that can successfully traverse the BBB, Alvarez-Erviti et al. [108] began to study targeting engineered EVs to the brain. They skillfully used DCs successfully transfected with LAMP-2B-modified pEGFP-C1 to secrete EVs expressing RVG, which were then electroporated with siRNA and administered to mice. A strong RNAi response was observed throughout the brain: the damaging β-amyloid 1-42 protein in the animal cerebral cortex was decreased by 55%, and the mRNA of the beta-site APP-cleaving enzyme 1, a protein implicated in the formation of Alzheimer's disease [136], was knocked down by 61%. Surprisingly, only a slight RNAi effect was detected in the liver and spleen. Importantly, there was almost no toxicity and very low immune response after systemic administration [108]. The efficacy of modified RVG in siRNA distribution was further supported by research employing hepatocyte growth factor (HGF) siRNA-loaded EVs from human embryonic kidney cells [137].

In comparison to siRNA, miRNA can cause target mRNA degradation or translation inhibition without completely binding to its target mRNA sequence. Nie et al. [138] exploited the EVs derived from BC cells that overexpressed integrin β4, as it could specifically act on surfactant C and be specifically internalized by cancer cells. When miRNA-126 was loaded, the PTEN/PI3K/AKT signaling pathway was blocked, and the growth and invasiveness ability of A3 lung cancer cells were significantly inhibited [138]. More specifically, miRNA's anticancer capabilities may be strengthened by EVs. As demonstrated by the Kuroda laboratory, engineered EVs loaded with miRNA are precisely targeted to BC cells overexpressing EGFR through peptide-targeting ligands, resulting in reduced growth of cancer cells [139]. Although still in the early stages, these studies together demonstrate the potential of engineered EVs in nucleic acid delivery therapy.

5.2 Drug delivery

One important argument for using engineered EVs as medication delivery carriers is their capacity to overcome drug restrictions and lessen toxicity in the human body. Additionally, EVs are a biological source, and clinical studies have shown that their immunogenicity is minimal [140, 141]. By loading drugs into EVs, we may not only reduce the limitation due to chemical properties of some drugs, such as hydrophobicity, but also improve the circulation time and efficacy in vivo and even reverse drug resistance in tumors. Curcumin, a phenolic compound with anti-inflammatory, anti-tumor, antioxidant, and chemopreventive activities [142, 143], has poor clinical availability due to its hydrophobicity, fast metabolism, and rapid systemic elimination [144]. However, the co-incubation of curcumin with EVs and subsequent delivery throughout the body has been demonstrated to retain the therapeutic activity of the drug, improve its solubility, increase its circulation time in vivo, and enhance its overall utilization [145]. A recent study suggested that loading doxorubicin into EVs secreted by mesenchymal stem cells (MSC-EXO) may be a superior option due to the excellent low immunogenicity, multi-directional differentiation, and homing ability of MSC-EXO [146]. Electroporation of doxorubicin into MSC-EXO not only reduces systemic dosing but also increases cytotoxicity, inhibits tumor growth [111, 147], and even overcomes drug resistance [148]. Additionally, compared to free drugs, drugs with engineered EVs demonstrated better internalization and longer circulation ability [149].

Aside from intravenous injection, oral administration is more convenient and suitable for patients. EV secretion in milk has been proven to be stable under the conditions of strong gastric acid and intestinal degradation, making it a more effective carrier for oral administration than free drugs [150]. Research has also found that drugs loaded with engineered EVs derived from milk have lower half maximal inhibitory concentration and do not cause immune response or inflammation in vivo, demonstrating better efficacy than free drugs [151].

6.1 Application of engineered EVs in tumor therapy

The formation of a tumor involves multiple factors and is a complex process with many characteristics, such as unlimited proliferation, immune escape, resistance to apoptosis, and gene mutation [152]. It needs to have a complete operating system against the body, and every factor that is not conducive to its growth will be suppressed. For example, missense mutations of the p53 gene occur in almost all progressive tumors, thus downregulating its function [153]. This is the so-called crackdown on dissidents and wooing partners. According to the “barrel principle,” as long as we attack and destroy the original balance of the tumor from one aspect, the growth of the tumor can be inhibited, which gives rise to a variety of treatment methods, such as immunotherapy for TME immunosuppression, RNAi directly acting on the molecular mechanism, and so on [154]. These drugs also face pharmacokinetic challenges in terms of poor targeting specificity and systemic circulation, cell uptake and endosomal escape. Engineered EVs can solve these problems because of their good biocompatibility, strong inclusiveness, and low immunogenicity (Figure 5).

6.2 Applications in other diseases

Numerous liver diseases, encompassing acute liver injury and chronic liver fibrosis, currently lack satisfactory treatment options, necessitating the search for alternative therapeutic interventions. In comparison to synthetic non-viral delivery vectors, vectors naturally present in the body possess the ability to circumvent causing immunogenicity and hepatotoxicity [173]. Wan et al. [62] proposed a novel method for treating liver diseases by utilizing the RNA-carrying capacity of engineered EVs. They developed EV ribonucleic acids and, upon systemic injection, observed a significant reduction in central lobular cell necrosis and hyperemia in a model of acute liver injury, as well as the inhibition of acetaminophen-induced acute liver injury and reduced mortality. Encouragingly, in a model of liver fibrosis, this method was also found to decrease the progression of chronic liver fibrosis. An additional study demonstrated that EVs modified with cationic amylopectin can more precisely target the liver and exhibit improved efficacy in treating liver injury [174]. These findings indicate that engineered EVs hold substantial therapeutic potential for the treatment of liver diseases.

The potential of peptide-modified EVs to treat cardiac tissues has been examined. EVs carrying cardiac tissue-specific peptides have shown improved cardiac targeting [175]. In another study, the ischemic myocardial targeting peptide was incorporated into the EV membrane protein LAMP-2B derived from MSCs through genetic engineering [176]. After being intravenously injected into mouse models, these modified EVs were shown to aggregate higher in the ischemic heart region than unmodified EVs. Additionally, in a mouse model of myocardial infarction, these modified EVs significantly reduced inflammation and cardiomyocyte apoptosis, promoted angiogenesis, and enhanced cardiac function (Figure 6) [176].