Anti-Tumor Effect of Targeted Drug Delivery by Engineered Exosomes
Cancers are now the leading cause of death in patients all over the world and their incidence has risen with each passing year. Tumours are currently treated with chemical drugs, with surgery, Chinese medicine and radiotherapy thrown in for good measure. Many anti-tumor drugs will kill normal cells and kill tumour cells in the process producing toxic and side effects. Blood vessels, immune cells, interstitial cells, etc., can interact with drugs to interfere with drug delivery and efficacy – which may make it difficult for drugs to penetrate the tumor or get eliminated. Thus, new treatments are desperately needed to have accurate treatment of tumours. Currently, several new delivery technologies have been developed one after the other, mainly by carriers or controlled release to disperse drugs to individual body areas in a targeted, stable and slow way, maximising drug efficacy and decreasing adverse events. Carriers such as synthesised liposomes, nanoparticles, dendritic polymers and micelles, for instance, enhance the therapeutic effect to some degree. But such carriers have flaws: they are not easily cleared by the human immune system, do not target very effectively, and their use is even more narrow.
Exosomes are uniquely biocompatible, bioavailable, biodegradable, and able to cross biological membranes, among many other advantages of being a carrier for drugs. They could be produced as precise delivery vehicles for tumours. Exosomes encapsulate drugs in their phospholipid bilayer layers for low clearance and long-term dispersion while targeting treatment via signal molecules expressed strongly on the surface. Exosomes are highly microbial and internalisable, almost immune inactivating, and highly biosafe. Exosomes can be loaded with small interfering RNA, antisense oligonucleotides, chemotherapeutics, immunomodulators and much more. But there are too many exogenous and endogenous impediments for exosomes to be useful. Thus, it’s loaded with drugs made from engineered exosomes for optimal, precise delivery to the tumour and minimal treatment-related adverse events. As tumor treatment technology keeps improving, engineered exosomes as new bio-nanocarriers are gradually emerging as promising candidates for anti-tumour therapies. By being precisely designed and modified, engineered exosomes can help target and tailor treatment, broaden the reach of anti-tumor therapy, and introduce new ways to treat tumors. As a novel drug delivery system, engineered exosomes not only boost the delivery of drugs but they help to explore and create novel anti-tumor medicines that allow drug scientists to develop novel multi-target treatment strategies.
Generation, Isolation and Characterization of Exosomes
Research on Exosomes
History When Chargaff and West found a coagulation factor (a cousin of thrombin) in anemia and haemophilia in 1946, this is considered the origin of the field of extracellular vesicle biology. WOLF published electron-microscopy photos of these particles around 20 years later. NUNEZ and GERSHON had found 1-10 nm-scale extracellular vesicles in the bat thyroid gland in 1974. Two papers in 1983 ratified intraluminal vesicles flowing from cells and designated them exosomes, the origin of this field. Not only have we learned about exosomes, but there are also researches into the properties of extracellular vesicles. JOHNSTONE et al. called exosomes “swaste disposal mechanisms” in the 1990s. As people’s curiosity about exosomes rose into the early 21st century, so did research on them, which multiplied exponentially. For instance, in 2001 cytokines were detected to be released via exosomes. Two years later, exosomes from immune cells also regulated the activity of the immune system. There was also the International Society of Extracellular Vesicles (ISEV) in 2011, which encouraged much exosome innovation and use. In 2019, single exosomes were found by new technologies, so exosome imaging has entered the single-exosome era in vivo. These findings rewrote how we thought of the informational communication between eukaryotic cells and the function of exosomes. Exosomes, as a natural transporter, also participate in the evolution of diseases like cardiovascular disease, nerve disease and cancer. They regulate target genes and signalling networks, particularly in cancer to influence major interconnections including tumor growth, death and angiogenesis. Not only will this groundbreaking finding expand the fields of tumor mechanism research, but it opens up new, novel strategies and methods for early diagnosis, treatment-predictive prognosis monitoring of tumours.
Formation of Exosomes
Exosomes are nanoscale vesicles released by cells under physiological and pathological conditions, with a diameter range of 40-160nm. The formation of exosomes includes four processes: budding, invagination, intracellular multivesicular body formation and secretion. First, the cell produces small vesicles through budding, and then the cell membrane invaginates to form clathrin-coated vesicles. These vesicles enter the cytoplasm to form early endosomes, which are rich in a large number of proteins, nucleic acids and other biologically active molecules. These early endosomes are specifically sorted and encapsulated to form multiple intraluminal vesicles, namely late endosomes. Subsequently, some late endosomes bind to lysosomes, and some of their contents and proteins are degraded. Finally, other late endosomes fuse with the cell membrane and release the intracellular vesicles to the outside of the cell in the form of exosomes.
Isolation of Exosomes
Today, ultracentrifugation is the exosome isolation gold standard. It is popular in both research and medicine because of its efficiency and robust separation effect. Meanwhile, current work has uncovered a series of new separation and purification technologies: ultrafiltration, immunoaffinity chromatography, size exclusion chromatography, immunoprecipitation and microfluidics. These separation methods could be effective in recovering exosomes but they were time-consuming, cumbersome, small and equipment-intensive, so the overall cost was high and preparation at large scale wasn’t possible. But scaling up is what will make exosomes work in clinical trials. It’s only with mass-produced and clinical application that exosomes’ efficacy and safety can be evaluated and scaled to clinical development. Exosomes have been difficult to use on a large scale because they’re expensive and complicated to perform, making them less accessible to the clinic. The question therefore becomes what can we do to reduce separation and purification costs, making operation easy, and bringing more yield.
Analytical Methods for Characterizing Exosomes
These days, exosomes are characterised by flow cytometry, protein blotting, nanoparticle tracking, dynamic light scattering, mass spectrometry and microscopy. Flow cytometry mostly quantitatively and qualitatively analyzes exosomes by marking proteins or other molecules on the exosome’s surface (CD9, CD63, CD81, etc.). You can also use protein blotting to measure how much and what kind of proteins are present in exosomes. This method is extremely sensitive and specific for exosome-associated marker expression (like TSG101 and Alix etc.). While we can identify and analyse the protein molecules of exosomes, mass spectrometry can also detect the lipid molecules of exosomes to find out its composition and concentration of all the other components. Electron microscopy can capture the morphological information of exosomes directly (such as their size, shape, membrane structure), and so it also captures the morphological information of exosomes. In addition, dynamic light scattering can determine the size distribution of particles by analyzing the scattering pattern of light, thereby understanding its characteristics in the sample. Nanoparticle tracking analysis can not only determine the size of exosomes, but also quantitatively analyze their concentration and distribution, providing important data support for the purification and quantification of exosomes. In actual research work, the above different methods should be combined to conduct comprehensive and accurate characterization and analysis of exosomes. Safety of exosomes in the biomedical field, exosomes, as an emerging drug delivery system, are gradually showing their unique advantages and potential. However, although exosomes have the characteristics of natural origin, good biocompatibility and the ability to cross biological barriers, their pharmacokinetic and toxicological properties in vivo still need to be further studied and fully verified to ensure the safety and effectiveness of exosomes as drug delivery systems.
Exosome Drug Loading Methods
Different methods for exosome drug loading. Common drug loading methods for engineered exosomes can be divided into two categories: 1. Direct drug loading method, drug-loaded exosomes are obtained from parent cells by transfection or co-incubation methods. Although these methods are highly reproducible and relatively simple, the loading efficiency is usually low and highly dependent on the parent cell type as well as the cargo characteristics and concentration gradient. 2.Indirect drug loading method, exosomes are extracted from cells of different sources and the cargo is introduced into extracellular vesicles by co-incubation, electroporation, sonication, extrusion, freeze-thaw cycles or transfection reagents. This strategy is more customizable than the former and minimizes the inclusion of other unnecessary substances.