Exosome isolation method

Abstract

Process of the invention, utilizes L-arginine-coated magnetic nanobeads for exosome isolation. pre-processing steps consisting of filtration and low-speed centrifugation enrich exosome- containing samples, allowing for more economical use of magnetic beads in subsequent stages compared to the initial sample volume.

Description

[0001] EXOSOME ISOLATION METHOD

[0002] Technical Field

[0003] The process of the invention, utilizes L-arginine-coated magnetic nanobeads for exosome isolation providing rapid, economical, and scalable method for high-purity, structurally intact, and high-concentration exosomes. Especially for clinical applications, it is critical that the isolated exosomes are of suitable purity for in vivo and ex vivo use.

[0004] State of the Art

[0005] Exosomes are nanovesicles with diameters of 40-160 nm, secreted by various cell types and playing important roles in intercellular communication. These structures have great potential as biomarkers for diagnostic purposes and as carriers of therapeutic agents. Exosomes have been used as drug delivery agents and in the treatment of neurodegenerative disorders, organ damage, degenerative processes, infectious diseases, regenerative procedures, cancer therapy, and immune function [1,2], However, the effective, easy, and high-purity isolation of exosomes remains one of the biggest challenges in research and applications in this field.

[0006] Existing exosome isolation techniques include ultracentrifugation, immunocapture-based methods, polymer-based precipitation kits, and size exclusion chromatography [3], While each of these methods has its unique advantages, they also have various limitations.

[0007] The oldest of these is ultracentrifugation isolation. This method requires long periods of very high-speed centrifugation (e.g., 100,000 xg), resulting in long, labor-intensive work hours and high energy consumption, as well as efficiency and purity problems. Polymer-based precipitation kits, using materials such as PEG (Polyethylene glycol), have achieved relatively higher efficiency and easier exosome extraction. However, they have problems such as high cost and the inability to use the collected exosomes directly in ex vivo or in vivo applications due to extra components added to the sample during purification. They also require long incubation times. Immunocapture-based kits are relatively less efficient and quite expensive. Due to the high cost of antibody content, limited amounts are used, resulting in relatively lower concentrations of exosome yield when using lower sample volumes to achieve exosome capture saturation. Also, only a specific subset of exosomes can be obtained. Size exclusion chromatography requires expensive equipment and has a long isolation time. When filtration is used alone, the obtained exosomes are contaminated with peptides such as albumin. Therefore, in this invention, filtration is not used alone but as a preliminary stage (filtration using sequential membranes). Various methods are compared as follows;

[0008] Ultracentrifugation Method:

[0009] • Advantages: Purer exosomes are obtained compared to methods such as polymer- based isolation.

[0010] • Disadvantages: Requires long-term (3-4 hours) and high-speed (100,000 xg) centrifugation, need for special equipment, low yield (5-25%), high energy consumption, and risk of disrupting the structural integrity of exosomes. The special tubes used in ultracentrifugation are consumables and are not inexpensive.

[0011] Immunocapture-Based Methods:

[0012] • Advantages: Ability to isolate specific exosome subtypes.

[0013] • Disadvantages: High cost, low yield, limited scalability, and isolation of only exosomes with certain surface proteins. Problem of releasing antibody-bound exosomes from antibodies after isolation.

[0014] Polymer-Based Precipitation Kits:

[0015] • Advantages: Easy application, no need for special equipment.

[0016] • Disadvantages: Low purity (contamination with proteins and other vesicles), long incubation times (usually overnight), obtained exosomes not suitable for direct in vivo or ex vivo use.

[0017] Size Exclusion Chromatography:

[0018] • Advantages: High purity and preservation of exosome integrity.

[0019] • Disadvantages: Low yield, long isolation time, expensive equipment requirement, and limited scalability.

[0020] Filtration Methods:

[0021] • Advantages: Fast and simple application.

[0022] • Disadvantages: When used alone, low purity (e.g., contamination with proteins such as albumin) and risk of exosomes sticking to the membrane.

[0023] The limitations of these methods highlight the need for new methods that can provide rapid, economical, and scalable isolation of high-purity, structurally intact, and high-concentration exosomes. Especially for clinical applications, it is critical that the isolated exosomes are of suitable purity for in vivo and ex vivo use. Brief Description of the Invention

[0024] The invention involves a method that employs L-arginine-coated magnetic nanobeads to isolate exosomes. Initial preprocessing steps, which include filtration and low-speed centrifugation, enhance the concentration of exosome-containing samples. This approach enables a more efficient use of magnetic beads in the later stages, compared to the volume of the original sample. The main principle of the invention is based on attracting exosomes with L-arginine- coated paramagnetic nanobeads. L-arginine has the ability to carry both positive and negative charges, making it amphoteric. Taking advantage of this property, the invention enables the isolation of negatively charged exosomes by first attracting and collecting them with positively charged L-arginine-coated nanoparticles, and then easily releasing them from these L-arginine- coated nanoparticles, which are readily made negative. This method provides efficient and easy exosome isolation without creating any artifacts / contamination or requiring the breaking of antibody-antigen bonds, as in antibody-based isolation methods.

[0025] The purpose of this invention is to provide a rapid and effective method for isolating exosomes with high purity, preserved structural integrity, and high concentration.

[0026] Figures

[0027] Figure 1 : Filtration process with Sequential Membranes:

[0028] Figure 2: Magnetic nanoparticles coated with L-arginine.

[0029] Figure 3 : Flow cytometry analysis of exosomes isolated with a commercial kit (A) and with the invention (B).

[0030] Detailed Description of the Invention

[0031] In the process of invention, exosome isolation is achieved by using L-arginine-coated paramagnetic nanobeads in combination with sequential membrane filtration and low-speed centrifugation methods. The membrane filtration method can be either filtration through a single membrane (e.g., 0.20 pm) or through membranes consisting of sequential filters with decreasing pore sizes at the top (Figure 1). This will cause the sample to be trapped between 200 nm and 30 nm. As shown in the example in the figure, the exosome-containing fluid can pass through the sequential membrane with a non-rapid centrifugal force, or this sequential membrane passage can be achieved with the pressure provided by the syringe or with a chip system. The pores of the membranes are arranged from high to low. Without being limited to these, for example, Membrane 1 : 0.450 pm; Membrane 2: 0.220 pm; Membrane 3: 0.150 pm; Membrane 4: 0.030 pm. However, it is not limited to these figures, provided that it is sequential and from large to small.

[0032] For example, sequential membranes can cause the sample to be trapped between 200 nm and 30 nm membranes with the help of any pressure-creating factor such as centrifugation or syringe (Figure 1). When using this system, centrifugation, syringe, or chip system can be utilized, and the sample can pass through the filters sequentially in a combined or separate manner. Thus, the exosome-rich fluid obtained from the large volume sample is mixed with L-arginine coated, cationic magnetic beads and attracted (as cationic nanobeads are positive and exosomes are negative, they will attract each other), and finally, the exosomes are eluted and released from the beads. This allows for obtaining high-purity exosomes with high concentration without any additional chemicals or residues.

[0033] The method consists of three main stages: (1) Pre-enrichment, (2) Magnetic capture, and (3) Exosome release.

[0034] Pre-enrichment Stage

[0035] This stage is designed to increase the exosome concentration in large-volume samples. It can consist of different options. However, basically, different samples such as plasma, serum, urine, and cell culture medium are centrifuged at low speed and then filtered. The membrane filtration method can be either filtration through a single membrane (e.g., 0.20 pm) or through membranes consisting of sequential filters with decreasing pore sizes at the top (Figure 1). For example, sequential membranes can cause the sample to be trapped between 200 nm and 30 nm membranes with the help of any pressure-creating factor such as centrifugation or syringe (Figure 1). When using this system, centrifugation, syringe, or chip system can be utilized, and the sample can pass through the filters sequentially in a combined or separate manner.

[0036] Low-Speed Centrifugation

[0037] • Different samples such as plasma, serum, urine, cell culture medium, cerebrospinal fluid (CSF), milk, plant juice are centrifuged at low speed. E.g., centrifuged at 5000 or 10,000 x g for 10-40minutes.

[0038] • This step removes remaining cellular debris and large vesicles. Sequential Membrane Filtration

[0039] • The sample is passed through a single membrane (e.g., 0.20 pm) or a series of membranes with gradually decreasing pore sizes.

[0040] • Typical membrane sequence (from large to small): pore sizes 0.45 pm, 0.22 pm, 0.15 pm, and 0.03 pm (Figure 1) can be also applied, optionally.

[0041] • These processes can be performed using centrifugation, syringe pressure, or a specially designed chip system.

[0042] Magnetic Capture Stage

[0043] In this stage, the exosome-rich sample obtained after pre-enrichment is treated with L-arginine coated magnetic nanobeads.

[0044] Preparation of L-arginine Coated Magnetic Nanobeads

[0045] • Iron oxide core nanobeads are synthesized together with L-arginine, and L-arginine- coated iron nanoparticles are obtained. These have paramagnetic properties. L-arginine is amphoteric.

[0046] • L-arginine-coated magnetic nanobeads can be made cationic by changing pH or with secondary coatings. These may include materials such as Polyethyleneimine (PEI), chitosan, Poly-L Lysine (PLL), and silica derivatives. Specific antibodies can also be added as a second layer to L-arginine-coated nanobeads.

[0047] Capture of Exosomes

[0048] • Prepared cationic L-arginine coated magnetic nanobeads are mixed with the exosome- rich sample.

[0049] • The mixture is shaken at room temperature.

[0050] • Nanobead-exosome complexes are collected by applying a magnetic field (e.g., using a neodymium magnet).

[0051] • The supernatant is removed, and the complexes are washed.

[0052] Exosome Release Stage

[0053] In this final stage, the captured exosomes are separated from the nanobeads, and a pure exosome suspension is obtained.

[0054] Changing the positively chargedNanobeads to negatively charged. L-arginine-coated nanobeads are made anionic (negatively charged) by buffering solutions (e.g. by changing the pH). Release of Exosomes

[0055] • The nanobeads that have become anionic repel the negatively charged exosomes.

[0056] • The mixture is gently shaken at room temperature.

[0057] • The magnetic field is applied again to remove the nanobeads.

[0058] • The supernatant is collected as a pure exosome suspension.

[0059] Final Purification (Optional)

[0060] • If necessary, the obtained exosome suspension can be passed through a 0.22 pm filter to remove any remaining nanobeads.

[0061] Cationized L-arginine-coated magnetic nanobeads (Figure 2) have been used for the first time in exosome isolation by the invention. Black dots are made of iron oxide, and the surrounding gray cloudy areas are made of L-arginine. Accelerating Voltage=20000 Volt; Magnification= 10000. The exosome-rich sample is first treated with cationic (positively charged) L-arginine-coated magnetic nanobeads. It is pulled with a magnet and separated from the liquid. Then, L-arginine-coated magnetic nanobeads are made anionic (negatively charged) with a solution, allowing the release of exosomes adhered to these nanobeads into the solution, and the exosomes are collected by removing the magnetic nanobeads again with a magnet.

[0062] Using cationized L-arginine coated nanobeads provides a highly pure, easy-to-apply, quick, and parallel processing of multiple samples for exosome isolation. These methods can provide an ex vivo extraction method that can meet large-scale clinical application demands.

[0063] The kit created with this invention yields exosomes suitable for in vivo and ex vivo applications (Figure 3). Exosomes were stained with CD9 and CD81 and analyzed on a Beckman Coulter Cytoflex device. In isolation with the commercial kit (A), the population in the FSC-SSC dot plot graph is wider and more dispersed. This indicates that there may be other cellular debris besides exosomes. Also, the P3 and P5 populations shown in brown in the CD9 and CD81 histograms indicate contamination. In contrast, in the exosome isolation performed with the method described in the invention (B), the cells are much more compact (FSC-SSC dot plot graph). This indicates a population consisting of only exosomes, without any other artifacts. The CD9 and CD81 histograms support this. Because there is no population in P3 and P5. These flow cytometry analyses show that highly pure exosomes are obtained with the invention. Although antibody-based magnetic nanobeads are quite effective in capturing exosomes, the efficiency of separating exosomes from antibody-bound beads may not be efficient. However, in this invention, it is easier to separate exosomes from nanobeads. For example, this separation can be achieved by simply adjusting the pH.

[0064] Thanks to the sequential filtration and centrifugation to be applied as a preliminary stage, relatively large volume samples can also be used.

[0065] • Structurally intact exosomes: As no ultrafiltration process is performed, the integrity of the exosomes will be preserved.

[0066] • Purity: As the exosomes are not treated with molecules used by other commercial kits such as PEG, Dextran, Silicon carbide, etc., they will be quite pure (Figure 3).

[0067] • Exosomes of suitable purity for in-vivo or ex-vivo applications can be obtained. Many existing commercial methods are not suitable for clinical applications due to limitations in terms of exosome purity (3).

[0068] • Rapid isolation: exosomes can be isolated in approximately 30 minutes. (For example, the time required for ultracentrifugation, which is considered the gold standard, is approximately 3 hours, and the recovery rate is 5-25%)

[0069] • Unlike polymer-based precipitation methods, it does not require overnight precipitation. Exosomes can be obtained very quickly and practically.

[0070] • High concentration of exosomes: Thanks to the sequential membrane trap applied, as the initial sample is turned into an exosome-rich sample, exosomes can be obtained in higher concentration.

[0071] This method provides rapid and effective isolation of high-purity and structurally intact exosomes. The entire process can be completed in approximately 30 minutes, and the obtained exosomes are suitable for direct use in in vivo or ex vivo applications.

[0072] This invention provides rapid and effective isolation of exosomes using L-arginine coated magnetic nanobeads. The potential application areas of this method include, but are not limited to, the following examples:

[0073] Cancer Diagnosis and Monitoring (e.g.Early Cancer Diagnosis with Liquid Biopsy)

[0074] • Exosomes isolated from patient blood are analyzed for cancer-specific biomarkers. • Method: Exosomes are isolated from 10 mL blood sample, their contents are subjected to proteomic and genomic analyses.

[0075] • Potential Outcome: Non-invasive diagnosis of early-stage cancers and determination of tumor type.

[0076] Monitoring Response to Cancer Treatment

[0077] • Exosomes are periodically isolated from the plasma of patients receiving chemotherapy.

[0078] • Method: Exosomes are isolated from blood samples taken before and during treatment, their contents are compared.

[0079] • Potential Outcome: Real-time monitoring of response to treatment and development of personalized treatment strategies.

[0080] Diagnosis of Neurodegenerative Diseases (e.g. Early Diagnosis of Alzheimer's Disease)

[0081] • Exosomes are isolated from cerebrospinal fluid (CSF).

[0082] • Method: Exosomes are isolated from 5 mL CSF sample, tau protein and beta-amyloid levels are measured.

[0083] • Potential Outcome: Diagnosis of Alzheimer's disease before clinical symptoms appear.

[0084] Regenerative Medicine Applications (e.g. Production of Stem Cell-Derived Exosomes)

[0085] • Therapeutic exosomes are isolated from mesenchymal stem cell culture medium.

[0086] • Method: Exosomes are isolated from 500 mL culture medium, stored under sterile conditions.

[0087] • Potential Outcome: High-purity exosome preparations that can be used for tissue regeneration and wound healing.

[0088] Drug Delivery Systems (e.g. Preparation of Exosome-Based Drug Carriers)

[0089] • Isolated exosomes are loaded with therapeutic molecules.

[0090] • Method: Isolated exosomes are loaded with small molecules, drugs or siRNA / shRNA using electroporation method.

[0091] • Potential Outcome: Production of biocompatible nano-carriers for targeted drug delivery.

[0092] Diagnosis of Infectious Diseases (e.g Rapid Diagnosis of Viral Infections) • Exosomes containing viral particles are isolated from the plasma of infected patients.

[0093] • Method: Exosomes are isolated from 5 mL plasma sample, viral nucleic acids are analyzed by RT-PCR.

[0094] • Potential Outcome: Rapid and sensitive diagnosis of viral infections, especially for viruses that are difficult to detect by conventional methods.

[0095] Biomarker Discovery (e.g. Identification of New Disease Biomarkers)

[0096] • The content of exosomes isolated from various patient groups is comparatively analyzed.

[0097] • Method: Exosomes are isolated from samples taken from different patient groups and healthy controls, proteomic and metabolomic analyses are performed.

[0098] • Potential Outcome: Discovery of new diagnostic and prognostic biomarkers for various diseases.

[0099] These application examples demonstrate the wide range of potential uses of the exosome isolation method using L-arginine coated magnetic nanobeads. Each example describes how the method can be applied and its potential outcomes.

[0100] References

[0101] 1- T., Naga, Aparna. (2024). Exosomes - naturally occuring drug delivery vehicles: an insight, doi: 10.58532 / v3bkpnl7p7chl

[0102] 2- Richard, J., Miron., Nathan, E, Estrin., Anton, Sculean., Yufeng, Zhang. (2024). Understanding exosomes: Part 2-Emerging leaders in regenerative medicine. Parodontologia, doi: 10.1111 / prd.12561

[0103] 3- Gao J, Li A, Hu J, Feng L, Liu L, Shen Z. Recent developments in isolating methods for exosomes. Front Bioeng Biotechnol 2022; 10: 1100892. doi:

[0104] 10.3389 / fbioe.2022.1100892

Claims

CLAIMS1. Exosome isolation method comprising using L-arginine coated magnetic nanobeads.

2. Method according to claim 1 comprising at least one membrane filtration of exosome- containing fluid before mixing with L-arginine coated magnetic nanobeads.

3. Method according to claim 2 comprising sequential membrane filtration of exosome- containing fluid before mixing with L-arginine coated magnetic nanobeads.

4. Method according to Claim 1 characterized in that mambran pore sizes are 0.45 pm, 0.22 pm, 0.15 pm, and 0.03 pm in order.

5. Method according to claim 1 or 2 characterized in comprising steps of a. Mixing L-arginine coated magnetic nanobeads with the exosome-rich sample b. Collecting nanobead-exosome complex by applying a magnetic field6. Method according to Claim 5 characterized in that said magnetic field is applied by neodymium magnet.

7. Method according to Claim 5 further comprises releasing the exosome by repelling negatively charged exosome by anionic L-arginine-coated nanobeads8. Exosome isolated by method of Claim 1.

9. Exosome according to Claim 5 for use as biomarker.

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