Yingtai: Application of Vacuum Freeze-Drying Technology in Exosome Research

Yingtai: Application of Vacuum Freeze-Drying Technology in Exosome Research

Exosomes are nanoscale extracellular vesicles that primarily regulate intercellular communication. They have significant potential in nanomedicine. However, the separation technologies for exosomes are limited by expensive equipment and reagents. Biomimetic nanotechnology provides a powerful platform for drug delivery, RNA interference (RNAi), protein delivery, and non-drug therapies, greatly impacting biomedical research. During the COVID-19 pandemic, nanotechnology played a key role in the global development of mRNA vaccines and diagnostic kits. However, the clinical translation of traditional nanomaterials has been hindered by poor target recognition ability and high costs. In this context, exosomes could provide a promising solution to these issues.

Exosome Separation Methods

There are several effective exosome separation methods, including ultracentrifugation (used in 80% of extracellular research), density gradient reagents with polyethylene glycol (PEG), size exclusion chromatography (SEC), and immunoprecipitation. However, these methods have certain limitations, such as the high cost of ultracentrifuges or commercial kits. Moreover, the purity, size, concentration, and functionality of separated exosome samples depend on the separation method used. Freeze-drying (vacuum freeze-drying) is now commonly used to extend the storage time of enriched exosomes. Its application in exosome separation is relatively new. Interestingly, most exosome storage buffers contain substances that help maintain their structure and function. For instance, 4% trehalose or 10% w/v sorbitol and sucrose are beneficial for the freeze-drying of exosome particles. Similarly, the ideal pH range is 6.5–7.5, and amino acids, glutamic acid, and 1% FBS (fetal bovine serum) are beneficial during the freeze-drying process to avoid freeze shock and preserve the structural integrity of exosome particles. Additionally, for cell culture media, Dulbecco’s Modified Eagle Medium (DMEM) enriched with glucose, pH 6.8–7.4, primarily L-glutamine, and supplemented with 10% FBS, protects exosomes in a similar manner to freeze-dried exosome storage media. Thus, cell culture media can protect exosomes. Therefore, freeze-drying-based technology can separate exosomes by freeze-drying aged cell culture media. However, traditional exosome separation techniques are still required for washing. Although SEC or ultracentrifugation can achieve this to some extent, no single separation method can fully purify exosome samples from protein contamination.

Advantages of Exosome Separation Technologies

Comparative studies of exosome separation technologies evaluate the efficiency, purity, and reproducibility of various commercial kits for exosome purification, as well as the quality of miRNA in plasma and serum samples. It was found that serum is more suitable for exosome separation and contains a wider variety of exosome miRNAs. The study also revealed that each separation method has certain limitations in terms of purity, exosome yield, and variety. In contrast, plasma is more favorable for exosome separation. For all types of exosome samples, ultracentrifugation, even compared to commercial kits, is limited by lower yields but relatively higher purity. Research shows that the detection and separation of exosome cargo completely depend on the separation protocol used. Freeze-drying-based exosome separation methods are an excellent choice due to their simplicity and cost-effectiveness.

Exosomes separated using freeze-drying methods can be characterized by size and shape using scanning electron microscopy (SEM). Additionally, nanoparticle tracking analysis (NTA) is used to accurately reveal the concentration and size of exosomes in the sample. The average size of extracellular vesicles from adherent and suspension cell lines is approximately 140 nm, which aligns with the reported size range for exosomes (30–200 nm). The protein-based concentration of exosomes is suitable for downstream applications. Common exosome surface protein markers are considered standard for characterization. For example, CD9, CD63, and CD81 are endosome-specific tetraspanins. CD9 was first identified on exosomes isolated from dendritic cells. TSG101 and HSP70 are components of the endosomal sorting complex required for transport (ESCRT), which play a key role in exosome biogenesis. Calnexin, an endoplasmic reticulum marker, is primarily evaluated as a negative control for exosomes, as it is mainly present in relatively larger vesicles, while CD63 is found in smaller vesicles. Western blotting confirmed that, apart from calnexin, all these important markers are present on the surface of the isolated exosomes. Exosomes have significant applications in nanomedicine for drug delivery and diagnostics. Researchers have assessed the drug-loading and release capabilities of these isolated exosomes by loading them with dasatinib and ponatinib (tyrosine kinase inhibitors used in Ph+ leukemia) and observed that exosomes can effectively load drugs and deliver them to target cells. Drug release studies were conducted in vitro by collecting samples at various time points. The optimal drug release occurred at 8 hours, while a decline was observed at 12 hours. These results were confirmed, as K562 is a leukemia cell line with a low pH, which triggers drug release from the exosomes.