Exosome Isolation Method

Abstract

The invention's process utilizes L-arginine-coated magnetic nanobeads for exosome isolation. Pre-treatment steps, consisting of filtration and low-speed centrifugation, enrich the exosome-containing samples and allow for more economical use of magnetic beads in subsequent stages compared to the initial sample volume.

Description

1 TARIFF EXOSOME ISOLATION METHOD Technical Field The invention process involves using L-argin-coated magnetic nanobeads for exosome isolation. rapid production of high-purity, structurally intact, and high-concentration exosomes. It enables the data to be obtained using an economical and scalable method. Especially in clinical applications, 5 It is critically important that the isolated exosomes are of suitable purity for both in vitro and ex vivo use. sah pt r. State of the Technology Exosomes are nanovesicles with a diameter of 40–160 nm and are secreted by various cell types. and play important roles in intercellular communication. These structures are used as biomarkers for diagnostic purposes. Exosomes have great potential as carriers of therapeutic agents. as an agent, in the treatment of neurodegenerative diseases, organ damage and degenerative processes, in infectious diseases, regenerative therapies, cancer treatment, and immunology. It has been used in its functions [1,2]. However, exosomes are effective, easy and high Isolation at a level of purity is one of the biggest challenges in research and applications in this field. br 15 It continues to be so. Current exosome isolation techniques include ultrasound centrifugation and immunohistochemistry. Basic methods, polymer-based precipitation kits and size-based separation chromatography [3]. While each of these methods has its own advantages, various There are also limitations. 20 The oldest of these methods is ultrasonic fugition isolation. This method uses very high speeds. This requires the application of centrifugation (e.g., 100,000 xg) over long periods; long and labor-intensive working hours, high energy consumption, and efficiency and purity. What problems does it cause? Polymer-based systems using materials like PEG (Polyethylene glycol). Precipitation methods have provided significantly higher yields and easier exosome extraction. 25 However, the collected material was affected by extra components added to the sample during purification. The inability to directly use exosomes in ex v vo or nv vo applications and their high cost GB problems exist. They also require long incubation periods. Immune capture. Basically, it has a lower efficiency and is quite expensive. High antibody content. The material is used in such limited quantities, and this situation affects exosome capture 30 2 When smaller sample volumes are used to reach saturation, the results are relatively lower. This leads to a concentration of exosomes. Furthermore, only a specific subset of exosomes is obtained. Size-based separation chromatography requires expensive equipment and isolation time. It is long. When filtration is used alone, the exosomes obtained are albumin. What happens to contamination with peptides? Therefore, in this invention, filtration alone is not the only step, but a preliminary stage. 5 It is used as a filtration method (using sequential membranes). Various methods have been compared as follows: Ultracentre fusing Method:  Advantages: Obtains purer exosomes compared to polymer-based isolation methods. ed l r. 10  Disadvantages: Long duration (3-4 hours) and high speed (100,000 xg) centrifugation. It requires special equipment, efficiency is low (5-25%), energy consumption is high, and There is a risk of disrupting the structural integrity of exosomes. Special methods used in ultracentrifugation. The tubes are consumables and not cheap. Immunocapturing-based methods: 15  Advantages: Ability to isolate specific exosome subtypes.  Disadvantages: High cost, low yield, limited scalability, and only for a specific number of people. Isolation of exosomes with surface proteins. After isolation, they are bound to antibodies. The problem of exosomes being released from antibodies. Polymer-Based Precipitation Kits: 20  Advantages: Easy application, no special additional software required.  Disadvantages: Low purity (contamination with proteins and other vesicles), long shelf life. Incubation periods (generally one night), the obtained exosomes are directly in vitro or exoskeletons. v vo is not suitable for use. Size Exclusion Chromatograph s: 25 • Advantages: High purity and preservation of exosome integrity.  Disadvantages: Low yield, long isolation period, expensive equipment requirements, and Limited scalability. Filtration Methods:  Advantages: Quick and simple application. 30 3  Disadvantages: When used alone, it has low purity (e.g., albumen, ngb). Protein contamination) and exosome adhesion risk to the membrane. The limitations of these methods include the need for high purity, structurally intact, and high-quality materials. rapid, economical and scalable isolation of concentrated exosomes It highlights the need for new methods that provide isolation, especially for clinical applications. It is very important that the exosomes obtained are of suitable purity for both in vitro and ex vivo use. Brief Description of the Invention The invention is based on the use of L-charge coated magnetic nanobeads to isolate exosomes. It includes a method involving initial pre-treatment including filtration and low-speed centrifugation. The steps increase the concentration of exosome-containing samples. This approach increases the concentration of the original sample by 10. Compared to the volume, this allows for more efficient use of magnetic beads in subsequent stages. The basic principle of the invention is to combine exosomes with L-arginine coated paramagnetic nanobeads. It is based on the fact that L-charge has the property of carrying both positive and negative charge. It is amphoteric. By taking advantage of this property, negatively charged exosomes are developed within the scope of this invention. First, the collection of L-charged nanoparticles coated with positive TF is performed by pulling them together, then 15 These L-argin-coated nanoparticles, which can be easily brought to a negative charge, are then easily released again. This ensures that it is released. This method is similar to anti-corrosion isolation methods. without requiring the breaking of antagonist-nuclear bonds and without any artifact / contamination It provides effective and easy exosome isolation without causing the formation of a new substance. The aim of this invention is to produce a high-purity, structurally intact, and highly concentrated 20 The goal is to provide a rapid and effective method for isolating exosomes. Shapes Figure 1: Filtration Process with Sequential Membranes. Figure 2: Magnetic nanoparticles coated with L-charge. Figure 3: Flow cytometry analysis of exosomes isolated with T car brkt (A) and invention (B). 25 Detailed Description of the Invention In the invention process, sequential isolation of exosomes using L-arginine-coated paramagnetic nanobeads was performed. By using membrane filtration and low-speed centrifugation methods in combination This is being carried out. Membrane filtration method uses a single membrane (e.g. 0.20 µm) This can be done through filtration, or in a way that the pore sizes gradually decrease at the top (30). This can also be done through membranes consisting of successive filters (Figure 1). This situation, 4 This will cause the sample to be held between 200 nm and 30 nm. In the example in the figure... As shown, the exosome-containing fluid passes through successive membranes by a slow centrifugal force. This successive membrane passage can be achieved with the pressure provided by the syringe or by a few other methods. This can also be achieved with the system. The pores of the membranes are from largest to smallest. These have been modified. Including but not limited to, for example; Membrane 1: 0.450 µm; Membrane 5 Membrane 2: 0.220 µm; Membrane 3: 0.150 µm; Membrane 4: 0.030 µm. However, these given in Figure 1... Not limited to values, but different values ​​provided they are consecutive and in descending order. It's possible. For example, successive membranes, centrifugation, or any pressure-generating device such as a syringe. With the help of factor 10, the sample is held between 200 nm and 30 nm membranes. It is possible (Figure 1). When using this system, centrifugation, syringe or pipe system can be used. The sample can be passed through the filters sequentially, either in combination or separately. Thus, Exosome-rich fluid obtained from a large volume ml sample, L-arginine coated, cationic mixed with magnetic k beads and attracted (cation k nano beads pos tf, exosomes (Because they are negatively charged, they will attract each other) and in the final stage, the exosomes will detach from the beads 15 It is released by elution. This results in a high concentration of substances without any additional chemicals or residue. It enables the production of exosomes in pure and high concentrations. The method consists of three main stages: (1) Pre-enrichment, (2) Magnetic capture and (3) Exosome release. Pre-enrichment Phase 20 This step aims to increase exosome concentration in large volume ml samples. It is designed. It can consist of different options. However, it basically consists of plasma, serum, urine, and cells. Different samples of the culture medium are centrifuged at low speed and then filtered. Membrane filtration is a method of filtration through a single membrane (e.g., 0.20 µm). It could be, as it consists of 25 successive filters with progressively smaller pore sizes at the top. This can also be done via membranes (Figure 1). For example, successive membranes can be used in centrifugation. or with the help of any pressure-generating factor such as a syringe, the sample is drawn between 200 nm and 30 This can cause the membranes to be trapped between the nm (Figure 1). When this system is used Centrifugation, syringe or pipe system can be used and sample sequentially passed through filters. It can be passed in combination or separately. 30 Low-speed centrifugal force spraying  Plasma, serum, urine, cell culture medium, cerebrospinal fluid (CSF), milk, and plant extracts. Different samples, such as sap, are centrifuged at low speeds. For example, at 5000 or 10,000 x g. The center is fuged for 10–40 minutes.  This step removes any remaining cellular debris and large particles. Sequential Membrane Filtration 5  The sample is placed through a single membrane (e.g., 0.20 µm) or membranes with progressively smaller pore sizes. brdz passes through the membrane.  T pk membrane dzs (from largest to smallest): Optionally 0.45 µm, 0.22 µm, 0.15 Pore ​​sizes of 0.03 µm and 0.03 µm (Figure 1) are also applicable.  These processes are carried out using a centrifuge, syringe pressure, or a specially designed internal pressure system. 10 It can be accomplished using... Magnetic Capture Phase At this stage, the exosome-rich sample obtained after pre-enrichment is L- It is processed with magnetic nanobeads coated with arj nn. Preparation of L-argin Coated Magnetic Nanobeads 15  Iron oxide core nanobeads are synthesized together with L-arginine and coated with L-arginine. Iron nanoparticles are obtained. These have paramagnetic properties. L-arginine amphoteric kt r.  L-arginine coated magnetic nanobeads, by changing the pH value or secondary They can be converted into cationic form with coatings. These include polyethylene (PEI), chitosan, and poly-20. Materials such as L-arginine (PLL) and its derivatives can be found. L-arginine coated. Specific antibodies can also be added to the nanobeads as a third layer. Exosome Capture  The prepared cationic L-arg n coated magnetic nanobeads are rich in exosomes. The sample is mixed. 25  Shake the mixture at room temperature.  Nano bead-exosome complexes are collected by applying a magnetic field (for example, (using neodymium magnets).  The supernatant is removed and the complexes are washed away. Exosome Release Phase 30 6 In this final stage, the captured exosomes are separated from the nanobeads, and a pure exosome is formed. Suspension is obtained. Converting positively charged nanobeads to negatively charged. L-arginine-coated nanobeads can be processed using buffer solutions (e.g., by changing the pH). The anion is converted to a negatively charged state. 5 Release of Exosomes  Nano beads that become anions transfer negatively charged exosomes.  The mixture is gently shaken at room temperature.  The magnetic field is applied again to disperse the nano-beads.  The supernatant is collected as a pure exosome suspension. 10 Final Treatment (Optional)  If necessary, the resulting exosome suspension can be used to remove any remaining nanobeads. It can be passed through a 0.22 µm filter. Within the scope of the invention, cation-rich L-arginine coated magnetic nanobeads (Figure 2) are exosomes. It was used for the first time in isolation. The black spots are made of iron oxide, and the surrounding nebula is 15. The fields consist of L-charges. Acceleration Voltage = 20000 Volts; Magnification = 10000 Volts. The rich sample in terms of exosomes is firstly coated with cationic (pose tf loaded) L-charge coated magnet It is processed with nanobeads. It is attracted by a magnet and separated from the liquid. Then, L-charge coated Magnetic nanobeads are converted into anionic (negatively charged) form with a solution, thus... Exosomes adhering to nanobeads are released into the solution, and the exosomes are magnetically activated. The nanobeads are collected again by removing them with a magnet. The use of cation-coated L-arginine nanobeads offers numerous advantages for exosome isolation. This allows for high-purity, easy-to-apply, fast, and parallel processing of the sample. methods, brex vivo extraction that can meet the demands of large-scale clinical application. It can provide a method. 25 This invention produces exosomes suitable for kt, nv vo and ex v vo applications (Figure 1). 3). Exosomes were stained with CD9 and CD81 and analyzed in a Beckman Coulter Cytoflex machine. ed lm şt r. In isolation with T car kt (A), the population in the FSC-SSC dot plot is wider. And it is more dispersed. This suggests that there may be other cellular debris besides exosomes. Additionally, the P3 and P5 populations, shown in brown on the CD9 and CD81 h-stograms, are 30 It shows contamination. In contrast, carried out by the method (B) described in the invention. 7 In exosome isolation, the cells are much more compact (FSC-SSC dot plot ğ). This, in other words... It shows a population consisting solely of exosomes, containing no artifacts. CD9 And CD81 h cytograms support this, because there is no population in P3 and P5. This flow The cytometric analyses show that high-purity exosomes were obtained with this invention. Anti-corrosion based magnetic nanobeads are quite effective at capturing exosomes, however 5 The efficiency of separating exosomes from antibody bound beads may not be high. However, this In this invention, it is easier to separate exosomes from nanobeads. For example, this separation process This can be achieved simply by adjusting the pH. By applying sequential filtration and centrifugation as a preliminary step, relatively large Samples in ml volume can also be used. 10  Structurally intact exosomes: Because no ultrafiltration process was performed. The integrity of the exosomes is preserved.  Purity: Exosomes, PEG, Dextran, Silicon carbide, etc. (in other commercial products) Since it is not processed with the molecules used, it will be quite pure (Figure 3).  Exosomes of suitable purity for in vitro or ex vivo applications can be obtained. The current 15 Many commercial methods have limitations in clinical applications due to exosome purity issues. It is not suitable for (3).  Rapid isolation: Exosomes can be isolated in approximately 30 minutes. (For example, gold) The standard time required for ultracentrifugation is approximately 3 hours, and furthermore... (The success rate is 5-25%) 20  Unlike polymer-based sedimentation methods, overnight sedimentation It is not required. Exosomes can be obtained very quickly and practically.  High concentration of exosomes: Thanks to the applied sequential membrane trap, the first Since the sample was converted into an NBR sample rich in exosomes, the exosomes were more It can be obtained in high concentrations. 25 This method allows for the rapid and effective extraction of high-purity and structurally intact exosomes. This allows for isolation. The entire process can be completed in approximately 30 minutes, and the result is obtained. ED LEN exosomes are suitable for direct use in NV VO or EXV VO applications. This invention utilizes L-arginine-coated magnetic nanobeads to rapidly and effectively target exosomes. This allows for isolation in this manner. Potential application areas of this method are illustrated with the following examples: 30 This includes, but is not limited to, the following: Cancer Diagnosis and Monitoring (e.g., Early Cancer Detection with Liquid Biopsy) 8  Exosomes isolated from patient blood were analyzed for cancer-specific biomarkers. ed l r.  Method: Exosomes are isolated from a 10 mL blood sample, and their contents are analyzed for proteomic and... The genome is subjected to analysis.  Potential Outcome: Non-invasive diagnosis of early-stage cancers and tumor type 5 determination. Monitoring Response to Cancer Treatment  Exosomes are periodically isolated from the plasma of patients undergoing chemotherapy.  Method: Exosomes are obtained from blood samples taken before and during treatment. The isolated contents are compared. 10  Potential Outcome: Real-time monitoring of treatment response and personalized treatment. development of strategies. Diagnosis of Neurodegenerative Diseases (e.g., Early Diagnosis of Alzheimer's Disease)  Exosomes are isolated from cerebrospinal fluid (CSF).  Method: Exosomes are isolated from 5 mL of CSF sample, tau protein and beta-amino acid 15 Levels are measured.  Possible Outcome: Diagnosis of Alzheimer's disease before clinical symptoms appear. Regenerative Medicine Applications (e.g., Production of Stem Cell-Derived Exosomes)  Therapeutic exosomes are isolated from mesenchymal stem cell culture medium.  Method: Exosomes are isolated from 500 mL of culture medium and incubated under sterile conditions for 20 minutes. It is hidden.  Potential Outcome: High potential for use in tissue regeneration and wound healing. Pure exosome preparations. Drug Delivery Systems (e.g., Preparation of Exosome-Based Drug Carriers)  Isolated exosomes are loaded with therapeutic molecules. 25  Method: Isolated exosomes were collected in small pieces using the electroporation method. The molecules are loaded with drugs or sRNA / shRNA.  Potential Outcome: Nano-compatible carriers for targeted drug delivery produce m. Diagnosis of Infectious Diseases (e.g., Rapid Diagnosis of Viral Infections) 30 9  Exosomes containing viral particles are isolated from the plasma of infected patients.  Method: Exosomes are isolated from a 5 mL plasma sample, and viral nucleic acids are analyzed using RT-PCR. le anal z ed l r.  Potential Outcome: Viral infections, especially those detected by traditional methods Rapid and accurate diagnosis of viruses that are difficult to detect. 5 Biobelt Discovery (e.g., Identification of New Disease Biobelt Agents)  Comparative analysis of the contents of exosomes isolated from various patient groups ed l r.  Method: Samples were taken from different patient groups and a healthy control group. Exosomes are isolated, and proteomic and metabolomic analyses are performed. 10  Potential Outcome: Discovery of new diagnostic and prognostic biomarkers for various diseases. These application examples use L-arginine-coated magnetic nanobeads to implant exosomes. The isolation method demonstrates its wide range of applications. Each example illustrates how the method works. It explains how it can be applied and its potential consequences. References 15 1- T., Naga, Aparna. (2024). Exosomes - naturally occurring ng drug del very veh cles: an ns ght. do : 10.58532 / v3bkpn17p7ch1 2- R chard, J., M ron., Nathan, E, Estr n., Anton, Sculean., Yufeng, Zhang. (2024). Understand ng exosomes: Part 2-Emerg ng leaders n regenerat ve med c ne. Parodontolog â, do : 10.1111 / prd.12561 20 3- Gao J, L A, Hu J, Feng L, L u L, Shen Z. Recent developments n solat ng methods for exosomes. Front B oeng B otechnol 2022;10:1100892. do : 10.3389 / fb oe.2022.1100892 30

Claims

REQUESTS 1. Exosome isolation method, characterized by the use of L-argin-coated magnetic nanobeads. It is the use of.

2. The method according to claim 1 is characterized by the fact that the exosome-containing fluid is placed in a magnetically coated L-charger. It must be passed through at least one membrane filtration before being mixed with nanobeads. 5 3. The method according to claim 2, and its characteristic is; L-charge coated magnetic nanobeads. sequential membrane filtration of the exosome-containing fluid before mixing passing is.

4. The method is based on claim 3 and its characteristic is that the membrane pore sizes are 0.45 µm, respectively. The thicknesses are 0.22 µm, 0.15 µm, and 0.03 µm. 10 5. The method is based on claim 1 or 2 and has the following characteristics: a. Exosome-rich L-arginine coated magnetic nanobeads mixing with the sample, b. Collection of nanobead-exosome complexes by applying a magnetic field. The process includes the following steps. 15 6. The method according to claim 5 is characterized by the fact that the magnetic field mentioned is in a neodymium magnet. It is implemented using...

7. The method according to claim 5 is characterized by; negatively charged L-charged nanobeads coated with anionic charge. Exosomes are released via the TMS pathway.

8. It is an exosome and its characteristic is that it was isolated using the method in Claim 1. 20 9. It is a bioreceptor and its characteristic is that it contains exosomes according to Claim 5. 30

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