Due to the low bioavailability of many drugs, uneven distribution in the body, and easy metabolic inactivation, the use of pure drugs alone often fails to meet the needs of clinical treatment or even produces serious adverse reactions. Multiple drug-delivery systems have been developed to address this phenomenon, including liposomes, nanoparticles, and micelles [1]. These traditional drug delivery systems have achieved a certain degree of success in enhancing drug efficacy and safety. However, they also have several shortcomings. For instance, there is insufficient targeting. Additionally, they may have poor biocompatibility. Toxicity and side effects can occur due to organic solvent residues in the preparation process. Moreover, the complexity of the production process makes it difficult to achieve large-scale production [2].

Small Extracellular Vesicles (sEVs) are double-membrane vesicle particles that are secreted by cells. They are rich in substances such as proteins, lipids, nucleic acids, and small molecular compounds [3]. They have a variety of biological activities and have promising applications. Specifically, EVs-mediated intercellular communication affects various physiological and pathological processes such as immune response, tissue regeneration, and tumor development. For example, EVs released by tumor cells can carry specific microRNAs that inhibit the expression of tumor suppressor genes in normal cells, thereby promoting tumor growth and metastasis; The EVs released by mesenchymal stem cells (MSCs) can promote the repair and regeneration of damaged tissues, such as those after heart or bone injury. EVs can also carry immune-related molecules, regulate the function of immune cells, and participate in the regulation of immune responses. For example, EVs released by antigen-presenting cells can present antigens to T cells, activating their immune response. In addition, EVs are also closely related to the occurrence and development of inflammatory diseases, neurodegenerative diseases, and infectious diseases [4], [5], [6]. At present, there are various methods for extracting extracellular vesicles (EVs), each with its advantages and disadvantages.

Ultracentrifugation is a classic method. However, it is time-consuming and has low purity.

Density gradient centrifugation has high purity. But the operation is complex.

Ultrafiltration is simple and fast. Nevertheless, it is prone to clogging the filter membrane.

Size exclusion chromatography has high resolution and causes minimal damage to EVs.

The immune affinity capture method has strong specificity. But it has a high cost.

The polymer precipitation method is simple and inexpensive. But the purity is relatively low.

Microfluidic technology enables high-throughput automation. But the technology is complex.

The selection method should be comprehensively considered based on factors such as experimental objectives and budget [7], [8], [9].

In contrast to traditional drug delivery systems, small extracellular vesicles (sEVs) offer notable advantages. While liposomes demonstrate remarkable benefits in micro drug delivery, sEVs present additional advantages, including enhanced biocompatibility, inherent targeting capabilities, and reduced immunogenicity [10], [11]:

Stronger penetration: sEVs, with their nanoscale tiny size, can easily penetrate the skin's stratum corneum, blood–brain barrier, and intestinal absorption barrier, while liposomes often face obstacles when crossing these physiological barriers due to their larger size.

Lower immunogenicity: sEVs are secreted by cells themselves and rarely trigger immune reactions in the body. In contrast, liposomes, as synthetic carriers, may be recognized by the immune system as foreign substances, leading to immune rejection.

Better targeting: sEVs carry specific molecules on their surface, which can spontaneously target specific cells or tissues, while liposomes typically require complex modification processes to achieve similar targeting effects.

Better stability: sEVs have a longer half-life in the body and can exist stably in the circulatory system, exerting sustained effects, while liposomes have poor stability and are easily cleared quickly.

They are widely used for loading and delivering drugs, and they are the popular direction of the research on novel nano-drug delivery carriers, which are expected to become the next generation of novel nano-drug delivery carriers. Two commonly used drug loading methods include pre-extraction drug loading and post-extraction drug loading [12], [13]. Pre-extraction drug loading is suitable for loading substances such as proteins, peptides, and small RNAs, which will not cause damage to the structure of extracellular vesicles, and there are two methods, one of which is to add the drug to the cell culture medium, where the drug enters the cells by passive diffusion and then is encapsulated into the sEVs. The other method is called endogenous drug loading, which is to construct a recombinant plasmid to transfect cells and induce them to overexpress a certain protein, peptide, or small RNA [14]. Post-extraction drug loading mainly consists of co-incubation, sonication, electroporation, freeze–thaw cycling, and extrusion [15].

Several common methods for loading drugs into small extracellular vesicles (sEVs) pose challenges. Techniques like sonication, electroporation, freeze–thaw cycling, and extrusion can compromise the structural integrity of sEVs, reducing their functional efficacy. In contrast, co-incubation offers milder conditions that preserve sEV structure, yet it struggles with limitations such as low drug loading efficiency, limited capacity, uneven distribution of encapsulated drugs, and reduced stability of the payload. These drawbacks highlight the need for optimized strategies to enhance sEV-based drug delivery systems while maintaining vesicle integrity and therapeutic potential. [16].

In conclusion, although there are many methods for drug loading of sEVs, all of them face some problems, and there is an urgent need to develop more advanced methods to improve the drug loading efficiency of sEVs while ensuring their structural integrity.

Methyl salicylate (MS) is a drug with a variety of pharmacological actions including anti-inflammatory, antibacterial, and analgesic [17], [18]. Methyl salicylate is irritating to the gastrointestinal tract and is usually used for dermal administration. During transdermal administration, MS is hydrolyzed by esterases in the skin to produce salicylic acid (SA). Both MS and SA have good anti-inflammatory activity. Currently, there are many commercially available dosage forms of methyl salicylate, including patches, gels, creams, and other traditional methyl salicylate dosage forms, but these dosage forms have problems such as skin irritation and poor transdermal permeability [19]. In order to reduce skin irritation and improve bioavailability and skin penetration, MS nano-emulsions and MS liquid crystals have gradually emerged. However, due to inherent defects such as residual organic solvents during carrier preparation, they still exhibit certain side effects [20]. Therefore, there is an urgent need for a safe and effective transdermal delivery carrier that can significantly improve the drug loading efficiency, transdermal permeability, and stability of MS without the need for surfactants or organic solvents.

As a naturally-derived nanoparticle, sEVs are ideal carriers for MS because of their wide range of sources, low immunogenicity, good biocompatibility, high drug-carrying efficiency, and the absence of surfactants or organic solvents. Considering that MS is a lipid-soluble drug, this study aims to utilize sEVs for co-incubation loading of MS. However, the co-incubation efficiency of ordinary sEVs is low. For example, researchers used tomato fruit-derived sEVs (TsEVs) to deliver calcitriol to enhance its anti-cancer effect. The encapsulation efficiency of calcitriol was 15.4 % (co-incubation), 34.8 % (freeze–thaw), and 47.3 % (ultrasound treatment). The co-incubation method has the lowest efficiency among them [21].

Carboxyesterase 1 is an important drug-metabolizing enzyme that can hydrolyze ester drugs [22]. It belongs to the serine hydrolase superfamily and is involved in the metabolic activation of over 85 % of ester drugs in the body [23]. The ester drugs that it can hydrolyze mainly include aspirin, oseltamivir phosphate, clopidogrel, and methyl salicylate. Among them, methyl salicylate, as a common aromatic ester compound, has been widely used as a model drug for carboxylesterase 1 (CES1) due to its simple structure (only one ester bond, which is easy to be recognized and hydrolyzed by CES1), easy synthesis, and clear metabolic pathway. MS can be hydrolyzed by CES1 to form SA after drug administration, and both SA and MS have anti-inflammatory activity, so based on the strategy of “esterase reaction activity loading” [24], this experiment obtained small extracellular vesicles engineered with carboxylesterase 1 through gene modification of sEVs, named CES1-sEVs. Currently, the research reports on the genetic modification of extracellular vesicles are mainly focused on mammalian cells, and there are fewer reports of studies on the modification of extracellular vesicles of yeast cells by using gene technology [25], [26], [27].

This experiment selected Pichia pastoris X33 (XPP) to modify extracellular vesicles, which is a commonly used low-level single-cell eukaryotic expression system with the following advantages.

XPP uses methanol induced AOX1 promoter, which has strong expression intensity, enabling it to efficiently express proteins.

XPP grows rapidly and is easy to cultivate in large quantities.

XPP does not produce endotoxins or inclusion bodies, and its genes are stable, making it easy to perform genetic manipulation.

Moreover, Pichia pastoris X33 (XPP) is considered a GRAS (Generally Recognised As Safe) strain, which is suitable for the biopharmaceutical field. So, Pichia pastoris X33 (XPP) was selected for the modification of extracellular vesicles in this experiment [28], [29]. The sEVs obtained from X33 were modified to obtain CES1-sEVs, which hydrolyze MS within the membrane to maintain concentration differences and promote MS diffusion to enhance drug loading efficiency. The anti-inflammatory activity of MS loaded sEVs (named MS-CES1-sEVs), was investigated on RAW264.7 cells (Murine macrophage cell line, RAW 264.7).