Preparation of extracellular vesicles encapsulating antitumor drugs via high-pressure homogenization for targeted tumor therapy

Extracellular vesicles (EVs) hold immense potential as drug delivery platforms due to their unique role in intercellular communication. However, numerous limitations—including low drug encapsulation efficiency and difficulties in scaling traditional methods—pose challenges for the clinical translation of EVs as delivery vehicles. Given the current success in preparing lipid nanoparticles, high-pressure homogenization (HPH) shows promise as an alternative method for the large-scale production of drug-encapsulated EV therapies. This study investigates the use of HPH for the large-scale preparation of EVs encapsulating the anticancer drug paclitaxel (PTX) and their application in targeted tumor therapy. Efficient encapsulation of PTX into milk-derived EVs (mEVs) was achieved by co-mixing distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), PTX, and mEVs prior to HPH treatment. Process conditions optimized in laboratory-scale equipment enabled the preparation of polyethylene glycol-modified paclitaxel-encapsulated microvesicles (PTX-mEVs) using large-scale industrial equipment. Furthermore, grafting cyclo(arginine-glycine-aspartic acid-D-phenylalanine-lysine) (cRGD) onto PTX-mEVs enhances the delivery of encapsulated paclitaxel to tumor cells. Furthermore, intravenous injection of cRGD-modified PTX-mEVs significantly suppressed tumor growth in subcutaneously injected Colon-26 tumor-bearing mice, demonstrating superior antitumor efficacy compared to PEG-modified PTX-mEVs. These results demonstrate for the first time the significant potential of high-pressure homogenization technology for large-scale preparation of drug-encapsulated extracellular vesicle (EV) therapies via a one-step drug preparation process. The resulting EV therapies show promise for tumor treatment.

Introduction

Extracellular vesicles (EVs) are nanoscale particles released by cells, composed of phospholipid bilayer membranes. EVs are categorized by size into small (diameter < 200 nm) and medium/large (diameter > 200 nm) types, and can be further subdivided into several subtypes, including apoptotic bodies. Extracellular vesicles transport functional cargo such as proteins and nucleic acids, participating in intercellular communication by transferring this cargo to nearby or distant cells. Furthermore, extracellular vesicles (EVs) have been reported to possess the ability to target specific tissues and cells, depending on the membrane proteins present on their surface, as well as their capacity to traverse endothelial barriers. Due to their unique properties, EVs hold promise for applications in disease therapy and drug delivery systems (DDS). Although EV applications in DDS remain limited (<10%), clinical trials are underway to leverage EVs for treating certain diseases such as tumors, ischemic stroke, and pneumonia.

Extracellular vesicles (EVs) possess the potential to deliver functional biomolecules to recipient cells, making them highly attractive drug delivery carriers. Extensive research is underway to demonstrate the practicality of EVs-based delivery systems by encapsulating small-molecule and macromolecular drugs. Direct incubation represents a straightforward method for therapeutic drug encapsulation, particularly suited for hydrophobic small molecules. Surfactants, extrusion, and repeated freeze-thaw cycles are also employed to temporarily disrupt vesicle membrane stability, thereby enhancing therapeutic molecule loading efficiency. Active physical treatments, including electroporation and ultrasonication, can further improve the encapsulation efficiency of therapeutic payloads into EVs by disrupting the lipid bilayer. However, these approaches present challenges such as insufficient encapsulation efficiency, limited scalability, risk of EV/drug aggregation depending on drug type, time-consuming processes, and poor reproducibility. To accelerate the clinical application of EVs as drug delivery carriers, scalable alternatives enabling efficient and straightforward drug encapsulation are required.

To achieve this goal, high-pressure homogenization (HPH) technology has been explored to enhance drug encapsulation efficiency in EVs and enable large-scale production. HPH has been employed for producing drug-encapsulated lipid nanoparticles (LNPs) due to its ease of scale-up from laboratory studies, short processing time, and absence of organic solvents. These advantages have driven the adoption of HPH technology for LNP preparation in pharmaceutical and cosmetic industries. Our previous research extended HPH technology to the functionalization of extracellular vesicles (EVs), demonstrating its capability to load the hydrophilic anticancer drug doxorubicin (DOX) into mouse macrophage-derived EVs. Concurrently, a one-step drug preparation process was employed to modify their surfaces using polyethylene glycol (PEG)-conjugated phospholipids. Furthermore, intravenous administration of prepared DOX-encapsulated EVs inhibited tumor growth in mice bearing subcutaneous Colon-26 tumors. The findings suggest that high-pressure homogenization (HPH) represents a promising alternative method for preparing extracellular vesicles (EVs) capable of encapsulating therapeutic drugs. However, it remains unclear whether HPH is suitable for large-scale production of drug-loaded EVs or for encapsulating therapeutic drugs with properties distinct from doxorubicin (DOX).

Extracellular vesicles (EVs) typically require costly and cumbersome steps to isolate from conditioned media of cultured cells, such as tumor cells. However, the difficulty in extracting and isolating large quantities of EVs remains a challenge for their application in therapeutics and drug delivery systems (DDS). In light of this, food has garnered significant attention as an alternative source of abundant extracellular vesicles. Extracellular vesicles derived from various foods (e.g., grapefruit, corn, and milk) have been demonstrated as cost-effective sources for drug delivery. Food-derived EVs present an attractive platform to address yield limitations, owing to the affordability and widespread availability of food sources. Among these, milk-derived extracellular vesicles (mEVs) have demonstrated practical utility due to their intriguing potential. This includes serving as drug carriers for oral administration and injection/oral absorption without immunogenicity, as well as exhibiting inherent therapeutic effects against certain diseases. Based on these advantages, mEVs are considered suitable for large-scale production of drug-encapsulated EVs via HPH, and for applying the prepared EV therapies to clinical treatments.

This study explores the potential of high-pressure homogenization (HPH) technology for the scalable production of micro-extracellular vesicles (mEVs) encapsulating anticancer drugs for targeted tumor therapy. We first employed laboratory-scale equipment to modify mEVs with DSPE-PEG and optimized the modification process. Subsequently, we investigated the application of HPH technology for encapsulating the hydrophobic anticancer drug paclitaxel (PTX) into mEVs. Previous studies have demonstrated that PTX can be encapsulated into EVs via alternative methods such as electroporation and ultrasonication; however, low encapsulation efficiency and increased particle size due to aggregation remain critical challenges. We also investigated the influence of HPH process parameters on PTX encapsulation and optimized the HPH method for PTX preparation, making it suitable for large-scale production using industrial-scale equipment. Furthermore, we developed tumor-targeting ligand-modified extracellular vesicles (mEVs) encapsulating PTX using the cyclic RGD (cRGD) motif (arginine-glycine-aspartic acid-D-phenylalanine-lysine) and evaluated their therapeutic efficacy in tumor-bearing mice. These results demonstrate for the first time that HPH technology can be used for large-scale preparation of extracellular vesicles encapsulating antitumor drugs, as well as for preparing tumor-targeted extracellular vesicle therapies for cancer treatment.

For detailed experimental procedures and results, please refer to the original text.

Results

Effects of High-Pressure Homogenization on the Physicochemical Properties of Miniature Extracellular Vesicles

As previously reported, extracellular vesicles derived from bovine milk were collected using a combination of acid precipitation and ultracentrifugation. The isolated mEVs exhibited an average particle size of 116.5 ± 7.0 nm, a zeta potential of −34.5 ± 3.0 mV, and a polydispersity index (PDI) of 0.12 ± 0.02. NTA analysis and the BCA assay determined the mEV particle concentration to be 5.2 × 10⁹ particles/μg mEV protein content. Western blotting revealed representative extracellular vesicle marker proteins—CD81, TSG101, and HSC70—in the isolated mEVs, which were undetectable in whey. Figure S1 demonstrates successful purification of extracellular vesicles (mEVs) from cow's milk. We first examined the effects of high-pressure homogenization (HPH) on the physicochemical properties of extracellular vesicles. Figure 1 displays the microfluidic device flow diagram (Figure 1). The HPH process pressure was set to 30,000 psi, the maximum capacity of the LV-1 microfluidic chip. After 5 and 10 HPH cycles, the average mEV particle size decreased to 107.3 ± 10.3 nm and 101.4 ± 6.9 nm, respectively (Figure 2A). After 10 HPH cycles, the PDI of mEVs remained below 0.3, indicating a monodisperse population (Figure 2B). Repeated HPH cycles had negligible effects on the ζ-potential of mEVs (Figure 2C). TEM images revealed spherical morphology of mEVs obtained via acid precipitation and ultracentrifugation (Figure 2D), consistent with previous reports. Notably, five cycles of HPH treatment at 30,000 psi had negligible impact on mEV spherical morphology while reducing their particle size (Figures 2A, E).

Figure 1. Equipment flow diagram of the HPH microjet apparatus.

Figure 2. Physicochemical properties of mEVs after high-pressure homogenization. (A–C) Particle size, polydispersity index (PDI), and zeta potential of mEVs after 30,000 psi high-pressure homogenization. Data represent mean ± standard deviation (n = 3). (D,E) Transmission electron microscopy (TEM) images of untreated (HPH(−)) and high-pressure homogenized (5 cycles) mEVs. Scale bar = 200 nm.