Microgel bead compositions and methods for extracellular vesicle isolation

Abstract

The present disclosure describes compositions comprising microgel beads. The microgel beads comprise chitosan-grafted poly-L-lysine (PLL), are crosslinked, have a surface charge that is tunable based on the concentration of PLL, and are capable of capturing and releasing one or more extracellular vesicles (EVs) via surface charge interactions. Methods of preparing the compositions are also described. Additionally, methods of isolating EVs using the compositions are described.

Description

[0001] Attorney Docket No. 29539-0843WO1

[0002] MICROGEL BEAD COMPOSITIONS AND METHODS FOR EXTRACELLULAR

[0003] VESICLE ISOLATION

[0004] PRIORITY CLAIM

[0005] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 683,343, filed on August 15, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0006] FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under grant numbers U18- TR003793. and R01-CA226871 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] The present disclosure describes compositions comprising crosslinked microgel beads comprising chitosan-grafted poly-L-lysine (PLL) having a surface charge that is tunable based on the concentrations of both PLL and chitosan. The disclosure also describes methods of isolating extracellular vesicles (EVs) from a sample using these compositions, which are capable of capturing and releasing one or more EVs via surface charge interactions. The methods of preparing the compositions are also described herein.

[0010] BACKGROUND

[0011] EVs are membrane-bound particles that are released into the extracellular space by all cell types, including cancer cells.[1]The unique molecular cargoes in EVs such as nucleic acids, proteins, lipids and small molecules reflect the physiological and pathological state of their cell of origin.!21This ability’ makes EVs ideal models for biomarker discovery in disease states such as cancer131, neurological disorders141, and cardiovascular disease151. Furthermore, EVs are recognized for their potential as minimally invasive biomarkers in cancer diagnosis, prognostics, and therapeutic monitoring.

[0012] Liquid biopsies involving analysis of EVs derived from blood, urine, saliva, and cerebrospinal fluid have all shown potential to enable early detection and monitoring of disease progression161. Of particular interest, urine emerged as a promising biofluid for non- invasive early detection and monitoring of genitourinary cancers, including bladder[7], prostate181, kidneys191, and reproductive tract1101, due to its accessibility and the potential for Attorney Docket No. 29539-0843WO1

[0013] EV recovery originating from these tissues. However, urine presents considerable analytical challenges due to its heterogeneous nature and fluctuating physicochemical parameters, including pH, conductivity, and solute concentration.

[0011]

[0014] Despite evidence that EVs have potential for therapeutic, diagnostic and prognostic purposes, clinical translation has been limited due to lack of EV isolation techniques that are compatible with complex biofluids such as urine, blood and saliva[12’13]. Conventional methods such as centrifugation

[0014] . filtration1151, and size exclusion chromatography (SEC)

[0016] are widely accepted but are often labor intensive, non-specific, compromised in integrity and retain contaminants. Techniques leveraging miniaturized platforms, such as acoustic, inertial, viscoelastic and electrochemical methods, offer an alternative for the isolation of EVs[17’21]. Despite their potential, these methods are often associated with complicated operational structures that can compromise the purity and structural integrity of EVs, and their utility for different types of biofluids remains restricted. In addition, electrochemically driven platforms are reliant on antibody conjugation and the application of strong electric fields, which is challenging for clinical application.

[0015] SUMMARY

[0016] The clinical translation of EV -based prognostics is constrained by challenges associated with their isolation from complex biological matrices such as urine. These isolation challenges include yield, specificity, and the preservation of EV structural integrity and functional molecular cargo, while urine as a biofluid introduces inter-individual variables including pH and temporal changes in dynamic composition and conductivity.

[0017] Certain aspects of the present disclosure are directed to methods of isolating EVs from a sample, the method comprising contacting the sample comprising the EVs with a composition comprising microgel beads comprising chitosan-grafted poly-L-lysine (PLL), wherein the microgel beads capture the EVs via surface charge interactions, resulting in the formation of microgel beads-EVs complexes; and releasing the captured EVs from the microgel beads-EVs complexes, thereby isolating the EVs from the biological sample.

[0018] In some embodiments, the biological sample is contacted with the composition comprising microgel beads for at least about 1 hour (h). In some embodiments, the composition comprises microgel beads at a concentration of about 0.5 x 106beads per milliliter (beads / mL) to about 1 x 106beads / mL. In some embodiments, the method achieves about 1.3 to about 1.7-fold higher yields than gold standard size exclusion chromatography. In some embodiments, the biological sample is a complex biofluid. In some embodiments, Attorney Docket No. 29539-0843WO1 the complex biofluid is one or more of blood, urine, saliva, cerebrospinal fluid, uterine lavage fluid, ascitic fluid, or a tear or lacrimal fluid sample. In some embodiments, the biological sample is urine. In some embodiments, the biological sample is cell culture media. In some embodiments, the methods further comprise purifying the microgel beads-EVs complexes by washing via centrifugation. In some embodiments, the releasing comprises adding a salt to the microgel bead-EVs complexes. In some embodiments, the salt is sodium chloride. In some embodiments, the methods further comprise concentrating the released EVs via centrifugation. In some embodiments, the methods further comprise washing concentrated EVs to remove residual salt. In some embodiments, the EVs are urinary' EVs. In some embodiments, the microgel beads-EVs complexes comprise small EVs with a diameter of about 65 nm to about 200 nm and large EVs with a diameter of about 200 nm to about 750 nm, and very large EVs, with a diameter about 750 nm to about 10 pm.

[0019] Certain aspects of the present disclosure are directed to a composition comprising microgel beads, wherein the microgel beads comprise chitosan-grafted poly-L-lysine (PLL), wherein the microgel beads are crosslinked, and wherein the surface charge of the microgel beads is tunable based on the concentration of PLL. and wherein the microgel beads are capable of capturing and releasing one or more extracellular vesicles (EVs) via surface charge interactions.

[0020] In some embodiments, the chitosan-grafted PLL comprises PLL at a concentration of about 0.01% to about 0.05% (v / v). In some embodiments, the chitosan-grafted PLL comprises PLL at a concentration of about 0.05% (v / v). In some embodiments, the diameter of the microgel beads is about -^50 microns (pm) to about 100 pm. In some embodiments, the microgel beads exhi bi t a zeta potential ranging from about 30 millivolts (mV) to about 100 mV, measured at a pH between about 3.0 to about 9.0. In some embodiments, the composition is shelf-stable for up to about one year. In some embodiments, the EVs are urinary' EVs.

[0021] Certain aspects of the present disclosure are directed to a method of preparing a composition comprising microgel beads, the method comprising: providing a dispersed phase comprising chitosan-grafted poly-L-lysine (PLL) and a continuous phase comprising a nonaqueous fluid; introducing the dispersed and continuous phases into a channel of at least one microfluidic device comprising at least one junction; flowing the dispersed and continuous phases through the at least one junction thereby forming microdroplets: collecting the microdroplets in a crosslinker solution; and crosslinking the microdroplets by’ heating the Attorney Docket No. 29539-0843WO1 microdroplets and the crosslinker solution, thereby generating the composition comprising pgBs.

[0022] In some embodiments, the dispersed phase comprises chitosan-grafted PLL dissolved in acetic acid. In some embodiments, the dispersed phase comprises about 1% to about 2% (w / v) chitosan-grafted PLL. In some embodiments, the dispersed phase comprises about 1.5% (w / v) chitosan-grafted PLL. In some embodiments, the non-aqueous fluid comprises a fluorinated oil and one or more polymeric surfactants. In some embodiments, the one or more polymeric surfactants comprise perfluoropoly ether-poly ethylene glycol-perfluoropolyether triblock copolymer (PFPE-PEG-PFPE), PFPE 5k, PEG 900, PFPE 5k, or a combination thereof. In some embodiments, the one or more polymeric surfactants are present at a concentration of about 1.5% to about 2.5% (w / v). In some embodiments, the one or more polymeric surfactants are present at a concentration of about 2% (w / v).

[0023] In some embodiments, the dispersed and continuous phases are introduced under controlled pressure conditions. In some embodiments, the dispersed phase is introduced at a pressure of about 160 millibar (mbar) to about 240 mbar. In some embodiments, the continuous phase is introduced at a pressure of about 80 mbar to about 120 mbar. In some embodiments, the formation of the microdroplets comprises forming microdroplets at a rate of about 30,000 droplets per minute to about 70,000 droplets per minute. In some embodiments, the crosslinker solution comprises mono-aldehyde, di-aldehyde, or a combination thereof. In some embodiments, the crosslinker solution has a concentration of about 2.0% (v / v) to about 10% (v / v).

[0024] In some embodiments, the crosslinker solution has a concentration of about 5% (v / v). In some embodiments, the composition comprising microdroplets and the crosslinker solution are heated to a temperature ranging from about 60 °C to about 70 °C. In some embodiments, the composition comprising microdroplets and the crosslinker solution are heated for about 30 minutes (min.) to about 12 h. In some embodiments, the methods further comprise, after crosslinking, destabilizing the continuous phase encapsulating the microgel beads and separating the microgel beads from the continuous phase. In some embodiments, destabilizing the continuous phase comprises adding a demulsifier.

[0025] Certain aspects of the present disclosure are directed to a method for detecting at least one cancer biomarker in a biological sample from a subject, the method comprising: obtaining the biological sample comprising extracellular vesicles (EVs); isolating the EVs from the biological sample using any of the isolation methods described herein; and detecting the presence of the at least one cancer biomarker comprising one or more of a protein, nucleic Attorney Docket No. 29539-0843WO1 acid, lipid, or metabolite associated with cancer, wherein the detection of the cancer biomarker in the EVs is indicative of the presence or progression of cancer in the subject.

[0026] In some embodiments, the cancer is bladder cancer.

[0027] The terms ‘‘subject” or “patient” as used herein refer to any mammal (e.g., a human or a veterinary subject, e.g., a dog, cat, horse, cow, goat, sheep, mouse, rat, or rabbit) to which a composition or method of the present disclosure may be administered, e.g.. for experimental, diagnostic, prophylactic, and / or therapeutic purposes. The subject may seek or need treatment, require treatment, is receiving treatment, will receive treatment, or is under care by a trained professional for a particular disease or condition.

[0028] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a microgel bead” includes mixtures of microgel beads, reference to “a microgel bead” includes mixtures of two or more such microgel beads, and the like.

[0029] As used herein, the expression “non-pharmaceutical carrier” refers to a composition that comprises ingredients compatible with other components of the diagnostic formulation and physiologically acceptable to a subject (e.g., a mammal such as a human), without inducing excessive undesirable physiological effects or causing a deleterious impact. In the context of diagnostic applications, such carriers may be used to stabilize, transport, or facilitate the processing of biological materials, such as extracellular vesicles, nucleic acids, proteins, or metabolites, for analytical or detection purposes. A composition as described herein may include one or more carriers, excipients, and / or diluents that support the integrity, recovery, or detection of diagnostic targets without exerting therapeutic activity.

[0030] As used herein, the term “shelf-stable” refers to an EV-based composition or formulation that maintains its diagnostic performance, structural integrity, and suitability for use over a defined period of time when stored under specified conditions (e.g., refrigeration). In some embodiments, shelf-stability includes the preservation of extracellular vesicle morphology and cargo, as well as the prevention of vesicle aggregation due to surface charge interactions or other destabilizing factors. A shelf-stable EV formulation may retain its ability to support biomarker detection, vesicle recover}’, and assay reproducibility without requiring reconstitution, specialized handling, or immediate use following preparation.

[0031] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when Attorney Docket No. 29539-0843WO1 values are expressed as approximations, by use of the antecedent “about;’ it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Furthermore, the use of the term “about,” as used herein, refers to an amount that is near the stated amount by about 10%, 5%, or 1%, including increments therein. For example, “about” can mean a range including the particular value and ranging from 10% below that particular value and spanning to 10% above that particular value.

[0032] As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0033] Where values are described in the present disclosure in terms of ranges, endpoints are included. Furthermore, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0034] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur according to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, Attorney Docket No. 29539-0843WO1 including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0036] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0037] DESCRIPTION OF DRAWINGS

[0038] FIGs. 1A-1D. Aims and modular design of the study. (A) Illustration of charge- mediated capture of EVs via pgBs. (B) Droplet-assisted microfluidics for the formulation of pgBs. The droplets containing precursor solutions, chitosan and / or chitosan and poly-L- lysine as building blocks are formed in the droplet microfluidic device and collected in a crosslinker solution. Heating to 60 °C triggers the cross-linking reaction, resulting in three- dimensional pgBs with a mesh-like structure after demulsification. The scale bar is 100 pm. (C) Chemical structures of chitosan and poly-L-lysine as building blocks. (D) Workflow for the isolation of EVs from biofluids with pgBs. The pgBs are incubated with biofluids including CCM and / or a urine sample from a BC patient. The highly positively charged pgBs rapidly captured EVs, and the pH w as controlled to ensure that non-EVs remained suspended in the solution, which were outside the charge stability range. The pgBs-EVs complexes were washed three times by centrifugation to purify the complexes. The EVs were released from the pgBs by neutralizing the charge interaction. The released EVs are separated from the pgBs by centrifugation. The purified EVs were concentrated and subjected to downstream analysis. Some of the illustrations in panel (D) were created with BioRender® (www. biorender, com) .

[0039] FIGs. 2A-2E Mechanistic elucidation of cross-linking pathways and comprehensive characterization of pgBs. (A) CS cross-links with mono-aldehyde. The -NH2 groups of chitosan reacts with mono-aldehyde to form imines (-C=N), which react with a different - NH2 group of another chitosan chain to form a cross-linked mesh-like structure. (B) Proposed mechanism for chitosan grafted poly-L-lysine pgBs using mono-aldehyde. A heterogeneous assembly of netw ork structures is likely present, incorporating both chitosan- poly-L-lysine and chitosan-chitosan crosslinks, resulting in a matrix of mixed networks. (C) Bright field microscopic image of demulsified chitosan pgBs fabricated with 1.5 w / v% chitosan. The scale bar is 50 pm. (D) Size distribution of chitosan pgBs. (E) Effect of pH on Attorney Docket No. 29539-0843WO1 the ^-potential of chitosan pgBs. The data are expressed as the mean ± SD (n=3). P-values were assessed using two-way ANOVA with correction for multiple comparisons.

[0040] FIGs. 3A-3H. Effect of precursor composition, crosslinker concentration and curing time on the surface properties of pgBs. (A) The zeta potential of chitosan and chitosan supplemented with three different concentrations of poly-L-lysine (0.01, 0.025 and 0.05 v / v%) as precursor. (B) Illustration of the increasing surface charge of chitosan grafted poly- L-lysine iigBs with increasing poly-L-lysine concentration. (C) Measurements of ^-potential of pgBs fabricated with 1.5% w / v chitosan supplemented with three different concentrations of poly-L-lysine (0.01, 0.025 and 0.05 v / v%). (D) Bright field microscopy image of demulsified chitosan grafted poly-L-lysine pgBs fabricated with 1.5 % w / v chitosan and 0.05 v / v % poly-L-lysine concentration. The scale bar is 50 pm. (E) Size distribution of chitosan grafted poly-L-lysine pgBs. (F) Illustration of the influence of crosslinker concentration on - NEL group availability. As the concentration of the crosslinker increases, the number of functional -NH2 groups decreases. At lower crosslinker concentrations, a greater number of - ML remain on the surface and become protonated to -NIL in acidic buffer conditions, leading to a higher positive surface charge on pgBs. In contrast, at higher crosslinker concentrations (e.g., 10%), entire -NEE are consumed during crosslinking. (G) Illustration of the influence of crosslinker concentration and curing time on the surface charge of chitosan grafted poly-L-lysine pgBs. (H) Measurements of the ^-potential of chitosan grafted poly-L- lysine pgBs fabricated under four different crosslinker concentrations (2.5. 5.0, 7.5, and 10 v / v%) with three curing times (0.5, 2, and 12 hr). The ^-potential was measured in pgB buffer, adjusted to pH 6.0. The data are expressed as the mean± SD (n=3). P-values were assessed using two-way ANOVA with correction for multiple comparisons.

[0041] FIGs. 4A-4E. Surface charge profiling of chitosan grafted poly-L-lysine pgBs and EV post-capture (A) Implementation of two parallel microfluidic droplet generators reduced water-in-oil (W / O) droplet collection time by about 50%, while significantly enhancing the surface charge of chitosan grafted poly-L-lysine pgBs (p < 0.05). pgBs were synthesized using 5% (v / v) mono-aldehyde crosslinker with a 30 min curing at 60°C. (B) Chitosan grafted poly-L-lysine pgBs (formulated with 1.5% w / v chitosan and 0.05% v / v poly-L- lysine) exhibited pH-responsive modulation of surface charge, as reflected in zeta potential measurements. (C) EVs-derived from PT-PC3 cells demonstrated similar pH-dependent shifts in surface charge, suggesting ionic microenvironment sensitivity. (D) Schematic illustrating chitosan grafted poly-L-lysine pgBs before and after EV capture. (E) ^-potential analysis revealed that the initially positive surface charge of chitosan grafted poly-L-lysine Attorney Docket No. 29539-0843WO1 pgBs became negative upon EV binding, indicating efficient capture. Data are expressed as mean ± SD (n = 3). P-values were assessed using two-tailed Student’s t-test and one-way or two-way AN OVA with corrections for multiple comparisons, as appropriate.

[0042] FIGs. 5A-5H. Comprehensive comparison of pgBs and SEC for EV isolation and characterization. (A) A schematic overview comparing the two EV isolation techniques, pgBs and SEC (B) Scanning electron microscopy (SEM) images of CS-grafted-PLL pgBs, highlighting their surface morphology, including the characteristic porous structure. (C) SEM images of EVs captured by pgBs, with EVs digitally color-enhanced (purple) in postprocessing to improve visual clarity. (D) SEM imaging of EVs isolated via SEC, providing insights into their structural integrity and overall morphology. (E) SEM analysis of EVs postrelease from pgBs. confirming the preservation of vesicular architecture. SEM of pgBs further reveals a 25-40% reduction in size when subjected to vacuum conditions (2x IO mbar), likely due to dehydration. (F) NTA demonstrates a broader EV size distribution for pgBs isolation relative to SEC, suggesting a more diverse EV population with vary ing sizes. (G) A comparative evaluation of EV yields between pgBs and SEC, highlighting differences in the efficiency of isolation between the two techniques. (H) Immunoblotting reveals a higher expression of EV markers in pgB-isolated EVs compared to both cell lysates and SEC -isolated EVs, suggesting enhanced EV yield and enrichment. The data are expressed as the mean± SD (n=3). P-values were assessed using two-tailed Student's t-test. Some of the illustrations in panel (A) were created with BioRender® (www.biorender.com).

[0043] FIGs. 6A-6G. Isolation and downstream characterization of uEVs from BC patients. (A) Schematic representation of uEV isolation from urine samples of BC patients utilizing the pgB method, benchmarked against the SEC. (B) Overview of the BC patient cohort included in this study, presented from left to right. (C) NTA demonstrates the size distribution profiles of uEVs isolated via pgBs (red) versus SEC (blue). Quantitative assessment indicates a significantly higher yield of uEVs using the pgBs method (p < 0.05). (D) Immunoblotting of uEVs isolated from 1.2 mL of patient urine (Pt2). The pgBs-isolated uEVs exhibit enhanced signal intensities for canonical EV markers, substantiating improved enrichment efficiency and higher particle recovery relative to both bulk urine and SEC- derived EVs. (E) Representative TEM image showing morphology and integrity of pgBs- isolated uEVs from a BC patient sample (Pt2), highlighting the presence of large uEV (>200 nm). (F) TEM image of uEVs isolated from the same patient sample via SEC, serving as a comparative reference. (G) Quantitative analysis of transcript levels for four target genes in uEVs. pgBs-isolated vesicles exhibit markedly elevated expression levels, underscoring the Attorney Docket No. 29539-0843WO1 potential utility of this platform for prognostic biomarker discovery and treatment monitoring in BC. The data are expressed as the mean ± SD (n=3). P-values were assessed using two- way ANOVA with correction for multiple comparisons. Some of the illustrations in panel (A) are created with BioRender® (www.biorender.com).

[0044] FIGs. 7A-7D. (A) Mechanistic elucidation of cross-linking pathways and comprehensive characterization of chitosan (CS) pgBs crosslinked with di-aldehyde. The amino group of chitosan reacts with di-aldehyde and forms imines (-C=N). which are bridged together by the presence of two aldehyde groups and form a cross-linked mesh-like structure. (B) Bright-field image of demulsified chitosan pgBs fabricated with 1.5 w / v% chitosan. (C) Size distribution of chitosan pgBs. (D) Zeta potential measurements of chitosan pgBs in pgB buffer with pH adjusted to 6.0. The data are expressed as the mean ± SD (n=3). The scale bar is 50 pm.

[0045] FIGs. 8A-8B. Effect of pH on the size and morphology of chitosan (CS) and chitosan grafted poly-L-lysine (CS-grafted-PLL) pgBs fabricated by droplet-assisted microfluidics. (A) Chitosan pgBs were resuspend in different pH value for overnight at 4°C. Notably, at pH 12.0. a pronounced volumetric contraction of about 55% was observed relative to pgBs maintained at acidic pH (3.0 or 6.0), concomitant with structural degradation of the pgB architecture. (B) In contrast, chitosan grafted poly-L-lysine pgBs exhibited enhanced resistance to alkaline-induced dimensional changes. Upon exposure to pH 12.0, these pgBs exhibited a reduced shrinkage of about 32%, with degradation still evident. The extent of shrinkage in chitosan grafted poly-l pgBs was approximately 23% less than that observed in chitosan pgBs under comparable conditions, indicating improved structural stability conferred by poly-L-lysine grafting.

[0046] FIG. 9. Raman spectra of chitosan and chitosan grafted poly-L-lysine pgBs. The increased peak intensities and noticeable shifts in the amide bond signals (from 1649 to 1639 cm and 1553 to 1550 cm '). as well as in the methylene bridge (from 1375 to 1369 cm ' ). indicating successful grafting of poly-L-lysine onto the chitosan grafted poly-L-lysine pgBs.

[0047] FIG. 10. Optimization of crosslinker concentration and curing time for the fabrication of chitosan grafted poly-L-lysine pgBs. Four different crosslinker concentrations (2.5, 5.0. 7.5 and 10 %v / v) and three different time points (0.5, 2.0 and 12 hr) are investigated to determine the optimum crosslinking conditions of pgBs correspond to surface charge. The scale bar is 50 pm.

[0048] FIG. 11. Stability analysis of the zeta potential of chitosan grafted poly-L-lysine pgBs. The data are expressed as the mean ± SD (n=3). P-values were assessed using two-way Attorney Docket No. 29539-0843WO1

[0049] ANOVA with correction for multiple comparisons. Data points exhibiting statistically nonsignificant variations (p > 0.05) were excluded for graphical representation.

[0050] FIGs. 12A-12F. Isolation of EVs from CCM and optimization of pgB buffer pH for EV recovery and molecular characterization (A) Schematic representation of the EV isolation workflow from CCM using size SEC. (B) NTA of EVs isolated under varying pH conditions of the pgB buffer, with IxPBS (pH 7.4) serving as the control. EV concentrations are normalized to the maximum EV count within each experimental dataset. (C) Quantitative comparison of EV yield across different pgB buffer pH values, highlighting the efficiency of isolation. (D) RNA concentration extracted from EVs, normalized to the corresponding maximum EV count at each pH, as quantified by automated electrophoresis. (E) Relative RNA expression levels of three canonical genes (ACTB, GAPDH, and EGFR) within EVs, demonstrating pH-dependent variation in transcript abundance. (F) Immunoblotting of EV- specific protein markers with band intensities reflecting expression changes in response to the pH of buffer. Data are presented as mean or mean ± SD (n=2-3, as appropriate. P-values were assessed using one-way or two-way ANOVA with correction for multiple comparisons. Illustrations in panel (A) are created with BioRender (www.biorender.com).

[0051] FIGs. 13A-13C. Bright-field and epi-fluorescence microscopy imaging of pgBs and EV-captured chitosan grafted poly-L-lysine pgB. (A) Bright-field image showing the pgBs before interaction with EVs, confirming their uniform morphology (B) Epi-fluorescence image under the TxRed filter, demonstrating minimal to no intrinsic autofluorescence of the pgBs. (C) Epi-fluorescence image of pgBs after charge-mediated capture of PT-PC3-derived EVs, showing robust red fluorescence, indicating successful EV binding and confirming the specificity and efficiency of the pgB-EVs interaction. The Scale bar is 20 pm.

[0052] FIG. 14. Scanning electron microscope images (as originally acquired) of chitosan grafted poly-L-lysine pgBs captured EVs. EV binding events (denoted by white arrows). A high-magnification image (highlighted by the yellow box) reveals regions enriched with densely clustered EVs.

[0053] FIG. 15. EV capture efficiency by chitosan grafted poly-L-lysine pgBs. The EV capture efficiency of pgBs w as quantitatively assessed across three distinct EV input concentrations, while maintaining a constant concentration of pgBs. The captured (bound) and unbound EVs was quantified using NTA. A statistically significant disparity was observed between the captured and unbound EV populations, with the capture process achieving an average recovery efficiency of about 90%. The data are expressed as the mean ± Attorney Docket No. 29539-0843WO1

[0054] SD (n=3). P-values were assessed using two-way ANOVA with correction for multiple comparisons.

[0055] FIG. 16. Urine pH of patients enrolled in this study.

[0056] FIGs. 17A-17B. Representative images of pgBs-isolated EVs isolated from patient sample (Pt2). (A) Urinary7EVs (uEVs) isolated via chitosan grafted poly-L-lysine pgBs. (B) uEVs isolated using SEC. The few black and white arrows show the EVs and lipoproteins, respectively. The scale bar is 2 pm.

[0057] FIGs. 18A-18B. Validation of BC subtype-specific gene expression signatures in BC cell lines and Corresponding EVs. (A) Quantitative assessment of transcript levels for a minimal residual disease (MRD) marker (CK19), basal-like subtype markers (KRT14, EGFR), and a luminal subtype marker (PPARG) was performed in both parental cell lines and their corresponding EVs. (B) The relative RNA expression ratio of EGFR to PPARG, serving as a surrogate indicator of basal versus luminal subtype, elevated in basal-like HT- 1376 cells and their EVs in comparison to luminal-like UMUC-1, supporting subtype-specific molecular profiling in EV cargo.

[0058] DETAILED DESCRIPTION

[0059] The compositions described herein comprise crosslinked microgel beads that comprise chitosan-grafted poly-L-lysine (PLL) and have a surface charge that is tunable based on the concentration of PLL. The microgel beads are capable of capturing and releasing one or more extracellular vesicles (EVs) via surface charge interactions. Methods of isolating EVs using the compositions described herein are also described herein. Furthermore, methods of preparing these compositions are also provided herein.

[0060] Some embodiments of the compositions and methods described herein may provide one or more of the following advantages.

[0061] As discussed in the Background section, despite evidence that EVs have potential for therapeutic, diagnostic and prognostic purposes, clinical translation has been limited due to lack of EV isolation techniques that are compatible with complex biofluids such as urine, blood and saliva. One of the key challenges from isolating EVs from these complex biofluids is their variable pH, which impacts surface charge and complicates the development of a method that works across the full pH range. Nano- and / or microbead-based methods, including the use of chimeric nanocomposites such as lactoferrin-conjugated 2.2- bis(methylol)propionic acid dendrimer-modified magnetic nanoparticles and superabsorbent microbeads have been used to isolate EVs from body fluids[22'23]. Although these methods Attorney Docket No. 29539-0843WO1 are selective, they often suffer from long processing time, low yield and challenges with downstream functional assays.

[0062] Recently, charged polymers have emerged as a promising label-free approach to isolate EVs by leveraging the intrinsic surface charge properties of EVs

[0024] . Owing to the presence of negatively charged phospholipids1251such as phosphatidylserine on the outer leaflet of the membrane, EVs typically have a negative surface charge ranging from -15 to - 55 mV modulated by the surrounding ionic environment^6’271. Significant progress has been made using bulk positively-charged polymers such as chitosan (CS)l28J, poly-L-lysine (PLL)l291and chromatography in combination with ion-exchange[301to enable isolation of EVs by leveraging their surface charge properties. These methods rely on entrapping the EVs in long polymer chain and / -or resin, resulting in lower yield, inferior purity, and complicated sample processing.

[0063] For example, some methods isolate EVs by precipitating them in a chitosan solution, where the polymer entraps the vesicles within its chains and the resulting complexes are collected via centrifugation.

[0028] Although EVs can be released from the polymer network using high salt concentrations, chitosan also interacts with membrane proteins and lipids through hydrophobic and hydrogen bonding — interactions that are not easily disrupted by salt. Chitosan remains soluble at acidic pH (<6.2), but begins to form a gel -like structure at higher pH levels. When introduced into biofluids with pH values above 6.5, this gelation can occur directly on the EV surface, potentially compromising membrane integrity, reducing purity, and interfering with downstream molecular characterization. Additionally, chitosan’s surface charge diminishes significantly across a broad pH range due to its pH-dependent protonation. At near-neutral pH, its positive charge becomes minimal, weakening electrostatic interactions with EVs and thereby reducing isolation efficiency. In some embodiments, despite the progress made with bulk positively charged polymers like chitosan, these limitations may hinder their utility7in isolating EVs from complex biofluids.

[0064] In another example, other systems and methods are based on a uniform coating with PLL layers (e.g., PLL-coated magnetic beads) to achieve sufficient positive surface charge for the isolation of EVs.

[0029] However, it remains difficult to achieve uniform coating across the entire surface of individual and aggregated beads. In addition, EV release is promoted by high salt concentrations, which degrades the PLL coating. PLL residues on the EV membrane can mask or alter the native surface proteins and ultimately impair the biological integrity and functionality of the isolated EVs. Attorney Docket No. 29539-0843WO1

[0065] Overall, in some embodiments, both PLL-coated magnetic beads and bulk chitosan systems lack the tunable surface charge properties that microgel-based systems offer, limiting their adaptability and efficiency for isolating EVs from diverse clinical biofluids.

[0066] In addition, the retention of excess polymer when using bulk solutions remains questionable, as it can lead to a change in molecular signatures and surface properties. However, a critical but often overlooked factor in EV isolation is the pH of biofluids, which is particularly challenging due to the large pH variations in biofluids such as urine and saliva131-32J. EV membranes and molecular cargoes are highly sensitive to pH fluctuations. During isolation, exposure to extremely acidic or alkaline environments potentially compromises the integrity of the lipid bilayer, leading to vesicle degradation. In addition, EV-associated biomolecules such as proteins and RNA are highly susceptible to pH-induced denaturation or degradation, so their natural physiological pH must be maintained within a narrow, stable range. Both isolation with chitosan under centrifugation and isolation with PLL-coated magnetic beads result in charge changes that can destabilize EVs by perturbing their surface charge environment. Therefore, traditional and current approaches are constrained in their applicability to complex biofluid matrices.

[0067] To address these challenges, the disclosed hybrid microgel beads (pgBs) approach for the on-demand capture and release of EVs from biofluids, as described in FIGs. 1A and 2D, was developed by harnessing the intrinsic surface charge of EVs generated by their phospholipid bilayer membranes. The tunable surface charge of pgBs enabled highly efficient sequestration of negatively charged EVs and their release by neutralizing the charge interaction. Compared to conventional isolation techniques such as precipitation of charged polymers

[0028] , size exclusion chromatography (SEC)[3?], affinity-based

[0034] and microfluidics[17‘ 19, 35-36] >approachofferssubstantially higher EV yield, including small EVs (sEVs) having a diameter of less than about 200 nanometers (nm) and large EVs (lEVs) having a diameter of greater than about 200 nm[L 371, with superior purity and universal adaptability.

[0068] The methods of fabricating pgBs described herein provide a modular design strategy to fabricate pgBs, utilizing droplet-assisted microfluidics in conjunction with biocompatible and biodegradable polymers. Through chemical cross-linking, these polymers form stable gel structures with precisely tunable surface charges. In some embodiments, the dynamic pH range of pgBs facilitates the precise and efficient isolation of EVs from both cell culture- conditioned media (CCM) and urinary EVs (uEVs). In some embodiments, this highlights the superiority of the pgB method over conventional isolation techniques and provides a robust and comprehensive approach for the efficient isolation of small and large EVs. In addition, in Attorney Docket No. 29539-0843WO1 some embodiments, the methods of isolating EVs disclosed herein enable a significantly higher yield of gene expression in isolated EVs. enhancing its potential utility in precision oncology by supporting clinical decision making and promoting personalized therapeutic strategies.

[0069] In some embodiments, the microgel bead-based methods for the isolation of EVs disclosed herein result in an efficient isolation of EVs from biofluids. The methods and compositions of the disclosure exploit the native surface charge of EVs that arises from their phospholipid bilayer membrane to facilitate rapid, label -free, and high yield of EVs using charge-tunable beads. As described in the Examples, microgel beads effectively isolated EVs from cancer cell lines and clinically annotated bladder cancer urine samples, achieving 1.3- and 1.7-fold higher yields than gold standard size exclusion chromatography. Thus, as described in the Examples, the methods and compositions described herein enabled enhanced molecular profiling of the isolated EVs, with higher RNA cargo yields along with an increase in detection of specific bladder cancer transcripts.

[0070] Methods of Isolating EVs

[0071] The present disclosure features methods of isolating EVs from a sample using the microgel bead compositions described herein. The methods include contacting the sample comprising the EVs with a composition comprising microgel beads comprising chitosan- grafted PLL. The chitosan-grafted PLL microgel bead compositions are further described herein in the section entitled “Compositions.” In some embodiments, the microgel beads capture the EVs via surface charge interactions, resulting in the formation of microgel beads- EVs complexes. The methods further include releasing the captured EVs from the microgel beads-EVs complexes, thereby isolating the EVs from the sample.

[0072] In some embodiments, the sample is a complex biofluid. In some embodiments, the complex biofluid is one or more of blood, urine, saliva, cerebrospinal fluid, uterine lavage fluid, ascitic fluid, or tear or lacrimal fluid. In some embodiments, the sample is urine. In some embodiments, the sample is cell culture media. In some embodiments, the EVs isolated by the methods described herein are urinary EVs. In some embodiments, the urinary EVs comprise EVs derived from a bladder tissue, a urethra, a prostate, renal epithelial cells, or any combination thereof.

[0073] In some embodiments, the EVs isolated by the methods described herein are EVs found in a blood sample. In some embodiments, the EVs found in a blood sample comprise EVs derived from platelets, erythrocytes, leukocytes, endothelial cells, tumor cells. Attorney Docket No. 29539-0843WO1 mesenchymal stem cells, hepatocytes, neural cells, adipocytes, apoptotic bodies, or any combinations thereof. In some embodiments, the EVs isolated by the methods described herein are EVs found in a saliva sample. In some embodiments, the EVs found in a saliva sample comprise EVs derived from salivary gland cells, oral epithelial cells, immune cells, neuronal cells, oral microbiota, blood-derived cells, and gastrointestinal tract cells, or any combinations thereof. In some embodiments, the EVs isolated by the methods described herein are EVs found in a cerebrospinal fluid sample. In some embodiments, the EVs found in a cerebrospinal fluid sample comprise EVs derived from neurons, astrocytes, glial cells, ependymal cells, choroid plexus epithelial cells, peripheral immune cells, or any combinations thereof. In some embodiments, the EVs isolated by the methods described herein are EVs found in a uterine lavage sample.

[0074] In some embodiments, the EVs found in a uterine lavage sample comprise EVs derived from endometrial epithelium. In some embodiments, the EVs isolated by the methods described herein are EVs found in an ascites fluid sample. In some embodiments, the EVs found in a ascites fluid sample comprise EVs derived from hepatocellular carcinoma cells, metastatic cancer cells originating from one or both intra-abdominal and extraabdominal primary' tumors, endothelial cells from peritoneal microvasculature, hepatocytes, peritoneal macrophages, gastrointestinal cancer cells (e.g., gastric cancer cells and / or colorectal cancer cells), or any combinations thereof. In some embodiments, the EVs isolated by the methods described herein are EVs found in a tear or lacrimal fluid sample. In some embodiments, the EVs found in a tear or lacrimal fluid sample comprise EVs derived from comeal epithelial cells, conjunctival epithelial cells, lacrimal epithelial cells, meibomian gland epithelial cells, conjunctival goblet cells, ocular surface immune cells, nen e-associated cells, or any combinations thereof.

[0075] In some embodiments, the captured EVs or the microgel beads-EVs complexes comprise one or more of small EVs, large EVs, or very large EVs. In some embodiments, the captured EVs or the microgel beads-EVs complexes comprise small EVs with a diameter of about 65 nm to about 200 nm (e.g., about 65 to about 75 nm, about 75 to about 85 nm, about 85 to about 95 nm. about 95 to about 105 nm. about 105 to about 115 nm, about 115 to about 125 nm, about 125 to about 135 nm, about 135 to about 145 nm, about 145 to about 155 nm, about 155 to about 165 nm, about 165 to about 175 nm, about 175 to about 185 nm, about 185 to about 195 nm, about 195 to about 200 nm, about 65 to about 110 nm, about 110 to about 155 nm, or about 155 to about 200 nm). Attorney Docket No. 29539-0843WO1

[0076] In some embodiments, the captured EVs or the microgel beads-EVs complexes comprise large EVs with a diameter of about 200 nm to about 2 pm (e.g.. about 200 nm to about 250 nm, about 250 nm to about 300 nm, about 300 nm to about 350 nm, about 350 nm to about 400 nm, about 400 nm to about 450 nm, about 450 nm to about 500 nm, about 500 nm to about 550 nm, about 550 nm to about 600 nm, about 600 nm to about 650 nm, about 650 nm to about 700 nm, about 700 nm to about 750 nm, about 750 nm to about 800 nm, about 800 nm to about 850 nm, about 850 nm to about 900 nm. about 900 nm to about 950 nm, about 950 nm to about 1000 nm, about 1000 nm to about 1050 nm, about 1050 nm to about 1100 nm, about 1100 nm to about 1150 nm, about 1150 nm to about 1200 nm, about 1200 nm to about 1250 nm, about 1250 nm to about 1300 nm, about 1300 nm to about 1350 nm, about 1350 nm to about 1400 nm. about 1400 nm to about 1450 nm, about 1450 nm to about 1500 nm, about 1500 nm to about 1550 nm, about 1550 nm to about 1600 nm, about 1600 nm to about 1650 nm, about 1650 nm to about 1700 nm, about 1700 nm to about 1750 nm, about 1750 nm to about 1800 nm, about 1800 nm to about 1850 nm, about 1850 nm to about 1900 nm, about 1900 nm to about 1950 nm. about 1950 nm to about 2000 nm, about 200 nm to about 300 nm, about 300 nm to about 400 nm, about 400 nm to about 500 nm, about 500 nm to about 600 nm, about 600 nm to about 700 nm, about 700 nm to about 800 nm, about 800 nm to about 900 nm, about 900 nm to about 1000 nm, about 1000 nm to about 1100 nm, about 1100 nm to about 1200 nm, about 1200 nm to about 1300 nm, about 1300 nm to about 1400 nm, about 1400 nm to about 1500 nm, about 1500 nm to about 1600 nm, about 1600 nm to about 1700 nm, about 1700 nm to about 1800 nm, about 1800 nm to about 1900 nm, about 1900 nm to about 2000 nm, about 200 nm to about 400 nm, about 400 nm to about 600 nm, about 600 nm to about 800 nm, about 800 nm to about 1000 nm. about 1000 nm to about 1200 nm, about 1200 nm to about 1400 nm. about 1400 nm to about 1600 nm, about 1600 nm to about 1800 nm, or about 1800 nm to about 2000 nm).

[0077] In some embodiments, the captured EVs or the microgel beads-EVs complexes comprise large EVs with a diameter of about 200 nm to about 750 nm (e.g., about 200 nm to about 250 nm, about 250 nm to about 300 nm, about 300 nm to about 350 nm, about 350 nm to about 400 nm. about 400 nm to about 450 nm, about 450 nm to about 500 nm. about 500 nm to about 550 nm, about 550 nm to about 600 nm, about 600 nm to about 650 nm, about 650 nm to about 700 nm, or about 700 nm to about 750 nm).

[0078] In some embodiments, the captured EVs or the microgel beads-EVs complexes comprise very large EVs with a diameter of about 2 pm to about 15 pm (e.g., about 2 pm to about 3 pm, about 3 pm to about 4 pm, about 4 pm to about 5 pm, about 5 pm to about 6 Attorney Docket No. 29539-0843WO1 pm, about 6 jam to about 7 un, about 7 pun to about 8 un, about 8 pun to about 9 pun, about 9 pun to about 10 pun, about 10 pun to about 11 pun, about 11 pun to about 12 pun, about 12 pun to about 13 pun, about 13 pun to about 14 pun, about 14 pun to about 15 pun, about 2 pun to about 7 pun, about 7 pun to about 12 pm, or about 12 pun to about 15 pun). In some embodiments, the captured EVs or the microgel beads-EVs complexes comprise very large EVs with a diameter of about 750 nm to about 10 pm (e.g., about 750 nm to about 1.25 pm, about 1.25 pm to about 1.75 pm, about 1.75 pm to about 2.25 pm, about 2.25 pm to about 2.75 pm, about 2.75 pm to about 3.25 pm, about 3.25 pm to about 3.75 pm, about 3.75 pm to about 4.25 pm, about 4.25 pm to about 4.75 pm, about 4.75 pm to about 5.25 pm, about 5.25 pm to about 5.75 pm, about 5.75 pm to about 6.25 pm, about 6.25 pm to about 6.75 pm, about 6.75 pm to about 7.25 pm, about 7.25 pm to about 7.75 pm, about 7.75 pm to about 8.25 pm, about 8.25 pm to about 8.75 pm, about 8.75 pm to about 9.25 pm, about 9.25 pm to about 9.75 pm, or about 9.75 pm to about 10 pm). In some embodiments, the very large EVs are oncosomes with a diameter of about 2 pm to about 15 pm.

[0079] The methods for the selective isolation of EVs from biofluids disclosed herein leverage the intrinsic surface potential of the EV lipid bilayers. The zeta potential of EVs at various pH conditions shows that their surface charge remains consistently negative even in an acidic environment, as shown in FIG. 4C. This sustained negative charge is primarily attributed to the presence of anionic phospholipids, particularly phosphatidylserine, which contribute to the stability and biophysical properties of the vesicle membrane.

[0080] In some embodiments, the methods include contacting the sample with the composition comprising chitosan-grafted PLL microgel beads for at least about 1 hour (h). In some embodiments, the methods include contacting the sample with the chitosan-grafted PLL microgel bead composition comprising about 0.5 x 106beads per milliliter (beads / mL) to about 1 x 106beads / mL (e.g., about 500,000 to about 550,000 beads / mL, about 550,000 to about 600,000 beads / mL, about 600,000 to about 650,000 beads / mL, about 650,000 to about 700,000 beads / mL, about 700,000 to about 750,000 beads / mL, about 750,000 to about 800,000 beads / mL, about 800,000 to about 850,000 beads / mL, about 850,000 to about 900,000 beads / mL, about 900.000 to about 950.000 beads / mL, or about 950.000 to about 1,000,000 beads / mL).

[0081] In some embodiments, the methods include contacting the sample with the chitosan- grafted PLL microgel bead composition comprising about 1.3 x 105beads / mL to about 1 x 106beads / mL (e.g., about 130.000 to about 180,000 beads / mL, about 180,000 to about 230,000 beads / mL, about 230,000 to about 280,000 beads / mL, about 280,000 to about Attorney Docket No. 29539-0843WO1

[0082] 330,000 beads / mL, about 330,000 to about 380,000 beads / mL, about 380,000 to about

[0083] 430,000 beads / mL, about 430.000 to about 480.000 beads / mL, about 480,000 to about

[0084] 530,000 beads / mL, about 530,000 to about 580,000 beads / mL, about 580,000 to about

[0085] 630,000 beads / mL, about 630,000 to about 680,000 beads / mL, about 680,000 to about

[0086] 730,000 beads / mL, about 730,000 to about 780,000 beads / mL, about 780,000 to about

[0087] 830,000 beads / mL, about 830,000 to about 880,000 beads / mL, about 880,000 to about

[0088] 930,000 beads / mL, about 930.000 to about 980.000 beads / mL, about 980,000 to about

[0089] 1,030,000 beads / mL, about 500,000 to about 550,000 beads / mL, about 500,000 to about 600,000 beads / mL, about 500,000 to about 650,000 beads / mL, about 500,000 to about

[0090] 700,000 beads / mL, about 500,000 to about 750,000 beads / mL, about 500,000 to about

[0091] 800,000 beads / mL, about 500.000 to about 850.000 beads / mL, about 500,000 to about

[0092] 900,000 beads / mL, about 500,000 to about 950,000 beads / mL, about 500,000 to about

[0093] 1,000,000 beads / mL).

[0094] In some embodiments, the sample is contacted with the chitosan-grafted PLL microgel bead composition at room temperature (e.g.. between about 18 °C to about 25 °C). In some embodiments, the after the EVs are placed in contact with the chitosan-grafted PLL microgel beads (e g., in the same vial), incubation is carried out under a gentle rocking condition. In some embodiments, after the 1 h incubation, three sequential washes are performed with a buffer (e.g., the pgB buffer described in the Examples) to remove unbound particles with comparatively lower negative surface charge. In some embodiments, the captured EVs or the microgel beads-EVs complexes are purified by washing via centrifugation.

[0095] In some embodiments, the efficiency of EV capture can be qualitatively evaluated by measuring the zeta potential of EVs captured by chitosan-grafted PLL microgel beads (also referred to herein as pgBs-EVs or microgel-beads -EVs complexes). In some embodiments, the zeta potential of microgel-beads-EVs complexes is less than the zeta potential of chitosan-grafted PLL microgel beads alone. This reduction in zeta potential is attributed to the capture of negatively charged EVs by the positively charged chitosan-grafted PLL microgel beads. Supporting data are shown in FIGs. 4D and 4E.

[0096] In some embodiments, a slightly acidic environment (e g., at a pH of about 6.0) optimally preserves EV integrity, yield, and cargo stability. Thus, in some examples, the methods of isolating EVs from a sample are performed at a pH of about 6.0. For example, in some embodiments, the pH of the sample can be adjusted to about 6.0 prior to contacting the sample with the chitosan-grafted PLL microgel bead compositions disclosed herein. In some Attorney Docket No. 29539-0843WO1 embodiments, the sample has a pH of about 6.0. In some embodiments, the sample has a pH of about 5.0 to about 6.0. In some embodiments, the sample does not require further adjustment of the pH if the pH ranges from about 5.0 to about 6.0.

[0097] In some embodiments, the methods of isolating EVs include releasing the captured EVs from the microgel-beads-EVs complexes by adding a salt to the microgel bead-EVs complexes to neutralizing the charge interactions. In some embodiments, the salt is sodium chloride. In some embodiments, the salt is added at a final concentration of about IM. In some embodiments, the salt is added at a final concentration of about 0.5 M to about 2 M. In some embodiments, the methods further include collecting and concentrating the released EVs via centrifugation. In some embodiments, the methods further include washing the concentrated EVs to remove any residual salt.

[0098] In some embodiments, the present disclosure features methods for detecting at least one cancer biomarker in a biological sample from a subject. In some embodiments, the methods for detecting the at least one cancer biomarker include obtaining the biological sample comprising EVs. In some embodiments, the biological sample can be any of the complex biofluids described herein. In some embodiments, the biological sample is one or more of blood, urine, saliva, cerebrospinal fluid, uterine lavage fluid, ascitic fluid, or tear or lacrimal fluid. In some embodiments, the method includes isolating the EVs from the biological sample using any of the methods described herein. Next, the method includes detecting the presence of the at least one cancer biomarker comprising one or more of a protein, nucleic acid, lipid, or metabolite associated with cancer. In some embodiments, the step of detecting the presence of the cancer biomarker includes analyzing expression levels of a nucleic acid (e.g., RNA). In some embodiments, analyzing such expression levels of the nucleic acid includes performing digital nucleic acid amplification (e.g., droplet digital PCR (ddPCR)). In some embodiments, the detection of the cancer biomarker in the EVs is indicative of the presence or progression of cancer in the subject. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is kidney cancer or prostate cancer. In some embodiments, the cancer is melanoma, glioblastoma, breast cancer, pancreatic cancer, liver cancer, prostate cancer, colorectal cancer, hematological malignancies, bladder cancer, kidney cancer, oral squamous cell carcinoma, head and neck cancers, medulloblastoma, endometrial cancer, ovarian cancer, gastrointestinal cancers, lung cancer, or any combinations thereof. Attorney Docket No. 29539-0843WO1

[0099] Compositions

[0100] The present disclosure features compositions comprising microgel beads that are capable of capturing and releasing one or more EVs in a sample via surface charge interactions. The microgel beads comprise chitosan-grafted poly-L-lysine (PLL), a hybrid polymer. The hybrid polymer is synthesized by grafting PLL onto chitosan moieties to create the chitosan-grafted poly-L-lysine structure, as shown in FIG. 2B. PLL functions as the co- polymeric component, contributing unique structural and functional properties to the hybrid material. The synergy between chitosan and PLL leads to an increase in surface charge of the microgel beads due to the dense structure of the primary amine groups and the ionic interaction between chitosan and PLL. In some embodiments, the chitosan-grafted PLL microbeads are capable of capturing EVs found in a complex biofl uid (e.g., urine or saliva) and then releasing the captured EVs. In some embodiments, the chitosan-grafted PLL microbeads are capable of capturing urinary EVs and then releasing the captured, urinary' EVs. In some embodiments, urinary EVs comprise EVs derived from a bladder tissue, a urethra, a prostate, renal epithelial cells, or any combination thereof.

[0101] In some embodiments, the microgel beads comprise chitosan-grafted PLL comprising chitosan at a concentration of about 1% (w / v) to about 2 % (w / v) (e.g., about 1% to about 1.5% or about 1.5% to about 2%). In some embodiments, the microgel beads comprise chitosan-grafted PLL comprising chitosan at a concentration of about 1.5% weight per volume (w / v).

[0102] In some embodiments, the microgel beads comprise chitosan-grafted PLL comprising PLL at a concentration of about 0.01% to about 0.05% volume per volume (v / v) (e.g., about 0.01% to about 0.015%, 0.01% to about 0.02%, 0.01% to about 0.025%, about 0.01% to about 0.030%, about 0.01% to about 0.035%, about 0.01% to about 0.040%, about 0.01% to about 0.045%, about 0.01% to about 0.05%, about 0.025% to about 0.030%, about 0.025% to about 0.035%, about 0.025% to about 0.04%, about 0.025% to about 0.045%, or about 0.025% to about 0.05%). In some embodiments, the microgel beads comprise chitosan- grafted PLL comprising PLL at a concentration of about 0.05% (v / v).

[0103] In some embodiments, the chitosan-grafted PLL microgel beads are crosslinked. In some embodiments, the chitosan-grafted PLL microgel beads are crosslinked with a monoaldehyde, a di-aldehyde, or a combination thereof. In some embodiments, the mono-aldehyde is formaldehyde. In some embodiments, the primary' amine groups of chitosan react with mono-aldehyde to form imines (-C=N), which react with a different primary amine group of another chitosan chain to form a cross-linked mesh-like structure. Thus, crosslinking of Attorney Docket No. 29539-0843WO1 chitosan and PLL via a mono-aldehyde crosslinker may yield a dense and heterogeneous network, potentially comprising diverse interaction patterns such as chitosan-chitosan and chitosan-PLL linkages, each exhibiting distinct spatial orientation.

[0104] In some embodiments, the surface charge, as measured by the zeta potential, of the chitosan-grafted PLL microgel beads is tunable based on the concentration of PLL in the chitosan-grafted PLL hybrid polymer. For example, the zeta potential of the microgel beads comprising chitosan-grafted PLL increases with increasing concentration of PLL, as shown in FIGs. 3A and 3C. In some embodiments, the surface charge, as measured by the zeta potential, of the chitosan-grafted PLL microgel beads is tunable based on the inclusion of PLL. For example, the zeta potential of the microgel beads comprising chitosan-grafted PLL is higher than the zeta potential of the microgel beads comprising chitosan alone, as shown in FIGs. 3A and 3C.

[0105] In some embodiments, chitosan-grafted PLL microgel beads with a higher, positive zeta-potential (e.g., at least 60 millivolts (mV) at pH 6.0) are desirable because the increased zeta-potential indicates greater stability than microgel beads with a lower zeta-potential, thereby increasing the applicability of the microgel beads in clinical samples having acidic to alkaline pH values (e.g., urine and saliva). In some embodiments, the greater stability is greater stability against aggregation due to charge stabilization attributable to electrostatic repulsion in colloidal systems.

[0106] In some embodiments, the chitosan-grafted PLL microgel beads have a zeta potential of about 60 mV to about 100 mV (e.g., about 60 mV to about 65 mV, about 60 mV to about 70 mV, about 60 mV to about 75 mV, about 60 mV to about 80 mV, about 60 mV to about 85 mV. about 60 mV to about 90 mV, about 60 mV to about 95 mV, about 60 mV to about 100 mV, about 70 mV to about 75 mV, about 70 mV to about 80 mV, about 70 mV to about 85 mV, about 70 mV to about 90 mV, about 70 mV to about 95 mV, about 70 mV to about 100 mV, about 80 mV to about 85 mV, about 80 mV to about 90 mV, about 80 mV to about 95 mV, about 80 mV to about 100 mV, about 90 mV to about 95 mV, about 90 mV to about 100 mV, or about 95 mV to about 100 mV) when measured at a pH between about 3.0 and about 6.0. In some embodiments, the chitosan-grafted PLL microgel beads have a zeta potential of about 100 mV when measured at a pH of about 6.0. In some embodiments, the chitosan-grafted PLL microgel beads have a zeta potential of about 50 mV when measured at a pH of about 6.0.

[0107] In some embodiments, the chitosan-grafted PLL microgel beads have a zeta potential of about 30 mV to about 40 mV (e.g., about 30 mV to about 31 mV, about 30 mV to about 32 Attorney Docket No. 29539-0843WO1 mV, about 30 mV to about 33 mV, about 30 mV to about 34 mV, about 30 mV to about 35 mV, about 30 mV to about 36 mV, about 30 mV to about 37 mV. about 30 mV to about 38 mV, about 30 mV to about 39 mV, about 30 mV to about 40 mV, about 35 mV to about 36 mV, about 35 mV to about 37 mV, about 35 mV to about 38 mV, about 35 mV to about 39 mV, or about 35 mV to about 40 mV) when measured at a pH of about 9.0. In some embodiments, the chitosan-grafted PLL microgel beads have a zeta potential of about 37 mV when measured at a pH of about 9.0.

[0108] In some embodiments, the chitosan-grafted PLL microgel beads have a zeta potential of about 50 mV to about 70 mV (e.g., about 50 mV to about 55 mV, about 50 mV to about 60 mV, about 50 mV to about 65 mV, about 50 mV to about 70 mV, about 65 mV to about 70 mV, about 60 mV to about 61 mV, about 60 mV to about 62 mV. about 60 mV to about 63 mV, about 60 mV to about 64 mV, about 60 mV to about 65 mV, about 60 mV to about 66 mV, about 60 mV to about 67 mV, about 60 mV to about 68 mV, about 60 mV to about 69 mV, or about 60 mV to about 70 mV) when measured at a pH of about 3.0. In some embodiments, the chitosan-grafted PLL microgel beads have a zeta potential of about 65 mV when measured at a pH of about 3.0.

[0109] In some embodiments, the chitosan-grafted PLL microgel beads have a zeta potential of about -40 mV to about -50 mV (e.g., about -40 mV to about -45 mV, or about -40 mV to about -50 mV) when measured at a pH of about 12.0. In some embodiments, the decrease in zeta potential when measured at a pH of about 12.0 is likely attributed to the extensive deprotonation of the abundant-NHv groups and the increased susceptibility of -OH groups to deprotonation under strongly basic conditions.

[0110] In some embodiments, the composition includes chitosan-grafted PLL microgel beads having a diameter ranging from about 50 microns (pm) to about 60 pm (e.g., about 50 pm to about 51 pm, about 50 pm to about 52 pm, about 50 pm to about 53 pm, about 50 pm to about 54 pm, about 50 pm to about 55 pm, about 50 pm to about 56 pm, about 50 pm to about 57 pm, about 50 pm to about 58 pm, about 50 pm to about 59 pm, about 50 pm to about 60 pm. about 56 pm to about 57 pm, about 56 pm to about 58 pm, about 56 pm to about 59 pm. or about 56 pm to about 60 pm) when measured at a pH of about 6.0. In some embodiments, the composition includes chitosan-grafted PLL microgel beads having a diameter of 56 pm when measured at a pH of about 6.0.

[0111] In some embodiments, chitosan-grafted PLL microgel beads exhibit enhanced resistance to alkaline-induced dimensional or size changes. In some embodiments, upon exposure to a pH of about 12.0, the chitosan-grafted PLL microgel beads exhibit a reduced Attorney Docket No. 29539-0843WO1 shrinkage (e.g., about 32%). In some embodiments, the extent of shrinkage in chitosan- grafted PLL microgel beads was less (e.g., about 23% less) than that observed in chitosan microgel beads under comparable conditions, indicating improved structural stability conferred by PLL grafting. In some embodiments, the chitosan-grafted PLL microgel beads exhibit pH-dependent structural contraction and / or reduction in size or diameter.

[0112] In some embodiments, the composition includes microgel beads having a diameter ranging from about 45 microns (pm) to about 65 pm (e.g., about 45 pm to about 50 pm, about 45 pm to about 55 pm, about 45 pm to about 60 pm, about 45 pm to about 65 pm, about 53 pm to about 55 pm, about 53 pm to about 56 pm, about 54 pm to about 56 pm, about 54 pm to about 57 pm, about 55 pm to about 57 pm, about 55 pm to about 58 pm. about 56 pm to about 58 pm, about 56 pm to about 59 pm, about 57 pm to about 59 pm. about 57 pm to about 60 pm, about 58 pm to about 60 pm, about 58 pm to about 61 pm, about 59 pm to about 61 pm, about 59 pm to about 62 pm, about 60 pm to about 62 pm, about 60 pm to about 63 pm, about 61 pm to about 63 pm, about 61 pm to about 64 pm, about 62 pm to about 64 pm, about 62 pm to about 65 pm, about 63 pm to about 65 pm) when measured at a pH of about 3.0. In some embodiments, the composition includes microgel beads having a diameter of 54 pm when measured at a pH of about 3.0.

[0113] In some embodiments, the composition includes microgel beads having a diameter ranging from about 50 microns (pm) to about 60 pm (e.g., about 50 pm to about 51 pm, about 50 pm to about 52 pm, about 50 pm to about 53 pm, about 50 pm to about 54 pm. about 50 pm to about 55 pm, about 50 pm to about 56 pm, about 50 pm to about 57 pm, about 50 pm to about 58 pm, about 50 pm to about 59 pm, about 50 pm to about 60 pm, about 56 pm to about 57 pm, about 56 pm to about 58 pm, about 56 pm to about 59 pm. or about 56 pm to about 60 pm) when measured at a pH of about 6.0. In some embodiments, the composition includes microgel beads having a diameter of 56 pm when measured at a pH of about 6.0.

[0114] In some embodiments, the composition includes microgel beads having a diameter ranging from about 40 microns (pm) to about 55 pm (e.g., about 40 pm to about 45 pm, about 40 pm to about 50 pm, about 40 pm to about 55 pm, about 48 pm to about 50 pm. about 48 pm to about 52 pm, about 48 pm to about 54 pm, or about 48 pm to about 55 pm) when measured at a pH of about 9.0. In some embodiments, the composition includes microgel beads having a diameter of 48 pm when measured at a pH of about 9.0.

[0115] In some embodiments, the composition includes microgel beads having a diameter ranging from about 30 microns (pm) to about 40 pm (e.g., about 30 pm to about 35 pm, Attorney Docket No. 29539-0843WO1 about 30 un to about 40 un, about 35 pun to about 36 pun, about 35 pun to about 37 pun, about 35 pun to about 38 pun, about 35 pun to about 39 pun, about 35 pun to about 40 pun. about 36 pun to about 37 pun, about 36 pun to about 38 pun, about 36 pun to about 39 pun, about 36 pun to about 40 pun) when measured at a pH of about 12.0. In some embodiments, the composition includes microgel beads having a diameter of 36 pm when measured at a pH of about 12.0.

[0116] In some embodiments, the composition includes microgel beads having a diameter ranging from about 45 microns (pm) to about 65 pm (e.g., about 45 pm to about 50 pm, about 45 pm to about 55 pm, about 45 pm to about 60 pm, about 45 pm to about 65 pm, about 50 pm to about 55 pm, about 55 pm to about 60 pm, or about 55 pm to about 65 pm) when measured at a pH between about 3.0 and 9.0. In some embodiments, the composition includes microgel beads having a diameter of 53 pm when measured at a pH between about 3.0 and 9.0.

[0117] In some embodiments, key factors for maintaining the high surface charge of chitosan-grafted PLL microgel beads include PLL concentration, crosslinker concentration, curing time, and high-throughput droplet formation, as further described in the section entitled “Methods of Preparing the Microgel Bead Composition.” In some embodiments, the surface charge of chitosan-grafted PLL microgel beads remains shelf-stable over a period of at least about 5 months (e.g.. about 1 to about 2 months, about 2 to about 3 months, about 3 to about 4 months, about 4 to about 5 months). In some embodiments, the surface charge of chitosan-grafted PLL microgel beads remains shelf-stable over a period of at least about 1 year. In some embodiments, the surface charge of chitosan-grafted PLL microgel beads remains shelf-stable over a period of up to about 1 year.

[0118] In some embodiments, the composition further includes a non-pharmaceutical carrier, formulation component, and / or additive. As used herein, the expression “non-pharmaceutical carrier” refers to a biocompatible material, composition, or vehicle for in vitro diagnostics that is in contact with the microgel beads. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be biocompatible in that it must be compatible with the other ingredients of the formulation and is compatible with biological samples (e.g., human biological samples) without interfering with diagnostic outcomes. Examples of non- pharmaceutical carriers include, but are not limited to, a solvent or dispersing medium containing, for example, water, pH buffered solutions (e.g., phosphate buffered saline (PBS), Attorney Docket No. 29539-0843WO1

[0119] HEPES, TES, MOPS, etc.), isotonic saline, Ringer’s solution, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), alginic acid, ethyl alcohol, and suitable mixtures thereof. In some embodiments, the non-pharmaceutical carrier can be a pH-buffered solution (e.g. PBS). In some embodiments, the non-pharmaceutical carrier can be a microgel bead buffer comprising about 0.1 mM Tris-buffered saline and about 0.001% (v / v) Triton™-X, sterile-filtered using a 0.2 pm membrane, with the pH adjusted to about 6.0. In some embodiments, the chitosan-grafted PLL microgel bead compositions are formulated for diagnostic use and / or for non-invasive prognostic use.

[0120] Methods of Preparing the Microgel Bead Compositions

[0121] The present disclosure features methods of preparing any of the microgel bead compositions. In some embodiments, the microgel bead compositions are prepared using a microfluidic device (e.g., a droplet microfluidic device). In some embodiments, droplet microfluidics provides a platform to generate water-in-oil (W / O) droplets with a high degree of uniformity. In some embodiments, at least one continuous droplet microfluidic device is used to produce the microgel beads disclosed herein. In some embodiments, two continuous droplet microfluidic devices are used in parallel to produce the microgel beads disclosed herein. In some embodiments, two or more continuous droplet microfluidic devices are used in parallel to produce the microgel beads disclosed herein. In some embodiments, the methods of preparing the microgel beads enable efficient and controlled fabrication of the microgel beads, which are shelf-stable for up to about five months when stored in a vial at about 4 °C.

[0122] In some embodiments, the microgel beads are produced at a production throughput of about 30.000 beads per minute (beads / min.) to about 60,000 beads / min. (e.g., about 30,000 to about 35,000 beads / min, about 35,000 to about 40,000 beads / min, about 40,000 to about 45,000 beads / min, about 45,000 to about 50,000 beads / min, about 50,000 to about 55,000 beads / min, about 55,000 to about 60,000 beads / min). In some embodiments, the microgel beads are produced at a production throughput about 60,000 beads per minute.

[0123] In some embodiments, a microfluidic device is configured to generate W / O microdroplets using a dispersed phase (e.g., an aqueous phase) and a continuous phase (e.g., an oil phase). In some embodiments, the device comprises a channel (e.g., a microchannel), a junction, two inlets, and an outlet. In some embodiments, the methods include providing the Attorney Docket No. 29539-0843WO1 dispersed phase comprising chitosan-grafted PLL polymer precursor and the continuous phase comprising a non-aqueous fluid.

[0124] In some embodiments, the dispersed phase comprises chitosan-grafted PLL dissolved in acetic acid. In some embodiments, the dispersed phase comprises about 1% to about 2% (w / v) chitosan-grafted PLL. In some embodiments, the dispersed phase comprises about 0.01% to about 2% (v / v) PLL (e.g., about 0.01% to about 0.05% or about 0.05% to about 2% (v / v)). In some embodiments, the dispersed phase comprises about 0.05% PLL. In some embodiments, the dispersed phase comprises about 1.5% (w / v) chitosan-grafted PLL. In some embodiments, the non-aqueous fluid comprises a fluorinated oil and one or more polymeric surfactants. In some embodiments, the one or more polymeric surfactants comprise perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer (PFPE-PEG- PFPE), PFPE 5k, PEG 900, PFPE 5k, or a combination thereof. In some embodiments, the one or more polymeric surfactants are present at a concentration of about 1.5% to about 2.5 % (w / v) (e.g., about 1.5% to about 2 % or about 2% to about 2.5 % (w / v)). In some embodiments, the one or more polymeric surfactants are present at a concentration of about 2% (w / v).

[0125] In some embodiments, the methods include introducing the aqueous phase through a first inlet into a channel of the microfluidic device and introducing the immiscible continuous phase through a second inlet into the same channel. In some embodiments, the dispersed and continuous phases are introduced into the channel under controlled pressure conditions. In some embodiments, the dispersed phase is introduced at a pressure of about 160 millibar (mbar) to about 240 mbar (e.g., about 160 to about 200 mbar, about 180 to about 200 mbar, about 200 to about 220 mbar, or about 200 to about 240 mbar). In some embodiments, the continuous phase is introduced at a pressure of about 80 mbar to about 120 mbar (e.g., about 80 to about 100 mbar, or about 100 to about 120 mbar). The fluids converge at the junction — such as a T-junction or flow-focusing geometry — within the channel.

[0126] At the junction, the continuous phase exerts shear forces on the dispersed phase, resulting in the formation of discrete microdroplets of the dispersed phase within the continuous phase. The generated droplets are conveyed downstream through the outlet. In some embodiments, the size, frequency, and uniformity of the microdroplets may be controlled by adjusting the flow rates of the respective phases, the geometry of the junction, and the physicochemical properties of the fluids.

[0127] In some embodiments, the methods include collecting the microdroplets in a crosslinker solution. In some embodiments, the crosslinker solution comprises mono- Attorney Docket No. 29539-0843WO1 aldehyde, di-aldehyde, or a combination thereof. In some embodiments, the crosslinker solution has a concentration of about 2.0% (v / v) to about 10% (v / v) (e.g.. about 2.0% to about 5% or about 5% to about 10% (v / v)). In some embodiments, the crosslinker solution has a concentration of about 5% (v / v).

[0128] In some embodiments, the methods include crosslinking the microdroplets by heating the microdroplets and the crosslinker solution, thereby generating the composition comprising the microgel beads. In some embodiments, the crosslinker (e.g., mono-aldehyde or di-aldehyde) induces gelation in chitosan and in mixtures of chitosan-grafted PLL precursors by imine cross-linking, resulting in the generation of monodisperse and highly stable chitosan and chitosan grafted-PLL microgel beads. In some embodiments, the composition comprising microdroplets and the crosslinker solution are heated to about 60 °C to about 70 °C to facilitate gelation. In some embodiments, the composition comprising microdroplets and the crosslinker solution are heated for about 30 min. to about 12 h. In some embodiments, the composition comprising microdroplets and the crosslinker solution are heated for about 30 min.

[0129] In some embodiments, polymerized microgel beads are purified following droplet formation and crosslinking. Initially, the upper oil phase (e.g., hydrofluoroether (HFE) 7500) and the lower crosslinker solution are removed from each reaction tube. Subsequently, about 500 pL of microgel bead buffer is added to each tube. In some embodiments, the microgel bead buffer comprises about 0. 1 mM Tris-buffered saline and about 0.001% (v / v) Triton™- X, sterile-filtered using a 0.2 pm membrane, with the pH adjusted to about 6.0.

[0130] In some embodiments, the methods further include, after crosslinking, destabilizing the continuous phase encapsulating the microgel beads to release and separate the microgel beads from the continuous phase. In some embodiments, destabilizing the continuous phase comprises adding a demulsifier (e.g., a fluorinated surfactant). For example, to release the polymerized microgel beads from the emulsion, about 500 pL of about 30% (v / v) perfluoro- 1 -octanol (PFO) is added to each tube, followed by vigorous vortexing for approximately 10 seconds and centrifugation at 5,000 x g for 30 seconds. In some embodiments, the microgel beads appear as a semi-transparent phase in the upper layer. In some embodiments, the lower demulsifier phase (e.g., PFO phase) is removed, and an additional 1 mL of 30% (v / v) PFO is added. In some embodiments, the vortexing and centrifugation steps are repeated. In some Attorney Docket No. 29539-0843WO1 embodiments, this washing step is performed three times, resulting in the microgel beads forming a translucent mass.

[0131] In some embodiments, following removal of excess demulsifier (e.g., PFO), about 1% (v / v) Span-80 prepared in n-hexane is added to facilitate settling of the pgBs. In some embodiments, the tubes are vortexed vigorously and centrifuged at about 5,000 x g for about 30 seconds. In some embodiments, this procedure is repeated twice. In some embodiments, afterward, about 500 pL of the microgel bead buffer is added to each tube, and the contents are transferred into a tube containing about 15 mL of the same buffer. In some embodiments, the mixture is vortexed thoroughly to ensure uniform dispersion and then centrifuged at about 3,000 x g for about 5 minutes at room temperature.

[0132] In some embodiments, the microgel beads sediment at the bottom of the tube, while residual n-hexane appears as a milky layer on the surface. In some embodiments, the upper solution is aspirated, and the washing step is repeated three times. Finally, in some embodiments, the purified microgel beads are suspended in the microgel bead buffer and stored at about 4 °C until further use.

[0133] EXAMPLES

[0134] Certain embodiments of the present disclosure are further described in the following examples, which do not limit the scope of any embodiments described in the claims.

[0135] Example 1 - Materials and Methods

[0136] The following materials and methods described in Example 1 were used in Examples 1-8 described below.

[0137] Materials

[0138] All reagents, chemicals, solvents and materials purchased commercially are used as received, unless otherwise specified. Chitosan medium molecular w eight (Cat. # 448877- 50G), Poly-L-lysine 0.1 % (w / v) (Cat. #P8920), sodium chloride (Cat. # S3014), DL- Dithiothreitol (Cat. #43815). Trichloro(lH,lH,2H,2H,2H-perfluorooctyl Silane (Cat. #448931), Triton™X-100 solution (Cat. # T8787), Amicon® Ultra-15 Centrifugal Filters 10 kDa MWCO (Cat.# UFC901024) and Amicon® Ultra - 2 mL Centrifugal Filters 100 kDa MWCO (Cat. # UFC210024) were purchased from Millipore Sigma. Tris-HCl pH 8.0 Solution (Cat. # 15568-025) and UltraPure™ Distilled Water were purchased from Invitrogen. HSP70 (Cat. # 4872S, Lot#4), Alix (Cat. # 92880, Lot#l, Clone: E6P9B), Attorney Docket No. 29539-0843WO1

[0139] Calnexin (Cat. # 2679, Lot#7, Clone: C5C9), Anti-rabbit IgG HRP -linked (Cat. #7074, Lot#33) and Anti-mouse IgG HRP -linked (Cat. #7076, Lot#38) antibodies were purchased from Cell Signaling Technology. CD63 antibody (Cat. # NB100-77913, Lot#539981, Clone: MEM-259) was purchased from Novus Biologicals.

[0140] CD9 antibody (Cat .# 312102, Lot# B433218, Clone: HI9a) was purchased from BioLegends. Ham's F-12K (Kaighn's) Medium (Cat. # 21127022), MEM medium (Cat. #11095080). Fetal Bovine Serum (FBS) (Cat. # 26140079). 10% heat inactivated FBS (Cat. # A5256701), Penicillin-Streptomycin, (Cat. #15070063), Trypsin-EDTA (Cat. # 25200056), PageRuler™ Prestained NIR Protein Ladder (Cat. # 26635), Phosphate-buffered saline (PBS) (Cat. # 10010-023) pH 7.4 Solution, n-Hexane, 99% (Cat. #AAL09938AU), SuperSignal™ West Atto Ultimate Sensitivity Substrate (Cat. #A38554). SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Cat. # 34579) and Pierce™ ECL Western Blotting Substrate (Cat. #32209) were purchased from Thermo Fisher Scientific. T-175 cm2flasks (Nunc™ EasYFlask™ Cell Culture Flasks, Cat. # 353112), T-75 cm2flasks (Nunc™ EasYFlask™ Cell Culture Flasks, Cat. # 353136) and Dulbecco's Modified Eagle Media with glutamine and 4.5 g L1glucose (Cat. #10-013-CV) was purchased from Coming Life Sciences. 4-20% Mini-PROTEAN® TGX™ Precast Protein Gels (Cat. # 4561094) and One-Step RT-ddPCR Advanced Kit for Probes (Cat. #864021) were purchased from Bio-Rad Laboratories.

[0141] 1H,1H,2H,2H-Perfluorooctanol, 97% (Cat. #B20156-18) was purchased from Alfa Aesar. PFPE-PEG-PFPE, PFPE 5k, PEG 900. PFPE 5k (Cat. # PPP-224) was purchased from Creative PEGWorks. MagMAX mirV ana Total RNA isolation kit (Cat. #A27828) was purchased from Applied Biosystems. RNeasy Micro Kit (QIAGEN #74004) was purchased from QIAGEN GmbH. qEV original 70 nm Gen 2 Column (Cat. # ICO-70) was purchased from Izon Science. HyBlot CL® Autoradiography Film (Cat. #1141 J51) was purchased from Thomas Scientific. Cole-Parmer pH electrode, Refillable / SJ / Glass / Extra- Long / Thin / 326x8mm (Cat. # EW-05990-40) was purchased from Cole-Parmer. Syringe filters, Acrodisc®, Supor® membrane, pore size 1.20 pm (Cat. # 28150-958) filters were purchased from VWR. Superfrost® Plus Microscope Slides (25mm x 75mm. Cat. # PO103) were purchased from FD NeuroTechnologies. Inc. 3M™ Novec™ 7500 Engineered Fluid was purchased from 3M™ Novec™ Sciences. SYLGARD™ 184 Silicone Elastomer Kit was purchased from Dow Inc. Attorney Docket No. 29539-0843WO1

[0142] Microfluidic Device Fabrication

[0143] The multi-step process consists of a computer-aided drawing (CAD) of a droplet microfluidic device, conventional photolithography and the fabrication of microchannels using poly dimethylsiloxane (PDMS). The design is printed on the film mask (Fineline Imaging), which is then used to produce a master mold, a Si wafer, with negative photoresist (SU-8, MicroChem) by photolithography, as described elsewhere

[0066] . The height of the device was set to 50 pm. To fabricate the device, the PDMS elastomer and the curing agent (SYTGARD 184 Silicon Curing Agent, Dow Inc) were thoroughly mixed at a ratio of 10: 1 (w / w) and then poured onto the master mold. The bubbles were removed by degassing under vacuum pressure and baked overnight at 55 °C in the oven. The PDMS was peeled off and cut into appropriate sizes, which were then bonded to a glass slide (Super Chip, 25 x 75 x 1mm, Cat. #22037213, Thermo Fischer Scientific) using a plasma etcher / cleaner (March Instruments PX250). The glass-bonded microchannel was heated to 85 °C for 1 min to strengthen the bond. To make the PDMS surface hydrophobic, the microchannel was coated with 2% trichloro(lH,lH,2H,2H-perfluorooctyl)silane prepared in Novec 7500 and kept for 10 min at room temperature. The excess silane was removed by applying a vacuum with a syringe to an outlet and heated at 110 °C for 1 hr. The PDMS devices were stored at room temperature in a dish sealed with parafilm.

[0144] Fabrication of Microgel Beads (pgBs) pgBs were fabricated in a microfluidic droplet focusing device (FIG. 1A). Two different types of pgBs were fabricated: chitosan and chitosan grafted poly-L-lysine pgBs. In chitosan pgBs, the dispersed (aqueous) phase is composed of 1.5% CS dissolved in 1% acetic acid whereas in chitosan grafted poly-L-lysine pgBs, the dispersed phase is a mixture of two different polymers: 1.5% chitosan dissolved in 1% acetic acid, supplemented with different concentration of poly-L-lysine. The continuous (oil phase) consists of Novec™ 7500 oil supplemented with 2% perfluoropoly ether-poly ethylene glycol-perfluoropolyether triblock copolymer (PFPE-PEG-PFPE), PFPE 5k, PEG 900, PFPE 5k. The pressure of the dispersed and continuous phase was approximately 200 mbar and 80 mbar respectively (may vary according to the experiment) and was controlled by a pressure-based flow control system Flow7control (Flow7EZ™). The disperse phase dissociates at the junction of the device and monodisperse water-in-oil (W / O) droplets formed at a rate of 60,000 droplets / min in a tw o parallel connected device system. Attorney Docket No. 29539-0843WO1

[0145] The monodisperse droplets were collected in 1.5 rnL Eppendorf tubes prefilled with varying concentrations of mono-aldehyde and / or di-aldehyde, which served as crosslinking agents, and then heated at 60 °C for different curing durations. The W / O emulsion is polymerized to highly cross-linked pgBs, which appear as a creamy phase. The upper oil phase (HFE 7500) and the lower crosslinker solution were removed and 500 pL of pgB buffer were added. The pgB buffer is composed of 0. 1 mM Tris-buffered saline and 0.001 % Triton™-X (sterile filtered with 0.2 pm membrane), pH adjusted to 6.0. To release the polymerized pgBs, 500 pL of 30% (v / v) perfluorooctanol (PFO) was added per tube and then vortexed vigorously for 10 sec followed by centrifugation at 5000xg for 30 sec.

[0146] The pgBs appeared as a semi-transparent phase in the upper layer. The lower PFO phase was removed and 1 mL of 30% (v / v) PFO was added and vortexed and centrifuged in the same way described before. The same step was repeated three times and pgBs appeared as a translucent mass. Subsequently, access PFO was removed and 1 % (v / v) Span® 80 prepared in n-hexane was added, which facilitated the settling of the pgBs. The pgBs containing Eppendorf vortexed vigorously and centrifuged at 5000xg for 30 sec. The procedure was repeated twice, adding 500 pL of pgB buffer to each tube, followed by transferring the solution into a 50 mL Falcon tube containing 15 mL of the same buffer.

[0147] The mixture was vortexed thoroughly to ensure proper mixing and then centrifuged at 3000xg for 5 min at room temperature. The pgBs sedimented, while traces of n-hexane appeared as a milky layer on the surface. The upper solution is aspirated, and the washing step is performed three times. Finally, the pgBs were suspended in pgB buffer and stored at 4°C until use.

[0148] Cell Culture

[0149] Human prostate cancer cells (PC3, ATCC- CRL-1435) cells were cultured in Ham's F-12K supplement with 10 % FBS and 1 % penicillin-streptomycin. Human embry onic kidney 293 (HEK-293T, ATCC-CRL-3216) cells were cultured in Dulbecco's Modified Eagle Media with glutamine and 4.5 g L1glucose supplemented with 10 % FBS and 1 % penicillin-streptomycin. Two types of human bladder cancer cell lines, basal type (HT-1376. ATCC- CRL-1472) and luminal ty pe (UMUC-1, Sigma- Aldrich, 6080301), were cultured in MEM medium containing 10% heat-inactivated FBS and 1% penicillin-streptomycin. All cell lines were cultured at 37 °C in a 5% CO2 environment. Attorney Docket No. 29539-0843WO1

[0150] Lentiviral Transductions

[0151] PC3 cells were transduced with a PalmtdTomato (PT) fluorescent reporter using lentivirus constructs as described before

[0035] . To do this, third generation lentiviruses were propagated under BSL2+ conditions according to the guidelines approved by the Mass General Brigham Institutional Biosafety Committee. The lentiviral packaging plasmids pMDLg / pRRE, pRSV-Rev and pVSV-G were combined with the lentiviral vector pCSCGW2-PalmtdTomato

[0067] and TransIT-Lenti (Mirrus. MIR6600) was used to transfect HEK-293T cells according to the manufacturer's protocol. After 48 hours, the lentiviral media was collected and filtered through a 0.45 pm filter. 1 pL of TransducelT transduction reagent (Mirrus, MIR6620) was added per 1 mL of lentiviral medium. Subsequently, 1.5 mL of the virus-containing medium was added to transduce the cells for 24 hr. After transduction, the transduced cells were selected for viral expression of PT by flow cytometry.

[0152] Extracellular Vesicles Generation from Cell Culture Media

[0153] PT- PC3, UMUC-1 and HEK-1376 cells were cultured in T-175 cm2flasks and / or T- 75 cm2flasks. At 80 % confluence of the cells, the supernatant was aspirated, and the monolayer of cells was washed three times with 15 mL of IxPBS to remove the FBS- containing media. The cells were then incubated for 48 hr at 37 °C with the serum-free cell culture media. CCM was collected and centrifuged at 300xg to remove dead cells. The collected supernatant was centrifuged again at 2000xg for 10 min to remove cell debris and apoptotic bodies. The medium was either used fresh or stored at -80 °C until further use.

[0154] Extracellular Vesicles Isolation from Cell Cultured-Conditioned Media

[0155] To isolate EVs by SEC, IZON columns were used. Prior to SEC, either 10 mL or 30 mL of CCM concentrated to a final volume of 500 pL using Amicon 15mL centrifugal filters at 4000xg for 20 min at room temperature. Columns were equilibrated by sequential washing with 17 mL of filtered milli-Q water followed by 17 mL of sterile IxPBS, in accordance with the manufacturer’s protocol unless specified otherwise. Subsequently, 500 pL of concentrated CCM was added and allowed to fully permeate the column, followed by the addition of 2.4 mL of IxPBS as a void volume. 1.6 mL EVs were collected from each outlet. The collected EVs were concentrated to 40-80 pL using Amicon 2 mL centrifugal filters by centrifugation at 4000xg for 20 min at room temperature, followed by downstream processing. Following EV collection, the SEC columns were regenerated by sequential Attorney Docket No. 29539-0843WO1 washing with 8 mL of 0.5 M NaOH, followed by 17 mL of 20% ethanol to ensure decontamination and preservation for subsequent use.

[0156] To isolate EVs with pgBs, CCM was concentrated as described above. After the first centrifugation, 5 mL of pgB buffer was added to the concentrated CCM, which was then centrifuged again under the same conditions to reduce the residual salt content. After centrifugation, the resulting sample was transferred to a conical tube containing 1 mL pgBs (0.5-1 x io6beads / mL) and allowed to sit on a rocker for 1 h at room temperature to facilitate EV capture. The samples were then centrifuged at 3000xg for 5 min at 4°C to pellet pgBs- EVs complexes. Thereafter, the supernatant was removed and the EVs captured on pgBs were resuspended in 3 mL of pgB buffer.

[0157] To further remove unbound EVs, the washing step was repeated three times under the same centrifugation conditions. After the last washing step, the pgBs-EVs complexes were resuspended in 1.5 mL of pgB buffer. To improve EV recovery, the supernatants from all wash steps were pooled, centrifuged simultaneously and any remaining pgBs-EVs complexes were collected by resuspending the pellet in 100 pL pgB buffer. These were then combined with the original pgBs-EVs suspensions. To release EVs from pgBs. IM NaCl (final concentration) was added, and the mixture was gently shaken on a rocker for 30 min at room temperature. The sample was then centrifuged at 3000xg for 5 min, and the supernatant containing purified EVs was collected. Finally, EVs were concentrated to a final volume of about 40-80 pL using Amicon centrifugal filters by centrifugation at 4000xg for 20 min. followed by a wash with 2 mL of pgB buffer under the same conditions to remove residual salts. The concentrated EVs were then subjected to downstream analyses.

[0158] Extracellular Vesicles Isolation from the Urine of Bladder Cancer Patients

[0159] Urine samples from BC patients were collected with patient's consent under the guideline of IRB protocol (MGB# 18-012 and 18-469) in a sterile container at Massachusetts General Hospital. The collected urine samples were stored in small aliquot at -80 °C until use. All preprocessing and isolation steps were performed at 4 °C unless otherwise specified. To isolate EVs, 1.2 mL of each urine sample was thawed on ice and centrifuged at 300xg for 5 min to remove urinary cells and cell debris. The supernatants were collected and centrifuged at 2000xg for 30 min. The supernatants were transferred to 1.5 mL Eppendorf to Attorney Docket No. 29539-0843WO1 isolate the EVs and placed on ice until further use. The pellet was gently dissolved by adding 200 pL of Dithiothreitol (DTT, 200 mg / mL) and incubated at 37 °C for 10 min.

[0160] The DTT released the EVs from the polymerized Tamm-Horsfall protein pellet (also known as uromodulin) by reducing the disulfide bond[611, thereby facilitating the release of entrapped EVs. The samples w ere centrifuged at 12000xg for 10 min to collect the released, non-purified EVs. The two fractions obtained were pooled and concentrated to about 500 pL (volume may vary) by centrifugation at 4000xg for 20 min at room temperature using Amicon 2 mL centrifugal filters. To remove excess DTT and excess salt contents, the collected solution was w ashed with 2 mL of IxPBS for subsequent isolation by SEC or with pgB buffer for EV isolation by the pgB method. Finally, the samples were processed according to the respective isolation protocols as described above.

[0161] Raman Spectroscopy

[0162] The chemical composition of chitosan and chitosan grafted poly-L-lysine pgBs w as characterized using a Raman spectrometer (XploRA™ PLUS, Horiba Scientific) at the Center for Nanoscale Systems (CNS) core facility, Harvard University. Measurements were performed using a near-infrared laser (785 nm, 41.8 mW and 600 diffractions grating). At least ten pgBs from each sample w ere used to acquire the spectral range from 0 to 2000 cm'1. For each pgB, the average spectrum was recorded at three different positions w ith an acquisition time of 5 sec.

[0163] Scanning Electron Microscopy (SEC)

[0164] The surface morphology of chitosan grafted poly-L-lysine pgBs, EVs captured on chitosan grafted poly-L-lysine pgBs and EVs isolated via SEC and pgBs were analyzed using a scanning electron microscope (Zeiss FESEM Ultra Plus) at the Center for Nanoscale Systems (CNS) core facility, Harvard University. For each sample, a drop of about 20 pL was loaded onto a coverslip (BRAND® cover glasses 18 x 18 mm). The samples w ere vacuum dried under 2xl0'3mbar and subsequently coated with a 5 nm pt / pd layer using a sputter coater (EM Quorum-EMS 150T S). After preparation, the samples were mounted onto a sample holder for imaging at varying resolutions.

[0165] Transmission Electron Microscopy

[0166] The morphology and purity of isolated uEVs were assessed using a transmission electron microscope (TEM) at the Microscopy Core of the Center for Systems Attorney Docket No. 29539-0843WO1

[0167] Biology / Program in Membrane Biology, Massachusetts General Hospital. For each sample, a formvar (polyvinyl formal) coated 100 mesh grid (EMS, Hatfield, PA) was placed on top of the EVs suspension and left for 5 min. The grids were carefully picked up and an excess solution was soaked up with Whatman filter paper. The grids were then quickly contrast- stained with 0.45 pm sterile-filtered 1% uranyl acetate. Excess uranyl acetate was immediately wicked up with the filter paper. The images were acquired with the JEM- 1400 flash transmission electron microscope (JEOL USA, Inc.) at 80 kV equipped with an AMT digital camera (Danvers, USA).

[0168] Zeta Potential

[0169] The surface charge of chitosan pgBs, chitosan grafted poly-L-lysine pgBs, isolated EVs, and EVs captured on chitosan grafted poly-L-lysine pgBs was characterized using a Zetasizer (Zetasizer Pro, Malvern Panalytical) at the Center for Nanoscale Systems (CNS) core facility' at Harvard University, as well as an in-lab nanoparticle analyzer (SZ-100V2 series, Horiba Scientific). A folded capillary zeta cell was used in conjunction with the Zetasizer Pro, while a disposable zeta cell with 6 mm carbon electrodes was used for measurements with the SZ-100V2 nanoparticle analyzer. For each sample, three independent measurements were taken at 10 sec intervals to ensure consistency of measurements.

[0170] Nanoparticle Tracking Analysis

[0171] The size distribution and concentration of the EVs isolated were measured with nanoparticle tracking analysis (NanoSight LM10, Malvern Panalytical). Samples were diluted in 1 xPBS and / or pgB buffer at various volume ratios. Samples were then loaded into the NanoSight viewing unit using a 1 mL syringe (BD™ Slip Tip Syringe sterile). Three measurements were taken at different times, and a 30 sec video was recorded with camera level: 12 and detection thresholds for each measurement. Finally, the videos were processed with the integrated software (NTA 3.4).

[0172] Immunoblotting

[0173] EVs were lysed in IxRIPA lysis buffer supplemented with Ixprotease inhibitors. To reduce disulfide bonds, the lysed samples were mixed with a final concentration of 1% Laemmle sample buffer containing 2-mercaptoethanol (350 mM) and heated to 95 °C for 5 minutes. Protein concentrations were quantified, and approximately 3-7pg of total protein per sample was resolved on precast 4-20% gradient SDS-PAGE gels under reducing conditions. Attorney Docket No. 29539-0843WO1

[0174] Electrophoresis was conducted at a constant voltage of 120V. The gels were then transferred to methanol-activated poly vinylidene difluoride (PVDF) membranes at 250 mA for 3 hr. Transferred membranes were blocked with 5% BSA (prepared in IxTris-buffered saline with 0.1% Tween® 20 (TBST)) for 45 mm.

[0175] Subsequently, the PVDF membranes were incubated overnight with the primary7antibodies at a dilution of 1 :500 (prepared in 5 % BSA containing 0.02% sodium azide). The following primary antibodies were used in this study: CD9. CD63, HSP70. Calnexin and Alix. On the following day, membranes were washed three rimes with IxTBST buffer and incubated for one hour with HRP -conjugated secondary7antibodies at a dilution of 1:5000: Anti-rabbit IgG and / -or Anti-mouse IgG depending on the source of the primary antibody. The membranes were washed three times with IxTBST, and the proteins were detected with three different chemiluminescent substrates (Pierce™ ECL Western Blotting Substrate, SuperSignal™ Western Blot Enhancer, and SuperSignal™ West Atto Ultimate Sensitivity7Substrate), chosen based on protein abundance. The images were taken on autoradiography films in the darkroom with different exposures.

[0176] RNA Extraction

[0177] RNA was extracted from about 1 x 105cells using the RNeasy Micro Kit. Cells were lysed in and homogenized in RLT lysis buffer by vertexing for 1 min. Subsequently, 75 pL of 70% ethanol was added and the mixtures were transferred to the RNeasy column. Centrifuged at 8000xg for 15 sec and discarded the flow through and DNase treatment was performed following the manufacturer's protocol. The purified RNA was collected in about 10 pL of RNase-free w ater and quantified using NanoDrop. All materials were used as supplied with the RNeasy isolation kit.

[0178] The isolation of RNA from EVs was carried out using the MagMAX™ mirVana™ Total RNA Isolation Kit, which uses a magnetic bead-based purification format. For each sample, 200 pL of lysis mixture (99 pL Lysis Buffer + 100 pL isopropanol + 1 pL f}- mer captoe thanol) was added, thoroughly mixed and incubated for 5 min at room temperature. The lysates were transferred to a 96-well plate and mixed with magnetic beads and binding enhancers. DNase treatment was then performed according to the manufacturer's protocol. The purified RNA w as eluted in pre-heated elution buffer according to the instructions given in the protocol. All materials w ere used as supplied with MagMAX mirVana Total RNA Isolation Kit. Attorney Docket No. 29539-0843WO1

[0179] One-Step Reverse Transcription and ddPCR

[0180] RNA transcript levels in cell and / or EV RNA were analyzed by droplet digital polymerase chain reaction (ddPCR) using the One-Step RT-ddPCR Advanced Kit for Probes. The predesigned primers / probes, as shown in Table 1 below, were used for each target gene (Integrated DNA Technologies, IDT). Each reaction was prepared with a volume of 20 pL in 96-well plates and comprised of isolated RNA, 500 nM primer / probe (final concentration) at a ratio of 4: 1 (primer: probe). 5 pL supermix, 15 mM DTT (final concentration) and 2U / pL reverse transcriptase. For ddPCR assays with BC molecular subtypes, a 1 ng / pl concentration of RNA from basal (HT-1376) and luminal (UMUC-1) cells was used. For EVs from HT- 1376 and UMUC-1 cell lines, RNA was extracted from EVs isolated from 30 mL of CCM and subsequently loaded according to the experimental conditions. For urine samples from BC patients, RNA extraction was performed from EVs isolated from 1.2 mL of urine using pgBs and / or SEC, with the extracted RNA loaded according to the experimental protocol.

[0181] Table 1. Attorney Docket No. 29539-0843WO1

[0182] Droplets were generated in the automated droplet generator (QX200 AutoDG, BioRad) and then amplified in the Cl 000 Touch Thermal Cycler (Bio-Rad) following the manufacturer's protocol. The amplified droplets were analyzed in a droplet reader (QX200 Droplet Reader, Bio-Rad) and analyzed using system-integrated software (QX Manager Software, Bio-Rad). All materials were used as supplied with the One-Step RT-ddPCR Advanced Kit for Probes.

[0183] Imaging Microgel Beads pgBs were imaged using a brightfield microscope (Nikon Eclipse Ti integrated with v711 camera). A clear, double-sided polyethylene film with a thickness of about 100 pm was used to make an observation chamber on a glass slide (Superfrost® Plus Microscope Slides, 25 mm x 75). A drop of 5-10 pL was pipetted onto the slide within the confined region and covered with a coverslip (BRAND® Coverslips, 18 x 18 mm). Images were taken with different fields of view, and the size distribution was calculated with ImageJ. An Epifluorescence microscope (Nikon Eclipse Ti) was used to visualize PT-PC3 derived EVs captured on chitosan grafted poly-L-lysine pgBs.

[0184] Statistical Analysis

[0185] Whenever possible, data are presented as mean ± standard deviation N=3). Statistical significance between two groups was determined using a two-tailed, unpaired t-test. For comparisons involving more than two groups, either a one-way ANOVA or a two-way ANOVA with correction for multiple comparisons was used. A P-value < 0.05 was considered statistically significant.

[0186] Example 2 - Continuous Droplet Microfluidics for Tunable Surface Charged Microgel Beads

[0187] Droplet microfluidics provides a promising platform to generate water-in-oil droplets wi th a high degree of uniformity. In this study, a continuous droplet microfluidic device was used to produce microgel beads (pgBs) with an average diameter of about 55 pm, achieving a production throughput of about 60,000 beads per minute. This approach allows for efficient and controlled fabrication of pgBs, which are shelf-stable for up to five months when stored in a vial at 4 °C. Specifically, two distinct types of pgBs were formulated with two different ty pes of building blocks: chitosan (CS) and chitosan grafted poly-L-lysine (CS-grafted-PLL). Attorney Docket No. 29539-0843WO1

[0188] For the fabrication of chitosan pgBs, CS (MW: 190-310 kDa, 75-85% deacetylated) was used as a precursor, whereas for hybrid chitosan grafted-poly-L-lysine pgBs, the precursor solution consisted of chitosan and poly-L-lysine (MW: 150-300 kDa). To synthesize chitosan pgBs, a 1 % acetic acid solution was first prepared, into which chitosan was gradually added until fully dissolved. To prevent clogging in the microchannels, the resulting solution was stirred for 4 hr at room temperature, then sonicated in a water bath for 30 sec. The precursors were drawn into microfluidic droplet devices, and a water-in-oil (W / O) emulsion containing the precursor solution was formed at the junction (FIGs. IB and 1 C). To generate stabilized and uniformly sized droplets, HFE-7500 oil supplemented with 2% w / v fluorinated surfactant was used

[0038] . It has been previously shown that various chemical crosslinkers and ionic crosslinkers were used to crosslink the chitosan and poly-L-lysine precursors[39-40]. Monoaldehyde (formaldehyde) was used as a crosslinking agent to initiate the transient interaction in the W / O emulsion followed by heating at 60 °C for 30 min to facilitate gelation. It was shown (FIG. 2C and FIG. 3D) that mono-aldehyde is capable of inducing gelation in chitosan and in mixtures of chitosan and poly-L-lysine precursors by imine cross-linking, resulting in the generation of monodisperse and highly stable chitosan and chitosan grafted-poly-L-lysine pgBs.

[0189] Example 3 - Fine Tuning of Microgel Bead Composition and Surface Charge

[0190] Fine-tuning the composition and surface charge of microgel beads is crucial, as these factors directly influence their stability, reactivity and interaction with biological or chemical environments. This precise control enables the production of materials that respond to specific external stimuli, such as changes in pH.

[0191] Composition and surface charge modulation through cross-linking chemistry

[0192] Droplet microfluidics-based pgB formulation offers versatility in customizing the composition of pgBs|41L Unique pgBs were developed with a tunable precursor composition that exhibit dynamic surface charge responses to pH as an external stimulus. To this end, 1.5% (w / v) chitosan was used to formulate chitosan pgBs by covalent crosslinking with 5% (v / v) mono-aldehyde (FIG. 2A). resulting in stable pgBs (FIG. 2C) after demulsification. The mono-aldehyde was chosen due to the simple reaction mechanism, which involves the formation of a Schiff base via a covalent bond between the amine group of the chitosan and the aldehyde group of the mono-aldehyde. This leads to a more uniform cross-linking and is expected to retain a significant amount of free amine groups (-NH2)

[0039] . The typically used dialdehyde (glutaraldehyde) crosslinker forms intermol ecul ar bridges due to the presence of Attorney Docket No. 29539-0843WO1 two aldehyde groups (FIG. 7A), thereby leading to over-crosslinking of the chitosan chains. However, it was found that the morphology of chitosan pgBs cross-linked with mono- and dialdehyde was the same (FIG. 2C; FIG. 7B), but a slight size reduction (about 8% in average diameter) is observ ed (FIG. 2D; FIG. 7C), when crosslinked with di-aldehyde. This can be explained by the fact that di-aldehyde can form strong, densely cross-linked networks that can lead to tight packing of pgBs.

[0193] In addition, the pH-responsive behavior of mono-aldehyde cross-linked chitosan pgBs under varying external pH conditions was assessed. For this purpose, the buffer pH was adjusted to 3.0, 6.0, 9.0, and 12.0, and the chitosan pgBs were incubated overnight at 4 °C. The following day. both morphology' and size distribution were characterized for each pH condition using bright filed microscopy. It was found that the alkaline conditions (pH 9.0 and 12.0) significantly influenced the size and stability of the chitosan pgBs. A size reduction of 32% and 55 % (FIG. 8A) was observed at pH 9.0 and 12.0, respectively. This is attributed to the deprotonation of the amino groups, which enhances hydrophobic interactions and induces the collapse of the chitosan chains, resulting in the shrinkage of the CS pgBs as water is expelled.

[0194] Under strongly alkaline conditions (pH 12.0), degradation of chitosan pgBs is also observed. The zeta potential (^-potential) was then measured for each pH value. As show n in FIG. 2E, the ^-potential increased when the pH was lower than the acid dissociation constants (pKa)

[0042] and increased to a value of +34.73 ± 1.88 mV. indicating the protonation of the free -NH2 group in a highly acidic environment (pH 3.0). The trend decreased when the pH was increased above the pKa limit, resulting in a ^-potential of -21.8 ± 2.30 mV in a strongly basic environment (pH 12.0). This shift is primarily attributed to the deprotonation of -NH3+groups, alongside the hydroxyl group (-OH) functionalities inherent to the chitosan structure.

[0195] In addition, there was an abrupt change in ^-potential from positive to negative (+16.23 ± 2.68 to -4.31 ± 1.18 mV) as the pH transitioned from pH 9.0 to 12.0. These results show that even the chitosan pgBs fabricated with mono-aldehyde have a ^-potential below the stability’ limit, except at pH 3.0. A ^-potential greater than +30 mV or less than -30 mV is generally considered stable against aggregation due to charge stabilization attributable to electrostatic repulsion in colloidal systems143(

[0196] Given that di-aldehyde cross-links the chitosan with a substantial amount of -NH21441. this behavior w as confirmed by measuring the ^-potential of chitosan pgBs fabricated in dialdehyde (FIG. 2E; FIG. 7D). The chitosan pgBs resuspended in buffer at pH 6.0 exhibited a 5.04-fold drop in ^-potential compared to those cross-linked in mono-aldehyde. Thus, Attorney Docket No. 29539-0843WO1 considering the limited pH working range and the ^-potential below the stability threshold, it might be concluded that the use of chitosan pgBs with biofluids such as urine is excluded, as pH plays a crucial role in maintaining the stability of biomolecular components such as proteins and RN A|45'461.

[0197] To mitigate this limitation, the design and fabrication of hybrid pgBs, achieved by grafting poly-L-lysine onto chitosan moieties to create chitosan grafted poly-L-lysine structure is presented herein. In this hybrid system, poly-L-lysine functions as the co- polymeric component, contributing unique structural and functional properties to the hybrid material!47!. This synergy' between chitosan and poly-L-lysine leads to an increase in surface charge due to the dense structure of the -NH2 group and the ionic interaction between chitosan and poly-L-lysine. Crosslinking of chitosan and poly-L-lysine via a mono-aldehyde crosslinker may yield a dense and heterogeneous network (FIG. 2B), potentially comprising diverse interaction patterns such as chitosan-chitosan and chitosan-poly-L-lysine linkages, each exhibiting distinct spatial orientation.

[0198] A statistically significant (p < 0.05) alteration in ^-potential (FIG. 3A) was observed when 1.5% v / v chitosan was supplemented with poly-L-lysine at concentrations of 0.01%. 0.025%, and 0.05% v / v, compared to the chitosan precursor alone. Notably, the addition of 0.05% v / v poly-L-lysine into 1.5% w / v chitosan increased the ^-potential by about 2.37-fold relative to 1.5% chitosan. To further elucidate the modulatory role of poly-L-lysine, chitosan grafted-poly-L-lysine pgBs were fabricated via droplet-assisted microfluidics using a fixed chitosan concentration (1.5% w / v) and varying poly-L-lysine concentrations (0.01 %, 0.025%, and 0.05% v / v), as described before. The ^-potential of pgBs fabricated by grafting 0.01, 0.025 and 0.05 % v / v poly-L-lysine into 1.5 % v / v chitosan at pH 6.0 was measured 1.09, 1.95 and 2.34-fold higher (FIG. 2E; FIGs. 3B and 3C), respectively, compared to chitosan pgBs fabricated with 1.5 % v / v chitosan. Therefore, further fabrication of pgBs were carried out with 1.5% v / v chitosan in combination with 0.05 % v / v poly-L-lysine, as the higher L- potential indicates higher stability and increases the applicability of pgBs in clinical samples having acidic to alkaline pH values such as urine1 ,21and saliva1'1L The chitosan grafted poly- L-lysine pgBs exhibited a morphology comparable to that of chitosan pgBs, with an average diameter of about 56 pm (FIGs. 3D, 3E). To confirm the grafting of poly-L-lysine in chitosan grafted poly-L-lysine pgBs (FIG. 9), Raman spectroscopy was performed. In the crosslinking reaction, the amide bond (-CONH) is formed between the amino group and the aldehyde group. In chitosan pgBs. the peak around 1650 cm'1(amide I) is a characteristic stretching of the carbonyl bond (-C=O), while the peak around 1550 cm’1(amide II) is an N- Attorney Docket No. 29539-0843WO1

[0199] H bending and a C-N stretching!48’491. The dense methylene bridges (-CH2) formed by the mono-aldehyde cross-linking result in C-H bending vibrations. These peaks are typically found between 1300-1450 cm’1and reflect the newly formed bonds between the chitosan molecules1491. The difference in the intensity7of the peaks with and without poly-L-lysine grafting provides semi-quantitative information. The higher peak intensity of amide I and II reflects additional -CONH bonds introduced by poly-L-lysine. Moreover, the peak shift of amide bonds (1649 to 1639 cm’1and 1553 to 1550 cm’1) can be attributed to the additional stretching of -C=O caused by the presence of two polymeric structures. The higher peak intensity and the significant shift from 1375 to 1369 cm’1of the methylene bridges are explained by changes in electron density' or steric interactions^01caused by the grafting of poly-L-lysine into chitosan grafted poly-L-lysine pgBs.

[0200] Influence of cross-linker concentration and curing time on microgel bead properties

[0201] The impact of crosslinker concentration and curing time on morphology', shrinkage, and especially on the surface charge of the pgBs was also investigated. For this purpose, chitosan grafted poly-L-lysine pgBs were synthesized with different crosslinker concentrations (2.0, 5.0, 7.5 and 10.0 % of mono-aldehyde). For each crosslinker concentration, three different curing times (0.5, 2.0 and 12 hr) were tested to evaluate its effects on the pgBs fabrication (FIG. 10), particularly the surface charge (FIGs. 3F-3H). It was found that 2.5% monoaldehyde could not crosslink the pgBs at curing times of 0.5 and 2 hr. Also, the chitosan grafted poly-L-lysine pgBs concentration was determined to be under 500 beads / mL, thereby leading us to assume that the ^-potential under these conditions is 0 mV. Extending the cross-linking duration to 12 hr facilitated the formation of pgBs; however, a significant depletion in ^-potential (FIG. 3H) to -37.63 ± 1.36 mV (at pH 6.0) was observed. This decrease is likely attributable to the complete depletion of free -NH2 groups, thereby inhibiting subsequent protonation and culminating in the manifestation of a net negative surface charge.

[0202] Furthermore, augmenting the cross-linker concentration to 5.0%, 7.5%, and 10% did not elicit any discernible alterations in the morphology and density of chitosan grafted poly- L-lysine pgBs under the specified experimental conditions. However, curing time emerged as a pivotal determinant in preserving the positive surface charge on pgBs. Extending the curing duration together with a simultaneous increase in the crosslinker concentration significantly- depleted the availability of free -NH2 groups. Consequently, it can be inferred that a crosslinker concentration of 5% together with a curing time of 30 min represents the optimal Attorney Docket No. 29539-0843WO1 conditions for the fabrication of chitosan grafted poly-L-lysine pgBs, yielding the highest positive ^-potential of +39.3 ± 2.30 mV. In addition, high-throughput water-in-oil (W / O) droplet formation was found to exert a substantial influence on surface charge.

[0203] Specifically, using a 1.5 mL Eppendorf tube prefilled with 500 pL of cross-linker solution, it required approximately 30-35 min to reach a final volume of 1.2 mL, with a droplet generation rate of 30,000 droplets per minute, resulting in the formation of about 0.5 to about 1 x 106chitosan grafted poly-L-lysine pgBs. Under these conditions, the ^-potential of chitosan grafted poly-L-lysine pgBs at pH 6.0 was determined to be 38.43 ± 3.57 mV. By integrating two microfluidic droplet generators in parallel, the fabrication time was reduced by a factor of 1.75, while the ^-potential showed a 1.25-fold increase (FIG. 4A).

[0204] Example 4 - pH-responsive Properties of Chitosan Grafted Poly-L-lysine Microgel Beads

[0205] The chitosan grafted poly-L-lysine pgBs demonstrated a markedly enhanced pH- responsiveness (FIG. 2E; FIG. 4B) in comparison to the chitosan pgBs. Specifically, at pH 3.0 and 6.0, the ^-potential of chitosan grafted poly-L-lysine pgBs were 1.84- and 2.94-fold higher, respectively, than those of chitosan pgBs. The most pronounced disparity' was observed at pH 9.0, where chitosan grafted poly-L-lysine pgBs exhibited a positive - potential of 36.57 ± 2.2 mV, in contrast to the negative (^-potential) of -4.31 ± 1.18 mV observed for chitosan pgBs. At a concentration of 0.05% w / v PLL. a significant fraction of the -NH2 groups in chitosan grafted poly-L-lysine pgBs remains protonated within an acidic buffer environment. Upon shifting to pH 9.0, the deprotonation of -NHv groups remain incomplete, leading to the retention of a net positive surface charge due to the pKa of poly-L- lysine being approximately 9.0

[0047] . At pH 12.0, chitosan grafted poly-L-lysine pgBs exhibit a 2.4-fold greater negative ^-potential compared to chitosan pgBs. This enhancement is likely attributed to the extensive deprotonation of the abundant-NH , groups and the increased susceptibility of -OH groups to deprotonation under strongly basic conditions. Overall, it can be concluded that poly-L-lysine concentration, crosslinker concentration, curing time and high-throughput droplet formation are key factors for maintaining the high surface charge of chitosan grafted poly-L-lysine pgBs. The surface morphology' of chitosan grafted poly-L- lysine pgBs was analyzed through scanning electron microscopy (FIG. 5B), revealing a Attorney Docket No. 29539-0843WO1 highly cross-linked three-dimensional (3D) structure. The pgBs pore size was found to about 1 pm in diameter.

[0206] Example 5 - Effect of Alkaline Buffers on Chitosan Grafted Poly-L-lysine Size

[0207] To assess any shrinkage of the pgBs in response to buffer pH (FIG. 8B), the pgB size relative to buffer alkalinity was measured. Compared to acidic conditions (pH 3.0), the average diameter of chitosan grafted poly-L-lysine pgBs decreased by approximately 11% at pH 9.0 and 33% at pH 12.0, indicating pH-dependent structural contraction. The chitosan grafted poly-L-lysine pgBs appear to be degraded at high alkaline buffer conditions, and the same effect was observed for chitosan pgBs (FIG. 8A). It is also found that the percentage shrinkage of chitosan grafted poly-L-lysine pgBs at pH 9.0 and 12.0 was significantly lower compared to chitosan pgBs. Specifically, the mean diameters of chitosan grafted poly-L- lysine pgBs at these pH values were 47.87 pm and 36.04 pm, corresponding to a 22% and 32% reduction in shrinkage, respectively, compared to chitosan pgBs under identical buffer conditions. This is likely due to the dense cross-linking and greater number of methylene bridges in chitosan grafted poly-L-lysine pgBs

[0050] .

[0208] These structural refinements provide the system with significantly improved robustness compared to chitosan pgBs, as evidenced by extended pH stability over a wide range, significantly reduced volume contraction and, most importantly, a pronounced increase in positive surface charge. Remarkably, the surface charge of chitosan-grafted poly- L-lysine pgBs remained stable over a five-month period (FIG. 11 ) and exhibited a sustained 1.5-fold increase over baseline measurements. This sustained increase is thought to be due to a dynamic interplay between protonation equilibria and matrix swelling phenomena, which together contribute to superior electrostatic stability and surface charge density.

[0209] Example 6 - Isolation of Extracellular Vesicles (EVs) from Biofluids

[0210] In EV isolation, pH critically regulates extracellular vesicle stability by modulating cargo preservation and surface charge dynamics

[0051] . Variations in pH alter zeta potential, affecting vesicle colloidal interactions, promoting aggregation, and compromising isolation efficiency, thereby fundamentally influencing EV function and analytical precision.

[0211] Optimization of pH-driven EV cargo stability

[0212] To identify the optimal pH conditions for EV isolation while preserving their unique charge properties-including surface and cytosolic proteins as well as RNA content, a range of Attorney Docket No. 29539-0843WO1 pH conditions was systematically evaluated. For this purpose, CCM derived from palmitoylated prostate cancer (PT-PC3) cells was used. Urine constitutes a biologically rich biofluid, harboring EVs derived from bladder tissue, the urethra, prostate, and renal epithelial cells making it a valuable source of clinically significant biomarkers[8, 52]. To test the impact of processing parameters, such as pH, on EV cargo, EVs were carefully isolated from PT- PC3 CCM using size exclusion chromatography (SEC), a gold standard method for EV isolation (FIG. 12A). The isolation process was performed in pgBs buffer (as described in Example 1 - Materials and Methods) over a pH range of 3.0, 6.0, 9.0 and 12.0, with Ixphosphate-buffered saline (PBS, pH 7.4) serving as the physiological control. The potential of the isolated EVs at each pH condition showed that their surface charge remained consistently negative even in an acidic environment (FIG. 4C). This sustained negative charge is primarily attributed to the presence of anionic phospholipids, particularly phosphatidylserine[261, which contribute to the stability and biophysical properties of the vesicle membrane. pH influence on EV surface charge and diameter

[0213] The data (FIG. 4C) indicate that the EV surface charge remains within the colloidal stability7threshold (^-potential < -30 mV) across a broad pH range (6.0, 9.0, and 12.0), suggesting stable dispersion under these conditions. In contrast, exposure to strongly acidic conditions (pH 3.0) results in a less negative ^-potential (about -16 mV), indicating compromised suspension stability and unsuitability for EV isolation. Furthermore, the effects of pH on EV size distribution and isolation yield were investigated (FIG. 12B,12C). Nanoparticle tracking analysis (NT A) revealed variations in both average EV size and yield at different pH values. An increase in EV size was observed under highly acidic conditions (pH 3.0), highlighted by a marked peak at about 270 nm. This is likely due to reduced colloidal stability7causing EV aggregation. This is supported by ^-potential measurements of EVs at pH 3.0 (FIG. 4C), which show values below the stability' threshold.

[0214] Influence of pH on EV RNA and protein cargo

[0215] Comprehensive downstream analyses of EV RNA and proteins across varying pH conditions were performed. RNA was extracted and quantified using automated electrophoresis (FIG. 12D), followed by droplet digital PCR (ddPCR) to evaluate the expression canonical housekeeping genes[53-551(EGFR, ACTB, GAPDH) (FIG. 12E). Immunoblotting (FIG. 12F) validated protein expression, with CD9 and CD63 as surface and Attorney Docket No. 29539-0843WO1

[0216] HSP70 and Alix as cytosolic markers (FIG. 12F). The results demonstrate that EV RNA and proteins remain stable at pH 6.0. whereas alkaline conditions impair RNA integrity via phosphodiester bond hydrolysis, corroborating with previous findings

[0056] . In addition, normalized RNA expression was significantly reduced at a pH of 3.0, presumably due to increased vulnerability to acid hydrolysis

[0045] . Furthermore, the attenuation of protein expression, especially at pH 9.0 and 12.0, emphasizes the profound impact of pH on differential stability

[0046] . Collectively, these findings establish that a slightly acidic environment (pH 6.0) optimally preserves EV integrity, yield, and cargo stability7.

[0217] Example 7 - Microgel Bead-Assisted Capture of Extracellular Vesicles

[0218] The disclosed charge-responsive microgel bead system, for the selective isolation of EVs from biofluids, leverages the intrinsic surface potential of their lipid bilayers. Precise pH modulation enabled efficient EV capture and downstream molecular cargo profiling. Validation in conditioned media demonstrates strong translational promise for clinical diagnostics and therapeutic monitoring.

[0219] Evaluation of chitosan grafted poly-L-lysine microgel beads for selective isolation of EV from cell culture media

[0220] To assess the efficacy of newly synthesized chitosan grafted poly-L-lysine pgBs. contrived samples (i.e. EVs isolated from cell cultured media) were first utilized to evaluate their capture efficiency (FIG. 5 A). For these experiments, 10 mL of cell culture media w as concentrated to about 500 pL prior to incubation with pgBs (as detailed in Example 1 - Materials and Methods). It w as estimated that the efficient capture of about IxlO10EVs (mean diameter about 200 nm) would require a minimum of about 1.3xl05pgBs (mean diameter about 55 pm). To ensure excess binding capacity and accommodate the presence of lEVs (>200 nm)[1’371, the total bead count was scaled to 0.5 x 106pgBs. After the EVs were placed in the same vial as the pgBs, incubation was carried out under gentle rocking for 1 hr at room temperature, followed by three sequential washes with pgB buffer to remove unbound particles with comparatively lower negative surface charge. The efficiency of EV capture was qualitatively assessed by measuring the ^-potential of pgBs captured EVs (hereafter referred to as pgBs-EVs) (FIGs. 4D, 4E).

[0221] The pgBs-EVs showed a 132.73% depletion of the ^-potential compared to pgBs (+55 mV), clearly validating the highly efficient capture of EVs. Additionally, bright field and fluorescence microscopy (FIG. 13) was used to confirm EVs capture on pgBs. Given that Attorney Docket No. 29539-0843WO1

[0222] EVs derived from PT-PC3 CCM exhibit intrinsic red fluorescence (excitation at 554 nm, emission at 581 nm). The presence of a strong red fluorescence signal on pgBs indicates a robust charge interaction between the negatively charged EVs and the positively charged pgBs. In contrast, pgBs devoid of EVs did not exhibit detectable fluorescence (FIG. 13B) under identical imaging conditions. Furthermore, the successful capture of EV on pgBs at the nanoscale was validated using scanning electron microscopy (SEM). The SEM analysis (FIG. 5C; FIG. 14) provided detailed morphological evidence of EVs attached to the pgBs. revealing a significant population of both individual EVs and clustered assemblies per pgB.

[0223] Molecular profiling of extracellular vesicles isolated using microgel beads

[0224] EVs captured on pgBs are released by neutralizing the charge interactions with IM sodium chloride (final concentration)1571and agitating for 30 min at room temperature. The released EVs are collected by centrifugation at 3000xg and characterized according to MISEV2023 guidelines1371. NTA analysis shows a about 30% higher EV yield compared to SEC (performed using the same starting volume of CCM), with a greater presence of sEVs (<200nm) and lEVs (>200 nm) (FIGs. 5F, 5G), which is consistent with the results of SEM analysis (FIGs. 5C-5E). Morphological analysis confirms that the pgBs preserve the native structure of EVs and comparable EVs isolated with SEC, demonstrating their suitability for the isolation of EVs from complex biofluids. Immunoblotting (FIG. 5H) was performed with canonical markers for EVs, including the tetraspanins CD9 and CD63, the cytosolic protein HSP70 and calnexin as a negative control indicating contamination of the endoplasmic reticulum.

[0225] The EV fractions showed significantly elevated band intensities for CD9, CD63 and HSP70 and an absence of calnexin compared to the corresponding whole cell lysates, confirming both the successful enrichment and the specificity of the isolated EVs. Notably, EVs isolated using pgB platform showed substantially higher expression levels of CD9 and CD63 compared to those isolated by SEC, highlighting the superior yield and enrichment efficiency offered by pgB method. Finally, EV recovery’ (FIG. 15) was rigorously assessed by incubating three discrete concentrations of purified EVs (5.1x10s, 7.2x10s, and 1.3xl09) with a fixed quantity of 0.5x10spgBs. Quantification of both bound and unbound EV populations was performed using NTA. Under all tested conditions, the system consistently exhibited high performance, with both capture and recovery efficiencies averaging approximately 90% underscoring the strong binding affinity’ and scalability of the pgB-based platform for robust and efficient EV isolation. Attorney Docket No. 29539-0843WO1

[0226] Example 8 - Microgel Bead-Assisted Isolation and Profiling of Urinary Biomarkers for Bladder Cancer Patient Samples pgB-based EV isolation provides an efficient, high-yield model for selectively capturing EVs. This method enables optimized recovery of both sEVs and lEVs, enhancing applications in molecular characterization and therapeutic research. Yet, the true translational potential lies in complex clinical biofluids such as urine, where EVs originate from different urogenital tract regions, including bladder, prostate and kidney. Urinary EV -based analyses have been explored for prognostic applications in patients undergoing treatment, but the molecular signatures that predict clinical outcome are still not well understood!581. A major challenge arises from the heterogeneous composition of urine, which includes non-EV contaminants such as proteins and metabolites, making it difficult to isolate and maintain the integrity of EVs for clinical use. In addition, the pH of urine varies from acidic to alkaline

[0059] , which significantly affects the yield, stability and molecular cargoes of EVs, including RNA and proteins that are often neglected in conventional isolation techniques1601. It is proposed herein that uEVs can serve as the foundation for a non-invasive prognostic platform. The improved yield of EV isolation and the presence of lEVs are expected to facilitate the correlation between highly expressed bladder cancer (BC) gene signatures and disease grade and stage, thus driving clinical applications.

[0227] To investigate this, a pilot study (FIG. 6B) with a cohort of five bladder cancer patient samples was conducted. Urine samples were obtained from patients diagnosed with BC at Massachusetts General Hospital in Boston. Patients consented under the guidelines of IRB protocol (MGB #18-012 and 18-469) with institutional oversight. All potential BC urine samples were collected from patients with a diagnosis of non-metastatic muscle-invasive bladder cancer, after the transurethral resection of bladder tumor (TURBT) procedure, but prior to initiation of definitive therapy. Urine was collected in a sterile container and aliquoted to small volumes before storage at -80°C prior to processing. EVs w ere isolated from 1.2 m of urine per patient sample. The urine pH ranged from 5.0 to 6.0 (FIG. 10), an acidic environment that facilitates EV isolation with pgBs by preserving the surface charge of the vesicles. To increase isolation efficiency, Tamm-Horsfall protein (THP / Uromodulin)1611. which is known to entrap uEVs and reduce sample purity, was enzy matically depolymerized (as described in the Materials and Methods). For each sample, about 0.5 x io6pgBs / mL were used for EV capture. For comparison, EVs were also isolated by SEC (FIG. 6A). NTA showed that the pgBs-based method resulted in an average 1.7-fold increase in EV efficiency compared to SEC (FIG. 6D). Of note, this approach consistently enriches sEVs (<200 nm) in Attorney Docket No. 29539-0843WO1 all patient samples, with significantly higher peak intensities than the SEC method. In addition, a distinct population of lEVs (>200 nm) was detected exclusively in pgBs-isolated fractions. Transmission electron microscopy (TEM) corroborated these results, confirming both the morphological integrity and the presence of a diverse range of EVs.

[0228] Representative TEM images (FIG. 6C) of pgBs-isolated uEVs revealed a heterogeneous population, including EVs with a size of about 450 nm. In contrast, the SEC- isolated EVs measure a size down to about 200 nm (FIG. 6E). Under optimized ionic conditions, non-EV impurities such as lipoproteins remained free in solution or carried an extremely low charge!62!, preventing their capture by pgBs. Low magnification TEM images showed minimal lipoprotein contamination in pgBs-isolated EVs, in stark contrast to SEC- isolated samples (FIG. 17). Furthermore, the pgBs-isolated samples exhibited a markedly higher abundance of EVs (FIG. 17 A), as evidenced by the increased density of vesicular structures.

[0229] To further validate the efficacy of the pgB method, the expression of EV markers was examined by immunoblotting (FIG. 6F). pgBs-isolated uEVs showed significantly higher levels of CD9. HSP70 and Alix than SEC-derived EVs. The elevated protein content in pgBs-isolated EVs, compared to both bulk urine and SEC-isolated EVs, can be attributed to both the increase in uEV yield and the substantial presence of large uEVs, which are known to carry a higher protein load.

[0230] The uEV RNA expression of four key genes with clinical significance in BC

[0063] was then analyzed. CK19 serves as a crucial marker of minimal residual disease, indicating active tumor progression. EGFR and KRT14 are highly expressed in basal BC subtypes, which are linked to aggressive tumor behavior and poor prognosis. In contrast, PPARG is characteristic of the luminal subtype, reflecting a hybrid molecular phenotype that may influence tumor progression and therapeutic response. These markers are essential for BC subtype classification, prognosis, and treatment stratification.

[0231] Prior to analyzing RNA level in uEVs, a validation workflow with BC cell lines representative of luminal and basal subtypes and their respective cell-derived EVs was established (FIG. 18A) using droplet digital PCR (ddPCR). Consistent with known BC molecular subtype characteristics, EGFR expression was markedly elevated in the basal subtype (HT-1376), whereas PPARG was predominant in the luminal subtype (UMUC-1), with an EGFR / PPARG ratio (FIG. 18A) of 5.48 and 0.98. respectively. Notably, EVs derived from these cell lines exhibited a similar trend, with EGFR / PPARG ratios of 7.48 in the basal Attorney Docket No. 29539-0843WO1 subtype and 0.62 in the luminal subtype. These findings support the clinical validity of these molecular markers for BC subtype differentiation.

[0232] Following successful validation of selected genes in cell-derived EVs, this approach was implemented in uEVs isolated from BC Patients. Comparative analysis of RNA expression levels (FIG. 6G) for the selected gene panel revealed a significantly higher signal when using the pgBs method compared to SEC. This improved response underscores the effectiveness of the technology descnbed herein, which was developed in clinical applications, and supports its potential as a non-invasive prognostic platform.

[0233] Example 9 - Future Directions and Clinical Utility

[0234] Our pgBs platform offers improved and rapid EV isolation from complex biofluids, including urine from BC patients, with high yield, purity, and integrity. Further, enhanced detection of RNA transcripts by ddPCR and stable EV protein expression observed through immunoblotting suggest that our approach preserves functional cargo stability7more effectively than traditional isolation protocols. uEVs serve as valuable biomarker reservoirs for non-invasive prognostics; however, clinical translation is still hampered by complex sample processing, pH variability, molecular charge stability and the challenge of high- throughput analysis with limited sample volume.

[0235] The current prognostic approaches for BC often rely on invasive methods

[0064] , including cystoscopy, biopsy, TURBT and lymph node biopsy, as they provide detailed and direct information about the stage, grade and extent of spread of the cancer. But real-time tumor surveillance of the molecular signature is riddled with challenges owing to the long waiting time for test outcomes. Recently , urinalysis-based methods have been considered to develop prognostic platforms for patients undergoing treatment, but there is limited knowledge of molecular signatures capable of accurately predicting clinical outcomes

[0058] . A preliminary7evaluation of the developed methodology7in clinically annotated urine samples from BC patients has shown promising BC-specific gene signature detection. Expanding the patient cohort with a focus on molecular subtyping of BC-associated genes might further increase the potential of this approach for the development of a non-invasive prognostic tool for cancer patients.

[0236] Example 10 - Conclusion

[0237] In conclusion, a versatile method to isolate EVs from biofluids was devised, including urine from BC patients, a step forward in developing a platform for non-invasive prognosis. Attorney Docket No. 29539-0843WO1

[0238] Microgel beads from biodegradable and eco-friendly polymers were developed and fabricated using high-throughput droplet microfluidics. This approach enables the formulation of uniform and stable microgel beads that are shelf stable (about 5 months at 4 °C) and can easily be accessible and used to isolate urinary' biomarkers and provide portable, device-free alternative for cancer prognosis.

[0239] This strategy also preserved the integrity of the EVs and maintained their unique charges in functional form, leading to higher expression of proteins and RNA for molecular selection. The rigorous downstream analyses performed show a 1.3-fold and 1.7-fold higher isolation yield in CCM and urine, respectively, along with higher purity' and a significant presence of lEVs. Using the pgB method, improved molecular profiling of bladder cancer- associated genes1651was achieve from minimal (about 1.2 mL) clinically annotated patient samples. Although clinical implications are still in their infancy, our data lay the foundation for the development of a non-invasive tool to monitor BC progression using uEVs. The expansion of the patient cohort to encompass diverse grades and stages of bladder cancer, along with the incorporation of additional cancer-associated genetic markers, will be critical for the development of a robust, non-invasive prognostic tool for cancer patients.

[0240] OTHER EMBODIMENTS

[0241] It is to be understood that while certain embodiments have been described within the detailed description, the present disclosure is intended to illustrate and not limit the scope of any embodiment defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

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Claims

Attorney Docket No. 29539-0843WO1WHAT IS CLAIMED IS:

1. A method of isolating extracellular vesicles (EVs) from a biological sample, the method comprising: contacting the sample comprising the EVs with a composition comprising microgel beads comprising chitosan-grafted poly-L-lysine (PLL). wherein the microgel beads capture the EVs via surface charge interactions, resulting in the formation of microgel beads-EVs complexes; and releasing the captured EVs from the microgel beads-EVs complexes, thereby isolating the EVs from the biological sample.

2. The method of claim 1, wherein the biological sample is contacted with the composition comprising microgel beads for at least about 1 hour (h).

3. The method of claim 1 or claim 2, wherein the composition comprises microgel beads at a concentration of about 0.5 x 106beads per milliliter (beads / mL) to about 1 x 106beads / mL.

4. The method of any one of claims 1-3, wherein the method achieves about 1.3 to about 1.7-fold higher yields than gold standard size exclusion chromatography.

5. The method of any one of claims 1-4, wherein the biological sample is a complex biofluid.

6. The method of claim 5, wherein the complex biofluid is one or more of blood, urine, saliva, cerebrospinal fluid, uterine lavage fluid, ascitic fluid, or a tear or lacrimal fluid sample.

7. The method of any one of claims 1-6, wherein the biological sample is urine.

8. The method of any one of claims 1-7, wherein the biological sample is cell culture media.

9. The method of any one of claims 1-8, further comprising purifying the microgel beads-EVs complexes by washing via centrifugation.Attorney Docket No. 29539-0843WO110. The method of any one of claims 1-9, wherein the releasing comprises adding a salt to the microgel bead-EVs complexes.

11. The method of claim 10, wherein the salt is sodium chloride.

12. The method of any one of claims 1-11, further comprising concentrating the released EVs via centrifugation.

13. The method of claim 12, further comprising washing concentrated EVs to remove residual salt.

14. The method of any one of claims 1-13, wherein the EVs are urinary EVs.

15. The method of claim 1, wherein the microgel beads-EVs complexes comprise small EVs with a diameter of about 65 nm to about 200 nm and large EVs with a diameter of about 200 nm to about 750 nm, and very large EVs, with a diameter about 750 nm to about 10 pm.

16. A composition comprising microgel beads, wherein the microgel beads comprise chitosan-grafted poly-L-lysine (PLL), wherein the microgel beads are crosslinked, and wherein the surface charge of the microgel beads is tunable based on the concentration of PLL, and wherein the microgel beads are capable of capturing and releasing one or more extracellular vesicles (EVs) via surface charge interactions.

17. The composition of claim 16, wherein the chitosan-grafted PLL comprises PLL at a concentration of about 0.01% to about 0.05% (v / v).

18. The composition of claim 17, wherein the chitosan-grafted PLL comprises PLL at a concentration of about 0.05% (v / v).

19. The composition of any one of claims 16-18, wherein the diameter of the microgel beads is about 50 microns (pm) to about 100 pm.

20. The composition of any one of claims 16-19, wherein the microgel beads exhibit a zeta potential ranging from about 30 millivolts (mV) to about 100 mV, measured at a pH between about 3.0 to about 9.0.Attorney Docket No. 29539-0843WO121. The composition of any one of claims 16-20, wherein the composition is shelf-stable for up to about one year.

22. The composition of any one of claims 16-21, wherein the EVs are urinary EVs.

23. A method of preparing a composition comprising microgel beads, the method comprising: providing a dispersed phase comprising chitosan-grafted poly-L-lysine (PLL) and a continuous phase comprising anon-aqueous fluid; introducing the dispersed and continuous phases into a channel of at least one microfluidic device comprising at least one junction; flowing the dispersed and continuous phases through the at least one junction thereby forming microdroplets; collecting the microdroplets in a crosslinker solution; and crosslinking the microdroplets by heating the microdroplets and the crosslinker solution, thereby generating the composition comprising pgBs.

24. The method of claim 23. wherein the dispersed phase comprises chitosan-grafted PLL dissolved in acetic acid.

25. The method of claim 23 or claim 24, wherein the dispersed phase comprises about 1% to about 2% (w / v) chitosan-grafted PLL.

26. The method of any one of claims 23-25, wherein the dispersed phase comprises about 1.5% (w / v) chitosan-grafted PLL.

27. The method of any one of claims 23-26. wherein the non-aqueous fluid comprises a fluorinated oil and one or more polymeric surfactants.

28. The method of claim 27, wherein the one or more polymeric surfactants comprise perfluoropolyether-poly ethylene glycol-perfluoropolyether triblock copolymer (PFPE-PEG-PFPE), PFPE 5k, PEG 900, PFPE 5k, or a combination thereof.Attorney Docket No. 29539-0843WO129. The method of claim 28, wherein the one or more polymeric surfactants are present at a concentration of about 1.5% to about 2.5% (w / v).

30. The method of claim 28, wherein the one or more polymeric surfactants are present at a concentration of about 2% (w / v).

31. The method of any one of claims 23-30. wherein the dispersed and continuous phases are introduced under controlled pressure conditions.

32. The method of claim 31, wherein the dispersed phase is introduced at a pressure of about 160 millibar (mbar) to about 240 mbar.

33. The method of claim 31, wherein the continuous phase is introduced at a pressure of about 80 mbar to about 120 mbar.

34. The method of any one of claims 23-33. wherein the formation of the microdroplets comprises forming microdroplets at a rate of about 30,000 droplets per minute to about 70,000 droplets per minute.

35. The method of any one of claims 23-34. wherein the crosslinker solution comprises mono-aldehyde, di-aldehyde, or a combination thereof.

36. The method of any one of claims 23-35, wherein the crosslinker solution has a concentration of about 2.0% (v / v) to about 10% (v / v).

37. The method of claim 36, wherein the crosslinker solution has a concentration of about 5% (v / v).

38. The method of any one of claims 23-37, wherein the composition comprising microdroplets and the crosslinker solution are heated to a temperature ranging from about 60 °C to about 70 °C.

39. The method of claim 38, wherein the composition comprising microdroplets and the crosslinker solution are heated for about 30 minutes (min.) to about 12 h.Attorney Docket No. 29539-0843WO140. The method of any one of claims 23-39, further comprising, after crosslinking, destabilizing the continuous phase encapsulating the microgel beads and separating the microgel beads from the continuous phase.

41. The method of any one of claims 23-40, wherein destabilizing the continuous phase comprises adding a demulsifier.

42. A method for detecting at least one cancer biomarker in a biological sample from a subject, the method comprising: obtaining the biological sample comprising extracellular vesicles (EVs); isolating the EVs from the biological sample using any of the methods described in claims 1-15; and detecting the presence of the at least one cancer biomarker comprising one or more of a protein, nucleic acid, lipid, or metabolite associated with cancer; wherein the detection of the cancer biomarker in the EVs is indicative of the presence or progression of cancer in the subject.

43. The method of claim 42. wherein the cancer is bladder cancer.

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