Golden Pen Award | Research Progress on the Application of Exosomes in Drug Delivery

Exosomes are specific membrane vesicles secreted by various types of cells under normal or pathological conditions, playing a vital role in intercellular communication and exchange of information. Widely present in various body fluids such as blood, urine, saliva, and breast milk, exosomes possess advantages such as good biocompatibility, biodegradability, non-toxicity, low immunogenicity, strong loading capacity, and the ability to cross biological barriers, thereby gradually gaining widespread research and application as an extremely valuable natural drug delivery carrier.

Advantages of Exosomes in Drug Delivery

As natural nanoscale extracellular vesicles, exosomes exhibit significant advantages over other traditional drug delivery systems. ① Compared to cellular therapies, exosomes are easier to store and possess higher safety. For instance, exosome-mediated gene drug delivery does not lead to immune rejection by depositing in different parts of the body. Furthermore, exosomes can be isolated from a patient's bodily fluids, modified, and then reintroduced into the same patient, significantly reducing the likelihood of immune reactions in clinical settings. ② Exosome-mediated drug delivery enhances drug stability. For example, exosomes can protect nucleic acids from nuclease hydrolysis during transport. Additionally, exosomes can directly enter the cytoplasm, bypassing metabolic elimination, thereby prolonging the drug's circulation time in the body. ③ As nanoscale molecules carrying cell surface substances, exosomes possess a strong ability to penetrate various biological barriers. ④ Exosomes exhibit natural targeting capabilities based on donor cells. For instance, exosomes derived from tumor cells carry tumor-specific antigens, proteins, and RNAs, which can elicit anti-tumor immune responses.

Exosome Drug Loading Methods

The natural properties of exosomes make them highly potential drug delivery carriers, capable of delivering various chemicals, proteins, nucleic acids, and more. Currently, methods for loading drugs into exosomes can be categorized into endogenous and exogenous loading approaches.

Endogenous loading involves modifying parental cells to secrete exosomes carrying functional molecules. This process integrates therapeutic drug molecules into exosomes during their biogenesis, turning the cells into living factories that directly produce the desired exosomes. Endogenous loading focuses on manipulating the cells that produce exosomes, often involving fusion expression of proteins/peptides, overexpression of genetic drugs, or other modifications based on the exosome biogenesis process, to enable them to produce exosomes carrying target molecules. Compared to exogenous loading, endogenous loading poses greater experimental challenges and requires a longer duration.

Exogenous loading, on the other hand, involves loading drugs into collected, naturally occurring exosomes after their separation. This process encompasses various methods such as direct co-incubation, ultrasonication, electroporation, extrusion, and repeated freeze-thaw cycles. Compared to endogenous loading, exogenous loading is simpler to perform, offers higher batch-to-batch consistency, and is more suitable for large-scale production.

① The direct co-incubation method involves placing exosomes directly with the drug for incubation, allowing the drug to fuse with the exosomes along a concentration gradient. The encapsulation efficiency depends on the polarity of the drug, with hydrophobic drugs exhibiting higher loading rates than hydrophilic drugs. Additionally, drugs with smaller molecular weights are more likely to penetrate the exosome membrane and enter the exosome. Although this loading method requires minimal equipment and is straightforward, its loading efficiency is relatively low, and the drug loading efficiency is susceptible to the molecular weight and hydrophilic/hydrophobic properties of the drug, thus limiting the application of the direct co-incubation method.

② The ultrasonication method involves mixing exosomes with small molecule or protein drugs and then disrupting the integrity of the exosome membrane through homogenization probe ultrasonication, thereby achieving drug encapsulation. The close structure of the phospholipid molecules on the exosome membrane limits the drug loading efficiency of exosomes. Ultrasound provides energy and mechanical force, temporarily creating pores on the exosome membrane, making it easier for drug molecules to enter the exosome. After ultrasonic treatment, the drug-loaded exosomes need to be incubated at a specific temperature for at least 1 hour to allow the exosome membrane structure to recover. The ultrasonication method has the advantages of high drug loading efficiency and sustained drug release, but it may also lead to exosome aggregation and affect the structure of their surface proteins.

③ Electroporation involves the use of an electric field to create reversible, tiny pores on the membrane of exosomes suspended in a conductive solution. These micro-pores allow drugs or nucleic acids, such as small interfering RNA (siRNA) or microRNA (miRNA), to diffuse into the interior of the exosomes. This method is straightforward and has been widely employed for the encapsulation of siRNA or miRNA. However, it may also lead to RNA precipitation or exosome aggregation, thereby reducing the efficiency of drug loading.

④ The extrusion method involves loading a mixture of exosomes and drugs into a lipid extruder equipped with a porous membrane (typically with pore sizes ranging from 100 to 400 nm). During the extrusion process, the drugs are encapsulated within the exosomes. This method boasts high drug-loading efficiency and produces exosomes with uniform particle sizes. However, the mechanical force applied during this process may alter the properties of the exosome membrane, such as its membrane potential and membrane protein structure.

⑤ The repeated freeze-thaw method involves incubating the drug with exosomes at room temperature for a period of time, followed by rapid freezing at -80°C or in liquid nitrogen, and then thawing. During the rapid freezing process, ice crystals form, which can cause ruptures in the lipid membrane of the exosomes. Subsequently, during the slow thawing process, the exposed lipids re-fuse, encapsulating the drug within. This process is repeated at least three times to ensure effective drug encapsulation. The method is straightforward, uses mild conditions that are unlikely to damage bioactive substances, and holds promise for large-scale production. However, it can induce aggregation of exosomes, and the encapsulation efficiency is generally lower than that achieved with sonication or extrusion methods.

Applications of Exosome-mediated Drug Delivery

1. Gene Therapy for Cancer

Studies have demonstrated that utilizing tumor-derived exosomes loaded with miRNA126 can be an effective treatment for non-small cell lung cancer. Due to the tissue-tropic nature of tumor exosomes, those loaded with miRNA126 can accumulate in lung cancer tissues, inhibiting tumor growth. Additionally, exosomes derived from natural milk sources have been loaded with siRNA targeting vascular endothelial growth factor (VEGF), EGFR, protein kinase B (AKT), mitogen-activated protein kinase (MAPK), and Kirsten rat sarcoma viral oncogene homolog (KRAS), effectively suppressing lung cancer metastasis. It has been found that encapsulating siRNA within exosomes significantly reduces its degradation, and in various tumor cells, the expression of target genes is markedly inhibited. Furthermore, exosomes derived from bone marrow-extracted mesenchymal stem cells (MSCs) have been loaded with miR-142-3p nucleotide inhibitors to suppress the expression of miR-142-3p in breast cancer cells. Research results indicate that these MSC-derived exosomes can rapidly deliver locked nucleic acid (LNA)-modified miR-142-3p inhibitor sequences to breast cancer cells within as little as 3 hours, downregulating miR-142-3p expression and thereby achieving therapeutic effects on the tumor.

2. Small Molecule Therapy for Cancer

Research has confirmed that anticancer drugs such as paclitaxel (PTX) and doxorubicin (DOX) can be successfully loaded into exosomes for use in antitumor therapy. In studies exploring the use of exosomes to deliver paclitaxel to specific tumor sites, it has been found that mesenchymal stem cells (MSCs) can autonomously home to the tumor microenvironment, enabling effective delivery of paclitaxel without the need for genetic manipulation and without compromising its biological activity. Even at low concentrations of vesicular proteins, this approach can induce apoptosis in tumor cells. In vitro experiments have demonstrated that the inhibitory effect on cancer cells is comparable to that achieved by using paclitaxel alone.

Scholars have utilized hyaluronic acid (HA) to modify exosomes and load them with doxorubicin for inhibiting CD44-positive tumor cells. This is because HA can highly specifically bind to the surface protein CD44 on tumor cells, and CD44 is overexpressed on the surfaces of tumor cells such as A549 and MDA-MB-231. As a result, HA-modified exosomes can specifically target tumor cells with high CD44 expression. Furthermore, exosomes loaded with doxorubicin derived from primary tumor cells have been employed to prevent tumor metastasis. Tumor cells that detach from primary tumors and enter the bloodstream can form metastatic sites at distant locations. Since exosomes from primary cancers carry the same targeting proteins, they can reach specific pre-metastatic sites. Leveraging this property, drug-loaded exosomes can specifically accumulate at pre-metastatic sites to interfere with tumor metastasis.

3. Exosome-mediated Drug Delivery for Other Diseases

Studies have reported that exosomes secreted by dendritic cells loaded with IL-10 (Interleukin-10) exhibit immunosuppressive effects, reducing T-cell proliferation. Research on a mouse model of collagen-induced arthritis has shown that a single dose of therapeutic exosomes can effectively inhibit disease progression and reduce the severity of arthritis. Similarly, dendritic cell-derived exosomes loaded with IL-4 (Interleukin-4) can alleviate arthritis symptoms and reduce inflammation in mouse models of arthritis.

Duchenne muscular dystrophy (DMD), a common X-linked disorder, has long lacked effective treatment options. Researchers have discovered that exosomes derived from mesenchymal stromal cells can deliver miR-29c, enhancing myoblast differentiation while reducing the expression of fibrogenic genes in DMD patient myoblasts, thereby holding significant clinical potential for DMD treatment.

Exosome-mediated drug delivery has also been applied in neuronal therapies. Using bone marrow mesenchymal stem cell-derived exosomes loaded with recombinant CD63-GFP lentiviral plasmids, studies have confirmed the uptake of exosomes by peripheral nerve endings and their retrograde transport of drugs to dorsal root ganglion (DRG) neurons and spinal motor neurons. This research presents a novel drug delivery method for neuronal diseases.

Liver fibrosis, a widespread liver disease caused by excessive deposition of fibrous scar tissue during chronic liver injury, can be addressed through the delivery of miR-214 to hepatocytes via hepatic stellate cell-derived exosomes. This reduces the expression of CCN2 protein, thereby decreasing liver fibrosis.

Curcumin, a yellow pigment extracted primarily from turmeric rhizomes, possesses anti-inflammatory, gastrointestinal motility-enhancing, anti-proliferative, anti-oxidant, anti-angiogenic, liver-protective, and detoxifying properties. However, curcumin's low water solubility and rapid metabolism limit its clinical application. Encapsulating curcumin and related compounds such as emodin within exosomes naturally enhances their solubility, stability, and bioavailability in aqueous environments.

Furthermore, the blood-brain barrier (BBB) poses a significant challenge for drug delivery to the brain. Exosomes, with their unique structure and specialized surface proteins, can traverse the BBB, delivering drugs directly to brain tissues. Studies have shown that exosomes loaded with catalase can successfully cross the BBB and enter the brain, improving the condition of Parkinson's disease.

References

[1] Zhang Yingying, Chen Liqing, Liu Xuan, et al. Research Progress on Exosomes as Drug Delivery Carriers [J]. Acta Pharmaceutica Sinica, 2019, 54(06): 1010-1016.

[2] Zhou Jianfen, Chai Zhilan, Xie Cao, et al. Research Progress on Exosomes as Drug Delivery Carriers [J]. Chinese Journal of Pharmaceutical Industry, 2020, 51(04): 425-433.

[3] Xie Xiaodong, Lian Shu. Research Progress on Exosome-Based Nanocarriers [J]. Journal of Minjiang University, 2022, 43(05): 56-67.

About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.