Exosome-Based Smart Drug Delivery for Cancer Treatment

Abstract

Advances in nanoscale materials have become indispensable for targeted drug delivery, early detection, and personalized approaches for cancer treatment. Among various nanoscale materials investigated, exosomes hold significant promise in drug delivery. Exosomes are nanoscale vesicles that are usually 30–150 nm in size and produced by cells for intercellular communication. Due to their unique composition and inherent tumor-targeting capacity, these particles are well suited for tumor-specific delivery systems. This chapter discusses exosome isolation, therapeutic loading methods, key roles of exosomes in the tumor microenvironment, current applications of exosomes in drug delivery, and possible clinical implications.

1. Introduction

Over the last few decades, there has been a significant increase in the development of innovative therapeutic drug delivery approaches. Although numerous approaches have been identified, exosome-based drug delivery has drawn significant attention. They are generally 30-150 nm-sized, membrane-bound nanovesicles with a variety of biologically active compounds [1, 2]. Intraluminal vesicles are generated by endocytosing various transmembrane proteins into the cell’s endosomes, which are then sorted and turned into intraluminal vesicles. These vesicles are discharged when an endosome merges with the cell membrane, delivering their contents into the extracellular space [3]. Tetraspanins (specifically CD9, CD63, and CD81) are among the most commonly found proteins on the surface of exosomes and are often used as markers specific to exosomes. It has been demonstrated that these proteins interact with other proteins, including integrins and major histocompatibility complexes [4].

Exosomes generally function as carriers for genetic and proteomic information, playing a crucial role in cellular communication [3, 5]. Exosomes have been associated with cellular communication in the tumor microenvironment. As a result, they are currently being studied as potential targets for therapy and as vehicles for delivering treatments [4, 6]. Targeted delivery of therapeutic drugs to cancer cells minimizes cytotoxic effects on healthy cells. Because of their unique composition and ability to receive various therapeutic substances, it is possible that exosomes can be used as a more precise targeting system for delivering drugs to tumors.

2. An overview of methods and strategies for exosome isolation

Ultracentrifugation is a widely used method for the isolation and purification of exosomes, small extracellular vesicles secreted by cells. This is currently the “gold standard” for isolating exosomes via a variety of centrifugation methods. The technique involves the use of high-speed centrifugation to separate exosomes from other cellular debris and contaminants based on their size and density. Though it is a standard approach for isolating exosomes, it results in a significant sample loss. The vesicles may be damaged by repeated centrifugation, and there is a chance that a highly immunogenic protein aggregate may also co-sediment [7, 8, 9]. Repeated ultracentrifugation operations resulted in decreased particle yields and a reduced total exosome recovery rate [9]. Ultracentrifugation combined with additional isolation techniques can result in a greater yield of exosomes. Payload concentration determination refers to the process of quantifying the concentration or amount of a target molecule or particle present in the sample after ultracentrifugation. Once the sample has been centrifuged and the pellet containing the payload has formed, it is necessary to determine the concentration of the payload accurately [10]. There are several methods to determine the payload concentration after ultracentrifugation. One common approach is to resuspend the pellet in a known volume of buffer solution and measure the optical density (OD) or absorbance at a specific wavelength using a spectrophotometer. By correlating the absorbance measurement with a standard curve generated from known concentrations of the payload, researchers can determine the concentration of the target molecule or particle in the sample. Another method is to measure the mass of the pellet after ultracentrifugation using a sensitive balance. By weighing the pellet and accounting for the resuspension volume, researchers can calculate the concentration of the payload in the sample. In addition to these methods, various analytical techniques such as immunoassays, PCR, or gel electrophoresis can be employed to determine the concentration or purity of specific components within the payload.

Ultrafiltration uses filters or membranes with defined pore sizes to selectively retain particles below a certain size, typically in the range of 10–100 nanometers. The exosomes, being smaller than the retention size, can pass through the membrane, while larger contaminants are retained. This technique allows for efficient separation and concentration of exosomes. Compared to ultracentrifugation, this method is expected to yield more exosomes, but it also has some potential drawbacks [9, 11]. To assess the concentration of payload during this process, the isolated exosomes are typically analyzed using various techniques, such as Western blotting, nanoparticle tracking analysis, or mass spectrometry. These methods allow researchers to assess the presence and abundance of specific cargo molecules within the exosomes, providing insights into their payload concentration [12].

Exosomes or proteins contained in exosomes could adhere to the membrane, which would hinder their retrieval for subsequent analysis [13]. Immunoaffinity capture methods rely on the specific binding between exosomal surface markers and antibodies, allowing for the selective capture of exosomes from a complex biological sample. The sample is typically pre-cleared to remove larger particles and debris, either through centrifugation or by using low-speed filtration. This step helps to eliminate background noise and increase the efficiency of immunoaffinity capture. Next, specific antibodies targeting exosomal surface markers are immobilized on a solid support, such as magnetic beads or a microplate. Antibodies against common exosome markers like CD9, CD63, CD81, or tissue-specific markers can be used. These antibodies have a high affinity for their respective target, allowing for efficient capture of exosomes. After capturing, the exosomes can be eluted from the solid support, usually with a solution that disrupts the antigen-antibody interaction. The eluted exosomes can then be collected, further purified if needed, and used for downstream applications such as characterization, analysis, or therapeutic purposes. The use of magnetic beads in an immunoisolation technique is one important variation of this strategy for quick isolation of exosomes. Utilizing magnetic beads coated with anti-EpCAM, this procedure was proven using exosomes secreted by LIM1863 colon cancer cells. To identify the concentration of the payload during immunoaffinity capture, researchers employ various techniques such as ELISA (enzyme-linked immunosorbent assay) or Western blotting. These methods utilize specific antibodies that can selectively bind to the target molecules of interest, allowing for quantification, and assessment of the payload concentration within the isolated exosomes.

Recent investigations identified a few improved isolation methods than ultracentrifugation [9]. Size exclusion chromatography (SEC) separates particles according to their size by using a porous column or resin through which the sample is passed. In SEC, the sample is loaded onto a column or resin composed of a porous material with specific pore sizes. The column is designed to allow smaller particles, such as exosomes, to enter the pores and interact less with the resin, resulting in faster elution. Larger particles, on the other hand, interact more with the resin and are retained longer within the column. It has been observed that this approach offers highly precise and reproducible outcomes when collecting exosomes. Nonetheless, conducting this separation technique takes a considerable amount of time due to the occurrence of gravity flow separation. Consequently, ultracentrifugation is often utilized to further intensify the concentration of the exosome sample [14, 15, 16]. In exosome isolation through size exclusion chromatography, the payload concentration of exosomes can be determined by analyzing the eluted fractions collected during the chromatographic process [17]. This can be done by quantifying specific cargo molecules, such as proteins or nucleic acids, present in the collected samples. Various analytical techniques like Western blotting, ELISA, or qPCR can be employed to measure the concentration of these cargo molecules, providing an estimation of the payload concentration in the isolated exosomes.

Polymer-based precipitation methods rely on the addition of polymers, such as polyethylene glycol, to the sample to precipitate exosomes, thereby facilitating their separation. A method of precipitating substances has been shown to potentially yield higher levels of exosomal RNA and protein purity compared to the widely accepted ultracentrifugation method. Exosomes isolated from ascites are the only source that can prove this, yet [18]. Polymer-based precipitation methods were regarded as a straightforward, quick, and scalable option to isolate and identify exosomes [11]. To determine the payload concentration, additional analysis techniques such as spectroscopy, mass spectrometry, or ELISA assays can be employed, targeting specific payloads of interest. These techniques can provide quantitative measurements and help to determine the payload concentration in the isolated exosomes. It’s important to note that the efficiency of polymer-based precipitation and the resulting payload concentration can vary depending on the specific experimental conditions and the quality of the starting sample.

Microfluidics-based devices are designed with nanostructured surfaces or channels that can selectively trap exosomes based on their size or surface markers. This technique allows for the isolation of exosomes without the need for extensive sample preparation, enhancing efficiency and reducing contamination. Additionally, this approach is more compatible with clinical laboratory practices than the exosome isolation practices that are currently in use. Furthermore, exosomes from serum can be separated using microfluidics devices in a single step as opposed to magnetic bead-based techniques [19]. To evaluate the payload concentration, microfluidics devices often utilize techniques such as fluorescence-activated sorting or single-particle analysis [20]. In fluorescence-activated sorting, the exosome sample is passed through a microfluidic channel where fluorescently labeled exosomes are detected and sorted based on their fluorescence intensity. This allows for the quantification of the payload concentration within the isolated exosomes. Single-particle analysis involves imaging individual exosomes within the microfluidic device. By capturing images of the exosomes, their cargo can be visually identified and analyzed. This provides insights into the payload concentration and can be used to study the heterogeneity of the cargo within the exosomes. These devices play a crucial role in the study of exosomes and their cargo, opening up exciting possibilities for research and potential applications in various fields such as diagnostics and therapeutics.

3. Advancements in drug loading strategies for exosomes

Electroporation (electric pulses to produce transitory pores in the lipid bilayer) facilitates the incorporation of therapeutic cargos such as drugs, nucleic acids, or proteins in exosomes. The electric pulses create temporary openings in the exosome membrane, enabling the efficient loading of therapeutic molecules into the exosomes. The therapeutic-loaded exosomes generated by electroporation methods have shown promise in various therapeutic applications. For targeted drug delivery applications, the loaded exosomes are engineered to specifically deliver therapeutic cargos to desired cell types or tissues. Additionally, these exosomes can be utilized in gene therapy, where exosomes with loaded nucleic acids can deliver genetic material to target cells for therapeutic purposes. Greco et al., [21] revealed that Mesenchymal stromal cells (MSC) and Human Embryonic Kidney 293 (HEK293) exosomes were suspended in an electroporation buffer containing several forms of siRNA at a specific concentration. Following that, the exosome-siRNA mixture was put into a cuvette and electroporated with a Bio-Rad® Gene Pulse XCell electroporation equipment. The authors were able to measure the exosome loading efficiency using siRNA that was allophycocyanin-labeled. The UMUC3 bladder cancer cells were incubated with the exosomes for 6 hours. They observed that the fluorescence intensity was more than 28-fold higher in the siRNA-loaded exosome-containing cell population. The enhanced fluorescence intensity demonstrated that electroporation was effective in transferring siRNA into exosomes, and consequently into the bladder cancer cell. A modified calcium chloride transfection technique is capable of introducing miRNA into exosomes [22]. This technique offers a versatile platform for loading a wide range of therapeutics. However, there is a chance that this approach will result in excessive aggregation or that the exosomes’ membrane integrity could be damaged. Electroporation also requires specific equipment to use, such as the Neon® Transfection System [22, 23]. It is important to note that further research is required to optimize electroporation methods for therapeutic loading in exosomes. Factors such as pulse parameters, cargo compatibility, and the impact of electroporation on exosome stability and functionality need to be carefully investigated.

Incubation methods for therapeutic loading in exosomes are another approach that researchers have explored to enhance the therapeutic potential of exosomes. This method involves incubating exosomes with therapeutic molecules, allowing for the passive uptake of these molecules by exosomes through diffusion or other mechanisms. The advantage of incubation methods is their simplicity and ease of use. They do not require complicated equipment or specialized techniques, which makes them accessible to a wide range of researchers. However, it is important to consider the limitations of incubation methods. The loading efficiency achieved through diffusion-based uptake can be relatively low, resulting in a fraction of exosomes being loaded with therapeutic cargo [24]. Curcumin-loaded exosomes have been used to demonstrate a simple incubation of exosomes with therapeutic payload. Curcumin and exosomes were mixed in PBS and incubated at 22°C for 5 min. The samples were subsequently centrifuged using a sucrose gradient for 1.5 hours at 20,000 × g, and HPLC analysis was used to determine the curcumin concentration [25].

Incubation of donor cells is often used as a method for packaging therapeutic cargo into exosomes. To load exosomes with therapeutic cargo, donor cells are first incubated with the desired cargo molecules. This can be achieved by treating the cells with specific drugs or by transfecting them with plasmids or viral vectors encoding the cargo of interest. The uptake and packaging of therapeutic cargo into exosomes occurs during the biogenesis of these vesicles. Mesenchymal stromal cells were treated with low-dose paclitaxel for 24 hours and then reseeded in new flasks. Exosomes containing paclitaxel and having a therapeutic effect on pancreatic cancer cells in vitro were isolated from media collected after growth [26]. Saponins are natural compounds found in certain plants and have been shown to facilitate the loading of therapeutic molecules into exosomes. This loading process involves incubating therapeutic agents together with exosomes and saponins, allowing them to interact and promote the encapsulation of the cargo inside the exosomes. Since saponin is thought to have a hemolytic impact, its concentration should be reduced to a minimum and, preferably, purified from the final product [24, 27, 28]. In the sonication approach, exosomes and cargo are sonicated using a homogenizer probe which leads to considerable deformation within the membrane, allowing for increased diffusion of drugs into the exosome [29].

Numerous studies have demonstrated the efficacy of the sonication method; in some instances, it has resulted in multiple layers of drug encapsulation, some of which incorporate inside the exosome and some within the membrane, resulting in a two-stage drug release, where the membrane-bound portion is released more quickly, and the internalized drug is released over a longer period of time [30]. Despite the fact that this approach may be advantageous for certain drugs, it has been shown to result in the aggregation and/or degradation of nucleic acids [28, 29]. In the extrusion method, exosomes are mixed with a drug, and the resultant mixture is passed through membranes ranging in size from 100 to 400 nm at a regulated temperature. This leads to a strong mixing of the exosomes and drug, resulting in membrane breakdown and drug loading. The effects of the strong mechanical forces caused by extrusion on exosomes are yet unknown [24, 29]. However, one study determined that exosome extrusion caused an altered zeta potential and cytotoxicity, but other loading methods did not cause cytotoxicity [27] for therapeutic loading in exosomes involve the use of specialized techniques to package therapeutic agents into exosomes, which are small vesicles secreted by cells. These methods aim to enhance the therapeutic potential of exosomes by incorporating specific drugs, proteins, nucleic acids, or other bioactive molecules.

Freeze-thaw cycling involves subjecting exosome samples to rapid freezing followed by thawing. This process helps to disrupt the integrity of the exosomes, allowing for enhanced cargo loading. During freezing, the exosome membrane becomes more permeable, which facilitates the uptake of therapeutic cargo molecules. Sato et al., demonstrated the utilization of freeze-thaw procedures to manufacture exosome-mimetic liposomal particles [31]. However, when compared to other methods of drug encapsulation, freeze-thaw cycling can promote exosomal aggregation and is often less successful than sonication or extrusion [24, 29].

Chemical transfection methods have been used to load exosomes with siRNA as shown in several studies [32, 33]. This approach may not be suitable due to a decreased loading efficiency as compared to electroporation. Furthermore, the Lipofectamine 2000 reagent was shown to make micelles, which could have impaired the quality of the exosomal preparation. Altogether, chemical transfection of exosomes is inefficient for loading drugs into exosomes [23]. Transfection of cells can indeed be used as a method for loading therapeutic cargo into exosomes. In this approach, instead of directly loading the cargo into exosomes, the cargo is introduced into the donor cells, which then release the cargo-loaded exosomes [23]. This method involves transfecting the donor cells with the desired therapeutic cargo, typically using transfection reagents or techniques such as electroporation. In a recent study by Katakowski et al., bone marrow stromal cells were utilized to transfect with miR-146b and resulting exosomes from these cells were isolated to treat 9 L gliosarcoma cells. The obtained exosomes from transfected cells revealed increased miR-146b expression and has been shown to inhibit glioma development in rat models [34].

4. Role of exosomes in tumor microenvironment

The role of exosomes in the tumor microenvironment is a fascinating and rapidly growing field of research [35, 36]. Exosomes are small extracellular vesicles that are released by various cell types, including cancer cells, into the surrounding environment [37]. They play an important role in intercellular communication between cancer cells and their tumor microenvironment, facilitating the exchange of molecular signals and genetic material, and have also been linked to a variety of physiological and pathological processes, including cancer development and progression [35, 38]. The microenvironment, in the context of cancer, refers to the cellular and non-cellular components surrounding the tumor, including stromal cells, immune cells, fibroblasts, the extracellular matrix, blood vessels, basement membrane, and endothelial cells [38]. These components actively interact with cancer cells and can influence tumor growth, metastasis, and response to therapy. Considering all these factors/components, there are four primary modes which exosomes from various sources can alter the TME: through facilitating immunological escape, drug resistance, increasing metastasis, and enhancing angiogenesis.

4.1 Exploring the role of exosomes in immune evasion

Exosome-mediated immune evasion occurs when these vesicles are utilized by cancer cells and pathogens to escape immune surveillance [39, 40]. These entities exploit exosomes to carry specific molecules that suppress the immune system, allowing them to evade detection and continue their malignant or infectious activities. The function of T cells can also be inhibited by these exosomes, and there are numerous ways to do this [41]. The activation of T cells’ immune systems to combat cancer cells is inhibited in one approach by the binding of programmed cell death ligand 1 (PDL1) to PD1 receptors on T cell membranes [42]. They also contribute to immune evasion through the transfer of immunosuppressive molecules. Cancer cells and pathogens package molecules such as transforming growth factor-beta (TGF-β), interleukin-10 (IL-10), and FasL into exosomes [43]. When these exosomes interact with immune cells, they can inhibit the activation and function of T cells, natural killer cells, and dendritic cells, subsequently undermining the immune response. Exosomes also participate in the establishment of an immunosuppressive microenvironment. They can influence the behavior of surrounding cells, including immune cells, by transmitting signals that promote an environment conducive to immune evasion. For instance, tumor-derived exosomes can induce the differentiation of regulatory T cells (Tregs), which suppress immune responses and foster tumor growth [44]. Exosomes associated with tumors have been shown to alter the phenotype of macrophages in multiple malignancies from M1 (tumor resistance, pro-inflammatory) to M2 (tumor promotion, anti-inflammatory) [45, 46, 47, 48]. As a result, exosomes derived from these altered M2 macrophages can promote migration and invasion further. This was shown in a recent study by Lan et al., who investigated that macrophage-derived exosomes contained high levels of miR-21-5p and miR-155-5p, which caused BRG1, a crucial component in colorectal cancer metastasis, to be downregulated [49]. Figure 1 summarizes these effects.

Figure 1.

Exosome-mediate tumor evasion.

Furthermore, exosome-mediated immune evasion can dampen the effectiveness of immunotherapies [43]. Immunotherapeutic strategies aim to enhance the immune system’s ability to recognize and eliminate cancer cells or pathogens [50]. However, exosomes produced by these entities can interfere with this process. They can either directly inhibit immune cells’ activity or carry molecules that counteract the effects of immunotherapy. Understanding this phenomenon is vital for the development of strategies to overcome immune evasion [51]. Researchers are exploring various approaches, including targeting specific molecules carried by exosomes, modulating exosome release, or utilizing exosomes for therapeutic purposes. By unraveling the mechanisms of exosome-mediated immune evasion, scientists hope to optimize immunotherapies and improve the prognosis for patients affected by diseases involving immune evasion mechanisms [43, 52]. Further research in this area promises to enhance our understanding of immune evasion mechanisms and pave the way for innovative therapeutic interventions.

4.2 Understanding how exosomes drive cancer progression

One important role of exosomes in the cancer-associated microenvironment is their ability to promote tumor progression and metastasis. Exosomes released by cancer cells can transfer oncogenic cargo (such as growth factors, cytokines, miRNA proteins, RNA, DNA, and lipids) to recipient cells in the microenvironment, altering their behavior and promoting a pro-tumorigenic state. Exosomes have been shown to promote EMT, stimulate cell growth, and even disrupt the ECM to facilitate invasion and metastasis from the initial site to distant sites in the body [53]. Furthermore, exosomes can confer invasive properties to cancer cells, leading to enhanced metastasis. Exosomes released by primary tumor cells can prepare distant sites for metastatic colonization by promoting pre-metastatic niche formation. They achieve this by influencing the extracellular matrix, remodeling, and preparing it for better adherence and colonization of metastatic cells. Wang et al., recently showed that miR-181-5p might be transferred by cancer-associated fibroblast exosomes to breast cancer cells, inhibiting CDX2, and accelerating EMT [25]. Previous studies by Hoshino et al., demonstrated that exosomes could establish at future metastatic locations, and the location of this could be determined in part owing to the combination of integrins situated on the exosomes [54]. Remarkably, a recent study from Yuan et al., suggested that bone metastases were more closely associated with breast cancer exosomes harboring miR-21 than non-metastatic tumors [55]. Moreover, exosomes can carry enzymes that facilitate the degradation of the extracellular matrix, allowing cancer cells to invade surrounding tissues and intravasate into blood or lymphatic vessels, ultimately spreading to distant organs [56]. By modifying the tumor microenvironment and enhancing the migratory and invasive abilities of cancer cells, exosomes contribute significantly to cancer progression [57].

Recent research on breast cancer exosomes containing miR-4443, which blocks tissue inhibitors of metalloproteinase 2 (TIMP2), suggested that the tumoral ECM may be degraded [58]. This study employed a mouse model to demonstrate that the release of these exosomes promoted metastasis and reduced metastases in vivo as a consequence of miR-4443 suppression [58]. Exosomes can carry different biomolecules, such as RNA, miRNA, DNA, and proteins, or which can influence different signaling pathways in other cells, according to numerous recent studies [59]. These may involve transfers between cancer cells and stromal cells or autocrine/paracrine actions on cancer cells [6]. A recent study demonstrated that lymph angiogenesis and enhanced metastasis through lymph nodes were induced by exosomes produced by bladder cells that contained the lncRNA LNMAT2 [60]. Another study revealed that EphA2, a kinase that influences ERK signaling to accelerate cancer growth, was abundant in exosomes isolated from drug-resistant breast cancer cells [60]. Exosomal SOX2 DNA was examined as a potential biomarker of glioblastoma cancer progression [61].

4.3 Exosomes as mediators of drug resistance in cancer

Drug resistance is a critical issue in the field of medicine, affecting the effectiveness of various treatments [62]. One emerging area of research that holds great promise in tackling drug resistance [63]. Exosomes have recently emerged as key mediators in the development and propagation of drug resistance in cancer and other diseases [64, 65]. Drug resistance is a complex phenomenon characterized by cells becoming less responsive or resistant to the effects of therapeutic drugs (Figure 2) [66]. This can occur through various mechanisms, including reduced drug uptake, increased drug efflux, altered drug metabolism, and the activation of cell survival pathways [66]. One-way exosomes contribute to drug resistance by transporting molecules involved in the resistance phenotype. For instance, exosomes secreted by cancer cells can carry multidrug resistance proteins, such as P-glycoprotein, which actively pump chemotherapeutic drugs out of recipient cells, reducing their efficacy [67].

Figure 2.

Exosome mediated chemo-resistance.

Additionally, exosomes may transfer genetic material, such as microRNAs and circular RNAs, that can alter cellular signaling pathways, leading to drug resistance [68]. Interestingly, exosomes themselves can be influenced by the presence of drugs, further affecting drug resistance. Studies have shown that chemotherapeutic drugs can modify the cargo composition of exosomes, making them more resistant to treatment [69]. This implies that exosomes contribute not only to drug resistance but also adapt to therapy, potentially amplifying resistance mechanisms. Examples of the second approach indicated above have been demonstrated in a variety of cancers. For example, breast cancer cells may directly sequester adriamycin in exosomes to increase resistance [70]. By using UV spectrophotometry, it was demonstrated that adriamycin was localized within the exosomes secreted by drug-resistant cells [70].

4.4 Exosomes as key players in angiogenesis

Several mechanisms have been proposed to explain how exosomes promote angiogenesis [71]. One mechanism involves the direct transfer of pro-angiogenic molecules from exosomes to recipient cells [72]. Exosomes have emerged as critical players in tumor angiogenesis [72, 73, 74, 75]. For example, exosomal transfer of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and matrix metalloproteinases (MMPs) can activate endothelial cells, promoting their sprouting and migration towards the site of angiogenesis [71]. Additionally, cancer cell-derived exosomes can influence the behavior of stromal cells in the tumor microenvironment, further enhancing angiogenesis and supporting tumor growth [76]. Moreover, exosomes can mediate angiogenesis by altering the expression of specific genes or proteins in recipient cells. Figure 3 illustrates these effects.

Figure 3.

Role of exosome in angiogenesis.

Exosomal cargo, such as microRNAs, can be transferred to endothelial cells and regulate gene expression, thereby modulating angiogenic processes [77]. The transfer of functional microRNAs, which can act as regulators of gene expression, provides an additional layer of complexity to the exosome-mediated regulation of angiogenesis. Understanding the mechanisms by which exosomes promote angiogenesis provides exciting opportunities for developing novel therapeutic strategies aimed at modulating angiogenesis in diverse contexts, ranging from tissue regeneration to cancer treatment. Further research in this field holds the potential to revolutionize the field of vascular biology and improve patient outcomes in a variety of diseases.

4.5 Emerging role of exosomal lncRNAs in tumor progression and metastasis

Exosomal long non-coding RNAs (lncRNAs) are important components of the tumor microenvironment [78, 79]. They have been reported to modify a variety of characteristics of the tumor microenvironment in the setting of cancer, including immune evasion, tumor development, angiogenesis, metastasis, and drug resistance [80]. Additionally, lncRNAs have the potential to be used as diagnostic or prognostic biomarkers in several cancer types [81], and can be extracted from a variety of biofluids, including urine, blood, and saliva offering a non-invasive way to track the progression of diseases. Studies have confirmed that exosomal lncRNAs exhibit differential expression in cancer patients as compared to healthy individuals, suggesting their potential as non-invasive diagnostic tools. Clinicians may be able to identify malignancies at an early stage by evaluating the expression of exosomal lncRNAs, enabling prompt treatment options and improving patient outcomes. One study revealed that PCAT-1, MALAT1, and SPRY4-IT1 were substantially concentrated in urine samples [82], while pCAT-1 and UbC H19 were elevated in the serum of bladder cancer patients [83, 84].

Patients with prostate cancer had elevated levels of P21 in their urine [85]. Numerous elevated lncRNAs have been identified through plasma analysis as potential biomarkers for various malignancies, including SAP30L-AS1 and SChLAP1 for prostate cancer [79], HOTAIR for breast cancer, the LNCV6 family for colon cancer, SOX2-OT for lung squamous cell carcinoma, and the LNCV6 family for colorectal cancer. Within the cervicovaginal lavage of cervical cancer patients, HOTAIR and MALAT1 were elevated while MEG3 was downregulated [86]. Upregulation of MALAT1 was seen in serum biomarker analysis for epithelial ovarian cancer [87], UEGC1 and HotTip for gastric cancer [88, 89, 90] HOTAIR for glioblastoma multiforme [90]. On the other hand, certain exosomal lncRNAs have been found to have tumor-suppressive effects, inhibiting tumor cell proliferation and metastasis. For instance, lncRNA Gas5 has been identified to be downregulated in non-small cell lung cancer patients’ serum, pointing to its potential function as a tumor suppressor [91]. In a previously described CRC animal model, carcinoma-associated fibroblasts (CAFs) were found to enhance stemness and chemoresistance by transferring exosomal H19 lncRNA, which in turn activated the beta-catenin pathway [92]. It has been demonstrated that the lncRNA RUNX2-AS1 contained in multiple myeloma exosomes interacts with the transcription factor RUNX2 to reduce the osteogenic potential of mesenchymal stem cells [93]. Under hypoxic conditions, the lncRNA UCA1 found in the exosomes of bladder cancer cells facilitated EMT and altered the tumor microenvironment [94].

The pro-oncogenic CCAT2, POU3F3, and HOTAIR in glioma cells, as well as exosomal lncRNAs, have been demonstrated to enhance angiogenic factors and hence promote invasion and metastasis [95, 96, 97]. Apoptosis suppression was indicated by an increase in Bcl2 expression and a decrease in Bax and caspase 3 [98]. The lncRNA MALAT1, which is derived from exosomes of epithelial ovarian cancer cells, has been identified to promote the pro-angiogenic genes VEGF-A, VEGF-D, IL-8, and angiogenin [87]. Another Study demonstrated that PCAT1 binds to miR-326 to enhance cell proliferation in esophageal squamous cell carcinoma while MALAT1 stimulates cell proliferation in breast cancer and non-small cell lung cancer [99, 100]. ZFAS1 stimulates cell cycle, apoptosis, and EMT in gastric cancer [59], whereas UCA1 has the same impact in bladder cancer [94]. By altering the expression of HNRNPK, 91H promotes metastasis in colorectal cancer [101]. H19 competes with miR-141 and turns on the β-catenin pathway, retaining tumor cell stemness and driving drug resistance [92]. RUNX2-AS1, LncRNA H19, FMR1-AS1, and Sox2ot have been examined for their functions in promoting tumor stem cells in various malignancies [92, 93, 102, 103].

Exosomal lncRNA has also been found to be a significant contributor to drug resistance in several malignancies. For example, UCA1 was the cause of tamoxifen resistance in breast cancer [104], cisplatin resistance in ovarian cancer [105], and cetuximab resistance in metastatic colorectal cancer [106]. For instance, AGAP2-AS1 and SNHG14 have reported trastuzumab resistance in breast cancer [91, 107]. Gefitinib resistance in esophageal squamous cell carcinoma was caused by Part 1 [108]. Increased lncRNA SBF2-AS1 activity in glioblastoma has been related to temozolomide resistance [68]. Erlotinib and gefitinib resistance in non-small cell lung cancer has been associated with RP11-838 N2.4 and H19 [98, 109]. Sunitinib resistance was increased by ARSR in renal cancer [110]. Moreover, exosomal lncRNAs can also modulate the tumor immune response. They can influence the activity and function of various immune cells, such as T cells, natural killer cells, macrophages, and dendritic cells [111]. By regulating immune cell functions, exosomal lncRNAs can impact immune surveillance, immune evasion, and immune suppression within the tumor microenvironment. In a nutshell, exosomal lncRNAs have emerged as important regulators of the tumor microenvironment. Their expression patterns have been linked to tumor aggressiveness, metastasis, and drug resistance. Therefore, exosomal lncRNAs may serve as prognostic indicators and therapeutic targets. Modulating the expression or function of exosomal lncRNAs could potentially interfere with tumor growth, reduce metastatic potential, and sensitize cancer cells to existing therapies.

5. Promising role of exosomes in enhancing drug delivery to specific cellular targets

Exosomes are being extensively explored for targeted delivery in cancer treatment. They offer several advantages in this context. One of the key benefits is their ability to specifically target cancer cells, reducing off-target effects and enhancing the effectiveness of therapy. Researchers have been able to modify the surface of exosomes to ensure their uptake by cancer cells. By loading these engineered exosomes with therapeutic agents such as drugs, siRNAs, or gene-editing tools, they can deliver the cargo directly to the tumor site. This targeted delivery enables the potent anticancer agents to act specifically on cancer cells, increasing their efficacy while minimizing adverse effects on healthy tissues. This was demonstrated by employing the delivery of siRNA to bladder cancer cells using exosomes that were obtained from HEK293 human embryonic kidney cells. Electroporation was used to introduce PLK1 siRNA to the exosomes before they were co-cultured with UMUC3 metastatic bladder cancer cells. According to an in vitro study, bladder cancer cells internalized HEK293 exosomes more than normal bladder cells, which resulted in an efficient knockdown of PLK-1 mRNA and protein [21].

Exosomes can also carry drugs that are otherwise challenging to deliver directly to tumor tissues. These vesicles have the advantage of being able to navigate through biological barriers, including the extracellular matrix and the blood-brain barrier, to reach their intended target. This ability makes them particularly useful for delivering therapeutics to tumors located in difficult-to-reach sites [112, 113]. A zebrafish model was used to examine the exosomes in vivo after they had been extracted, characterized, and loaded with chemo agents via incubation. In comparison to the basic drugs, the loaded exosome systems demonstrated substantially better CNS delivery capability [114]. Moreover, exosome-mediated chemotherapeutic administration has been found to increase anti-cancer effects in a number of studies [115, 116, 117, 118]. Doxorubicin, another of the most potent anti-cancer drug, is employed to treat leukemia, lymphoma, and a variety of solid tumors. It has been observed that exosomes can carry chemotherapeutic drugs like doxorubicin and paclitaxel. However, because of its poor biocompatibility and substantial side effects such as bone marrow suppression and cardiotoxicity, the clinical usage of doxorubicin is extremely limited. Many nanoparticle technologies are being used to improve doxorubicin’s biocompatibility and anti-cancer properties, but they also cause immunological response and oxidative damage [118, 119]. Exosome-mediated anti-cancer therapy has extensively investigated doxorubicin because of its easy-to-track intrinsic fluorescence [118]. The optimized cholesterol endocytosis process and phospholipid composition of exosomal membranes allow exosomes to target cancer cells better than liposomes [120]. Since doxorubicin frequently causes cardiotoxicity, loading doxorubicin into exosomes prevents doxorubicin from reaching cardiac endothelial cells. As a result, this lowers the risk of cardiotoxicity [121]. It has been revealed more recently that the cellular absorption rate and anticancer effect of doxorubicin in osteosarcoma could be improved by exosomes produced from mesenchymal stem cells [122]. In cisplatin-resistant patients, paclitaxel is frequently used to overcome drug resistance [123]. Mesenchymal stromal cells treated with paclitaxel were able to generate paclitaxel-loaded exosomes, which exhibited powerful anti-cancer effects in human pancreatic cancer [26]. MDR stands for multiple drug resistance, and it is one of the main challenges to effective cancer treatment. Exosomes have demonstrated efficacy in overcoming multidrug resistance in cancers. In MDCK MDR1 cells, paclitaxel-loaded macrophage-derived exosomes demonstrated higher cell uptake and a lower IC50 than free paclitaxel, circumventing the P-glycoprotein drug efflux transporter [30]. Exosomes produced from U-87 MG cells have the potential to deliver paclitaxel and overcome MDR, which could lead to an enhanced therapeutic effect in glioblastoma multiforme [124]. Furthermore, the use of exosomes in cancer treatment holds promise for personalized medicine. Exosomes can be isolated from a patient’s own cells, loaded with specific therapeutic agents, and then reintroduced back into the patient. This approach takes advantage of the unique characteristics of each patient’s tumor, tailoring the treatment to their specific needs. While the use of exosomes in targeted delivery for cancer treatment shows great potential, there are still challenges that need to be addressed. These include optimizing exosome production and cargo loading techniques, ensuring the stability of exosomes during storage and transportation, and further improving their targeting efficiency. In addition, the exosomes were native to the animal and were small, so they naturally avoided phagocytosis, which lowered the immunological response [125]. Zhou et al., [126] showed that exosomes from bone marrow mesenchymal stem cells were loaded with siRNA and oxaliplatin and used for the treatment of pancreatic cancer in a rat model. In vitro and in vivo, exosomal delivery increased the uptake of these compounds, indicating a greater therapeutic effect than a free drug.

Exosomes have also been explored as potential diagnostic and prognostic biomarkers in cancer. The analysis of exosomes obtained from body fluids, such as blood or urine, can provide valuable information about the presence of specific genetic alterations or the expression of tumor-related molecules. This non-invasive approach has the potential to revolutionize cancer diagnosis and monitoring, allowing for earlier detection and personalized treatment strategies. Qambrani et al., [127] demonstrated that cancer-cell-derived exosomes can function as both a drug delivery system and a potential fluorescent biomarker, as shown by HeLa-derived exosomes loaded with doxorubicin and silver nanoclusters [127]. Recently, there has been growing interest in using exosomes derived from bovine milk as a drug delivery vehicle [128, 129]. For example, Li et al., [130] recently demonstrated that doxorubicin uptake and therapeutic impact against cancer cells in vitro were considerably improved by first isolating milk exosomes, coating them with hyaluronan for CD44-targeting, and then loading them with the drug. Figure 4 depicts the appearance of an isolated exosome and exosome-specific biomarker analysis in the experimental context [131].

Figure 4.

(A) A flowchart depicting the isolation of exosomes from human peripheral blood neutrophils (N-ex). (B) Transamination electron microscopy (TEM) analysis of N-ex. (C and D). Morphological study of N-ex using atom force microscopy (AFM). N-ex nanoparticle tracking assay (NTA) for size determination particle size (E), zeta potential (F). (G) DLS was used to measure the PDI of N-ex. (H) Western blot studies of exosomal biomarkers (CD63, CD81 CD9, and Alix) and ER markers (calnexin) in N-ex. (I and J) fluorescence confocal laser microscopy and imaging flow cytometry were used to investigate DiR-labeled N-ex untacking in the gastric cancer cell (HGC27). The nuclei of the cells were stained with nuclear dye DAPI. Scale bars, 20 μm. Adopted from ref. [131].

In a recent study, Carobolante et al., delineated that milk-derived exosomes had a less efficient uptake when compared to exosomes produced by Caco-2 epithelial cells. Additional modifications are necessary for milk-derived exosomes to be utilized as a drug delivery system [132]. Another study confirmed that paclitaxel-loaded exosomes (milk derived) inhibited tumor growth more effectively than free paclitaxel, with fewer systemic side effects [133]. In one instance of drug delivery, exosomes containing curcumin were used to treat inflammatory disease. In a clinical trial, exosomes formed a complex with curcumin that increased its effectiveness compared to free curcumin. In another study, curcumin was loaded onto exosomes derived from EL-4 murine tumor cells via incubation. In vitro experiments revealed that exosomal curcumin substantially decreased inflammatory cytokine levels when compared to curcumin in its native form [134, 135, 136].

Exosomes have been extensively studied for their ability to deliver genetic therapies including small interfering RNA (siRNA) and microRNA (miRNA), because they naturally carry nucleic acids like DNA and RNA [3, 5], as previously mentioned [117]. The expression levels of target genes can be downregulated or disrupted using siRNA as a therapeutic agent. Typically, siRNA is unstable and rapidly destroys in the bloodstream. Exosomes serve as a delivery vehicle for these RNA molecules, protecting them from destruction in systemic circulation [117]. Kamerkar et al., used kRAS siRNA-loaded exosomes in pancreatic cancer mice models [137]. This study proves that compared to siRNA-loaded liposomes; the exosome group showed a higher reduction in tumor growth as well as a lower clearance from the body. Wahlgren et al., [32] reported that exogenous siRNA was inserted into exosomes delivered to human blood cells after exosomes were isolated from lung cancer cells, suggesting the potential of utilizing exosomes for gene therapy. Nevertheless, the advancements in exosome research provide a promising avenue for improving cancer treatment outcomes and minimizing side effects associated with conventional therapies. Ongoing studies continue to explore the full potential of exosomes in targeted delivery for cancer therapy.

6. Challenges and limitations in implementing exosome-based therapies in clinical practices

Exosomes have gained significant attention in the field of medicine due to their potential clinical applications. One of the most promising applications of exosomes is in regenerative medicine. Exosomes derived from stem cells have shown great potential in promoting tissue repair and regeneration. They can transfer bioactive molecules such as proteins, nucleic acids, and lipids to target cells, stimulating healing processes. As previously discussed, exosomes, could deliver drugs to target areas and cross barriers when compared to other nanoparticles. Exosomes’ drug efficacy and half-life were well maintained when they were injected into the recipient cell, according to the research findings. Because the exosomes are endogenous mediators, they possess natural cell permeability, which allows them to pass physical barriers and even avoid lysosomal breakdown and endosomal pathways [138]. Macrophage-derived genetically engineered exosomes can transport drugs without being rejected [139]. These targeting characteristics can be tailored to malignancies or other disorders with specific markers or proteins. They can also cross the BBB, allowing CNS-active drugs to be delivered [125].

Exosomes also hold promise as diagnostic markers for several cancer types and as an early detection tool in many clinical studies. This has the potential to revolutionize disease diagnosis and monitoring, enabling earlier detection and personalized treatment approaches. For instance, the goal of the colon cancer clinical study NCT04523389 is to create diagnostic markers. As seen on Clinicaltrials.gov, accessed on Aug 8, 2023, there are currently dozens of trials when searching for the terms “cancer” and “exosomes.” While most focus on the use of exosomes as a diagnostic marker, some have a specific interest in using exosomes as a delivery system. However, despite the progress made in exosome research, several challenges remain. Standardization of methods for exosome isolation, characterization, and quantification is crucial for ensuring consistent and reliable results. Additionally, the large-scale production of exosomes and their scalable purification for clinical use need to be addressed [125]. Overall, the clinical use of exosomes is an exciting and rapidly evolving field. With further research and development, exosomes hold immense promise for advancing regenerative medicine, diagnostics, and drug delivery, potentially transforming the way we approach various diseases and conditions in the future. Table 1 highlights recent clinical trials demonstrating the various ways to exploit exosomes as a natural drug delivery system.

Cancer Type	Source	Drug Delivery System	Clinical Phase	ClinicalTrials.gov ID	Status
Metastatic Pancreatic Adenocarcinoma	Mesenchymal stem cell	KrasG12D siRNA (iExosomes)	Phase I	NCT03608631	Recruiting
Pancreatic Cancer	Exosome-mediated Intercellular Signaling			NCT02393703	Recruiting
Head and Neck Cancer	Plant	Grape exosomes	Phase I	NCT01668849	Completed
Advanced Hepatocellular Carcinoma	Cell-derived exosomes	exoASO-STAT6 (CDK-004)	Phase I	NCT05375604	Terminated
Colon Cancer	Plant	Curcumin	Phase I	NCT01294072	Unknown
Non-Small Cell Lung Cancer	Dendritic cells	Vaccination With Tumor Antigen-loaded Dendritic Cell-derived Exosomes	Phase II	NCT01159288	Completed
Malignant Glioma	Tumor cells	IGF-1R/AS ODN)-exosomes	Phase I	NCT02507583; NCT01550523	Completed

Table 1.

The recent clinical trials of exosomes as a drug delivery vehicle in cancer.

7. Conclusion

In summary, exosomes have emerged as promising nanoscale vesicles for targeted drug delivery in cancer treatment. Their unique composition, small size, and inherent tumor-targeting ability make them valuable vehicles for delivering therapeutic substances to tumors. This chapter has discussed the isolation methods, therapeutic loading techniques, and the key role of exosomes in the tumor microenvironment. The use of exosomes as drug delivery systems holds great potential for minimizing cytotoxic effects on healthy cells, while effectively delivering treatments to cancer cells.

8. Future prospective

There are numerous prospective implications for exosome-based smart delivery of drugs in cancer treatment. First, further research is needed to optimize the isolation and purification techniques of exosomes to ensure consistent quality and scalability for clinical applications. Second, exploring alternative methods for loading therapeutic agents onto exosomes, such as genetic engineering or bioconjugation strategies, could enhance drug encapsulation efficiency and therapeutic efficacy. Third, understanding the mechanisms of exosome-mediated cellular communication within the tumor microenvironment can open avenues for designing more targeted and personalized treatments. Additionally, exosome’s potential as diagnostic tools for early cancer detection and monitoring treatment response may also be investigated. Overall, with continued advancements in exosome research, we anticipate exosome-based smart drug delivery systems to revolutionize cancer treatment by providing more effective and tailored therapies in the near future.

Acknowledgments

This work was partially funded by Start-up from Department of Immunology and Microbiology, School of Medicine, University of Texas Rio Grande Valley, and NIH grants (R01 CA210192 and R01 CA206069). Authors thank the CPRIT (RP210180 and RP230419) and UT-System Star Award.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

Original draft preparation: S.M. and M.S., Editing: S.K. and M.M.Y., Final proofreading and supervision: S.C.C. All authors have read and approved the final manuscript.