Composition and methods for exosome isolation from large and small volume human biofluids using exosome precipitation agent

Abstract

Compositions and methods comprising EXO-PEG-TR precipitation agent for extracellular vesicle (especially exosome) isolation from various biologic fluid samples are provided. The compositions and methods are capable of efficient extracellular vesicle and exosome isolation with high purity and high quality suitable for downstream analysis.

Description

Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025COMPOSITION AND METHODS FOR EXOSOME ISOLATION FROM LARGE AND SMALL VOLUME HUMAN BIOFLUIDS USING EXOSOME PRECIPITATION AGENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 736,341 filed December 19, 2024 the specification of which is incorporated herein in its entirety by reference.FIELD OF THE INVENTION

[0002] The present invention relates to biofluid-precision diagnostics and extracellular vesicle isolation methods. The present invention also relates to compositions comprising exosome precipitation agents, and compositions and methods for exosome isolation from large and small volume human biofluids using exosome precipitation agents.BACKGROUND OF THE INVENTION

[0003] Extracellular vesicles (EVs) are membrane bound vesicles produced and secreted by many cells and are present in nearly all biological fluids - blood, plasma, urine, saliva, peritoneal fluid, ascites, pleural fluid, CSF, etc. EVs comprise a diverse category of vesicles, such as microvesicles, exosomes, and apoptotic bodies. Recently, EVs have garnered significant attention due to their role in intercellular communication and the transfer of biological cargo between cells. It has been well established that EVs, specifically exosomes, play a critical role in a variety of physiologic processes and pathologic conditions like cancer, infectious diseases, endocrine disorders, and neurodegenerative disorders.

[0004] Exosomes are small membrane-bound nanovesicles, about 30 to 200nm nm in size, and are present in various biologic fluids, including blood (plasma / serum), urine, saliva, breast milk, CSF, semen, bronchial lavage, pleural fluid, bile, peritoneal lavage, and tears. Exosomes play a crucial role in intercellular communication by transferring proteins, lipids, and nucleic acids between cells. As a result, exosomes have drawn significant attention from both basic and clinical research domains. Exosomes provide insights into disease mechanisms and could enable early diagnosis, and even serve as vehicles for targeted therapy.

[0005] The knowledge about exosomes in disease states and the ubiquitous presence of exosomes in various biologic fluids have made them an ideal target for next-generation liquid biopsy. However, a significant hurdle in harnessing exosomes for clinical applications is the lack of standardized, efficient, and scalable isolation techniques. Isolation of exosomes has beenReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 fraught with many challenges; time, labor, equipment, and purity. The commonly used techniques for exosomes isolation are ultracentrifugation, sucrose gradient isolation, size exclusion chromatography, and precipitation. Ultracentrifugation is considered the gold standard but is the least convenient as it is labor-intensive, time-consuming (requires >2 days), and equipment-dependent and hence not suitable for clinical scalability. On the other hand, precipitation techniques using commercially available precipitation liquid, although less cumbersome, are expensive due to the cost of the liquid, and such techniques do not yield high-purity7exosomes. When isolating exosomes from biofluids using precipitation technique, there is a risk of co-isolating other extracellular vesicles, lipoproteins, and protein aggregates. These contaminants affect the purity of the isolated exosomes and the results of subsequent analysis. There is a critical need for reliable and scalable exosome isolation protocols using precipitation agents to expand exosome discoveries into clinical practice.BRIEF SUMMARY OF THE INVENTION

[0006] The study of exosomes in biofluids for research and clinical purposes requires a reliable and easy method of isolation and preservation of exosomes. This invention features a new precipitation agent, EXO-PEG-TR, and a specific protocol utilizing this reagent for the isolation of exosomes from biofluids (small and large volumes). It is an objective of the present invention to provide compositions, including those comprising of EXO-PEG-TR, and methods that allow for the efficient isolation of exosomes from biofluids, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0007] The present invention features methods of EV and exosome isolation using a newly designed precipitation reagent named EXO-PEG-TR, which contains Polyethylene Glycol - Trehalose. Methods described herein offer an easy and efficient technique to separate high quality, high purity exosomes from all biologic fluids with minimal time and effort. The separation reagent uses the hydrophilic polymer polyethylene glycol in combination with Trehalose. This allows for high-efficiency separation to be achieved, without the need for specialized ultracentrifugal equipment. Furthermore, the reagent also preserves the exosome membrane during storage of biologic fluids for future analysis.

[0008] This exosome isolation protocol allows for exosome isolation using a simple three-step process with minimal centrifugation to produce a homogenous population of exosome-sized extracellular vesicles from biofluids. The newly developed EXO-PEG-TR reagent facilitates theReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 preservation of exosomes in biofluids and isolation of high purity and high quality with maximal recovery from any biofluid. The proposed isolation method with EXO-PEG-TR is scalable and easily adoptable for research and clinical use.

[0009] Compared to the exosomes isolated using commercially available precipitation liquid, exosomes isolated using the present new isolation technique(s) with EXO-PEG-TR are of higher quantity and quality. The exosomes isolated using ultracentrifugation are “leaky” exosomes, due to the membrane damage from the sheer force exerted by ultracentrifugation for long periods of time. In comparison, the membrane integrity of exosomes isolated using EXO-PEG-TR are preserved. EXO-PEG-TR can be used to isolate high purity and high quality exosomes and exosome subpopulations for further downstream diagnostic and therapeutic applications. It can be further used for study of exosomes- genomics, transcriptomics, proteomics, lipidomics, and metabolomics. Furthermore, EXO-PEG-TR also serves as a preservative as it preserves the quality and quantity of exosomes in biological fluids stored under various conditions.

[0010] In some embodiments, the present invention comprises a novel exosome precipitation reagent, EXO-PEG-TR, to overcome the issues of current exosome isolation. PEG-P2139 (25 gm 8kDa) and Trehalose dihydrate (405 mg TO 167) are combined with 50ml (W4502) of nuclease free water and ultrasonicated with full waves for 30-90 minutes to achieve a homogeneous aqueous solution. The solution is centrifuged at 5000g for 20 minutes at +4°C. The supernatant is filtered through a stericup (S2GPU05RE) 0.45pm filter to obtain the EXO-PEG-TR precipitation reagent (50%, g ml-1). The resultant precipitation reagent can be stored at room temperature for 3 months or at +4°C for a year. This long shelf life is also another advantage of EXO-PEG-TR. EXO-PEG-TR protocol for isolation of exosomes, the newly designed EXO-PEG-TR precipitation reagent was used for isolation of exosomes from large and small volume human biofluids. The protocol for isolation of exosomes from small and large volume biofluids and exosome precipitates are shown.

[0011] One of the unique and inventive technical features of the present invention is EXO-PEG-TR. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for greater purity, quality, quantity, and speed, while allowing usage of quantities of biological fluid that would not otherwise be clinically feasible to isolate exosomes from. Furthermore, without wishing to limit the invention to any theory or mechanism, it is also believed that the present invention allows for isolation of exosomes of all different sizes, thereby capturing subsets of exosomes that wereReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 previously difficult if not impossible to isolate. Prior to the present invention, EVs have also been difficult to store for long periods of time (> 1 week) for future analysis, due to degradation of contents, membrane damage, and loss of quantity. These issues have resulted in significant challenges for conducting research on EVs. With the present invention, EVs isolated by EXO-PEG-TR and stored at -80C are also well preserved for long periods of time. None of the presently known prior references or works have the unique inventive technical feature of the present invention.

[0012] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary7skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0013] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0014] FIG. 1A-1B shows synthesis of EXO-PEG-TR exosome precipitation reagent. FIG. 1A shows a flow chart depicting the chemical composition of EXO-PEG-TR for EV isolation. FIG. IB shows a schematic of the 5-step, streamlined synthesis process for EXO-PEG-TR reagent using polyethylene glycol, trehalose, and nuclease-free water. Polyethylene glycol (PEG-P2139, 25gm, 8kDa, Sigma) and Trehalose dihydrate (405 mg T0167) are combined with 50ml (W4502) of nuclease free water and ultrasonicated with full waves for 30-90 minutes to achieve a homogeneous aqueous solution. The solution is centrifuged at 5000g for 20 minutes at 4°C. The supernatant is filtered through a stericup (S2GPU05RE) 0.45pm filter to obtain the EXO-PEG-TR precipitation reagent (50%. g ml-1). The reagent can be stored at room temperature for 3 months or at 4°C for 1 year. In some embodiments, the steps for the formulation synthesis of EXO-PEG-TR precipitation reagent are as follows: Step 1 : Mix 25g PEG-P2139 with 405 mg Trehalose dihydrate and add 50 ml millipore molecular biology' grade nuclease free water (W4502). Step 2: Ultrasonicate full waves for 30-90 minutes to achieve homogeneous aqueous solution. Step 3: Centrifuge the solution at 5000g for 20 minutes at 4°C to remove undissolved PEG particles and collect the supernatant. Step 4: Filter supernatant through stericup 0.45pm. Step 5: Store in a suitable container EXO-PEG-TR precipitation reagentReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025(reagent can be stored at room temperature for 3 months or at 4°C for 1 year in 50ml sterile falcon tubes.

[0015] FIG. 2 shows the chemical composition of EXO-PEG-TR.

[0016] FIG. 3 shows isolation of exosomes using EXO-PEG-TR reagent from small-volume human biofluids (e.g., plasma / serum). A stepwise protocol for isolating EVs from small volume samples, including step-1 and step-2 precipitation steps for plasma samples using EXO-PEG-TR reagent (1:4 reagent / sample ratio). The isolation is detailed for small volume human biofluid as follows: Step 1 : Collect fresh or frozen plasma / serum. Step 2: Dilute plasma with IX Dulbecco's modified phosphate buffer solution (D-PBS) (1:9) ratio. For example, mix 500pl of plasma / serum with 4.5ml 1X-PBS to achieve 5 ml final volume. Step 3: Filter the diluted plasma / serum with a 0.45pm filter to deplete serum residual contaminant. Step 4: First precipitation: Add EXO-PEG-TR to the diluted plasma / serum 1:4 (reagent / sample) ratio. For example, add 1.25ml of EXO-PEG-TR to the 5ml diluted plasma / serum or 63pL to the 250 pL undiluted plasma / serum samples. Mix and incubate without shaking for 30 minutes to 2h or leave overnight at 4°C. Step 5: Centrifuge the mixture at 3000 x g for 15 minutes at 4°C to obtain the 1st exosome pellet. Step 6: Discard the supernatant and resuspend the 1st exosome pellet with 10ml lx D-PBS. Step 7: Second precipitation: Add 2.5 ml of EXO-PEG-TR reagent 1:4 (reagent / sample) ratio to deplete residual contaminants. For smaller exosome pellet quantities, use 4ml PBS and 1ml EXO-PEG-TR. Mix and incubate without shaking for 2-12h or leave overnight at 4°C. Step 8: Centrifuge the mixture at 3000 x g for 15 minutes at 4°C to obtain the 2nd exosome pellet. Step 9: Discard the supernatant and resuspend the exosome pellet in mixture of 1ml lx D-PBS-TR (2mM Trehalose) buffer and store at -80°C for further downstream applications like subpopulation exosome fractionation, genomics, transcriptomics, and proteomics analysis. Note that dilution of plasma / serum with IX PBS (3-5 folds) and filtration with a 0.45pm filter are, in some embodiments, important steps for extracting a pure exosome population by depleting albumin and other contaminants, thus increasing sedimentation, and maximizing the recovery of the exosomes with EXO-PEG-TR isolation method.

[0017] FIG. 4 shows exosome isolation from large volume (e.g., peritoneal lavage, ascites, and urine) samples using EXO-PEG-TR. A stepwise protocol for isolating EVs from large volume samples, including an initial filtration step-1 for peritoneal lavage and urine samples using EXO-PEG-TR reagent (1 :4 reagent / sample ratio). Fresh or frozen large volume (e.g., ascites, peritoneal lavage, and urine) samples are collected for exosome isolation as follows: Step 1 : Centrifuge the large volume biofluid at 3000 x g for 15 minutes at 4°C to remove cellular debrisReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 and collect the supernatant. Step 2: Filter supernatant with a 0.45pm filter to deplete residual protein contaminant and collect the filtrate. Step 3: First precipitation: Add EXO-PEG-TR to the filtrate large volume fluid at 1 :4 (reagent / sample) ratio. For example, add 10ml of EXO-PEG-TR to 40ml large volume samples. Mix and incubate without shaking for 2-12h or leave overnight at 4°C. Step 4: Centrifuge the mixture at 3000 x g for 15 minutes at 4°C to obtain the 1st exosome pellet. Step 5: Discard the supernatant and resuspend the exosome pellet with 40ml IX D-PBS. Additional, optional steps (not pictured) are as follows: Step 6: Second precipitation to deplete residual contaminants for fluids such as ascites: Add 10ml EXO-PEG-TR reagent to the resuspended exosome pellet at 1 :4 (reagent / sample) ratio. Mix and incubate without shaking for 2-12h or leave overnight at 4°C. Step 7: Centrifuge the mixture at 3000 x g for 15 minutes at 4°C to obtain the 2nd exosome pellet. Step 8: Discard the supernatant and resuspend the exosome pellet in mixture of 1ml 1X-PBS-TR (2mM Trehalose) buffer and store at -80°C for further downstream applications like subpopulation exosome fractionation, genomics, transcriptomics, and proteomics analysis.

[0018] FIG. 5 shows isolation of exosomes from small volume human biofluids (e.g., plasma and serum).

[0019] FIG. 6 shows isolation of exosomes from large volume human biofluids (e g., large volume ascites, peritoneal lavage, urine).

[0020] FIGs. 7A-7C show exosome pellets from small volume (plasma) and large volume samples (plasma, peritoneal lavage, ascites, and urine), with all tubes showing pure white exosome pellets at the bottom of each tube. Exosomes were isolated from cohorts of cancer and healthy small and large volume samples by using EXO-PEG-TR reagent to determine the application of this isolation method in different biofluids. FIG. 7A shows representative images of isolated exosomes from 500pl small volume biofluid (plasma) with exosome pellets at the bottom of a 15 ml tube. FIG. 7B shows an exosome pellet for the large volume samples of 40ml peritoneal lavage. FIG. 7C shows an exosome pellet for the large volume samples of 40ml urine samples.

[0021] FIG. 8 shows a comparison of EXO-PEG-TR with ultracentrifugation and commercial precipitation reagent. Exosomes were isolated from small (plasma) and large volume (ascites, peritoneal lavage, and urine) biofluids, subjecting each sample to isolation using ultracentrifugation, commercially available exosomes precipitation reagent and EXO-PEG-TR. First. EXO-PEG-TR demonstrated high yield and high-quality exosomes even within 30 minutes as compared to ultracentrifugation which had low yield of exosomes even after 16h. EVs isolatedReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 by EXO-PEG-TR were of significantly beter quality, with well preserved membranes, as compared to ultracentrifugation, which resulted in damaged membranes and leaky EVs. Compared to commercial precipitation reagent, EXO-PEG-TR resulted in isolation of a high quantity of sEVs with very' minimal residual contaminants. The RNA quantity of the sEVs isolated by EXO-PEG-TR was also significantly higher compared to the commercial precipitation reagent. These results show the superior performance of EXO-PEG-TR compared to commonly used, traditional EV isolation techniques. Additional comparisons of EXO-PEG-TR to ultracentrifugation and commercial precipitation reagent is available in FIG. 22A-22R.

[0022] FIG. 9 shows further advantages of EXO-PEG-TR. Benefits of EXO-PEG-TR demonstrated herein are as follows: High purity and yield: EXO-PEG-TR minimizes contaminants, delivers superior sEV purity, and achieves significantly higher yields. Cost-effectiveness: EXO-PEG-TR has superior reagent cost as compared to commercial kits, decreases overall experimental expenses, and removes financial barriers for large-scale studies. Efficiency and simplicity: EXO-PEG-TR streamlines isolation workflow, reduces hand-on time and labor, and enables rapid clinical translation. Preservation of sEVs: EXO-PEG-TR maintains sEV integrity and quantity, is stable under various storage conditions, and is ideal for biobanking and longitudinal studies. Broad applicability: EXO-PEG-TR is compatible with diverse biofluids, and is effective for use in at least the critical biofluid categories of peritoneal lavage fluid, urine, and plasma human biofluid samples. Comprehensive molecular profiling: EXO-PEG-TR is fully compatible with multi-omics analyses, supports genomics, transcriptomics, proteomics, lipidomics, and metabolomics, and accelerates precision medicine applications.

[0023] FIG. 10 shows exosome Nano tracking Analysis (NTA) for healthy and cancer large volume (ascites, peritoneal lavage) by EXO-PEG-TR. showing the bin size distribution of 50.5nm to 500.5nm scale along the x-axis and concentration of the exosomes along the y-axis. Exosomes were isolated from large volume samples from patients with cancer and non-cancer / healthy patients using EXO-PEG-TR. Samples included ascites (n=12), cancer peritoneal lavage (n=65), and non-cancer / healthy peritoneal lavage (n=25) samples. The data shown here is a representative example based on 8 healthy peritoneal lavage, 1 ascites, and 4 cancer peritoneal lavage samples. NTA showed the most prevalent bin size distribution of 150nm in healthy peritoneal lavage samples with concentration of 1.4x106 particles / ml. On the other hand, the ascites and cancer peritoneal lavage samples exosome NTA represented the most prevalent bin size distribution of 50nm size in lavage with concentration of 3.3 x!06 particles / ml respective bins. These findings show the feasibility7of isolation of exosome subpopulations fromReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 biofluids and differences in subpopulations based on the clinical conditions (healthy vs. cancer).

[0024] FIG. 11 shows exosome RNA concentration in small volume (plasma) and large volume (peritoneal lavage) samples by EXO-PEG-TR and commercial-EXO-Precipitation reagent (Comm-Exo-ppt-Reag). RNA was extracted from the isolated exosomes using EXO-PEG-TR isolation protocol from small volume plasma (n=25) and large volume cancer peritoneal lavage (n=65). A representative set of samples show the differences in RNA concentration from exosomes isolated by EXO-PEG-TR protocol as compared to the commercial precipitation reagent. RNA concentrations were measured using nanodrop. The EXO-PEG-TR exosome RNA yield was 3S. I 2ng / pl and 29.4ng / pl for plasma and peritoneal lavage samples which was significantly higher as compared to 11.77ng / yl and 17.14ng / pl from exosomes isolated using commercial exosome precipitation agent. These measurements help in assessing the quantity of RNA extracted from the small volume plasma and large volume fluids, which is crucial for downstream qPCR and differential gene expression (DEG) applications.

[0025] FIG. 12 further shows exosomes RNA concentration and integrity isolated from small volume (plasma) and large volume (peritoneal lavage) biofluid samples using nanodrop.

[0026] FIG. 13 shows transmission electron microscopy images of exosomes isolated from small volume (plasma) and large volume (peritoneal lavage) samples by EXO-PEG-TR and commercial-exosome precipitation reagent. Transmission electron microscope images (scale bar of lOOnm) of exosomes isolated from peritoneal lavage and plasma samples using EXO-PEG-TR demonstrate intact exosomes with minimal to no residual contaminants. Also shown are exosomes isolated by commercial exosome precipitation reagent with residual contaminants.

[0027] FIG. 14 shows comparative analysis by cryo-electron microscopy of exosome structural integrity in large volume (peritoneal lavage) samples by EXO-PEG-TR vs Ultracentrifugation. Cryo-EM images of exosomes isolated from lavage samples showing clear intact, round shape exosomes by using EXO-PEG-TR are shown. In contrast, exosomes isolated by ultracentrifuge demonstrate membrane damage with leakage of intravesicular contents.

[0028] FIG. 1 shows cryo-electron microscopy of exosomes in healthy and cancer large volume (peritoneal lavage) samples isolated by EXO-PEG-TR. Cryo-EM images of exosomes isolated from healthy and cancer lavage samples show clear intact, round shape exosomes isolated using EXO-PEG-TR.

[0029] FIG. 16 show s transmission electron microscopy of exosomes in healthy and cancer small volume (plasma) samples isolated by EXO-PEG-TR. TEM images of exosomes isolated from healthy and cancer plasma samples show- clear intact, round shape exosomes by usingReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025EXO-PEG-TR.

[0030] FIG. 17A-17B show data relevant to plasma exosome parent gene analysis. FIG. 17A shows plasma sEVs parent gene expression analysis using conventional qPCR analysis from small volume plasma samples. FIG. 17B further shows plasma exosomes parent gene expression using two-step qPCR analysis by EXO-PEG-TR.

[0031] FIG. 18 shows nCounter® PanCancer Progression Panel differential gene signature expression analysis for sEVs isolated with EXO-PEG-TR from large volume peritoneal lavage samples.

[0032] FIGs. 19A-19D show exosome protein expression patterns isolated by EXO-PEG-TR. The Bruker biotyper MALDI-TOF mass spectrometry system was used for identifying exosome protein patterns by Exo-PEG-TR. The mass spectral fingerprints are analyzed to find unique exosome protein patterns for clinical applications. The compared patterns are used to find the protein spectral mass databases for unique identification of exosome-based protein finger printing patterns. FIG.19A shows MALDI-TOF protein standards (lkDa-7kDa). FIG.19B shows small volume (plasma) results. FIG. 19C shows large volume (peritoneal lavage) samples by EXO-PEG-TR, showing the unique exosome protein patterns with different molecular weights of the exosome protein patterns. FIG. 19D shows western blot protein analysis for exosome-specific protein TSG101 (~45kDA) isolated from large volume and small volume (urine, plasma, peritoneal lavage and ascites) samples are shown to validate the presence of exosomes precipitated by EXO-PEG-TR.

[0033] FIG. 20A-20K show optimization of filtration and precipitation steps for using the present invention with human biofluids. FIG. 20A shows a filter comparison, more specifically a nanoparticle tracking analysis (NTA) of average size of EVs isolated by EXO-PEG-TR in large volume fluids (peritoneal lavage / ascites, n=10) using 0.22pm (nylon) and 0.45pm (PVDF) membrane filters, with significant differences in concentration precipitated for EVs < 200nm and 200 - 500nm (p=0.0089 and p=0.0161, respectively). FIG. 20B shows an NTA of the EVs isolated from peritoneal lavage (n=10) after step-1 precipitation, showing a peak concentration of particles measuring 197nm with an average size of 171.53 ± 2.8nm. FIG. 20C shows transmission electron microscopy images of EVs precipitated from peritoneal lavage following step-1 precipitation. FIG. 20D shows an NTA of the EVs isolated from urine (n=10) after step-1 precipitation, showing a peak concentration of particles measuring 191nm with an average size of 183.70 ± 6.7nm. FIG. 20E shows transmission electron microscopy images of EVs precipitated from urine samples following step-1 precipitation. FIG. 20F shows BCA protein assay results,Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 demonstrating there is a significantly higher amount of residual proteins in EXO-PEG-TR step-1 than step-2 in plasma samples (n=7) (p<0.0001). FIG. 20G shows an NTA of the EVs isolated from plasma (n=7) after step-1 precipitation, showing peak concentration of particles measuring 208nm with average size of 215.8 ± 2.3nm. FIG. 20H shows transmission electron microscopy images of EVs precipitated from plasma samples following step-1 precipitation. FIG. 201 shows an NTA of the EVs isolated from plasma (n=7) after step-2 precipitation, showing peak concentration of particles measuring 154nm with average size of 173.9 ± 2.0nm. FIG. 20J shows transmission electron microscopy images of EVs precipitated from plasma samples following step-2 precipitation. FIG. 20K shows the collective bin size distribution of EVs < 200nm and ranging between 200-500nm for step-1 and step-2 EXO-PEG-TR isolation, demonstrating significant precipitation of EVs <200nm in second precipitation of plasma (n=7) (p<0.0001). Note that, when comparing FIG. 20C, 20E, 20H, and 20J, the transmission electron microscopy images of EVs precipitated from peritoneal lavage, urine and plasma (FIG. 20C) peritoneal lavage and (FIG. 20E) urine samples (FIG. 20H and FIG. 20 J) following step-1 and step-2 precipitation, it is evident that there is less protein residual contaminant after second precipitation in plasma samples (lOOnm scale bar).

[0034] FIG. 21A-21L show sEVs pellets isolated by EXO-PEG-TR and characterization in a separate set of samples to show validation and reproducibility. FIG. 21 A shows white pellets, located at the bottom of conical tubes, precipitated from large volume (peritoneal lavage) human biofluid samples after application of EXO-PEG-TR. FIG. 21B shows Nanoparticle tracking analysis (NTA) assessing the peak particle concentrations observed were 81.5nm for large volume peritoneal lavage samples with average particle size 165.7 ± 17.3nm (n=3). FIG. 21C shows precipitation with EXO-PEG-TR results in significantly more EVs measuring < 200nm than EVs measuring 200 - 500nm from large volume human biofluids (peritoneal lavage) (n=3, pO.OOOl). FIG. 21D, FIG 21H, and FIG 21L shows cryo-electron microscopy images of EVs isolated from large and small volume human biofluids. The membrane structure of the EVs are maintained showing the ability of EXO-PEG-TR protocol to isolate EVs with high structural integrity. The scale bars in the electron microscope images represent lOOnm, providing a reference for the size of the EVs. FIG. 21E shows white pellets, located at the bottom of conical tubes, precipitated from large volume (urine) human biofluid samples after application of EXO-PEG-TR. FIG. 21F shows NTA assessing the peak particle concentrations observed were HOnm for large volume urine samples with average particle size 165.1 ± 3.2nm (n=3). FIG. 21G shows precipitation with EXO-PEG-TR results in significantly more EVs measuring < 200nmReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 than EVs measuring 200 - 500nm from large volume human biofluids (urine) (n=3, p<0.0001). FIG. 21H shows cryo-electron microscopy images of EVs isolated from large and small volume human biofluids. The membrane structure of the EVs are maintained showing the ability of EXO-PEG-TR protocol to isolate EVs with high structural integrity. The scale bars in the electron microscope images represent lOOnm, providing a reference for the size of the EVs. FIG. 211 shows white pellets, located at the bottom of conical tubes, precipitated from small volume (plasma) human biofluid samples after application of EXO-PEG-TR. FIG. 21J shows NTA assessing the peak particle concentrations observed w ere 152nm for large volume urine samples with average particle size 143.76 ± 1.9nm (n=3). FIG. 21K shows precipitation with EXO-PEG-TR results in significantly more EVs measuring < 200nm than EVs measuring 200 - 500nm from small volume human biofluids (plasma) (n=3, p<0.0001). FIG. 21L shows cryo-electron microscopy images of EVs isolated from plasma. The membrane structure of the EVs are maintained showing the ability of EXO-PEG-TR protocol to isolate EVs with high structural integrity. The scale bars in the electron microscope images represent lOOnm, providing a reference for the size of the EVs.

[0035] FIG. 22A-22R shows a comparison of EXO-PEG-TR with ultracentrifugation and commercial precipitation reagent FIG. 22A and FIG. 23C show' nanoparticle tracking analysis (NTA) comparing EV isolation via EXO-PEG-TR precipitating agent (FIG. 22A) and differential ultracentrifugation (dUC) (FIG. 22C) of large volume fluid (peritoneal lavage) with enrichment of smaller EVs with EXO-PEG-TR (average size of 85.0 ± 1.3nm) (FIG. 22A) than dUC (average size 183.9 ± 14.2nm) (FIG. 22C). Higher concentrations of EVs were isolated utilizing EXO-PEG-TR (FIG. 22A) than dUC (FIG. 22C) by ten-fold. FIG. 22B shows cry o-electron microscope images showing intact membrane(s) of sEVs isolated from peritoneal lavage fluid by EXO-PEG-TR in black arrows. FIG. 22D shows, in red arrows, membrane damage of sEVs isolated from peritoneal lavage fluid using dUC. FIGs. 22E, 22F, 22H, and 221 show NTA peak(s) of EVs in large volume (peritoneal lavage fluid; FIG. 22E and FIG. 22F) and small (plasma; FIG. 22H and FIG. 221) isolated by EXO-PEG-TR (FIG. 22E and 22H) and commercial precipitation reagent (FIG. 22F and FIG. 221). FIG. 22G and FIG. 22J show collective bin size analysis for EXO-PEG-TR vs commercial precipitation reagent, demonstrating significantly higher amount of small EV (< 200nm) enrichment (p<0.0001) in plasma samples (n=5) and larger EV (200 - 500nm) in peritoneal lavage samples (n=5, p<0.0001). FIG. 22K, 22L, 220, and 22P show transmission electron microscopy images of sEVs isolated from large volume (peritoneal lavage; FIG. 22K and FIG. 22L) and small volume plasma samples (FIG. 220 andReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025FIG. 22P) by EXO-PEG-TR in comparison to commercial precipitation reagents. FIG. 22L, showing large volume (peritoneal) and FIG. 22P, showing small volume (plasma) data, show more residual contaminants along with EVs. FIGs. 22M and FIG. 22Q show BCA protein assay(s), showing significantly less residual protein in EXO-PEG-TR than commercial precipitation reagent in peritoneal lavage (n=5) and plasma (n=5) human biofluid samples (p<0.0001 and p<0.0001, respectively). FIG. 22N and FIG. 22R show the sEVs RNA concentration in small (plasma) and large volume (peritoneal lavage) samples by EXO-PEG-TR and commercial precipitation reagent. RNA concentrations were significantly higher in the EXO-PEG-TR for both large volume and small human biofluids compared to the commercial precipitation reagent (p=0.0005 and p=0.031, respectively).

[0036] FIG. 23A-23D show information regarding peritoneal lavage samples stored at -80°C for 1 year, using both EXO-PEG-TR (FIG. 23A and FIG. 23B) and commercial precipitation reagent (FIG. 23C and FIG. 23D). FIG. 23A and FIG. 23C show the size distribution of EVs determined by NTA for large volume peritoneal lavage human biofluids. FIG. 23C and FIG. 23D show the cryo-electron microscopy images of EVs to comparison of recovery of EVs stored at -80°C for 1 year isolated by EXO-PEG-TR compared to commercial precipitation reagent in which there was membrane degradation and lower concentration with more residual proteins of EVs. The scale bars for all the electron microscopy images represent 50-100nm.DETAILED DESCRIPTION OF THE INVENTION

[0037] Referring now to FIGs. 1A-23D, the present invention features a diagnostic reagent comprising: polyethylene glycol; trehalose; and water; wherein the diagnostic reagent is useful for isolating extracellular vesicles from biological fluids.

[0038] In some embodiments, the present invention features a method of producing a diagnostic reagent comprising: mixing polyethylene glycol, trehalose, and water together to create a pre-diagnostic reagent mixture; exposing the pre-diagnostic reagent mixture to ultrasonication to create an ultrasonicated pre-diagnostic reagent mixture; exposing the ultrasonicated pre-diagnostic reagent mixture to centrifugation to create a centrifuged ultrasonicated pre-diagnostic reagent mixture; and filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture to create a diagnostic reagent; wherein the diagnostic reagent is useful for isolating extracellular vesicles / exosomes from biological fluids.

[0039] In some embodiments, the present invention features a method of isolating extracellularReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 vesicles from biological fluids comprising: obtaining a biological fluid sample; exposing the biological fluid sample to a diagnostic reagent comprising polyethylene glycol, trehalose, and water; and incubating the biological fluid sample with the diagnostic reagent for a period of time; wherein incubating the biological fluid sample with the diagnostic reagent results in isolation of extracellular vesicles from the biological fluid sample.

[0040] In some embodiments, the polyethylene glycol is an 8 kilodalton polyethylene glycol. In some embodiments, the polyethylene glycol is anywhere from about 4,000 daltons to about 9,500 daltons. In some embodiments, the polyethylene glycol is anywhere from about 400 daltons to about 60,000 daltons. In some embodiments, the polyethylene glycol is a 6-12 kilodalton polyethylene glycol. In some embodiments, the polyethylene glycol is less than 6 kilodaltons. In some embodiments, the polyethylene glycol is greater than 12 kilodaltons.

[0041] In some embodiments, the trehalose is trehalose dihydrate.

[0042] In some embodiments, the water is purified water. In some embodiments, the water is molecular water.

[0043] In some embodiments, the ratio of trehalose to polyethylene glycol to water is about 0.4 to 25 to 50. In some embodiments, the ratio of trehalose to polyethylene glycol to water is about 0.2 to 15 to 25. In some embodiments, the ratio of trehalose to polyethylene glycol to water is about 0.6 to 30 to 75. In some embodiments, the ratio of trehalose to polyethylene glycol to water is about in the range of 0.2-0.6 to 15-30 to 25-75. In some embodiments, the ratio of trehalose to polyethylene glycol to water is about 0.8 to 25 to 50. In some embodiments, the ratio of trehalose to polyethylene glycol to water is about 0.2-1.0 to 10-100 to 20-250.

[0044] In some embodiments, the pre-diagnostic reagent mixture is exposed to ultrasonication for about 1 hour. In some embodiments, the pre-diagnostic reagent mixture is exposed to ultrasonication for less than 1 hour. In some embodiments, the pre-diagnostic reagent mixture is exposed to ultrasonication for more than 1 hour.

[0045] In some embodiments, the ultrasonicated pre-di agnostic reagent mixture is exposed to centrifugation for about 20 mins at about 5000 g at about 4 degrees C. In some embodiments, the centrifugation occurs for less than about 20 minutes. In some embodiments, the centrifugation occurs for more than about 20 minutes. In some embodiments, the pre-diagnostic reagent mixture is ultrasonicated, wherein the pre-diagnostic reagent mixture is ultrasonicated for between 15Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 minutes and 4 hours. In some embodiments, the centrifugation occurs at less than about 5000 g. In some embodiments, the centrifugation occurs at more than about 5000 g. In some embodiments, the centrifugation occurs at less than about 4 degrees C. In some embodiments, the centrifugation occurs at more than about 4 degrees C. In some embodiments, the centrifugation occurs for about 10 minutes to 30 minutes. In some embodiments, the centrifugation occurs with a force of about 2500 g to about 7500 g. In some embodiments, the centrifugation occurs with a force of about 2500 g to about 7500 g. In some embodiments, the centrifugation occurs at about 20 degrees C. In some embodiments, the sonicated pre-diagnostic reagent mixture is centrifuged for between about 5 minutes to about 120 minutes at about 1500 g to about 10.000 g at about 4 degrees C to about 25 °C.

[0046] In some embodiments, filtering the centrifuged sonicated pre-diagnostic reagent mixture comprises filtering the centrifuged sonicated pre-diagnostic reagent mixture using an about 0.22 micron filter to an about 0.45 micron filter. In some embodiments, filtering the centrifuged ultrasonicated pre-di agnostic reagent mixture comprises filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture using a 0.45 micron filter. In some embodiments, filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture comprises filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture using a filter less than 0.45 micron. In some embodiments, filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture comprises filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture using a filter greater than 0.45 micron. In some embodiments, filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture comprises filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture using 0.22 micron filter. In some embodiments, filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture comprises filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture using a filter less than 0.22 micron. In some embodiments, filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture comprises filtering the centrifuged ultrasonicated pre-diagnostic reagent mixture using a filter greater than 0.22 micron.

[0047] EXAMPLE 1

[0048] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0049] Synthesis of Exo-PEG- TR Reagent: 25 g of PEG-P2139 (8kDa, Sigma) was added to the 50mL, Ambion (AM9937) Nuclease Free water with Trehalose dihydrate T0167 (405mg) andReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 ultrasonicated with full waves for Ih to obtain a homogeneous aqueous solution. The solution was centrifuged at 5000g for 20 min at +4°C and the supernatant was filtered through a Stericup (S2GPU05RE) 0.45pm filter, which produced the Exo-PEG-TR (50%, g mL-1, similarly hereinafter). This newly-developed reagent was used for the following exosome isolation protocols. The biologic fluid of interest was subject to initial centrifugation to remove any cellular components. The supernatant was then incubated at +4°C with the appropriate ratio of Exo-PEG-TR (50ml / 12ml Exo-PEG-TR). The duration of incubation may be as short as 30 minutes to as long as 12 h based on the objective of the analysis and ty pe of biofluid. After incubation for the appropriate amount of time, the samples were centrifuged at 3000g for 10 mins. The supernatant was discarded and the exosome pellet was collected for further analysis (two-step Exo-PEG-TR based precipitation). The precipitated exosomes appeared as a white pellet at the bottom of the tubes. For further enhancing the purity of isolated exosomes and to deplete any residual proteins in the exosome pellet, an additional wash step may be incorporated. For this step, the exosome pellet was washed with 10ml D-PBS IX without disturbing the pellets and suspended in 9 ml PBS. Then, the Exo-PEG-TR was added once again in the samples to a final PEG concentration of 10% with 9ml D-PBS and 1ml Exo-PEG-TR. After incubation at +4°C for 2 h, the samples were centrifuged again at 3000g for 10 min to obtain the purified exosomes pellets. The supernatant was discarded completely, and the exosomes pellets were harvested for downstream biological analysis by Two-step based precipitation Exo-PEG-TR methods.

[0050] EXAMPLE 2

[0051] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0052] Comparison of performance of Exo-PEG-TR with commercial exosome precipitation reagent (Exoquick). To assess the performance of Exo-PEG-TR, a series of experiments were conducted, focused on the purity, quantity and RNA content of isolated exosomes. The performance of Exo-PEG-TR w as compared with Exoquick, one of the commercially available precipitation reagents used for exosome isolation for research purposes. Exosome isolation from various biologic fluids (peritoneal lavage, ascites, plasma, serum) was performed by subjecting each sample to exosome isolation with both Exoquick and Exo-PEG-TR. The isolated exosomes were evaluated using Nanoparticle tracking analysis (NTA) to assess the size and quantity. RNAReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 was isolated using standard RNA isolation techniques and the quantity of RNA was assessed using Nanodrop. Finally, the appearance and size of exosomes were evaluated using transmission electron microscopy. The experiments were repeated at least three times and the aggregate of the results are presented below. First, NTA showed the size of the most prevalent EVs isolated by Exo-PEG-TR isolation protocol was 94 nm compared to 124 nm with Exoquick. The relevance of this finding is rooted in the evidence that smaller exosomes have more functional significance in disease states, particularly in cancer. The average size of EVs was also statistically significantly smaller in the Exo-PEG-TR group compared to the Exoquick group. Second, the amount of EVs isolated (parti cles / ml) was also higher with Exo-PEG-TR compared to Exoquick, indicating high efficiency of Exo-PEG-TR. Third, the RNA content of the exosomes was also significantly higher with Exo-PEG-TR both in peritoneal lavage 28.05ng / l) vs. 17.09 ng / 1) and plasma (38.1 ng / l)vs. 11.77 ng / 1) compared to Exoquick. The high-yield of RNA from highly pure exosomes may have a major impact in downstream transcriptomic analysis. Finally, transmission electron microscopy of exosomes clearly demonstrates the membrane bound vesicles and lack of contamination with Exo-PEG-TR compared to Exoquick.

[0053] EXAMPLE 3

[0054] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0055] Downstream transcriptomic Analysis of Exosomes isolated by EXO-PEG-TR: RNA was isolated from exosomes isolated by EXO-PEG-TR from cancer peritoneal lavage / ascites (n=10) and healthy lavage fluid (n=10). Gene expression analysis was performed using Nanostring Pan cancer progression panel (770 genes). The RNA quantity was adequate for successful gene expression analysis in all 20 samples. There was statistically significant differential gene expression between cancer and healthy lavage samples indicating that the isolation technique with EXO-PEG-TR is suitable for dow nstream "omics" analysis.

[0056] EXAMPLE 4

[0057] Efficient Isolation of Small Extracellular Vesicles from Human Biofluids Using Novel Precipitation Agent EXO-PEG-TR: Despite the recognition of the critical role of small extracellular vesicles (sEVs), in various diseases, the clinical application of sEV-basedReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 diagnostics and therapeutics is dampened by the lack of standardized and clinically scalable isolation techniques from a wide spectrum of human biofluids. To overcome this critical barrier, a new sEVs isolation reagent, EXO-PEG-TR, was designed, developed, and optimized. EXO-PEG-TR allows for highly efficient isolation of sEVs, with high purity and integrity; from both small- and large-volume human biofluids. EXO-PEG-TR based isolation resulted in significantly higher concentration of sEVs with less protein contamination than routinely used ultracentrifugation and commercial precipitation agents. Additionally, EXO-PEG-TR based-sEV isolation and long term storage at -80°C resulted in recovery7of significantly higher number of sEV with intact membranes even after a year of storage, outperforming commonly used methods. Collectively, EXO-PEG-TR provides a clinically scalable, standardized sEV isolation method for both large and small volume human biofluids and has the potential to improve research and clinical applications of sEV.

[0058] Extracellular vesicles (EV) are lipid bilayer-bound vesicles that are produced by all cells and range in size from nano- to micrometers. EV contain rich biologic cargo and play a critical role in various pathophysiologic processes. Currently, EV are categorized based on their size and origin. EV that are smaller than 200nm are commonly referred to as small EV (sEV) as per the Minimum Information for Studies of Extracellular Vesicles (MISEV2023) guidelines. sEV include exosomes, a distinct group of EV with endogenous origin from the internal compartments of the cell and released into the extracellular compartment by the fusion of multivesicular bodies with the cell membrane. sEV can be found in various biological fluids, including plasma, serum, ascites, peritoneal lavage, CSF. saliva, amniotic fluid, and urine. sEV, particularly exosomes, provide insights into disease mechanisms, enable early diagnosis, and even sen e as vehicles for targeted therapy. However, a significant hurdle in harnessing sEV for clinical applications is the lack of standardized, efficient, and scalable isolation techniques.

[0059] Differential ultracentrifugation (dUC) is considered gold standard for isolation of sEV however, it is not feasible to use this approach for small volume human biofluids (ex. plasma, serum, saliva, urine, CSF, etc.) due to the amount of fluid required for dUC. Due to this limitation, researchers often pool human biofluids to isolate sEV by dUC. This is not an ideal method for isolation of sEV for the purposes of precise clinical diagnostics. Furthermore, dUC is cumbersome, time-consuming and is not scalable for clinical applications. While sEV isolation based on precipitation techniques using commercially available isolation kits are straightforward, they are fraught with concerns about purity, reliability, and cost. Other methods of isolation suchReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 as sucrose gradient cushion, size exclusion chromatography, and immuno- or affinity precipitation each have their limitations, ranging from low yield, labor and time-intensive, lack of purity, and prohibitive costs. There is a critical need in the field of EVs for isolation techniques that are efficient, reliable, reproducible, and clinically scalable to isolate sEV from both small and large volume human biofluids.

[0060] An EV isolation reagent, EXO-PEG-TR, was developed, along with an associated protocol for isolation of EV from human biofluids that enriches for sEV. EXO-PEG-TR comprises polyethylene glycol and trehalose. Polyethylene glycol is a precipitation agent used to separate nanoparticles from liquids. Trehalose is a non-toxic, natural disaccharide that is commonly used in food and cosmetic industries as a stabilizer and texturizer. Trehalose may be used as a cryo preservative for vaccines, protein drugs, liposomes, and cells as it stabilizes protein and cell membranes and prevents intracellular ice formation during freezing. Trehalose also reduces EV aggregation and loss when used for storage at -80°C. The properties of PEG and trehalose in EV isolation and storage have been leveraged by combining these reagents to develop a novel EV precipitation agent, EXO-PEG-TR.

[0061] A highly efficient isolation of sEV with high purity and integrity using EXO-PEG-TR is demonstrated herein, as compared to ultracentrifugation and commercial precipitation agent. The isolated sEV populations were validated using nanoparticle tracking, cryo- and transmission-electron microscopy, western blotting, gene signature analysis, qPCR and MALDI-TOF mass spectrometry analysis. Additionally. EXO-PEG-TR is ideal for storage of sEV at -80°C for longer periods, ensuring high integnty and maximal yield without aggregation. The proposed sEV isolation method with EXO-PEG-TR is scalable for both small and large volume biofluid samples and is easily adaptable for any downstream applications in sEV-based precision diagnostics.

[0062] Synthesis of EXO-PEG-TR: EXO-PEG-TR was developed by combining Polyethylene Glycol (PEG) 8 kDA and Trehalose, a natural disaccharide.

[0063] The composition and synthesis of EXO-PEG-TR are shown in FIG. 1A and FIG. IB, respectively. Briefly, PEG-P2139 (25gm, 8kDa, Sigma) and Trehalose dihydrate (405 mg T0167) are combined with 50mL (W4502) of nuclease free water, ultrasonicated with full waves for 30-90 minutes and then centrifuged at 5000g for 20 minutes at 4°C to achieve a homogeneous aqueous solution. The supernatant is filtered through a Stericup® (S2GPU05RE) 0.45pm filter toReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 obtain the EXO-PEG-TR precipitation reagent (FIG. IB). The reagent can be stored at room temperature for 3 months or at 4°C for up to 6 months.

[0064] Optimization and Development of EXO-PEG-TR sEV Isolation Protocol: EXO-PEG-TR-based EV isolation protocol was created after extensive study and optimization of EV isolation from 225 patient samples that included both large volume (peritoneal lavage n=130, urine n=25, and ascites n=20) and small volume (plasma n=50) biofluid samples. The optimization of various steps are described as below.

[0065] Filtration of Human Biofluids

[0066] The majority of previously published EV isolation protocols are based on cell culture media and utilize a 0.22 pm nylon filter for the initial filtration step. However, frequent clogging issues were encountered when human biofluids were filtered through the 0.22pm nylon filter, as it was more viscous and contained larger amounts of protein than cell culture media. Hence, a 0.45pm polyvinylidene fluoride (PVDF) filter was selected instead. The time required to filter the human biofluids through the 0.45pm PVDF filter was reduced by half compared to the 0.22pm nylon filter (5 minutes vs. 10 minutes) and the filter clogging issue was completely eliminated. To assess if change in filter pore size affected the enrichment for sEV, the EV quantity and bin size distribution was compared between 0.22pm nylon and 0.45pm PVDF filters. In both filter groups, the most abundant EV were sEV (< 200) and there was significantly more sEV in the ,45pm PVDF filter compared to the 0.22pm nylon filter (p=0.0089) (FIG. 20A). Based on the above observations and validations, the 0.45pm PVDF filter was selected as the filter for isolation and enrichment of sEV from human biofluids.

[0067] Precipitation steps for sEV isolation from large and small volume fluids: Next, to optimize the EXO-PEG-TR based isolation protocol for enrichment of sEV with minimal residual contaminants, various human biologic fluids were tested, such as peritoneal lavage, urine, ascites, and plasma, as they are inherently different in their physical properties and composition. Typical EV isolation by precipitation technique consists of a single precipitation step in which the biofluid is incubated with the precipitation reagent for 2-f2h and later centrifuged to retrieve the EV pellet.

[0068] A single precipitation step was utilized, and the quantity and purity evaluated of EV isolated from peritoneal lavage, urine and plasma by NTA and TEM. The single precipitationReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 step resulted in enrichment of sEV in peritoneal lavage (n=10) and urine (n=10) samples, with peak concentration particle size of 197nm and 191nm and average EV size of 171.5 ± 2.8nm and 183.7 ± 6.7nm respectively (FIG. 20B, FIG. 20D). On TEM imaging, there were very minimal residual contaminants associated with the EV isolated from peritoneal lavage and urine (FIG. 20C, FIG. 20E). On the other hand, single precipitation of plasma showed high residual protein contamination on TEM. Since plasma contains large amounts of protein compared to fluids such as peritoneal lavage, and urine, a single precipitation step may not be enough to adequately remove all the protein contaminants. Hence, a second precipitation step was performed in which the EV pellet obtained by first precipitation from plasma was resuspended with EXO-PEG-TR and centrifuged after an incubation period to obtain the second EV pellet. The second precipitation resulted in minimal protein residue. To objectively demonstrate the difference between single and double precipitation steps, NTA and TEM analysis of the EV pellet was conducted after single and double precipitation steps from the same plasma samples (n=7). BCA protein assay was performed, which demonstrated that the amount of protein residue was significantly lower with the second precipitation step compared to the single precipitation step (p<0.0001) (FIG. 20F).

[0069] Then, to comparatively visualize the difference in residual contaminants, TEM images were performed after 1stand 2ndstep precipitation (FIG. 20H, FIG. 20J) and there were minimal residual contaminants with the 2ndstep precipitation. NTA of the EVs isolated from 1ststep (FIG. 20G) and 2ndstep (FIG. 201) precipitation showed peak concentration particles size of 208nm and 154nm with average size of 215.8 ± 2.3nm and 173.9 ± 2.0nm, respectively. Furthermore, when assessing the bin size distribution of the nanoparticles, categorized as <200nm vs. 200-500nm, it was observed that the proportion and quantity of sEV in plasma (n=7) significantly increased after the second precipitation step (pO.OOOl, FIG. 20K). Therefore, as a result of the low amount of residual protein and high enrichment of sEV after a second precipitation, it was determined a second precipitation step may be ideal for plasma.

[0070] EXO-PEG-TR - EVs Isolation Protocol for Large and Small Volume Human Biofluids: Based on these observations, a standardized, clinically scalable sEV isolation protocol is provided for both large and small volume human biofluids (FIG. 3, FIG. 4). As depicted in FIG. 4, the isolation protocol for large-volume samples (40 mL) of peritoneal lavage and urine requires filtration followed by a single precipitation step. In contrast, the isolation protocol for small-volume biofluids such as plasma (500 pL) involves dilution with D-PBS in a 1:9 ratio,Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 followed by initial filtration and subsequent two step precipitation with EXO-PEG-TR (FIG. 3). The EXO-PEG-TR-to-sample ratio is optionally kept constant at 1 :4 during precipitation steps regardless of the type of biofluid. The duration of centrifugation is also minimal, in this case, 15 minutes at 3000g between the filtration and precipitation steps.

[0071] EV Isolation and Characterization based on EXO-PEG-TR Isolation protocol: Utilizing the above described EXO-PEG-TR-EV isolation protocol, EV were isolated from large and small volume biofluids to demonstrate reproducibility.

[0072] To optimize the EXO-PEG-TR based isolation protocol for enrichment of sEV with minimal residual contaminants, the EV pellet(s) was / were analyzed by NTA and TEM from peritoneal lavage, urine and plasma. Representative sample results are shown in FIG. 21. The isolation method resulted in a white EV pellet from a spectrum of large and small volume fluid samples as shown in Fig. 21 A, FIG. 21E, FIG. 211). EV characterization by NTA showed that the peak concentration particle size and average EV sizes in peritoneal lavage, urine, and plasma were 81.5nm, HOnm, and 152nm, and 165.7 ± 17.3nm, 165.1 ± 3.2nm, and 143.8 ± 1.9nm respectively (FIG. 21B, FIG. 21F, FIG. 21J) . Bin size distribution of EV showed that in all three sample types the predominant population of EV were <200 nm indicating enrichment for sEV (p<0.0001, Fig. 21C, FIG. 21G, FIG. 21K). Cryo-EM images of the isolated EVs also showed intact membrane-bounded vesicles (FIG. 2 ID, FIG. 21H, FIG. 211).

[0073] Comparison of EXO-PEG-TR with Ultracentrifugation and a Commercial Precipitation Reagent: Next, the efficiency and effectiveness of EV isolation with EXO-PEG-TR was compared to the commonly used isolation techniques, namely dUC and precipitation using a commercially available precipitation reagent. sEVs were isolated from large (peritoneal lavage, ascites, and urine) and small (plasma) volume biofluids, subjecting each sample to isolation using EXO-PEG-TR and a commercially available sEVs precipitation reagent or dUC.

[0074] EXO-PEG-TR vs. Ultracentrifugation: First, EXO-PEG-TR demonstrated high yield of sEVs as compared to ultracentrifugation which had low yield of sEVs even after 16h of UC (FIG. 22A, FIG. 22C). Cryo-EM images of sEVs isolated from lavage samples showed intact membrane EVs (black arrows) with EXO-PEG-TR (FIG. 22B). In contrast, sEVs isolated by ultracentrifugation demonstrated membrane damage with leakage of sEV contents (red arrows) (FIG. 22D).Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025

[0075] EXO-PEG-TR vs. Commercial Precipitation Reagent: NTA analysis of EVs isolated from peritoneal lavage and plasma samples isolated by EXO-PEG-TR (FIG. 22E, FIG. 22H) and commercial precipitation reagent (FIG. 22F, FIG. 221) showed that EXO-PEG-TR yielded a higher quantity of EVs, and these were predominantly within the 10-200nm range, as compared to the commercial precipitation reagent (FIG. 22G, FIG. 22J). TEM images of sEV isolated using EXO-PEG-TR from peritoneal lavage (FIG. 22K) and plasma (FIG. 220) samples revealed intact sEV with minimal to no residual contaminants. This is in contrast to sEVs isolated by the commercial precipitation reagent, which showed abundant residual contaminants (FIG. 221, FIG. 22P). This was quantitatively confirmed by protein assay which showed significantly higher concentrations of residual protein in the EV isolated with the commercial precipitation reagent (p<0.0001) compared to EXO-PEG-TR, for the same quantity of EV (FIG. 22M, FIG. 22Q). Furthermore, the RNA concentrations of sEVs isolated by EXO-PEG-TR, both from the large and small volume biofluids, were also significantly higher than from the EVs isolated using the commercial precipitation agent (peritoneal lavage 422.2ng vs. 217.6ng(p=0.031) and plasma 318.9ngvs. 176.2ng(p=0.0005) (FIG. 22N, FIG. 22R).

[0076] Recovery of sEVs Stored at -80°C: Given that one of the important needs in the field of EV research is to freeze, store and recover isolated EV for future downstream analysis, a storage experiment was conducted in which sEV isolated by EXO-PEG-TR and commercial precipitation reagent were retrieved after one year of storage at -80°C, and the quality and quantity of recovered sEV were compared. NTA demonstrated that there was a higher concentration of sEV retrieved from the EXO-PEG-TR group compared to the commercial precipitation reagent quantitatively in large volume (peritoneal lavage) (FIG. 23B). Furthermore, for EV isolated by commercial precipitation reagent, there was evidence of membrane degradation (FIG. 23D). In contrast, the EV isolated with EXO-PEG-TR showed intact membranes indicating that the integrity of the EV is maintained even after prolonged storage for a year at -80°C .

[0077] EXO-PEG-TR sEVs Downstream Applications: To investigate if sEV isolated by EXO-PEG-TR is suitable for downstream genomics, transcriptomic, and protein expression analysis gene expression, RT PCR, western blot, and mass spectrometry' experiments were conducted.

[0078] sEVs RNA Gene Expression by EXO-PEG-TR: Gene expression analysis was performed in peritoneal lavage samples (n=8) using Nanostring nCounter® PanCancer Progression Panel (XT PGX HuVI CancerProg CSO XT-CSO-PROG1-12) which showed highly differentiallyReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 expressed genes across all cancer peritoneal lavage samples (FIG. 18). The EV-derived gene of interest expression in plasma samples (n=4) is shown as the relative quantity of expressed (0.6-1) RNA fold change (FIG. 17A).

[0079] EXO-PEG-TR sEV Protein Expression: Western blot was performed on large (peritoneal lavage, urine, ascites) and small volume (plasma) biofluid EV.TSG101 was found to be prominently expressed in both small and large volume samples, indicating that the isolation technique using EXO-PEG-TR enriched for exosomes. (FIG. 19D). The MALDI-MS EV protein patterns from previously published studies were used as a reference standard for comparing the sEV protein patterns of small and large volume samples. The protein patterns displayed a broad range of molecular weights (lkDa-7kDa) (FIG. 19A), indicating that a diverse range of proteins were present in the isolated sEV. The sEV surface finger printing protein patterns in and large (peritoneal lavage) small (plasma) (FIG. 19E, FIG. 19F) volume samples show consistent sEV surface protein patterns.

[0080] Discussion: The growing importance of sEV in a wide range of diseases has made them attractive targets for next-generation liquid biopsy diagnostics and therapeutics. Hence, developing isolation methods that provide high-efficiency and clinically scalable isolation of sEV with integrity and purity are necessary to advance the field. A novel EV precipitation reagent, EXO-PEG-TR, has been developed, that is simple to prepare and extremely practical to isolate sEV from a broad-range of small and large-volume human biofluids, and that has the potential to fulfill this need.

[0081] The creation of EXO-PEG-TR leverages the applications of polyethylene glycol and trehalose to develop an isolation reagent that offers an easy and efficient technique to separate high quality, high purity sEV from small and large volume human biofluids with minimal time and effort. The performance of EXO-PEG-TR is superior to dUC and a commercially available EV isolation kit in terms of EV yield, quality, and integrity. There are important methodological innovations and protocol modifications that have led to the superior results seen with EXO-PEG-TR. First, EXO-PEG-TR contains trehalose in addition to PEG, which is a unique difference from the commercially available isolation kits. Although PEG is capable of causing precipitation of nanoparticles including EV from liquids, there are concerns about EV aggregation and co-precipitation of protein aggregates. Addition of trehalose provides a distinct advantage as it reduces EV aggregation and results in a better yield of sEVs. In addition, trehalose stabilizes EV membranes resulting in higher membrane integrity of isolated EVs, andReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 this integrity is maintained even after prolonged storage. On the other hand, dUC, a commonly used isolation technique for EV research, led to damaged and leaky EVs as the ultra-high speed used in dUC leads to extreme mechanical forces resulting in membrane damage. This may result in compromised reproducibility. As demonstrated in this study, EXO-PEG-TR overcomes all of the disadvantages of using dUC, including low yield; the inability to use individual small human samples; damaged EV; and the time and labor required. This makes EXO-PEG-TR an attractive new EV isolation method for research. EXO-PEG-TR also outperformed commercial precipitation agents, yielding a significantly higher concentration of sEV, with minimal residual contaminants. Furthermore, it has also been demonstrated that the storage of EV isolated using EXO-PEG-TR leads to better recovery of EVs after freezing compared to other isolation and storage methods. This is an important advantage as effective storage and retrieval of EV are necessary' for generating reproducible results in the clinical research setting. EXO-PEG-TR based isolation of EV is suitable for a range of downstream analyses, including genomics, transcriptomics, and proteomics, which positions it as a valuable tool for both research and clinical applications, particularly in the development of predictive and diagnostic assays.

[0082] Indeed, the isolation of sEVs is a critical step in leveraging their potential for diagnostic and therapeutic applications. EXO-PEG-TR offers a promising solution to streamline the sEVs isolation process, reducing time and labor while enhancing purity and yield. EXO-PEG-TR is effective for EV isolation across various biofluids, including blood, plasma, urine, ascites and peritoneal lavage, making it versatile for a broad range of clinical and research applications. The reagent's ability to preserve the quality and quantity of sEVs under different storage conditions is particularly beneficial for longitudinal studies and biobanking. EXO-PEG-TR is also more cost-effective than existing commercial reagents and could eliminate the financial barriers for EV research. The benefits of EXO-PEG-TR are summarized below.

[0083] Conclusion: EXO-PEG-TR precipitation provides an efficient, standardized and clinically scalable protocol for sEV isolation with high purity and integrity from a wide range of human biofluids, and is superior to commonly used isolation methods. EXO-PEG-TR based isolation of EVs is also superior for long term frozen storage of EVs. EXO-PEG-TR represents a significant advancement in sEV isolation technology. By addressing the limitations of current methods, it provides a scalable, efficient, and cost-effective solution that can enhance the research and the clinical applications of sEV.Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025

[0084] Methods: Preparation of EXO-PEG-TR EVs Precipitation Reagent: Mix PEG-P2139 (25 g 8kDa), Trehalose dihydrate (405 mg) with 50ml - Nuclease Free water. Ultrasonicate the solution with full waves for 30-90 minutes to achieve a homogeneous aqueous solution. Centrifuge the solution at 5000g for 20 minutes at +4°C and filter the supernatant through a Stericup - 0.45pm filter to obtain the EXO-PEG-TR precipitation reagent.

[0085] EV Characterization: EVs were isolated from small (plasma) and large volume (peritoneal lavage, ascites, and urine) biofluids samples using EXO-PEG-TR following a detailed protocol, reported here. The pellet was then resuspended in 1ml of 2mM Trehalose in 0.45pm filtered PBS for further dilutions and other downstream analysis.

[0086] Nanoparticle Tracking Analysis (NTA): The NanoSight NS300 (Malvern, Inc., Malvern, UK) was used to validate the isolation of sEVs for size and concentration, using the following instrument settings: NanoSight NTA 3.2 software, camera levels 12-14, detection threshold 5, 60-second capture time, 5 captures, and 20 flow rates. For precise size analysis, vesicles were diluted 1:75 Dilution in filtered Nano pure water to obtain the best concentrations of homogenous particle distribution. After 1-2 cycles of 1-2 seconds of sonication in a sonicating water bath, the sample was then loaded into the NanoSight. NanoSight measures the nanoparticles’ Brownian motion utilizing a laser to measure the particle’s velocity over time and, therefore, the particle size and concentration.

[0087] Cryo-Electron Microscope (cryo-EM): Initially, 200 mesh EM grids with lacey carbon were glow discharged to enhance hydrophilicity. For the preparation of the grid, 3pL of purified EVs solution was applied to both grids. Next, the grid was blotted and quickly plunged into the precooled liquid ethane. The frozen samples were then clipped under liquid nitrogen, placed into an autoloader cassette, and subsequently transferred to a Thermo Fisher Scientific Glacios microscope for imaging. The specimen grid was examined under low-dose conditions using EPU software (Thermo Fisher Scientific) to reduce electron radiation damage to the specimen by restricting exposure to 20 electrons / 2. Images were captured using a Ceta camera (Thermo Fisher Scientific). The area of each exosome was measured from these high-resolution images using ImageJ software (National Institutes of Health) to measure the diameter of the EVs. The magnification of the microscope was 57,000x with a pixel size of 0.255 nm angstrom at specimen space

[0038] .Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025

[0088] Negative- Stain Transmission Electron Microscope (TEM): Highly purified sEVs preparations (lOpL) were first fixed using an equal volume of 4% paraformaldehyde in PBS at room temperature (RT) for 30 minutes. The fixed samples were subsequently transferred onto a glow-discharged carbon-coated 200-mesh copper grid and incubated at RT for 10 minutes. After the samples adhered to the grid, they were quickly and softly dabbed with filter paper, then immersed in 10 pL of 2% uranyl acetate at RT for 5 minutes, followed by drying on filter paper. The morphology of the sEVs on carbon films was examined using a JEOL JEM-21 OOF microscope (JEOL USA), and the images were recorded with a Gatan OneView CCD camera using digital micrograph software. The captured images were then analyzed by imageJ software (National Institutes of Health) to determine the length of the sEVs.

[0089] Ultracentrifugation (UC): Biofluid samples were centrifuged at 3,000 x g for 15 minutes to separate floating cells and then filtered through a 0.45pm PVDF Stericup® Millipore® filter to eliminate cell debris. Then, supernatants were diluted with ice-cold PBS to a final volume of 90 ml and transferred to ultracentrifuge tubes (#345776, Beckman Coulter, Brea, CA, USA) with a titanium spacer (38 mm; #342697, Beckman Coulter, Brea, CA, USA) to perform the ultracentrifugation round at 100,000 x g (4°C) for 16h using a Type-45 Ti fixed-angle titanium rotor (#342697 Beckman Coulter, Brea, CA, USA).

[0090] RNA Isolation: Total RNA, including small RNAs (17-200 nucleotides), was isolated from lavage and plasma samples using the Direct-zol™ RNA Miniprep kit following the modified TRIzol reagent method. Agilent Bioanalyzer High Sensitivity RNA Analysis and Small RNA Analysis were utilized to assess the quantity and quality of the isolated RNA from the EVs. The quality of the RNAs were calculated using the RNA Integrity Numbers (RIN) and samples were included in downstream analysis, such as quantitative PCR (qPCR) and NanoString, if they were within the range of 1-2.

[0091] sEVs Parent Gene Amplification by Real-Time Quantitative PCR (qPCR): Reverse transcription was performed using the first strand cDNA synthesis kit (iScript™ cDNA Synthesis Kit, (1708890), Bio-Rad, USA). The RT reaction was performed using treated total RNA and the RT primer TSLP or USLP. The amplification of the target parent gene is amplified using the iTaq™ Universal SYBR® Green One-Step Kit, (1725150) as per the Bio-Rad manufacturer’s instructions. The intron spanning assays was performed with reaction composition including iTaq-lOul, iScript-RT-0.25pL, forward primer (FP)-lUl(lOpm), reverse primer (RP)-lul-(lO pm), RNA 50ng, and molecular grade H2O-variable to a total reaction mix of 25pL. sEVs parentReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 gene specific primers were used to avoid any false amplifications and the real time qPCR was performed using the CFX-1000 at 50°C for lOminutes, 95.0°C for 15 minutes initially and 95°C for 15 seconds and 60°C for 45 seconds for 35 cycles and melt cure was obtained at 55°C to 95°C with 5 seconds increments per plate read.

[0092] NanoString Gene Expression Analysis: The standard protocol for Nano String’s nCounter PanCancer Progression Panel (Hamburg, Germany) was performed using 120ng of isolated EV’s RNA from the human biofluid samples using EXO-PEG-TR method. Then reads are then independently verified for sample integrity, checked using the violin plots interquartile range (IQR). The data are typically analyzed using the housekeeping (HSK) genes as the reference across all plates using Rosalind nCounter platform. The raw data was exported for raw genes counts from the digital analyzer. Then differential gene expression analysis was computed using fold change for each GOI namely collagen type III alpha 1 chain (COL3A1), osteoglycin (OGN), complement regulatory protein (CD46), mitogen-activated protein kinase kinase kinase 7 (MAP3K7), von Hippel-Lindau tumor suppressor (VHL), AKT serine / threonine kinase 2 (AKT2), fibroblast grow th factor receptor 4 (FGFR4), serpin family H member 1 (SERPINH1) which have been identified in exosomes in various studies.

[0093] Western Blot Analysis for EVs Isolated by EXO-PEG-TR: EV Sample Preparation and SDS-PAGE: EV protein lysates from plasma, peritoneal lavage, ascites, and urine were isolated using EXO-PEG-TR. Samples were thawed on ice and centrifuged at 3000g for 10 minutes. Protein concentrations were determined by the BCA assay. Samples were lysed with RIPA buffer containing protease and phosphatase inhibitors (1:4 ratio). Loading dye was added (1:4 ratio). 25 pL samples were heated at 75°C for 10 minutes and separated on 10% SDS-PAGE gels. Tris-glycine buffer was used at 120V for 15 minutes, then 75V for 60-90 minutes. Fisher Bioreagents molecular weight markers (7 pL EZ-RUN, 180-10 kDa) provided size reference.

[0094] Protein Transfer and Immunoblotting: Transfer sandwiches were assembled with PVDF membranes equilibrated in methanol (20 seconds), water (1-2 minutes), and Tris-glycine transfer buffer with methanol (2-3 minutes), with wet transfer performed with IX Tris glycine transfer buffer at 25V overnight at 4°C. Membranes were stained with Ponceau S to verify complete transfer, washed in IX phosphate-buffered saline with Tween 20 detergent (PBST), blocked with 5% protease free bovine serum albumin (BSA) for 3-6 hours at 4°C, and incubated with TSG-101 primary7antibody (1: 1000 in 5% BSA-PBST) overnight at 4°C with gentle agitation. Following 5 minutes PBST washes, membranes were incubated with species-appropriateReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 secondary horseradish peroxidase (HRP) conjugated streptavidin antibodies (1: 1000 in 5% BSA / PBST) for 1-3 hours at room temperature, washed three times with PBST, and developed using Ultra TMB substrate for 15-25 minutes. Membranes were photographed and stored at 4°C, with reprobing performed using stripping buffer (20 minutes at room temperature or 37°C for 10-15 minutes) to assess additional EV markers and evaluate EXO-PEG-TR isolation efficiency across small and large volume samples.

[0095] Bruker Biotyper MALDI-TOF Mass Spectrometry for sEVs Protein Pattern Finder: The Bruker Biotyper® MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time-of-Flight) mass spectrometry system is utilized for sEVs protein isolation using EXO-PEG-TR method. The isolated sEVs are lysed to release their protein content. Protein extracts are then mixed with a suitable matrix for MALDI-TOF analysis and allowed to cry stallize. The plate is then introduced into the MALDI-TOF instrument. The resulting mass spectrum represents a fingerprint to find exosome protein patterns. Software provided with the Biotyper® can be used to compare the spectrum against known databases for protein identification or to generate exosome protein patterns.

[0096] Statistical Analysis: NTA peak size was calculated as mean ± SEM. and EV concentrations were measured across all small (plasma) and large (peritoneal lavage, ascites and urine) volume samples; the concentrations were reported as standardized E5 particles / ml. Particle size distribution was analyzed in two groups for collective bin size distributions: <200 nm and 200-500 nm. Protein quantification, including EV native proteins and residual protein contaminants, was performed using BCA assay and reported as pg / ml concentration. Statistical comparisons and data were analyzed using a two-way7repeated measures analysis of variance (ANOVA) to examine the effects of EXO-PEG vs EXO-PEG-TR on small (plasma) and large (peritoneal lavage and urine) volume human biofluids across 5 different runs for EV bin and concentration measurements. This analytical approach was selected to account for the within-subjects design where each subject was measured under multiple conditions, thereby controlling for individual differences and increasing statistical power. Normality7of the samples was assessed and found present. EXO-PEG-TR was treated as a fixed effect and sphericity was corrected with the Geisser-Greenhouse correction. Statistical significance was set at a = 0.05 for all tests. Results are reported as p = p-value. All analyses were performed using GraphPad Prism 10.4.1 (GraphPad Software, San Diego, CA, USA). Methods adhered to MISEV2023 guidelinesReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 for extracellular vesicle research reporting standards for NTA and EV bin distribution <200 and 200-500nm analysis performed.

[0097] EMBODIMENTS

[0098] The following are non-limiting embodiments of the present invention. It is to be understood that said embodiments are not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0099] Embodiment 1 : A diagnostic reagent for isolating extracellular vesicles from biological fluids, the diagnostic reagent comprising: polyethylene glycol; trehalose; and water.

[0100] Embodiment 2: The diagnostic reagent of embodiment 1, wherein the polyethylene glycol is an 8 kilodalton polyethylene glycol.

[0101] Embodiment 3: The diagnostic reagent of embodiment 1, wherein the polyethylene glycol is a 6-12 kilodalton polyethylene glycol.

[0102] Embodiment 4: The diagnostic reagent of any one of embodiments 1-3, wherein the trehalose is trehalose dihydrate.

[0103] Embodiment 5: The diagnostic reagent of any one of embodiments 1-4, wherein the water is purified water.

[0104] Embodiment 6: The diagnostic reagent of any one of embodiments 1-4. wherein the water is molecular water.

[0105] Embodiment 7: The diagnostic reagent of any one of embodiments 1-6, wherein the ratio of trehalose to polyethylene glycol to water is about 0.4 to 25 to 50.

[0106] Embodiment 8: The diagnostic reagent of any one of embodiments 1-6, wherein the ratio of trehalose to polyethylene glycol to water is about 0.8 to 25 to 50.

[0107] Embodiment 9: The diagnostic reagent of any one of embodiments 1-6, wherein the ratio of trehalose to polyethylene glycol to water is about 0.2-1.0 to 10-100 to 20-250.

[0108] Embodiment 10: A method of producing a diagnostic reagent for isolating extracellular vesicles from biological fluids, the method comprising: mixing polyethylene glycol,Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 trehalose, and water together to create a pre-diagnostic reagent mixture; sonicating the pre-diagnostic reagent mixture to create an sonicated pre-diagnostic reagent mixture; centrifuging the sonicated pre-diagnostic reagent mixture to create a centrifuged sonicated pre-diagnostic reagent mixture; and filtering the centrifuged sonicated pre-diagnostic reagent mixture to create a diagnostic reagent.

[0109] Embodiment 11: A method of isolating extracellular vesicles from biological fluids comprising: obtaining a biological fluid sample; exposing the biological fluid sample to a diagnostic reagent comprising polyethylene glycol, trehalose, and water; and incubating the biological fluid sample with the diagnostic reagent for a period of time; wherein incubating the biological fluid sample with the diagnostic reagent results in isolation of extracellular vesicles from the biological fluid sample.

[0110] Embodiment 12: A method of isolating and storing extracellular vesicles from biological fluids comprising: obtaining a biological fluid sample; exposing the biological fluid sample to a diagnostic reagent comprising polyethylene glycol, trehalose, and water; incubating the biological fluid sample with the diagnostic reagent for a period of time; wherein incubating the biological fluid sample with the diagnostic reagent results in isolation of extracellular vesicles from the biological fluid sample; and storing the isolated extracellular vesicles at a temperature at or below about zero degrees Celsius.

[0111] Embodiment 13: The method of any one of embodiments 10-12, wherein the polyethylene glycol is an 8 kilodalton polyethylene glycol.

[0112] Embodiment 14: The method of any one of embodiments 10-12, wherein the polyethylene glycol is an 6-12 kilodalton polyethylene glycol.

[0113] Embodiment 15: The method of any one of embodiments 10-14, wherein the trehalose is trehalose dihydrate.

[0114] Embodiment 16: The method of any one of embodiments 10-15, wherein the water is purified water.

[0115] Embodiment 17: The method of any one of embodiments 10-15, wherein the water is molecular water.Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025

[0116] Embodiment 18: The method of any one of embodiments 10-17, wherein the ratio of trehalose to polyethylene glycol to water is about 0.4 to 25 to 50.

[0117] Embodiment 19: The method of any one of embodiments 10-17, wherein the ratio of trehalose to polyethylene glycol to water is about 0.8 to 25 to 50.

[0118] Embodiment 20: The method of any one of embodiments 10-17, wherein the ratio of trehalose to polyethylene glycol to water is about 0.2- 1.0 to 10-100 to 20-250.

[0119] Embodiment 21 : The method of any one of embodiments 1-20, wherein the pre-diagnostic reagent mixture is ultrasonicated, wherein the pre-diagnostic reagent mixture is ultrasonicated for about 1 hour.

[0120] Embodiment 22: The method of any one of embodiments 1-20, wherein the pre-diagnostic reagent mixture is ultrasonicated, wherein the pre-diagnostic reagent mixture is ultrasonicated for between 15 minutes and 4 hours.

[0121] Embodiment 23: The method of any one of embodiments 1-22, wherein the sonicated pre-diagnostic reagent mixture is centrifuged for about 20 mins at about 5000 g at about 4 °C.

[0122] Embodiment 24: The method of any one of embodiments 1-22, wherein the sonicated pre-diagnostic reagent mixture is centrifuged for between about 5 minutes to about 120 minutes at about 1500 g to about 10,000 g at about 4 degrees C to about 25 °C.

[0123] Embodiment 25: The method of any one of embodiment 1-24, wherein filtering the centrifuged sonicated pre-diagnostic reagent mixture comprises filtering the centrifuged sonicated pre-diagnostic reagent mixture using a .45 micron filter.

[0124] Embodiment 26: The method of any one of embodiments 1-24, wherein filtering the centrifuged sonicated pre-diagnostic reagent mixture comprises filtering the centrifuged sonicated pre-diagnostic reagent mixture using an about 0.22 micron filter to an about 0.45 micron filter.

[0125] As used herein, the term ‘’about” refers to plus or minus 10% of the referenced number.

[0126] Although there has been shown and described the preferred embodiment of theReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase ‘'comprising” includes embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

Claims

Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025WHAT IS CLAIMED IS:

1. A diagnostic reagent for isolating extracellular vesicles from biological fluids, the diagnostic reagent comprising: a) polyethylene glycol; b) trehalose; and c) water.

2. The diagnostic reagent of claim 1, wherein the polyethylene glycol is an 8 kilodalton polyethylene glycol.

3. The diagnostic reagent of claim 1, wherein the polyethylene glycol is an 6-12 kilodalton polyethylene glycol.

4. The diagnostic reagent of claim 1, wherein the trehalose is trehalose dihydrate.

5. The diagnostic reagent of claim 1, wherein the water is purified water.

6. The diagnostic reagent of claim 1, wherein the water is molecular water.

7. The diagnostic reagent of claim 1, wherein the ratio of trehalose to polyethylene glycol to water is about 0.4 to 25 to 50.

8. The diagnostic reagent of claim I , wherein the ratio of trehalose to polyethylene glycol to water is about 0.8 to 25 to 50.

9. The diagnostic reagent of claim 1, wherein the ratio of trehalose to polyethylene glycol to water is about 0.2-1.0 to 10-100 to 20-250.

10. A method of producing a diagnostic reagent for isolating extracellular vesicles from biological fluids, the method comprising: a) mixing polyethylene glycol, trehalose, and water together to create a pre-diagnostic reagent mixture; b) sonicating the pre-diagnostic reagent mixture to create an sonicated pre-diagnostic reagent mixture; c) centrifuging the sonicated pre-diagnostic reagent mixture to create a centrifuged sonicated pre-diagnostic reagent mixture; andReference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 2025 d) filtering the centrifuged sonicated pre-diagnostic reagent mixture to create a diagnostic reagent.

11. A method of isolating extracellular vesicles from biological fluids comprising: a) obtaining a biological fluid sample; b) exposing the biological fluid sample to a diagnostic reagent comprising polyethylene glycol, trehalose, and water; and c) incubating the biological fluid sample with the diagnostic reagent for a period of time; wherein incubating the biological fluid sample with the diagnostic reagent results in isolation of extracellular vesicles from the biological fluid sample.

12. A method of isolating and storing extracellular vesicles from biological fluids comprising: a) obtaining a biological fluid sample; b) exposing the biological fluid sample to a diagnostic reagent comprising polyethylene glycol, trehalose, and water; c) incubating the biological fluid sample with the diagnostic reagent for a period of time; wherein incubating the biological fluid sample with the diagnostic reagent results in isolation of extracellular vesicles from the biological fluid sample; and d) storing the isolated extracellular vesicles at a temperature at or below about zero degrees Celsius.

13. The method of any one of claims 10-12, wherein the polyethylene glycol is an 8 kilodalton polyethylene glycol.

14. The method of any one of claims 10-12, wherein the polyethylene glycol is an 6-12 kilodalton polyethylene glycol.

15. The method of any one of claims 10-12, wherein the trehalose is trehalose dihydrate.

16. The method of any one of claims 10-12, wherein the water is purified water.

17. The method of any one of claims 10-12, wherein the water is molecular water.

18. The method of any one of claims 10-12, wherein the ratio of trehalose to polyethylene glycol to water is about 0.4 to 25 to 50.

19. The method of any one of claims 10-12. wherein the ratio of trehalose to polyethylene glycol to water is about 0.8 to 25 to 50.Reference No.: UCI 24.13 PCTInventor’s last name: Senthil & RadhakrishnanDocument Date: December 19, 202520. The method of any one of claims 10-12. wherein the ratio of trehalose to polyethylene glycol to water is about 0.2-1.0 to 10-100 to 20-250.

21. The method of claim 2, wherein the pre-diagnostic reagent mixture is ultrasonicated, wherein the pre-diagnostic reagent mixture is ultrasonicated for about 1 hour.

22. The method of claim 2, wherein the pre-diagnostic reagent mixture is ultrasonicated, wherein the pre-diagnostic reagent mixture is ultrasonicated for between 15 minutes and 4 hours.

23. The method of claim 2, wherein the sonicated pre-diagnostic reagent mixture is centrifuged for about 20 mins at about 5000 g at about 4 °C.

24. The method of claim 2, wherein the sonicated pre-diagnostic reagent mixture is centrifuged for between about 5 minutes to about 120 minutes at about 1500 g to about 10,000 g at about 4 degrees C to about 25 °C.

25. The method of claim 2, wherein filtering the centrifuged sonicated pre-diagnostic reagent mixture comprises filtering the centrifuged sonicated pre-diagnostic reagent mixture using a .45 micron filter.

26. The method of claim 2, wherein filtering the centrifuged sonicated pre-diagnostic reagent mixture comprises filtering the centrifuged sonicated pre-diagnostic reagent mixture using an about 0.22 micron filter to an about 0.45 micron filter.

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