1 Introduction
Cancer is a life-threatening public health problem, accounting for approximately 10 million deaths worldwide in 2020. The most commonly used treatment for cancer patients is chemotherapy. Although chemotherapeutic agents can inhibit the development of cancer, they also have many serious side effects. Systemic infusions of chemotherapy drugs cause cardiomyopathy, vomiting, fatigue, and hair loss because they cannot be targeted to the cancerous tissue of the body alone. Therefore, there is an urgent need for new and effective tumor-targeted therapies.
Extracellular vesicles (EVs), including Exosomes, Microvesicles (MVs), and apoptotic bodies, are important mediators of cell-to-cell communication and play important roles in physiological and pathological processes, including stem cell maintenance, tissue repair, immune regulation, and tumor growth
[1⇓~3]. Exosomes are one of the major types of extracellular vesicles and have received considerable attention
[4]. Exosomes are nanometer-sized (30 – 150 nm) extracellular vesicles secreted by a variety of cells
[5]. At first, it was thought that exosomes were an excretion pathway of cells. Further studies found that exosomes were also a medium for information exchange and material transport between cells, carrying proteins, lipids and nucleic acids of host cells
[6][7⇓~9]. And the exosomes have small size, good biocompatibility, low toxicity and long half-life. As a carrier for drug delivery, it can solve the problems that limit the efficacy of traditional chemotherapeutic drugs, such as poor drug solubility, short half-life, low efficiency of drug delivery to tumor cells leading to drug resistance, and significant local and systemic toxicity. In addition, the delivery of anticancer drugs contained in exosomes has better tumor targeting and higher in vivo anticancer activity compared with free drugs
[10,11]. Therefore, this paper summarizes the design strategy of exosomes as drug delivery carriers and their application in cancer therapy, mainly introducing exosomes and their isolation methods, the design strategy of tumor-targeted exosomal drug delivery carriers, the research progress of exosome drug delivery to solid tumors, and the shortcomings of exosomes in clinical application.
2 Exosomes and their properties
2.1 Composition of exosomes
Exosomes contain an array of lipids, proteins, and nucleic acids and can be naturally taken up by cells to deliver these contents to recipient cells
[12][13]. The lipid bilayer of exosomes is rich in cholesterol and diacylglycerol
[14]. Lipids such as sphingomyelin and monosialotetrahexosylganglioside (GM3) in exosomes determine the stiffness of exosomes. In addition to these lipids, some bioactive lipids are present in exosomes, such as prostaglandins, leukotrienes, and enzymes (e.g. Phospholipid trafficking enzymes are expressed from phosphatidylserine)
[8,15]. The protein content in exosomes mainly depends on the cellular source of exosomes, mainly including the following types of proteins: heat shock proteins (Hsp60, Hsp70, Hsp90), which mainly carry out the intracellular assembly and transport of exosomes; Transporters (ATP7A, ATP7B, MRP2, SLC1A4, SLC16A1, CLIC1) and receptor proteins (CD46, CD55), performing their communication and cargo transport functions; Tetramer family (CD9, CD81, CD82), which mediates signaling, cell fusion, and migration; Targeting proteins and fusion proteins (e.g., tetrammine proteins, lactoadherin, and integrins)
[16,17]. Nucleic acid is an important component of exosomes. Messenger RNA (mRNA) mediates the transfer of genetic information in exosomes
[18]; microRNA has the function of cell targeting and gene silencing
[19]. Exosomes also contain non-coding RNAs, which are mainly involved in carcinogenesis and cancer progression. Circulating DNA (cDNA) reflects mutations, rearrangements, and amplifications in tumor tissue
[20,21]. Other types of RNAs have been found in exosomes, such as viral RNAs, Y-RNAs, transport RNAs (tRNAs), small nuclear RNA, small nucleolar RNA, and pi-RNA.
2.2 Occurrence of exosomes
The occurrence of exosomes is a process that involves the double invagination of the plasma membrane and the formation of intracellular multivesicular bodies containing intraluminal vesicles (Fig. 1). The first invagination of the plasma membrane forms a cup-like structure in which cell surface proteins and soluble proteins associated with the extracellular environment are encapsulated, which leads to the formation of early endosomes (ESEs),ESEs mature into late endosomes (LSEs) under the control of membranous organelles such as the Golgi apparatus and the endosomal protein sorting and transport apparatus (ESCRT). The second plasma membrane invagination of LSEs results in intracellular multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs). MVBs can either fuse with lysosomes or autophagosomes to be degraded, or fuse with the plasma membrane to release the contained ILVs, the exosomes
[3,22,23].
2.3 Physiological function of exosomes
Exosomes contain a variety of nucleic acids, proteins and lipids in cells, which play a vital role in intercellular communication through the transfer of substances between cells. In these processes, exosomes can modulate the biological responses of target cells, which may have beneficial or detrimental effects on the organism. Exosomes are involved in fundamental physiological processes such as neuronal communication, antigen presentation, immune response, and reproductive processes
[24][25][26][27]. They are also involved in several pathological processes, including cancer progression, progression of central nervous system-related diseases, joint disease, cardiovascular disease, and inflammation, and even favor viral infections
[28][29][30][31][32][33].
2.4 Isolation of exosomes
With the development of exosome research, its potential application value has been explored. Reproducible isolation and enrichment of exosomes will facilitate the assessment of their biological function. However, the morphology of exosomes is similar to other extracellular vesicles, with overlapping size ranges and extremely small volumes, making exosomes difficult to isolate. To achieve high-recovery, high-purity, and high-throughput separations, researchers have employed various methods, each with its advantages and disadvantages (Table 1), with ultracentrifugation, immunoisolation, polymer precipitation, and size-exclusion chromatography being the most commonly used (Figure 2).
2.4.1 Ultracentrifugation
The most common separation technique is centrifugation. Ultracentrifugation achieves separation based on the difference in sedimentation coefficient between exosomes and other extracellular contents. Ultracentrifugation can be further divided into differential ultracentrifugation and gradient density ultracentrifuge. Differential ultracentrifugation involves sequential differential centrifugation at low and high speeds, followed by ultracentrifugation. Upon low speed centrifugation, cell debris is removed and cells are separated. Upon high speed centrifugation, large extracellular vesicles, apoptotic bodies, and microvesicles are separated and removed. Ultracentrifugation was finally used, and the exosomes were pelleted and collected. However, the purity of this method is low, but it can be overcome by gradient ultracentrifugation. Gradient ultracentrifugation uses two or more gels or solutions of different densities to separate extracellular vesicles according to their density, thus achieving a high separation rate. However, this method is very time consuming because it takes some time to reach the equilibrium of the solution
[34,35].
2.4.2 Immunoisolation
The principle of immunoisolation is based on the capture of exosomes by antigen-antibody reaction. Immunoseparation can lead to similar results with a smaller sample size than ultracentrifugation. Immunoisolation can also be used for qualitative and quantitative determination of exosomes. It has strong specificity, high sensitivity, high purity and high yield, but because it involves special reagents and cell-free samples, its cost is too high to be suitable for large-scale isolation of exosomes
[36,37].
2.4.3 Polymer precipitation
The polymer precipitation method usually uses polyethylene glycol (PEG) as a medium to harvest exosomes under centrifugal conditions by reducing the solubility of exosomes. The polymer precipitation method has the advantages of simple operation and short analysis time, and is suitable for the treatment of large dose samples. However, the purity and recovery rate are low, the specificity is low, and the polymer produced is difficult to remove, which is not conducive to the subsequent experimental analysis
[38].
2.4.4 Size exclusion chromatography
This technique is mainly based on the size difference between exosomes and other components in biological samples. The separation principle of size exclusion chromatography (SEC) is that macromolecules cannot enter the gel pores and are eluted along the gaps between the porous gel and the mobile phase, while small molecules remain in the gel pores and are finally eluted by the mobile phase. The method has high cost, high purity and high yield
[39].
2.5 Drug loading mechanism of exosomes
Drug loading into exosomes is one of the key steps in the development of exosome drug delivery systems. The main factors to consider when loading drugs onto exosomes are: how to achieve better encapsulation or loading efficiency, how to maintain the structural integrity of exosomes and how to maintain the activity of drugs. In this article, we briefly discuss several ways of drug loading in exosomes (Table 2).
2.5.1 Electroporation
Electroporation is the process of applying an external electric field to form hydrophilic pores, which can increase the permeability of the cell membrane and allow chemicals, DNA, RNA, or drugs to enter the cell
[41]. Exosomes are cell-generated vesicles that inherit the same phospholipid membrane of the cell membrane, so electroporation is widely used for exosome drug loading
[42]. The integrity of the exosomal membrane is quickly restored after the drug is loaded into the interior of the exosome through the temporary pore.
2.5.2 Incubation method
The incubation method is relatively common and simple, and when the drug and exosomes are incubated together, the drug will be fused into the exosomes along the concentration gradient. The loading efficiency depends on the hydrophobicity of the drug molecule, and the hydrophobic drug can interact with the lipid layer of the vesicle membrane
[43]. The loading efficiency of the incubation method is relatively low, especially for some large miRNAs, so the incubation method is often combined with other methods to improve drug loading.
2.5.3 Ultrasonic method
Ultrasound method is to mix exosomes from donor cells with drugs or proteins, sonicate them by a probe sonicator, and diffuse the drugs into the exosomes during membrane deformation. In some cases, the drug is not only encapsulated inside the exosome, but also attached to the outer layer of the membrane. It was observed that drug release had two phases: the first burst release phase was due to the release of drug attached to the outer layer of exosomes, and the second release phase was the slow release of drug encapsulated within exosomal vesicles. Ultrasound can improve the loading efficiency and is suitable for small ribonucleic acid, but it is easy to cause membrane deformation.
2.5.4 Click chemistry
Click chemistry can directly attach molecules to the surface of exosomes through covalent bonds. Copper-catalyzed azide alkyne cycloaddition, known as click chemistry, is ideal for binding molecules to the surface of exosomes
[44]. This reaction is fast and efficient compared with traditional cross-linking reactions, such as maleimide-thiol coupling, and provides better control over the binding site. It is also applicable to the modification of biological macromolecules because it can occur in aqueous media
[45]. Moreover, the click chemistry method is relatively mild and does not affect the structure or function of exosomes.
3 Tumor targeting strategy of engineered exosomes
Exosomes have good biodistribution, biocompatibility, and low immunogenicity, making them a highly effective drug delivery system. Exosomes are very permeable and can cross most biological membranes
[3]. Compared with liposomes, exosomes are more effective in delivering cancer therapeutic drugs because of their good biocompatibility, strong protection against loaded drugs, and higher tumor homing ability, although the drug loading of exosomes is slightly worse than that of liposomes
[46]. Furthermore, as nano-sized particles, exosomes were shown to be able to cross the tumor vasculature with enhanced permeability and retention (EPR), and exosomes could increase nanoparticle accumulation in brain tumors by hijacking transendocytosis, allowing brain cells to take them up to cross the BBB
[47][48]. In order to develop more effective tumor targeting vectors, researchers have taken various measures to improve the tumor targeting of exosomes, which we will discuss separately in this section.
3.1 Exosomes from different cells
Exosomes are divided into animal-derived exosomes and plant-derived exosomes. Because exosomes can be secreted by both normal and tumor cells, animal-derived exosomes are further divided into normal cell exosomes and tumor cell exosomes.
Exosomes are produced by almost all types of normal cells, such as human umbilical vein endothelial cells, mesenchymal stem cells, T cells, B cells, macrophages, dendritic cells (DCs), and natural killer (NK) cells
[44,49,50]. Exosomes from different cells show different characteristics and functions
[51].
3.1.1 Tumor-derived exosomes
tumor-derived exosomes (TEXs) play a key role in tumor development and progression by establishing or altering the tumor microenvironment, promoting tumor angiogenesis and invasion, inhibiting host anti-tumor response, and mediating immune escape and multidrug resistance
[52].
Tumor-derived exosomes are used to deliver therapeutic drugs and immunotherapeutic vaccines because of their many advantages. Exosomes extracted from tumor cells were able to successfully target tumor cells and were efficiently taken up by parental cells. A large number of tumor cells and tumor exosomes can be found in malignant effusions, and tumor exosomes have been shown to carry tumor-associated specific antigens and MHC-I molecules. Tumor exosomes can also deliver antigens to dendritic cells, thereby inducing T cell-mediated immune responses against tumor cells
[53]. In addition, exosomes from melanoma patients have been shown to enhance the production of myeloid-derived suppressor cells (MDSCs), an important mechanism by which tumors avoid immune recognition
[54]. Most importantly, tumor exosomes can suppress effector T cells by expressing apoptosis-inducing ligands, such as FasL, TRAIL, or PDL-2
[55]. Many research groups have investigated the use of tumor cell-derived exosomes to deliver chemotherapeutic drugs or other anticancer drugs. Yong et al. Developed a biocompatible tumor cell exosome biomimetic porous silicon nanoparticles (PSiNPs) as a drug carrier for targeted cancer chemotherapy (Fig. 3), and the exosome-encapsulated doxorubicin-loaded PSiNPs (DOX @ E-PSiNP) showed enhanced tumor aggregation and could penetrate deep into the tumor after intravenous administration
[56]. Qiao et al. Found that in vitro exosomes from tumor cells preferentially fuse with the tumor cells from which they are derived
[57]. Systemic injection of tumor-derived exocrine will target the tumor tissue. In addition, drug-loaded exosomes showed stronger retention in tumor tissue and more efficient tumor elimination in nude mice than Doxil alone.
3.1.2 Macrophage-derived exosomes
Macrophages play a role in inflammation, immunomodulation, wound healing, and angiogenesis. It is widely distributed in the body and can polarize into M1 or M2 macrophages
[58]. Studies have shown that macrophage-derived exosomes can regulate colorectal cancer cell migration and invasion, inhibit inflammation and wound healing, inhibit angiogenesis, inhibit cardiac healing, and regulate insulin sensitivity in vitro and in vivo
[59⇓⇓~62]. Macrophage-derived exosomes can also serve as drug carriers. Wang et al. Isolated M1-Exos from M1-macrophages by ultracentrifugation and characterized by transmission electron, nanoparticle tracking analysis, dynamic light scattering, and Western blotting
[63]. The M1-Exos was then used as a paclitaxel (PTX) carrier to prepare a nanoformulation (PTX-M1-Exos), and the drug delivery system (PTX-M1-Exos) was prepared using an ultrasonic method. In vivo experiments in tumor-bearing mice showed that M1-Exos as a carrier could deliver PTX to tumor tissues and enhance the anti-tumor effect of chemotherapeutic drugs on tumor-bearing mice.
3.1.3 Mesenchymal stem cell-derived exosomes
mesenchymal stem cells (mesenchymal stem cells MSCs) are pluripotent stem cells with the ability of self-renewal and multi-directional differentiation. MSCs can not only adapt to the tumor microenvironment, but also have strong paracrine activity and secrete a large number of exosomes
[64]. Studies have shown that MSC-derived exosomes have the functions of immunomodulation, bone regeneration, anti-inflammatory, anti-aging and wound healing
[65,66]. MSC-derived exosomes have a tropism for tumors, making them potential vehicles for targeted delivery of antitumor drugs. Pascucci et al. Found that paclitaxel-mediated MSCs had a powerful antitumor effect, and they could uptake the drug and subsequently package it in their secreted EVs
[67]. Katakowski et al. Transfected MSCs with mir-146b expression plasmid and harvested exosomes released by MSCs
[68]. Intratumoral injection of MSC-derived exosomes expressing mir-146 was then found to significantly reduce glioma growth in a rat model of primary brain tumor.
3.1.4 Dendritic cell-derived exosomes
Dendritic cells (DCs), as antigen-presenting immune cells, play a central role in initiating antigen-specific immunity and tolerance
[69]. In cancer, DCs serve as an initial link between tumorigenesis and the host immune system and can eliminate cancer cells by activating T cells
[70]. Dendritic cell-derived exosomes (Dex) contain functional MHC peptide complexes, as well as other immunostimulatory components, which together promote immune cell-dependent tumor rejection, so they have attracted much attention as immunotherapeutic anticancer drugs
[71]. Studies have shown that Dex, as a drug carrier, can play a dual role of immunity and anti-tumor therapy in cancer therapy
[72]. In addition, Dex can overcome biological barriers such as the blood-brain barrier (BBB), making it more attractive for future drug delivery
[73]. Morse et al. Prepared a MAGE-tumor antigen-loaded Dex and injected four doses of Dex every other week into nine pretreated patients with advanced MAGE-expressing non-small cell lung cancer
[74]. The results suggest that Dex vaccine is well tolerated in patients with advanced non-small cell lung cancer and that Dex is promising as a cancer vaccine candidate.
3.1.5 Bacteria-derived exosomes
Outer membrane vesicles (OMVs) are produced in vitro and in vivo by Gram-negative bacteria. OMVs are nanometer-sized spherical carriers with lipid bilayer membranes containing a variety of components derived from the mother bacteria. Because of their presence of bacterial antigens, pathogen-associated molecular patterns (PAMPs), adhesins, various proteins, and vesicular structures, OMVs have been developed for use in bacterial vaccines, adjuvants, cancer immunotherapeutics, drug delivery vehicles, and anti-bacterial adhesion agents
[75].
An important role of OMVs in nature is to deliver biomolecules to specific distant sites of their parent bacteria. Based on this phenomenon, researchers have proposed the possibility of developing drug delivery systems for OMVs. OMVs drug delivery vehicles have multiple advantages. The first advantage is the ease of targeted drug delivery to target cells. Because OMVs are derived from bacteria and contain many different PAMPs, they are readily detected and internalized by neutrophils and macrophages, and thus they can be used to specifically deliver drugs to these cells. Second, the size of OMVs allows them to passively accumulate in the tumor via the EPR effect, an inherent advantage that AIDS in drug delivery to the tumor. OMVs can also induce an immune response, which would be beneficial for tumor therapy.
Gujrati et al. Loaded siRNA into OMVs by electroporation
[76]. To reduce the endotoxicity of lipopolysaccharide, they genetically engineered an E. coli strain with a mutation in the msbB gene that removes toxic lipopolysaccharide to express human epidermal growth factor receptor 2 (HER2) for cancer cell targeting. The siRNAs were then loaded into HER2 gene-expressing OMVs by electroporation. Injecting mice with siRNA-loaded HER2 gene-expressing OMVs silenced spindle kinesin, resulting in increased tumor accumulation of siRNA and significant inhibition of tumor growth, confirming the effectiveness of OMVs as tumor-targeting delivery vehicles. Therefore, OMVs have potential applications as an efficient tumor-specific drug delivery vehicle.
3.1.6 Exosomes from other sources
Exosomes are also widely distributed in various biological fluids, such as milk, saliva, plasma, urine, amniotic fluid, cerebrospinal fluid, ascites and so on
[77]. Milk exosomes are promising candidates for the development of new therapies for various diseases, including cancer. In the application of milk exosomes for cancer therapy, researchers have paid extensive attention to the delivery of anti-tumor drugs and therapeutic nucleic acids
[78][79,80].
The potential role of food-derived exosomes (FDEs) in the treatment of diseases, as well as in the regulation of intestinal flora, has been documented
[81,82]. In addition, FDEs may provide biocompatible carriers for the delivery of anticancer drugs, such as paclitaxel
[83]. Ginger granules can prevent the development of liver-related diseases, while grape, carrot, grapefruit and ginger-extracted exosome-like nanoparticles (ELN) have anti-inflammatory effects and can maintain intestinal homeostasis
[84][85,86].
3.2 Surface modification of exosomes
Tumor targeting can be improved by surface modification of exosomes. Surface modification of exosome vectors is divided into chemical bonding and genetic engineering.
3.2.1 Chemical bonding modification
Chemical bonding modifications of exosome carriers fall into four categories (Fig. 4): targeting cell adhesion molecules (iRGD, CRGDKGPDC
[87]; LFA-1, lymphocyte function-associated antigen-1), targeted cell receptor (AA-PEG, aminoethylamide polyethylene glycol
[88][89]; GE11, amino acid sequence YHWYGYTPQNVI
[90]; RVG, rabies virus glycoprotein), enhanced antigen presentation (GALA, a pH-sensitive fusion peptide), and targeting T cells (MHC, major histocompatibility complex)
[91][92][93]. For example, GALA peptide-modified exosomes developed through streptavidin-biotin interactions can be used to control tumor cell-derived exosomes to facilitate release and intracellular transport in an acidic tumor environment, thereby enhancing tumor antigen presentation capacity through MHC-I class molecules
[92]. CP05 peptide-based surface modification also provides a simple approach for exosome surface expression of targeted or therapeutic moieties. In addition, the hydrophobic interaction between liposomes and exosomes is another new method, in which the liposomal membrane is functionalized with peptides, antibodies, or polyethylene glycol (PEG), and then the exosome and liposomal membranes are fused by the freeze-thaw method, resulting in an exosome-liposome hybrid product with effective tumor-targeting ability
[94].
3.2.2 Genetic engineering modification
Compared with chemical bonding modification, which is complex to manipulate and difficult to control, exosomes can be modified by genetic engineering to improve delivery capacity and targeting specificity. And exosome-based drug delivery systems may have unique advantages over other systems in delivering nucleic acids, including low immunogenicity, good stability, good biocompatibility, and possibly fewer off-target effects due to the propensity of exosomes to act on specific target cells. But the complexity of exosome components may also increase its off-target effects, as not all components of exosomes are required for their proper function. These vesicles can be artificially mimicked to create drug delivery vehicles with low complexity, good targeting, and low off-target effects by assembling liposomes containing only the key components of natural exosomes. Another approach is to improve targeting and reduce off-target effects through surface modification and smart drug delivery systems
[96]. Ohno et al. Achieved targeting of cancer tissues by engineering donor cells to express the platelet-derived growth factor receptor fused to the transmembrane domain of the GE11 peptide
[90]. Intravenously injected exosomes delivered the let7a miRNA into EGFR-expressing transplanted breast cancer tissue in RAG2 −/− mice. The results showed that GE11-let7a-Exos obtained by genetic engineering had good targeting to breast cancer tissues. Leonard et al. Used a genetic engineering method to combine the targeting peptide with a glycosylation sequence (GNSTM), and found that the glycosylation-stable peptide enhanced the targeted delivery of exosomes to neuroblastoma cells
[97].
3.3 Stimuli-responsive exosome
Stimulus-responsive exosome is an intelligent carrier system, which can respond to exogenous stimuli to achieve the sustained release effect of loaded drugs. Exogenous stimuli are mainly physical signals such as magnetism, light, heat and ultrasound.
3.3.1 Magnetic-responsive exosome
It has been found that exosomes can be targeted to disease sites according to the reaction mechanism of magnetic nanoparticles to external magnetic fields. Superparamagnetic colloidal nanoparticle clusters (SMCNCs) have superparamagnetic properties, and SMCNC-modified drug carriers can be effectively separated and purified from solution or blood by using a moderate magnetic field, and the targeting of drug carriers can be improved. Qi et al. Developed a bifunctional exosome-based superparamagnetic nanoparticle cluster as a targeted drug delivery vehicle for cancer therapy (Fig. 5)
[98]. Through in vivo experiments, it was found that the delivery of drug-loaded exosome vector enhanced the tumor targeting under the external magnetic field, which realized the targeted drug delivery of exosomes for tumor therapy and inhibited the growth of tumors. Zhan et al. Designed a multifunctional combinatorial delivery system for highly abundant and safe blood exosomes by manipulating the exosomal membrane, in which doxorubicin (DOX) and cholesterol-modified miRNA21 inhibitor (miR-21 I) were co-embedded in the lipid bilayer of exosomes, and magnetic molecules and endolytic peptide L17E were bound to the exosomal membrane through ligand-receptor coupling and electrostatic interaction, respectively
[99]. Studies have shown that this engineering strategy not only retains its inherent characteristics, but also easily integrates many characteristics such as tumor targeting, efficient transfection and gene/chemotherapy combination therapy into blood exosomes. The lipid bilayer structure of exosomes allows them to co-load DOX and miR-21i at high loadings. Moreover, benefiting from the integration of the magnetic molecule and L17E polypeptide, the engineered exosomes exhibited stronger tumor aggregation and better endosomal escape ability, thereby specifically and efficiently delivering the encapsulated drug to tumor cells. It was found that significant inhibition of tumor growth was observed in tumor-bearing mice without significant side effects.
3.3.2 Photothermal responsive exosomes
Exosomes are used to deliver drugs because of their good biocompatibility, low immunogenicity, long circulation, and non-toxic properties. However, the controlled release and tumor targeting of exosomal drugs are still great challenges. As a new cancer therapy, photothermal therapy has received much attention in recent years, not only because it can use the high heat generated by photothermal agents under near-infrared (NIR) laser irradiation to ablate tumor cells, but also because it can effectively control the release of drugs
[100][101]. Therefore, researchers have begun to focus on designing functionalized exosomes that can achieve photothermal therapy. Wang et al. designed a functionalized exosome (Fig. 6) based on the tumor-targeted chemotherapy/photothermal therapy strategy of gold nanorods (AuNRs) and exosomes. The results showed that the functionalized exosome not only retained the inherent functions of natural exosomes, but also acquired multiple abilities of efficient tumor targeting, controlled release and hyperthermia like synthetic nanocarriers
[102]. Wang et al. Prepared a targeted extracellular vesicle ICG/PTX @ RGE-EV loaded with indocyanine green (ICG) and paclitaxel (PTX) by electroporation and click chemistry using Raw264.7 cells extracted to exosomes
[103]. Then a glioma model was established in nude mice, and it was found that ICG/PTX @ RGE-EV had good targeting and anti-tumor effects in vivo by chemotherapy combined with hyperthermia.
3.3.3 Ultrasound-responsive exosomes
Sonodynamic therapy (SDT) is an ultrasound (US) -based method that is easy to implement and has the high tissue penetration inherent to US
[104,105]. Possible underlying mechanisms of SDT action include ultrasonic cavitation, free radical production, and apoptosis
[106]. Sonosensitizer is the key factor of SDT. Traditional sonosensitizers usually have the disadvantages of low water solubility, low chemical and biological stability, poor tumor accumulation and rapid clearance from the bloodstream. In order to solve these problems, people have focused on exosomes. The endogenous source of exosomes provides good biocompatibility and the ability to avoid uptake by the reticuloendothelial system (RES), thus protecting the encapsulated content until it is delivered to the lesion site, and is a potential vehicle for SDT. Liu et al. Designed a functionalized smart nanosonosensitizer (EXO-DVDMS) to load porphyrin sensitizer (DVDMS) with potential therapeutic and imaging applications onto tumor cell-derived exosomes (Fig. 7)
[107]. The results show that tumor cell-derived exosomes exhibit high stability and specificity for homotypic tumors, while controlling ultrasound-responsive drug release and promoting the generation of reactive oxygen species (ROS) to enhance SDT. In addition, functionalized exosomes can facilitate simultaneous imaging and inhibition of tumor metastasis by 3- and 10-fold over the free form, respectively.
4 Application of Exosomes as Drug Delivery Carriers in Cancer Therapy
4.1 Lung cancer
Lung cancer has been the leading cause of cancer death worldwide for decades
[108]. Although researchers have developed drugs for the prevention and inhibition of lung cancer progression, there has been a lack of suitable drug carriers. Therefore, there is an urgent need for drug delivery systems that can specifically deliver drugs to tumors. Exosomes have good bioavailability, low toxicity and good tumor targeting, which make them a potential drug carrier for targeting lung cancer
[109].
Berrylike compounds, such as aglycones and anthocyanins, have antioxidant, antiproliferative, antiapoptotic, and anti-inflammatory properties and are useful in lung cancer therapy. However, these compounds are not effectively utilized due to their poor bioavailability and retention. In a recent study, Munagala et al. Attempted to encapsulate these compounds in the exosome lumen and showed good therapeutic effects in cancer cells and nude mouse lung cancer xenograft models, indicating the advantages of using exosomes as drug delivery vehicles in the treatment of lung cancer
[110]. Celastrol (CEL), a plant-derived triterpenoid, is an inhibitor of Hsp90 and NF-κB activation pathways, and is considered to have important therapeutic effects on a variety of cancers. Aqil et al. Found that CEL-loaded exosomes had a better inhibitory effect on lung cancer compared with free CEL. In addition, researchers have attempted to use exosomes modified with the Sigma receptor ligand aminoethylamide (AA) to treat lung cancer metastasis. Kim et al. Used PEG-AA to modify paclitaxel (PTX) -loaded exosomes, and showed that systemically administered PEG-acrylic acid-modified exosomes had better lung tumor targeting and higher in vivo therapeutic efficacy than unmodified exosomes and paclitaxel
[89].
4.2 Pancreatic cancer
Adenocarcinoma is the seventh most common malignancy and the fourth and sixth leading cause of cancer-related death in the United States and China, respectively
[111]. The 5-year survival rate of patients is only 5% to 10%, and most patients with pancreatic cancer do not develop significant symptoms until they reach an advanced stage of the disease
[111,112]. Treatment for pancreatic cancer is often followed by short-term recurrence after surgical resection, and most drug-targeted therapies fail to provide substantial efficacy due to the limited delivery of cytotoxic drugs. Exosomes have good targeting and are ideal drug carriers. MSCs can be packaged by exosomes to deliver PTX, which can significantly inhibit the proliferation of pancreatic cancer cell lines
[67]. Meanwhile, exosomes have also been used to deliver functional nucleic acids and proteins to avoid side effects and improve targeted efficacy. Kras
G12D is a common gene mutation in glandular adenocarcinoma. Kamerkar et al loaded KRAS
G12DsiRNA into exosomes and found that the tumor of mouse pancreatic cancer model was inhibited and the overall survival was greatly improved
[34].
4.3 Breast cancer
Breast cancer has been identified as one of the leading causes of cancer death in women, and there is still no effective treatment for breast cancer, and almost 20% of patients still suffer from recurrence of the disease
[113]. Exosomes, a well-targeted, non-toxic and non-immunogenic drug delivery technology, have gained great attention as a potential targeted drug delivery carrier in the treatment of breast cancer.
The target of many antineoplastic chemotherapeutics is intracellular, so chemotherapeutics need to cross the cell membrane before they can exert their effects. Chemotherapeutic drugs are difficult to achieve the desired effect and have serious side effects due to poor water solubility or premature degradation. Exosomes are characterized by low immunogenicity, which makes them less side effects. In addition, the lipid bilayer of exosomes can protect the drug loaded in exosomes
[114]. Exosomes also readily enter cells through the interaction of membrane proteins with recipient cells. Therefore, exosomes are the best natural carriers of chemotherapeutic drugs
[115]. Tian et al. Found that DOX-loaded exosomes could significantly inhibit the growth of breast tumor cells and were not toxic to mice
[116]. Combining dendritic cell-derived exosomes with specific iRGD peptides, the exosomes are endowed with the ability to target breast cancer more effectively than chemotherapeutic agents alone. Circulating tumor cells (CTCs) play an important role in tumor metastasis. Chemotherapy is difficult to eliminate CTCs. There are various specific surface adhesion molecules on the membrane of tumor-derived exosomes, which mediate targeting to homologous tumor cells. Wang et al. Co-encapsulated ROS-sensitive monothioether-linked paclitaxel-linoleic acid prodrug (PTX-S-LA) and cucurbitacin B into PEG-PCL polymer to prepare dual drug-loaded micelles, and further modified with tumor cell-derived exosomal membrane to prepare exosome-like sequential activation biomimetic prodrug nanoplatform (Fig. 8)
[1]. The formulation showed excellent CTCs targeting ability, enhanced prodrug activation, prolonged in vivo circulation time, more tumor accumulation and deeper intratumoral penetration, and showed excellent primary tumor inhibition and anti-metastasis activity in both orthotopic and ectopic MDA-MB-231 tumor models, providing a new strategy for anti-breast cancer metastasis.
Nucleic acid drugs such as siRNA and miRNA have high application value in cancer therapy, but the lack of suitable delivery systems limits their clinical translation. Ohno et al. Used the GE11 peptide as a ligand for EGFR expressed on breast cancer cells
[90]. For this purpose, HEK293 cells were transfected with the synthetic let7a gene and the pDisplay vector encoding GE11, resulting in GE11-let7a-Exos. Luciferase-transfected breast cancer cells, HCC70-luc, were transplanted into the mammary fat pad of RAG2 −/− mice, followed by intravenous injection of GE11-let7A-Exos. The results suggest that exosomes can be used for nucleic acid drug therapy targeting EGFR-expressing cancer tissues.
4.4 Colon cancer
Colorectal cancer (CRC) is the third most deadly cancer in the world, with high aggressiveness, poor prognosis, and a lack of targeted therapies
[117]. 5-Fluorouracil (5-FU) -based chemotherapy plays an important role in the treatment of colorectal cancer. However, long-term use of 5-FU causes multidrug resistance (MDR), which severely weakens the therapeutic effect
[118].
The expression of miRNA plays an important role in CRC drug resistance, for example, miR-21 can induce 5-FU resistance by down-regulating human DNA MutS homolog2 (hMSH2) in CRC
[119]. And restoring dysregulated miRNAs can effectively overcome drug resistance.
There are mRNAs, miRNAs and proteins in exosomes of different cells, which can mediate cell-to-cell communication. Exosomes can effectively deliver biological drugs, and can target and identify tumor cells through molecular pathways to achieve precise drug delivery. Therefore, researchers have considered using exosomes to co-deliver functional RNAs and chemotherapeutic drugs to reverse colon cancer drug resistance and eliminate tumor cells. Liang et al. Utilized engineered exosomes to simultaneously deliver the anticancer drug 5-FU and miR-21 inhibitor (miR-21i) to Her2-expressing cancer cells
[120]. The results showed that the engineered exosome-based 5-FU and miR-21 I co-delivery system could effectively promote cellular uptake and significantly down-regulate the expression of miR-21 in 5-FU-resistant HCT-1165FR cells. The combined delivery of miR-21i and 5-FU with engineered exosomes effectively reversed the resistance of 5-FU to colon cancer and significantly enhanced its cytotoxicity compared with treatment with miR-21i or 5-FU alone.
4.5 Glioblastoma
Glioblastoma (GBM) is the most common and aggressive primary brain tumor, and the current standard of care for patients with GBM is maximal surgical resection followed by radiotherapy and chemotherapy with temozolomide (TMZ). However, even with this combination therapy, less than 10% of patients survive 5 or more years after diagnosis
[90]. Therefore, more effective treatment strategies are urgently needed.
The blood-brain barrier prevents toxic compounds from entering the brain, thereby limiting the delivery of most drugs for brain-related diseases. Exosomes have been found to transport drugs across the blood-brain barrier
[121]. Yang et al. Found that exosome-delivered anticancer drugs crossed the blood-brain barrier and entered the brain
[122]. In a brain cancer model, exosome-delivered anticancer drugs significantly reduced the fluorescence intensity of xenograft cancer cells and tumor growth markers.
Without specific cell source selection and membrane modification, exosomes lack natural targeting ability, so modification is necessary
[91]. Many studies have confirmed that exosomes with surface modification have stronger targeting ability
[123]. Jia et al. Loaded superparamagnetic iron oxide nanoparticles (SPIONs) and curcumin (Cur) into exosomes, and then combined the exosomal membrane with NRP-1 to obtain glioma-targeted exosomes with imaging and therapeutic functions (Fig. 9)
[124]. These engineered exosomes can smoothly cross the blood-brain barrier to achieve targeted imaging and therapy of glioma. In addition, SPIONs-mediated magnetic flow hyperthermia (MFH) and Cur-mediated chemotherapy have also shown strong synergistic anti-tumor effects. The diagnosis and treatment effect of glioma is obviously improved, and the side effect is reduced. This novel glioma-targeting exosome, which can carry both nanomaterials and chemical agents to target glioma, provides a potential way to improve the diagnosis and treatment of intracranial tumors.
5 Summary and Prospect
Chemotherapy and targeted therapy in the course of cancer treatment, due to various defects, it is difficult to achieve the desired efficacy, and patients may also have various side effects due to a large number of drug intake. As a drug delivery carrier, exosomes have great advantages such as low immunogenicity, long-term safety and no cytotoxicity
[125]. In recent years, researchers have made many outstanding achievements in the study of exosomes as drug carriers for targeted therapy of tumors. However, there are still many challenges to give full play to its potential as a drug carrier: ① Tumor cell-derived exosomes contain tumor-supporting components, which can promote tumor growth, invasion and metastasis, so tumor exosomes as a tool for cancer treatment may be a double-edged sword with certain security risks
[126]. ② If exosomes are to be used for drug delivery on a large scale, high-purity exosomes must be isolated on a large scale. Currently, there is no clear and ideal technique for the isolation of highly pure exosomes. Existing isolation techniques can only produce a small amount of exosomes, which is expensive to produce on a large scale. ③ Exosomal drugs are targeted, but their targeting stability has not been studied in depth, and there may be off-target phenomenon. ④ The criteria of manufacturing, loading efficiency, purification, storage, use, stable duration and dosage of exosomes as carriers remain to be studied. ⑤ Exosomes from different sources have different biological effects. Different cell types and culture conditions may also alter key regulatory factors in some exosomes in vivo. Therefore, there is an urgent need to standardize the use of exosomes.
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