Drugs are not simple transporters through the body. All drug carriers will deteriorate the bioavailability of drugs because of biocompatibility on the way in. DDS is a drug delivery technology invented in the last several decades to improve drug performance and address the problems of poor bioavailability of naked drugs, poor in vivo targeting effect and some toxic side effects. The exosomes are thin-walled vesicles released by cells with a diameter of 40160nm, which are key messengers between cells. Exosomes are very tolerant and biocompatible, not immunogenic, they are engineerable, and they are localized. They have been commercially deployed as carriers today for the cure of cardiovascular conditions, kidney failure, immune disorders, nervous-system disorders and cancer.
Exosome Biogenesis
Extracellular vesicles are bits that get released from cells, held apart by a double lipid shell, and don't themselves reproduce. Every cell makes extracellular vesicles and there are several kinds of extracellular vesicles and, to date, no standard. From the biogenesis of vesicles, they can roughly be classified into exosomes made by the endocytic system, microvesicles made by budding of plasma membranes, and apoptotic bodies made by cell death. Then there are studies that break up exosomes into exosomes, matrix vesicles, oncosomes, etc. because of where they come from, their size, their biology and their behaviour. Plasma membrane invasion invaginates exosomes. The cell membrane invades and makes a cup-like object, then the invaded cup-like object becomes an intracellular vesicle (that is, an early endosome). In the invagination step, early endosome can have cell membrane surface proteins and soluble proteins in the extracellular domain. The early endosome then develops into a late endosome. In this time, the Golgi apparatus inside the cell and endoplasmic reticulum also enriches the endosome. The late endosome also grows into a multivesicular body (MVB) that includes some intraluminal vesicles (ILVs), future exosomes too. Multiversicular bodies before release can join lysosomes or autophagosomes to be degraded, and undegraded multivesicular bodies with the cell membrane to expel intraluminal vesicles into exosomes.
Mechanism of Exosome Biogenesis
ILVs are formed by sprouting from the endosome, and there are two ways they happen: ESCRT- and non-ESCRT-dependent. The ESCRT system is located in the cell and mediates vesicle budding and content sorting for membrane remodeling. The process depends on five subunit complexes: ESCRT-0, -I, -II, -III and Vps4. The first, the most important subunit of ESCRT-0, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), grabs ubiquitinated cargo, identifies it and sorters it into endosomal locations high in PI3P. Then ESCRT-0 pulls in ESCRT-I through an interaction with the ESCRT subunit tumor susceptibility gene 101 (Tsg101), and ESCRT-I and ESCRT-II trigger inward growth of endosomal domains on ubiquitinated protein clusters. The charged multivesicular protein-6 (CHMP6) subunit of ESCRT-III then attaches to ESCRT-II and grabs CHMP4, which spins into a coil around the neck of the developing ILV pocket, attaches to CHMP3, buds and is broken down to produce ILVs. Finally, ESCRT-III is broken down by ATP with Vps4. Beyond the ESCRT-dependent process, ILV biogenesis involves other processes as well: lipids, the tetraspanins. Complex lipids like ceramide in the lipid pathway will self-form raft-like structures and allow the membrane to bud's inwards to form intraluminal vesicles; tetraspanins are ubiquitous in exosomes and often used as exosome biomarkers that can influence exosome biogenesis and structure. The tetraspanin CD63 binds to apolipoprotein E and modulates pre-melanosome loading and ILV sorting during melanogenesis (this happens without ESCRT).
Isolation and Extraction of Exosomes
Ultracentrifugation centrifuges a solution's components. Cells, particles, proteins, etc.), separated by size, shape, density, and viscosity of medium. The larger the density difference, the further apart. Ultracentrifugation is the "new gold standard" of exosome purification, but usually needs more than 100,000 g centrifugal force Differential centrifugation and density gradient centrifugation are ultracentrifugation. It is separated into differential centrifugation of the extracellular particles, whose density, size and shape are also separated. Performance is based on the time of centrifugation, temperature and dilution of samples. That's usually more time to divorce and more to work. This is the snag between density gradient centrifugation and differential centrifugation: with density gradient centrifugation you will have to prepare the density gradient separation medium in the centrifuge tube. Separation media: Two of the most common separation media are sucrose and iodixanol. The disadvantages of this approach are that it takes a long time to separate, produces low exosome numbers, and the sample size is huge.
Figure 1. Schematic diagram of the exosome isolation techniques. (Wang X, et al.; 2023)
- Ultrafiltration and Size Exclusion Chromatography
These two steps, ultrafiltration and size exclusion chromatography, both separate exosomes by size. The concept of ultrafiltration is to feed samples through pore size varying membrane filters and filter by particle size and molecular weight. Ultrafiltration is easy to handle, does not involve expensive, special instruments, is energising and time-saving. Size exclusion chromatography does the same thing with size of particles, so the bigger particles go off first and are removed, the smaller ones come later and are removed.
This is done by precipitating the solution with polyethylene glycol (PEG) and separating the exosomes using low speed centrifugation at 1500g. This technique is easy to use and appropriate for high number of samples but the separated samples are not as pure.
- Immunoaffinity Chromatography
It's based on the antigen molecules that sit on the surface of exosomes, separated by binding to antibodies. It comprises immunomagnetic beads, exosome kits and monoclonal antibody techniques. It's very easy to use, quick, efficient, and highly specific, but comes with disadvantages such as low exosome production and the lack of complete removal of antibodies from the sample.
Drug Loading Methods of Exosomes
Exosome drug loading process are divided into exogenous drug loading and endogenous drug loading. The exogenous drug loading is the in-direct loading of drug molecules on to purified exosomes and includes electroporation, incubation, ultrasound, extrusion, freeze-thaw, surface chemical modifying etc.; whereas endogenous drug loading is the release of drugs in the exosomes by altering the mother cell that exosomes are a product of such as cell transfection, viral infection, etc. Exogenous and endogenous loading has its pros and cons. During the real experimental session, the right procedure needs to be selected based on the nature of the drug loaded and exosomes' origin.
Exosome Delivery of Other Forms of Drug
- Nucleic Acid Drug Delivery
The old-fashioned small molecule drugs and antibody drugs usually have their efficacy in the treatment of human disease, acting on the proteins downstream of the disease gene; most pathogenic proteins aren't currently targetable by small molecules or antibody drugs. A more precise and effective therapy – nucleic acid drugs – has been progressively rolled out into clinical practice over the past few years. Nucleic acid therapeutic agents that are already in use are mRNA, miRNA, siRNA, ASO, and RNA aptamers. The bodies' targets of action are usually intracellular and highly tolerant of body-generated ribozymes. This way a delivery mechanism will shield the drug, which is the solution for making the drug more effective. Exosomes as a natural delivery system is finding a lot of applications for nucleic acid drug delivery.
Small interfering RNA (siRNA) is a 21-23 nucleotide double-stranded RNA molecule that can also complementally attach to mRNA of the target protein, so as to "silencing" the expression of certain genes. SiRNA can go after any gene, in principle, but we have to figure out how to safely and efficiently deliver these molecules to the cells where they're needed. Over the past few years, more and more experiments have been conducted to deliver siRNA with exosomes.
miRNA is a non-coding RNA molecule, which can be as long as 20–25 nucleotides. They've observed that miRNA expression alters in cancer, autoimmune disease, infectious diseases and neurodegenerative disorders. There are miRNA medications currently in clinical trials, and miRNA has advanced much in disease management. But clinical use of miRNA is still limited by the challenges of biological bottlenecks and body kinematics. We have been increasingly implementing exosomes as carriers of drug therapies because they are safe and non-immunogenic. For many diseases, now exosomes infused with miRNA are used.
- Protein/Peptide Drug Delivery
Exosomes' surface is packed with membrane proteins including endosome proteins (Alix, Tsg101, Rab proteins), tetraspanins (CD63, CD81, CD82, CD53 and CD37), lipid raft proteins, and cholesterol-, sphingomyelin- and glycerophospholipid-rich lipids. In the past couple of years, scientists have layered protein/peptide drugs like antibody fragments, vaccine antigens, Cas9, etc. with exosome proteins to enable exosome targeting or drug delivery. Interleukin 3 (IL3) receptor is overexpressed in hematopoietic cells of patients with chronic myeloid leukemia (CML) – and as such, it could be targeted as a receptor for the delivery of cancer drugs. In CML patients, the translocation of chromosomes generates chimeric Bcr-Abl oncoprotein with constitutive tyrosine kinase activity. Bcr-Abl kinase can phosphorylate and activate downstream substrates that are involved in cell signalling to promote growth, death and change cell adhesion. The researchers built a HEK293 cell exosome scaffold protein and IL3 fusion protein – Lamp2b-IL3, so that IL3 was distributed on the 293 cell exosome surface, and siRNA to the Bcr-Abl fusion gene was added to the 293 cell exosomes. Bcr-Abl siRNA-containing exosomes that would be sent to CML cells could prevent cancer cells from multiplying and reduce the size of the tumour in vivo and in vitro. CREKA consists of a polypeptide, cyanine-arginine-glutamic acid-lysine-alanine (CREKA), that can be grafted onto the fibrin-fibronectin structure, and the DMPE-PEG polyethylene glycol conjugated phospholipid derivative, which is an amphiphilic molecule that can be used for some modifications on the plasma membrane.
- Small Molecule Drug Delivery
Exosomes can also be a perfect transport vehicle for small molecules. Exosomes have also been discovered to increase the efficacy of small molecule drugs like curcumin, paclitaxel, doxorubicin and doxorubicin. The natural polyphenol curcumin extracted from the rhizome of turmeric is anti-inflammatory, anti-tumor, antioxidant and chemopreventive. Curcumin was packed into mouse lymphoma cell exosomes. Curcumin delivered by exosomes was stable relative to free curcumin in a mouse septic shock model, and mice survived better. The in vitro anti-inflammatory effect of exosome-encapsulated curcumin was greater. A second, in vitro incubation had enclosed the chemotherapy agents paclitaxel and docetaxel into milk exosomes. The tumour inhibitory power of exosome-loaded chemotherapy drugs was much higher than in the free drug group.
References
- Wang X, et al.; Recent progress in exosome research: isolation, characterization and clinical applications. Cancer Gene Ther. 2023, 30(8):1051-1065.
- Liang Y, et al.; Engineering exosomes for targeted drug delivery. Theranostics. 2021, 11(7):3183-3195.
- Sadeghi S, et al.; Exosome engineering in cell therapy and drug delivery. Inflammopharmacology. 2023, 31(1):145-169.
