Exosomes are nanoscale extracellular vesicles secreted by cells. They have recently emerged as promising drug delivery vehicles. These vesicles feature a lipid bilayer membrane that encapsulates biologically active substances such as proteins, lipids, and nucleic acids. They exhibit high biocompatibility and low immunogenicity, which helps reduce immune clearance and supports targeted drug delivery. However, the clinical application of natural exosomes faces several limitations. These include a short circulating half-life, variability in cell sources, and insufficient drug-carrying capacity. To address these challenges, researchers have used bioengineering techniques to develop engineered exosomes. These modified exosomes are designed to enhance drug encapsulation and targeting capabilities. Generally, engineered exosomes can prevent aggregation, adsorption, and rapid clearance. They also maintain higher drug loading and stability while enabling efficient customization of drug delivery systems. This approach has become a popular research direction in the fields of biomedicine and biotechnology^[1].

Bioengineering techniques can enhance exosome production, stability and purity. Engineered exosomes generated by genetic modification of parental cells are promising carriers for drug delivery. Exosomes are first isolated from body fluids and then engineered to carry specific cargoes or express ligands on their surface. The drug-carrying capacity of engineered exosomes needs to be evaluated before exploring their potential clinical use (Figure 1). In addition, engineered exosomes offer advantages such as drug protection, enhanced stability and bioavailability, reduced toxicity, and improved targeting capabilities^[2].

Drug encapsulation methods for engineered exosomes can be divided into two main technology types: endogenous encapsulation and exogenous encapsulation. Endogenous encapsulation mainly utilizes CRISPR/Cas9 technology to achieve drug encapsulation in cells. Exogenous encapsulation methods include incubation, electroporation, sonication, freeze-thaw cycles, and extrusion after exosome isolation.

This method employs genetic engineering techniques to modify donor cells. These cells are transfected to express desired therapeutic substances. Subsequently, exosomes containing these biologically active components are isolated from the donor cell culture supernatants. For instance, the CRISPR/Cas9 system holds significant potential for cancer treatment. However, its clinical translation is hindered by challenges such as immunogenicity. These limitations can be addressed through the use of engineered exosomes. When exosomes are loaded with the CRISPR/Cas9 system, they can effectively bind to cancer cell membranes. This enables highly efficient, non-invasive gene editing. In one supporting study, exosomes carrying CRISPR/Cas9 successfully induced apoptosis in ovarian cancer cells. This finding underscores their promising role in precision anti-cancer therapy.

In addition, exosomes are being investigated as potential cancer vaccines. Compared to conventional chemotherapy, exosome-based vaccines offer several advantages, including high specificity, controllability, and low toxicity. Recent studies have further explored their potential. For example, exosomes released from M1-polarized macrophages have been co-administered with cancer vaccines in mouse models. This combination was shown to enhance vaccine immunogenicity and strengthen anti-tumor immune responses. These findings highlight the therapeutic promise of engineered exosomes. They reveal their potential role in the development of optimized anti-cancer vaccines.

In this method, exosomes are first isolated and then externally incorporated with drugs or bioactive substances using various techniques. The exogenous encapsulation method has many advantages over the endogenous encapsulation method. It provides easier handling, higher stability and better scalability for large-scale production of engineered exosomes.

Common methods of exogenous drug encapsulation include co-incubation, electroporation, sonication, freeze-thaw cycling and extrusion. These techniques provide a versatile and efficient way to encapsulate therapeutic agents into exosomes for targeted delivery.

Surface engineering aims to confer cell-type target specificity to exosomes. This field employs two main modification strategies: genetic engineering and chemical modification. Genetic engineering utilizes biofusion expression to achieve this goal. In this approach, the gene sequence of a target peptide is fused to that of selected exosomal membrane proteins. This fusion allows for the efficient presentation of the target peptide on the exosome surface. Chemical modification offers an alternative pathway. It can be used to display various natural and synthetic ligands through click reactions or lipid assembly. However, the complexity of the exosome surface can pose challenges. It may reduce reaction efficiency or even damage the structure and function of the vesicle. Currently, numerous studies favor the use of biofusion expression for modifying exosome surface proteins^[4].

Exosome membranes naturally contain proteins such as tetraspanins, integrins and lectins. These proteins can influence exosome biodistribution through interactions with target cell receptors. The targeting ability of engineered exosomes can be significantly enhanced by genetically modulating parental cells to overexpress or knock down specific proteins.

Cell-binding motifs can be displayed on the surface of exosomes by protein engineering methods that typically include the use of viral targeting motifs, cell-penetrating peptides, and protein transfection domains. Notably, each of these elements plays a unique role in enhancing the ability of exosomes to target and penetrate cells, making them valuable tools in the fields of targeted drug delivery and disease treatment.