33 Proteomics, Genomics & Microarrays


Future Technologies and Applications


Biomarkers With the potential ability to read the messages cells pass to other cells, EVs can be a great source for diagnostics. Currently, there is one EV product clinically approved for non-invasive screening for prostate cancer. This test uses urine taken from at-risk males 50 years or older for high grade prostate carcinoma (HGPCa). Urine samples are taken from these individuals to isolate exosomal RNA creating an EPI risk score. This determines if a further biopsy test is needed.


Due to the increased interest in precision medicine, new biomarkers have been an area of important research and development for pharmaceutical companies. The potential use of EVs as a biomarker to monitor the microenvironment in disease states can provide information on drug effi cacy, enabling better precision-based medical treatment.


Figure 2. Illustration of cells secreting exosomes. Therapeutics Characterisation Methods


Due to the numerous characterisation targets available for EVs, multiple characterisation techniques should be used during an EV study. Some characterisation targets include population size distribution, number of EVs, proteins, DNA, RNA, lipid structure, and more.


Basic tools used in EV analysis include: light scattering techniques such as nanoparticle tracking analysis (NTA) to determine the number of EVs and the size distribution of your population, identifying the amount of protein with a bicinchoninic acid (BCA) assay, and carrying out specifi c protein identifi cation with western blotting.


Additional tools for EV characterisation include ELISA and fl ow cytometry. ELISA kits can identify and quantify specifi c protein markers of interest found on the surface of EVs. This method uses antibodies to specifi cally target markers of interest on EVs, then fl uorescently activated markers will be added to identify the markers of interest.


The use of fl ow cytometry has increased in popularity among EV researchers as it gives the ability to quantify different subpopulations of EVs. By fl uorescent labelling of target proteins with different colours in isolated EV populations, single cells can be analysed and compared to highlight differences in EV populations.


Recognising and analysing nucleic acids associated with EVs is another key area of importance for accurately characterising isolated EV populations. First, commercially available kits are used to lysate the EVs open and extract the nucleic acid from the solution. Then species of nucleic acid are sequenced and analysed. This can be done using a variety of different methods, including RNAseq.


Finally, mass spectrometry is widely used to analyse and characterise lipid content of EV membranes. Lipids have an important structural role in exosomal membranes as the lipid content of EVs are enriched in cholesterol, sphingomyelin, glycosphingolipids, and phosphatidylserine. They are also essential players in EV formation and release to the extracellular environment.


Custom Service


EV studies can be diffi cult, need a lot of scientifi c experimental nuance knowledge, and require a specifi c set of tools for completing a successful study. This is why companies like AMSBIO help customers by offering a variety of custom EV research services to help researchers with their exosome-related projects. These services include EV isolation, characterisation, and quantifi cation, as well as custom EV production and engineering. Also provided are RNAseq and proteomics analysis.


The EV isolation services offered by AMSBIO include: ultracentrifugation, size-exclusion chromatography, and precipitation methods. These methods can be customised to fi t the specifi c needs of the researcher’s project and can be used to isolate exosomes from a variety of sample types such as blood, urine, and cell culture media.


AMSBIO also offers EV characterisation services which include exosome size and concentration analysis, as well as protein and RNA analysis. These services can be used to identify the specifi c exosomes present in a sample and to understand their biological properties.


Custom exosome production and engineering services are also available at AMSBIO, which can be used to generate exosomes with specifi c characteristics such as specifi c protein or RNA content. These custom exosomes can be used as standards or as tools for exosome-based therapeutics and diagnostics.


Overall, EV research services can be tailored to fi t the specifi c needs of the researcher’s project. These services can help researchers to isolate, characterise and quantify exosomes, as well as to generate and engineer custom exosomes for various applications.


Currently, there are no FDA approved EV based therapies on the market. Direct Biologics has entered phase 3 clinical trials for ExoFlo to treat all moderate to severe causes of acute respiratory distress syndrome (ARDS). ExoFlo is isolated from human bone marrow mesenchymal stem cells (MSCs) containing both growth factors and EVs for the treatment of ARDS.


Another exciting clinical application for exosomes is using them as a drug delivery vehicle as their membrane is biodegradable and has a high payload capacity. EVs can also improve biodistribution and can offer a controlled drug release. There are different techniques that can be used to load exosomes, including electroporation, sonication, transfection of cells that will then produce exosomes, treating exosomes with surfactants, and dialysis. EVs can be used for specifi cal drug delivery, by including targeting ligands specifi cally aimed at tumour cells.


Finally, EVs can also be used in imaging applications. For this, they need to be labelled with fl uorescent dyes, nanoparticles or proteins. Additionally, bioluminescent molecules, such as luciferase can be used to track exosomes. For whole body imaging, gold or iron labelled nanoparticles need to be used to overcome detection issues.


Conclusion


Extracellular vesicles (EVs) hold enormous promise in the clinical landscape with their unique ability to serve as a vehicle for targeted delivery of therapeutic molecules and their use as diagnostic biomarkers. Although challenges remain in standardising isolation and characterisation protocols, the potential clinical benefi ts of EVs are undeniable. With further research and advancements in understanding EVs’ biology, EV-based therapeutics and diagnostics could emerge as a game-changing clinical tool in personalised medicine, delivering safer, more effective, and precise treatments to patients.


References


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