Light-induced extracellular vesicle and particle adsorption for label-free capture and quantification of molecular cargo

Abstract

Disclosed are devices, systems, and methods related to the characterization and / or analysis of extracellular particles (EPs) and interactions therewith. The devices, systems, and methods can include a micropatterned substrate defining a binding region and a passivation region. The binding region includes a photoetched charged surface to non-specifically adsorb a population of EPs from a sample.

Description

[0001] LIGHT-INDUCED EXTRACELLULAR VESICLE AND PARTICLE ADSORPTION FOR LABEL-FREE CAPTURE AND QUANTIFICATION OF MOLECULAR CARGO

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 560,984, filed March 4, 2024, and U.S. Provisional Application No. 63 / 734,374, filed December 16, 2024, each of which is incorporated by reference herein in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under Grant No. TR003807 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Extracellular particles (EPs) include a variety of heterogeneity of particles secreted by cells and present in different biofluids. For example, extracellular vesicles (EVs) are micro- and nanoscale lipid-enclosed packages that are released by all cells and are found in various biofluids. Other particles released by cells include lipoproteins, exomeres, supermeres, that can be present in biofluids. Due to their ability to relay bioactive cargo over short and long distances or deliver therapeutic payloads, there has been increasing attention directed to better understand their dynamic roles in health and disease. Current techniques for observing EPs often require the use of complementary characterization tools to classify EPs based on their surface antigens, internal contents, or even physical and mechanical properties. For example, single EV characterization using fluorescence requires efficient EV capture using antibodies, other specific ligands, or nonspecific lipoplexes to effectively concentrate EVs on surfaces. Thus, there is a need for a versatile platform using a tunable and nonspecific approach for EP micropatterning to enable the effective study of EVs.

[0008] SUMMARY

[0009] In accordance with the purposes of the disclosed materials, compounds, compositions, articles, devices, and methods, as embodied and broadly described herein, the disclosed subject matter relates to compositions and methods of making and using the compositions. In a specific aspect, disclosed herein is a micropatterned substrate defining a binding region and a passivation region, wherein the binding region comprises a photoetched charged surface to non-specifically adsorb a population of extracellular particles (EPs) (e.g., extracellular vesicles (EVs), lipoproteins, viruses, non-vesicular particles, etc.) from a sample.

[0010] In some aspects, the charged surface comprises a surface functionalized with a cationic polymer (e.g., a cationic protein). In some aspects, the cationic polymer comprises a polyamine. In some aspects, the cationic polymer includes an aminosilane. In some aspects, the cationic polymer comprises poly-L-lysine (PLL). In some aspects, the passivation region comprises a photocleaveable antifouling coating. In some aspects, the antifouling coating comprises methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA), polyether, polyethylene glycol, and / or poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG). In some aspects, the binding region of the micropatterned substrate is monolithically etched into the passivation region.

[0011] In some aspects, at least a portion of the binding region is arranged in a gradient pattern. In some aspects, the gradient pattern comprises a linear gradient, an exponential decay gradient, a Gaussian distribution, and / or any user-defined mathematical function. In some aspects, the binding region is arranged in an array (e.g., an addressable array). In some aspects, the binding region comprises a plurality of distinct geometric features. In some aspects, the binding region is patterned in a migrasome-mimetic trail.

[0012] In some aspects, the population of EPs comprises large EVs (lEVs). In some aspects, the population of EPs comprises small EVs (sEVs). In some aspects, the population of EPs comprises migrasomes. In some aspects, the population of EPs comprises lipoproteins. In some aspects, the population of EPs comprises viruses. In some aspects, the population of EPs comprises non-vesicular particles. In some aspects, the population of EPs comprises a labeling group. In some aspects, the population of EPs comprises a plurality of labeling groups. In some aspects, the population of EPs comprises non-vesicular particles. In some aspects, the population of EPs comprises fluorescently labeled EPs.

[0013] Also described herein are systems including the device according to any of the aspects described herein; and an imaging element positionable about the device to collect images of adsorbed EP.

[0014] Further described here are methods for analyzing and / or characterizing extracellular particles (EPs) (e.g., extracellular vesicles (EVs)), comprising: contacting a sample comprising a population of EPs (e.g., isolated EPs) with the device according to any of the aspects described herein; and obtaining images via an imaging element positionable about the device.

[0015] Additional advantages of the disclosed subject matter will be set forth in part in the description that follows, and in part will be obvious from the description, or can be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] The accompanying Figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the invention and together with the description serve to explain the principles of the invention.

[0018] Figure 1 shows Light-induced Extracellular Vesicle Adsorption (LEVA) according to Example 1. (Panel A) LEVA schematic showing coating of an oxygen plasma-cleaned coverslip with PLL, mPEG-SVA, and a high concentration PLPP photoactivator. (Panel B) Example patterning template of EV biogenesis and (Panel C) resulting EV micropattern using WGA-labeled U-87 MG sEVs (scale bar = 50 pm). (Panel D) EVs patterned as an EV crosssection, with magnified regions showing nearly single EV resolution patterning and (Panel E) an example of multi-EV patterning showing the initial EV pattern on the left (red) and the second EV pattern on the right (green).

[0019] Figure 2 shows a study of large and small EV binding kinetics in LEVA. (Panels A- B) NTA size distribution and TEM images of bioreactor-produced U-87 MG large EVs and small EVs. (Panel C) Kinetic energy and (Panel D) particle density over time based on COMSOL Multiphysics particle simulations and quantification of time-lapse TIRFM images of large and small EV-sized particles. Time-lapse TIRFM microscopy images and COMSOL Multiphysics simulations results of LEVA for (Panels E-F) large EVs and (Panels G-H) small EVs (scale bar = 10 pm).

[0020] Figure 3 shows LEVA resolution test using various EV sources. (Panel A) Resolution test template for circle array. (Panel B) Resolution test template for microtrack array. TIRFM images of successful LEVA micropattems for (Panel C) U-87 MG small EVs, (Panel D) U- 87 MG large EVs, (Panel E) E. coli OMVs, and commercially available GFP-EV standards from (Panel F) Vesiculab and (Panel G) Sigma. Figure 4 shows LEVA gradient test using various EV sources. (Panel A) (left to right) Logarithmic gradient template and resulting patterns for E. coli OMVs, U-87 large and small EVs, and commercially available GFP-EVs from Vesiculab and Sigma. (Panels B-C) Linear and Gaussian gradient templates and resulting TIRFM images for various EVs in a manner similar to (Panel A). (Panel D) Quantification of desired EV patterning density and normalized fluorescence from TIRFM images for logarithmic, linear, and Gaussian gradients for large and small U-87 EVs. (Panel E) Digital titration of anti-CD63-AF488- and miR-21 molecular beacon co-labeled U-87 small EVs and (Panel F) a magnified portion of the image (Panel E).

[0021] Figure 5 shows glioblastoma cell migration on migrasome-mimetic trails. (Panel A) Template of 20 pm microtracks used for engineering surface of cover slip bottom 96-well plate and (Panel B) TIRFM image of resulting migrasome-mimetic trails made of U-87 large EVs (scale bar = 20 pm). (Panel C) Time-lapse images of U-87 MG cell migration on migrasome-mimetic trails (scale bar = 60 pm).

[0022] Figure 6 shows a study of OMV-mediated neutrophil swarming. (Panel A) NTA size distribution and TEM of E. coli OMVs. (Panel B) Phase contrast images of (1) native neutrophils, (2) neutrophils with dye, and (3) neutrophils with OMVs (scale bars = 5pm) with percentage activation of each condition on the right (* denotes p < 0.0001 Tukey’s HSD, n = 3 0.45mm2FOV). (Panels C-D) (left to right) Patterning template of star (above) and square (below) (both patterns are approximately 2830pm2), OMVs filled into the pattern, and timelapse images of OMV-mediated neutrophil swarm at 15, 30 and 60 minutes, respectively (phase contrast and cell nucleus is the glow, scale bars = 10pm). (Panel E) (left to right) Endpoint (90 minutes) wide FOV (2500pm x 2500pm) of Control, 30pm diameter OMV pattern neutrophil swarms (17x17), and 60pm OMV diameter pattern neutrophil swarms (6x6) (nucleus is cyan hot and scale bars = 400pm) and neutrophil accumulation over time on 30pm and 60pm OMV patterns (n=25 swarms). (Panel F) Circle and star OMV pattern with 400pm center-to-center spacing (both patterns are approximately 2830pm2) time-lapse images of the OMV-mediated neutrophil swarming at 15, 30, and 60 minutes (phase contrast and cell nucleus is the glow and scale bars = 30pm). (Panel G) Neutrophil migratory tracks after 20 minutes of swarming where the tracks are displayed either on the star half of the FOV (red) or circle half of the FOV (blue) and the neutrophil’s chemotactic index overtime either towards the circle OMV pattern (blue) or star OMV pattern (red).

[0023] Figure 7 shows the LEVA resolution test comparing dye-only control. Small EVs and dye-only control were imaged using TIRFM with constant LUT values to demonstrate minimal background fluorescence. Resolution test results for 5 and 10 gm circle array template and 2, 5, and 10 pm microtrack array template shown for (Panel A) WGA-labeled U-87 MG small EVs and (Panel B) WGA dye-only control. Exponential, linear, and gaussian gradient LEVA tests for (Panel C) WGA-labeled U-87 MG small EVs and (Panel D) WGA dye-only control.

[0024] Figure 8 illustrates EV Isolation Schematic for U-87 MG Large and Small EVs, showing collection of roughly 15 ml of conditioned media from CELLine Adherent Bioreactor cell chamber followed by centrifugation steps to remove cells and large debris, differential ultracentrifugation to isolate crude large and small EVs, and density gradient ultracentrifugation (DG) and size exclusion chromatography (SEC) to isolate large and small EVs, respectively, from other contaminants. The Optiprep DG used to isolate large EVs follows the “Isolation of migrasomes from serum samples” protocol established by Zhao et al., and the SEC protocol uses a 35 nm qEV Original column with fractions 1-4 pooled, as detailed in Hisey et al.

[0025] DETAILED DESCRIPTION

[0026] The compounds, compositions, articles, devices, and methods described herein can be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples and Figures.

[0027] Before the present compounds, compositions, articles, devices, and methods are disclosed and described it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0028] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0029] General Definitions

[0030] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings. As used herein, the article “a,” “an,” and “the” means “at least one,” unless the context in which the article is used clearly indicates otherwise.

[0031] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0032] Ranges can 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 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. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0033] As used throughout, by a "subject" (or a “host”) is meant an individual. Thus, the "subject" can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject can be a mammal such as a primate or a human. The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0034] The term “extracellular particle” broadly refers to the collection of lipoproteins (LPs), extracellular vesicles (EVs), viruses, outer membrane vesicles (OMVs), and non-vesicular extracellular particles. “Extracellular vesicles” (EVs) are a specific type of extracellular particles that include cell-derived membranous structures, such as exosomes, microvesicles, virus-like particles, macrovesicles, oncosomes, gesicles, and apoptotic bodies. These extracellular vesicles are typically categorized based on their size, specific markers, cellular origin and biogenesis processes. For example, extracellular vesicles can be categorized as small EVs (sEVs) which includes EVs that are 200 nm in size or less, while the term large EVs (IEVS) is used for particles greater than 200 nm.

[0035] The term “adsorption” and the like refers to the process by which a material, such as a cell, organism, virus, pathogen, polypeptide, polynucleotide, chemical molecule, or biological molecule adheres to the surface of a material.

[0036] The terms “label” or “tag”, as used herein, refer to a composition capable of producing a detectable signal indicative of the presence of the target in an assay sample. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like.

[0037] As used herein, the term “functionalized” refers to the formation of a chemical bond, such as a covalent, coordinative, hydrogen bond, ionic, or dispersive (van-der-Waals) bond between a structure and a functional group.

[0038] In various examples disclosed herein, the term “image”, refers to a two- or three- dimensional representation of a sample that contains spatial information. Images can include, for example, any visual representation, such as a photo, a video frame, streaming video, as well as any electronic, digital, or mathematical analogue of a photo, video frame, or streaming video. References to the term images includes both optical images and non-optical images. Optical images refers to images obtained from an optical microscope. Non-limiting examples of optical microscopy techniques that may be used with the present methods and systems include those configured for super-resolution microscopy, such as total internal reflection fluorescence microscopy (TIRFM), stimulated emission depletion (STED), reversible saturable optical linear fluorescence transitions (RESLOFT), ground state depletion (GSD), saturated structured illumination microscopy (SSIM), fluorescence photoactivation localization microscopy (FPALM), photoactivation localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). Likewise, a non-optical image refers to images obtained from a non-optical microscope. Non-limiting examples of non-optical imaging techniques include, but are not limited to, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), scanning tunneling microscopy (STM), Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS) and the like.

[0039] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

[0040] Devices, Systems, and Methods

[0041] Disclosed herein is a device comprising: a micropatterned substrate defining a binding region and a passivation region, wherein the binding region comprises a photoetched charged surface to non-specifically adsorb a population of extracellular particles (EPs) (e.g., extracellular vesicles (EVs)) from a sample.

[0042] In various aspects, the device includes a microfluidic device. In the present disclosure, the term “microfluidic device” refers to a device having features of micrometer or submicrometer dimensions, and which can receive and process small amounts of fluid (e.g., biological fluid). The EPs from the sample can include, for example, a biological or non- biological fluid. In various aspects, the sample is obtained from a mammalian subject. For example, the sample can be obtained from a human. In some aspects, the sample includes blood, urine, semen, milk, sputum, mucus, a buccal swab, a vaginal swab, a rectal swab, an aspirate, a needle biopsy, a section of tissue obtained for example by surgery or autopsy, plasma, serum, spinal fluid, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, tumors, or organs that include EPs. In various aspects, the sample includes isolated EPs.

[0043] The device includes a micropatterned substrate defining a binding region and a passivation region. In some aspects, the charged surface comprises a surface functionalized with a cationic polymer (e.g., a cationic protein). The charged surface can include, for example, electrostatic proteins or polypeptides, including salts of poly-L-omithine, poly-L- arginine, poly-L-lysine, poly-D-lysine, polyallylamine and polyethyleneimine. In some aspects, the cationic polymer comprises poly-L-lysine (PLL). In some aspects, the cationic polymer includes a polyamine. In some aspects, the cationic polymer includes an aminosilane. In some aspects, the passivation region comprises a photocleavable antifouling coating. In some aspects, the antifouling coating comprises methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA), polyether, polyethylene glycol, and / or poly(L-lysine)-grafted- poly(ethylene glycol) (PLL-g-PEG). In some aspects, the binding region of the micropatterned substrate is monolithically etched into the passivation region. For example, the binding region can be monolithically formed by selectively etching (e.g., using a photoactivator) portions of the photocl eavable antifouling coating. The photoetched binding region can be formed using a photoactivator to selectively cleave portions of a coating on the substrate. In some aspects, the photoactivator comprises 4-benzoylbenzyl- trimethylammonium chloride (PLPP).

[0044] In the present disclosure, the term “micropatterned substrate” and the like generally refer to a substrate that has at least one surface which has an intended plurality of features that define a profile characterized by raised portions and recessed portions which are raised or recessed relative to a datum surface. The features of the micropatterned substrate (e.g., the arrangement of the binding region relative to the passivation region) are sufficiently identifiable such that a distinguishable profile can be observed in collected images. In some aspects, at least a portion of the binding region is arranged in a gradient pattern. In some aspects, the gradient pattern comprises a linear gradient, an exponential decay gradient, a Gaussian distribution, and / or any user-defined mathematical function. In some aspects, the binding region is arranged in an array (e.g., an addressable array). In some aspects, the binding region comprises a plurality of distinct geometric features. In some aspects, the binding region is patterned in a migrasome-mimetic trail.

[0045] In some aspects, the population of EPs comprises large EVs (lEVs). In some aspects, the population of EPs comprises small EVs (sEVs). In some aspects, the population of EPs comprises migrasomes. In some aspects, the EPs include outer membrane vesicles (OMVs) derived from the outer membrane of bacteria. In some aspects, the population of EPs comprises lipoproteins. In some aspects, the population of EPs comprises viruses. In some aspects, the population of EPs comprises non-vesicular particles. In some aspects, the population of EPs comprises non-vesicular particles. In some aspects, the population of EPs further comprises a labeling group. For example, the labeling group can be a polypeptide, a nucleic acid, and / or an aptamer, such as a luminescent, fluorescent, fluorogenic, chromogenic, magnetic, radioactive or other type of detectable label. In some aspects, the labeling group includes a fluorophore, dye, or fluorescent protein, such as a fluorescent- conjugated antibody and / or chromophore. A wide variety of different types of fluorophores are readily available and applicable to the disclosed devices, systems, and methods and include fluorescein, or rhodamine-based dyes, cyanine dyes and the like. A variety of such dyes are commercially available and include the Cy dyes available from GE Healthcare (Piscataway, N.J.), such as Cy3, Cy5, and the like, or the Alexa® family of dyes available from Invitrogen / Molecular Probes (Carlsbad, Calif.), such as Alexa 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, and 750. It is appreciated that fluorophores may be present as individual fluorophores or they may be present in interactive pairs or groups, e.g., as fluorescent resonant energy transfer (FRET) pairs. Other labeling strategies may employ inorganic materials as labeling groups, such as fluorescent or luminescent nanoparticles, e.g. nanocrystals, i.e. Quantum Dots, that possess inherent fluorescent capabilities due to their semiconductor make up and size in the nanoscale regime (See, e.g., U.S. Pat. Nos. 6,861,155, 6,699,723, 7,235,361). By way of example, nanocrystal materials are generally commercially available from, e.g., Invitrogen, Inc., (Carlsbad Calif.). In some aspects, the population of EPs comprises a plurality of labeling groups (e.g., 1, 2, 3, 4, 5, etc.). In some aspects, the population of EPs comprises fluorescently labeled EPs.

[0046] Methods of preparing and separating EPs from a biological sample are generally known. For example, separation of these particles may be achieved by leveraging differences in size, density, buoyancy, etc. In some aspects, centrifugation or filtration may be performed to isolate the EPs. By way of illustrative example, separation and isolation can be achieved using ultracentrifugation (e.g., density gradient ultracentrifugation and / or differential ultracentrifugation), size exclusion chromatography, and combinations thereof.

[0047] Also described herein are systems including the device according to any of the aspects described herein; and an imaging element positionable about the device to collect images of adsorbed EP.

[0048] As noted above, the present system can use optical images and / or non-optical imaging elements. In some aspects, the present system utilizes an optical imaging element. Nonlimiting examples of optical microscopy techniques that may be used with the present methods and systems include those configured for super-resolution microscopy, such as total internal reflection fluorescence microscopy (TIRFM), stimulated emission depletion (STED), reversible saturable optical linear fluorescence transitions (RESLOFT), ground state depletion (GSD), saturated structured illumination microscopy (SSIM), fluorescence photoactivation localization microscopy (FPALM), photoactivation localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). In some aspects, the present system utilizes a non-optical imaging element. Non-limiting examples of suitable non-optical imaging techniques include, but are not limited to, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM) and scanning tunneling microscopy (STM), Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS) and the like.

[0049] In some aspects, the system further includes a processor. The processor of the described system, for processing information associated with the process(es) or method(s) described herein, can include, for example, any computer processor is known in the art capable of performing calculations and directing functions for interpreting and / or performing input, output, calculation, and display of data in accordance with the disclosed methods. The processor may comprise any type of processing unit, such as typical computer processor(s), controller(s), microcontroller(s), microprocessor s), and / or programmable logic controllers (PLCs). The information to be processed by the processor may include, for example, information contained in analog or digital signals and / or translated signals and / or information contained in a data storage. Processing of the information may involve, for example, performing calculations on received signals such as, but not limited to, vector analysis, picture identification, pattern recognition, frequency analysis / Fourier transforms, numerical computations, machine learning, or, as described herein, applying predetermined or recursively fit correlative algorithms to received sensor data. In some embodiments, the system comprises more than one processor, and the reference herein to “processor” includes reference to multiple processors and vice versa. In various examples, the processor is configured to receive user-generated instructions (e.g., the target location and / or the target orientation).

[0050] In an aspect, the processor is in communication with both the imaging element and a power source. In another embodiment, the processor may also be in communication with data storage(s), and, optionally, display(s). The components of the system, such as the imaging element, power supply, sensors, computing device(s), processor(s), feedback controller(s), data storage(s), and / or display(s), and any other components of system, device or computing device, may communicate using any electronic wired or wireless means or protocols for communication known in the art, including but not limited to Ethernet™, Bluetooth™, WiFi™, infrared, near-field communications (NFC), radio-frequency identification (RFID), WiMAX™ (fixed or mobile), cellular communications protocols such as GSM, EDGE, GPRS, CDMA, EMTS, LTE, LTE-A, IMS, and any other cellular communications protocols including, but not limited to, up to and including 5G protocols as established under the 3 GPP, for example, and any other communications protocols suitable for the method(s) and system(s) described herein, including any proprietary protocols. Components of the system may exist on the same network or on separate networks, and the network(s) may include any type of network suitable for the system(s) and method(s) described herein, including but not limited to wired or wireless personal area networks (PANs), local area networks (LANs), mesh or ad hoc networks, wide area networks (WANs), metropolitan area networks (MANs), virtual private networks (VPNs), and any other suitable network type, as well as any suitable network configuration or topology (e.g., token ring, star, bus, mesh, tree, etc.). The presently described system(s) further includes any components necessary to affect the communication and / or network type employed, such as wireless or wired routers and access points.

[0051] The presently described methods and systems may be implemented on a secure network to which access may be limited to authorized users by any known means and which may be protected by known security measures, such as by the use of firewalls. In some embodiments, authentication may be required before granting access to authorized users, such as where autologous cell manufacturing and / or patient data is involved and / or where compliance with government regulations is mandated (such as Title 21 of the U.S. Code of Federal Regulations). Such authentication may be implemented for any one or more of the system components, such as for access to a computing device or machine housing the processor, access to data storage, access to a database of the data storage, access to any of the sensors or sensor readings of the device or imaging element, access to a graphical user interface (GUI) of the system, access to the device or imaging element, etc. Security of the presently described methods and systems may be further provided for by encrypting communications among system components by any means or protocols known to persons skilled in the art, such as Internet Protocol Security (IPSec), Transport Layer Security (TLS), Secure Sockets Layer (SSL), etc., in order to reduce the potential for the tampering with or corruption of the preprogrammed correlative algorithm(s).

[0052] The presently described system may also include a data storage for storing information associated with the described methods. The data storage may include, for example, various types of local or remote memory devices such as a hard disk or hard drive (of any type, including electromechanical magnetic disks and solid-state disks), a memory chip, including, e.g., random-access memory (RAM) and / or read-only memory (ROM), flash memory, optical memory such as CD(s) and DVD(s), floppy disks, and any other form of optical, physical, electronic, and / or magnetic memory devices in or on which information may be stored. The data storage may comprise non-volatile memory. In some embodiments, the data storage may only be accessed via secure data transfer, which may be accomplished using one or more known server platforms and security protocols. The information to be stored in the data storage may comprise, for example, one or more predetermined algorithms for correlating sensor inputs to various outputs and records of such predicted determinations along with associated actions (such as feedings) and / or associated timestamps, unique identifiers, authorized users and associated authentication information for use in authenticating users for authorized access to the data storage or any other system component, and any other pertinent information. In operation, the data storage is in communication with the processor.

[0053] The system may also include a display (which may be co-located with the processor, e.g., where the processor and display are part of a computer or server used for carrying out the method steps described herein) for visually presenting information associated with the described methods. The display may comprise, for example, a computer monitor (e.g., LCD, a CRT monitor, a projection (e.g., heads-up display (HUD) laser), etc. In some embodiments, the visual display may comprise, for example, that of a mobile device such as a tablet computer, cellular phone, smartphone, personal digital assistant (PDA), personal computer (PC), laptop computer, augmented reality display (e.g., Google™ Glass™ or Microsoft™ HoloLens™), etc. The information presented on the display may include any other information collected in the course of carrying out the methods described herein, prompts for information entry associated with one or more steps of the described methods, and / or any predetermined formulae or algorithms, as previously described. The display may also be capable of receiving input (such as, e.g., where the display includes a touch-screen and is capable of receiving touch input and accordingly transmitting information to the processor).

[0054] Further described here are methods for analyzing and / or characterizing extracellular particles (EPs) (e.g., extracellular vesicles (EVs), lipoproteins, viruses, non-vesicular particles, etc.), comprising: contacting a sample comprising a population of EPs (e.g., isolated EPs) with the device according to any of the aspects described herein; and obtaining images via an imaging element positionable about the device. In some aspects, the disclosed devices, systems, and methods are used for EV digital titration wherein EVs pre-labeled with a plurality of binding agents are characterized by observing colocalization effects of various binding agents and the kinetics of particle binding. In some aspects, the micropattemed substrate is arranged to form a migrasome-mimetic trail to characterize a cellular response (e.g., cellular migration behavior). In some aspects, the disclosed devices, systems, and methods are used to produce a neutrophil swarming cascade. EXAMPLES

[0055] The following examples are set forth below to illustrate the methods, compositions, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

[0056] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0057] Example 1: Light-induced Extracellular Vesicle Adsorption

[0058] Introduction.

[0059] This example details the development of a versatile platform technology called Light- induced Extracellular Vesicle Adsorption (LEVA). LEVA enables the high-resolution, nonspecific, tunable, scalable, and rapid micropatterning of EVs from diverse sources for a wide range of applications in human health. Initially, LEVA was optimized using large EVs (lEVs) and small EVs (sEVs) from bioreactor cultures and commercially available GFP-EV standards to produce well-defined shapes and gradient patterns at subcellular scale. COMSOL simulations and time-lapse total internal reflection fluorescence microscopy (TIRFM) imaging were then used to better understand the underlying EV adsorption kinetics and its dependence on EV size. Next, LEVA was applied to several EV applications including the first reported examples of digitally titrated single EV characterization by protein and RNA fluorescence colocalization, patterning of glioblastoma (GBM) migrasome-mimetic trails followed by single GBM cell migration, and E. coli OMV-mediated neutrophil swarming. As LEVA exploits relative increases in attractive forces between the engineered regions of surfaces and innate EV properties, it permits nonspecific adsorption rather than antibody or other specific ligand-based capture. Thus, it can be applied at scale to many other types of applications with little modification to the reported workflow. Finally, such precise and tunable control of the location and concentration of EVs on surfaces should ultimately enhance rigor and reproducibility within this rapidly growing field.

[0060] Methods

[0061] Light-induced EV Adsorption. LEVA was developed by repurposing the previously reported Light-induced molecular adsorption of protein (LIMAP) technique

[0065] , with careful considerations and adaptations needed for the EV field including EV production, isolation, characterization, labeling, and application-relevant patterning. First, glass coverslips were cleaned with ethanol followed by deionized (DI) water by sonication for 3 min each. The coverslip was oxy gen-plasma-treated for 1 min to activate and clean the surface. A silicone gasket (7 mm x 7 mm, 16 wells) was placed on the functionalized coverslip. 50 pL of 0.01 % (w / v) poly-L-lysine (PLL) was added to the desired wells for 1 h. The remaining PLL was rinsed three times with 0.1 M HEPES. 20 pL of 100 mg / mL of methoxy-poly(ethylene glycol) succinimidyl valerate (mPEG-SVA) diluted in 0.1 M HEPES was incubated at room temperature for Ih. Following the incubation, the coverslip was rinsed with DI water and dried with a nitrogen airflow. 4-benzoylbenzyl-trimethylammonium chloride (PLPP) was then added as a photoactivator prior to UV illumination in a manner dependent on the specific EV application (PLPP gel was diluted in 96% ethanol in most exposure cases).

[0062] The functionalized coverslips were photoetched using a DMD optical module (Primo, Alveole) on an automated inverted microscope (Nikon TE2000-S). Grayscale images were transformed into high-resolution UV light, allowing for a maskless illumination of different UV intensities corresponding to grayscale values. UV doses of 30 mJ / mm2were used for LEVA. The photoetched coverslip was rinsed with DI water and dried by nitrogen flow. The photoetched coverslip was incubated in PBS for 5 min and the EVs were allowed to adsorb for up to 10 min following 10 washes with PBS.

[0063] LEVA Simulations. The LEVA simulations were conducted using a COMSOL Multiphysics version 6.0 license provided by the Ohio State Supercomputer Center at The Ohio State University, Columbus, USA. The study utilized the time-dependent Lagrangian Particle Tracing for fluid flow module to gain insights into the dynamics of EVs on the engineered glass surface. The glass surface measured 80 pm x 80 pm and featured nine equidistant 10 pm patterns. These patterns were made positively charged, while the rest of the surface remained neutral and nonreactive. The EVs were considered as suspended particles in a PBS solvent with physical properties including a density of 1.13 mg / mL and a surface tension similar to PBS. Two types of samples were analyzed: sEVs with a mean size of 100 nm and a standard deviation of 50, and larger EVs with a mean size of 135 nm and a standard deviation of 45. The zeta potential of the EV particles was found to be negative, allowing them to bind with the positively charged patterns on the glass surface.

[0064] The simulations incorporated Lennard-Jones particle interactions coupled with Newtonian laws of motion for particle propagation. The initial boundary condition was set at time 0 sec, with all particles grid mesh-based release onto the entire surface. The walls were treated as diffusion scattering to ensure varied angles of reflection, providing a closer approximation to reality. The influence of gravity was neglected, as Brownian motion dominated the nanoscale particle movements. For the Finite Particle Tracing (FPT) simulations, the study used a physics-controlled extremely fine mesh. The time-dependent equations were solved using 20 vertex elements, 560 boundary elements, and a total of 28,834 domain elements. These computations were distributed across 4 nodes with 112 cores and took 13 hours to calculate trajectories of 2.78E8 particle concentration per mL with an output time of up to 60 seconds, in steps of 0.0001. Post-processing involved calculating the average surface density of particles and the surface integral of velocity magnitude. Particle trajectories at various time intervals were collected and analyzed to determine particle density at the binding sites.

[0065] Commercial EV Standards. Commercially available GFP-EV standards were used for the final validation of LEVA. The lyophilized GFP-EV standards were reconstituted by carefully following the specific manufacturer’s instructions. Vesi-Ref EVs (Vesiculab) and fluorescent exosome standards (SAE0193, Sigma) were resuspended in 100 ul of ultrapure water, avoiding bubbles. Vesi-Ref EVs were then vortexed for 60 seconds, briefly centrifuged, and briefly mixed again via pipetting. Concentrations of both commercial EV standards were then normalized by NTA measurement prior to patterning.

[0066] Bioreactor Production of Cell Line EVs. CELLine AD 1000 bioreactors (Wheaton) were used for EV production following an established adaptation and EV collection and isolation protocol [66-68], Briefly, bioreactors were adapted from complete media with 10 % fetal bovine serum (FBS) supplementation, then adapted to CDM-HD (Fibercell). EVs were collected in around 15 ml of conditioned media three times per week from the cell chamber, and the media chamber was refreshed once per week.

[0067] EV Isolation and Labeling. Cells and large debris were first removed using differential centrifugation via a 200 x g for 5 min followed by a 2000 x g for 10 min. lEVs and sEVs were then isolated using a combination of differential ultracentrifugation and either density gradient ultracentrifugation or size exclusion chromatography. Crude lEVs were isolated by ultracentrifugation (Beckman XP-100, Ti 70 rotor) at 20,000 x g for 30 min, and the resulting supernatant was then ultracentrifuged (Beckman XP-100, Ti 70 rotor) at 100,000 x g for 60 min to isolate crude sEVs. The 1EV pellets were further fractionated using an iodixanol (OptiPrep, D1556-250ML) density gradient containing 500 «L layers of 30 %, 25 %, 19%, 15 %, 12 %, 10 %, 8 %, and 5 % with the lEVs contained in the top layer. Following ultracentrifugation (Beckman XP-100, SW 28 Ti rotor) at 150,000 g for 4 h, lEVs were isolated from seventh fraction 7 by washing twice with PBS by centrifugation at 20,000 xg for 30 min each wash. The sEVs were isolated by pooling fractions 1-4 following size exclusion chromatography using a 35 nm qEV Original column coupled with an Automated Fraction Collector (AFC, Izon). All EVs were characterized using basic NTA (Nanosight NS300). Given previously reported successes in labeling migrasomes with WGA

[0069] , lEVs and sEVs were incubated with l «g / ml wheat germ agglutinin (WGA kit, W7024, Thermo) for 10 min at room temperature. Excess dye was removed via ultrafiltration using 100 kDa filters (Vivaspin 500, Sartorious) at 10,000 * g. The solution was fully washed with PBS three times following the same procedure, including a dye-only control.

[0068] Production of GFP containing OMVs from E. coli. The pETZEGFP-strep-his plasmid used to express the green fluorescent protein (GFP) containing outer membrane vesicles (OMVs) was acquired from the Wood Lab strain collection (Ohio State University, OH, USA). Enhanced green fluorescent protein (EGFP) has a monomerizing single A206K mutation

[0070] , pETZEGFP-strep-his was miniprepped according to the manufacturer’s instructions (Qiagen) and transformed into competent Escherichia coli BLR (DE3) cells. The primary culture (10 mL Luria Broth media supplemented with 100 ug / mL ampicillin) was inoculated with a single transformed colony and grown at 37°C and 225 rpm for 18 h. The 10 mL primary culture was used to inoculate one liter of Terrific Broth (TB) media (1 % v / v inoculum) supplemented with 100 ug / mL ampicillin in a 2.5 L Thompson Ultra Yield™ flask (Oceanside, CA, USA). The culture was grown to an OD600 of 3 ± 0.5 at 37°C and 275 rpm and then induced with 0.5 mM (final concentration) of isopropyl / LD-1 -thiogalactopyranoside (IPTG). The culture was left shaking for 16 hours at 16°C and then harvested at 8,000 ref for 20 minutes. The supernatant containing the OMVs (conditioned media) was collected and frozen at -20°C until ready for use.

[0069] To begin concentrating the OMVs, the conditioned media was thawed at 4°C and 0.2 «m sterile filtered using a vacuum flask (Coming, Cat#430769). The supernatant was then 10 times enriched through tangential flow filtration, which used polysulfone hollow fiber cartridges (Molecular weight cutoff: 300kDa, Repligen, Cat#D02-S500-05-N) as the filter. The enriched supernatant was then further purified through size exclusion chromatography in the same manner as bioreactor-produced U87 MG sEVs (qEV Original, Izon). Importantly, initial resolution testing of LEVA patterning with OMVs was performed by WGA-488 labeling, whereas neutrophil swarming experiments were performed with GFP-OMVs to reduce any potential confounding issues caused by the WGA dye.

[0070] Human Neutrophil Isolation and Labeling. The morning of an experiment, human blood was collected in I<2-ethylenedi aminetetraacetic acid (K2-EDTA), purple top, tubes (BD Vacutainer, Fisher Scientific) according to protocol #2018H0268 approved by the Biomedical Sciences Committee Institutional Review Board (TRB) at The Ohio State University. The fresh blood was added to a 10 mM sodium bicarbonate, 0.1 mM EDTA, and 150 mM ammonium chloride solution of pH 7.4 at a 1 :20 ratio for 5 minutes to lyse the red blood cells. The solution was then centrifuged at 350 x g to isolate the white blood cells and remove the red blood cell debris. Neutrophils were retained in solution through negative immunomagnetic separation through the EasySep™ Human Neutrophil Isolation Kit (STEMCELL Technologies, Vancouver, Canada). Neutrophils were incubated in Iscove’s Modified Dulbecco’s Medium (IMDM, ThermoFisher), and stained with 20 «g / mL Hoechst 33342 (Thermofisher) for 10 minutes. The neutrophils were then washed by adding phosphate buffer solution (PBS) to 5 times the original cell volume and centrifuged at 1000 RPM. The final neutrophil suspension was at a concentration of 1.2 x 106cells per ml and contained IMDM, 20% fetal bovine serum (FBS) (Gibco, Thermofisher), and 1% penicillinstreptomycin (PS) (Gibco, Thermofisher).

[0071] EV Characterization. EVs were characterized primarily using NTA and TEM (Tecnai). For NTA, 5 x 30-second videos were recorded (acquisition: screen gain 1, detection threshold) at low flow rates and analyzed using the v3.3 software (screen gain 10, detection threshold 5). For negative staining TEM, on a strip of parafilm, 20 p droplets of water for injection (WFI) and two 20 pL droplets of negative stain (UranyLess EM stain, Electron Microscopy Sciences) were placed. The TEM grid was subjected to plasma treatment for 1 min. 10 pL of the pooled EV solution was placed onto the treated surface of the grid then was incubated for 1 min and blotted with filter paper to remove excess. The grid was then submerged into the WFI droplet and blotted dry using filter paper, and this process was repeated with the second WFI droplet. The grids were stained via submersion into the negative stain, blotted, and then submersion into the second droplet of the negative stain. The grid was incubated in the stain for approximately 22 seconds and excess solution was again wicked away using filter paper. Grids were stored overnight in a grid box to ensure thorough drying. TEM imaging was performed using a Tecnai TF-20 microscope (FEI Company, Hillsboro, OR) operating at 200 kV.

[0072] Image Acquisition. Images of the EVs were captured with TIRFM (Nikon Eclipse Ti Inverted Microscope System, Melville, NY) with a 100* oil immersion lens. The TIRFM images were analyzed through ImageJ.

[0073] Results

[0074] Based on the initial template design, the DMD photochemistry process exposes certain regions of the functionalized surface to UV illumination for durations that scale with the template’s grayscale values at nearly single-micron resolution (detailed in the Methods section). This results in well-defined regions on the surface with predictable levels of nonspecific EV adsorption. A representative schematic of a typical experimental workflow is presented in Fig. 1, panel A, including an optional iterative approach for patterning multiple types of EVs. An example of a grayscale template and the resulting EV micropattern following the LEVA process are shown in Fig. 1, panels B and C, respectively. In these templates, black regions represent areas, which are not exposed to UV light and will fully retain PEG at their surface, whereas white / gray regions are exposed for a duration that scales with their grayscale values. An additional image of EVs patterned as an EV crosssection image, with magnified regions showing nearly single EV resolution patterning is shown in Fig. 1, panel D, and an example of multi -EV patterning is shown in Fig. 1, panel E.

[0075] The stark contrast in binding affinity between these different regions, combined with the ability to rapidly produce gradients by dynamically modulating UV exposure, ultimately enables the selective adsorption of EVs with high predictability and customization. Furthermore, the PEG-functionalized regions greatly reduce the initial nonspecific binding of EVs and allow rapid removal of most of the undesired EVs with a simple wash step, without significant loss of EVs in the desired regions. Notably, by not employing antibodies or other ligands, the platform greatly reduces cost and increases scalability compared to any previously reported technologies.

[0076] LEVA Kinetics and Simulations. To better understand the LEVA process, COMSOL Multiphysics simulations and time-lapse TIRFM were used to model and experimentally measure EV binding kinetics on the engineered surfaces. Fig. 2, panels A and B show the size distributions of U-87 MG bioreactor-derived lEVs and sEVs, respectively, which were characterized using nanoparticle tracking analysis (NTA) and negative staining TEM to verify their expected size and morphology [6], It should be noted that the optimization and development of this process were at least partly attributable to the production of vast quantities of EVs using CELLine AD 1000 bioreactors.

[0077] Simulations were performed based on known property estimates of these two general EV subpopulations, revealing an exponential increase in particle density at the positively charged defined surfaces, while density decreased over the rest of the glass surface where the charge remained neutral. Notably, sEVs (mean = 100 nm, standard deviation = 50 nm) exhibited a higher rate of particle binding capability compared to lEVs (mean = 135 nm, standard deviation= 45 nm). Without wishing to be bound by theory, this was believed to be attributed to the increased effect of Brownian motion on sEVs compared to lEVs, resulting in greater mobility and a higher probability of contacting the patterned regions on the surface. sEVs took approximately 8 sec to sufficiently bind and settle on the positively charged surface, whereas larger EVs took around 58 sec. Increasing the average size of the EVs resulted in even longer binding times. Smaller EVs also bound more closely to the positively charged surface, allowing for a clear delineation of the surface boundary, while larger EVs exhibited slower binding velocities, impacting their kinetic energy. Fig. 2, panels C and D show the kinetic energy and particle density over time for both large and sEVs.

[0078] To validate the simulation results experimentally, bioreactor-produced U87 MG lEVs and sEVs were labeled with WGA-488, free dye was removed by repeated ultrafiltration and washing, EVs were normalized by concentration using NTA, and EVs were finally added instantaneously to the patterned arrays of 10 «m circles in a manner consistent with the simulations. Time-lapse TIRFM was used to monitor the EV adsorption in real-time. Remarkably, the quantified fluorescence intensity results from TIRFM videos agree with the simulation results, showing slower binding kinetics of lEVs compared to sEVs shortly after the addition of the EV solutions. As illustrated in Fig. 2, while the rapid TIRFM acquisition of images resulted in some photobleaching of the EVs, there is clear and rapid initial adsorption of EVs in the UV-illuminated regions with minimal nonspecific binding to the unexposed areas.

[0079] Resolution Test. To determine the resolution limits of LEVA, several diverse types of fluorescently labeled EVs were patterned using the same templates and general workflow. To ensure that the reported results are from EV binding and not simply dye contamination, dye-only controls (Fig. 8) and commercially available GFP-EV standards were also used. The initial templates for resolution testing included 1, 2, 5, and 10 «m diameter circles as shown in Fig. 3, panel A, and 1, 2, 5, and 10 «m microtracks as shown in Fig. 3, panel B. Generally, consistent EV patterning results were seen for 5, and 10 «m diameter circles, while 2, 5, and 10 «m microtracks were consistently patterned successfully. EVs could also be patterned on 2 «m circles on occasion, but 1 «m circles and microtracks were rarely visible despite repeated attempts. TIRFM images of successful EV patterning are shown in Fig. 3 for (panel C) U-87 sEVs, (panel D) U-87 lEVs, (panel E) E. coli OMVs, and commercially available GFP-EV standards from (panel F) Vesiculab and (panel G) Sigma. Clearly, depending on the EVs used, the fidelity of the EV micropatterns can vary, but successful EV micropatterns were produced for all the samples tested in the desired regions, with minimal nonspecific binding in the unexposed regions.

[0080] EV Digital Titration. The study further attempted to use gradient templates to micropattern EVs from various sources. Three gradients were chosen to determine the adsorption differences, including a linear gradient to visualize chromatic increases in free energy, an exponential decay gradient to visualize drastic changes in gradation, and a Gaussian distribution to simulate the stochastic accumulation of particles towards a central line. Generally, the sEVs produced more homogeneous patterns, whereas lEVs produced more variable patterns with higher rates of non-specificity. For the exponential decay gradient shown in Fig. 4, panel A, the sEVs demonstrated a better fit (Spearman’s coefficient, p = 0.9983 for sEVs and p = 0.9749 for lEVs, p < 0.0001) and for the linear gradient shown in Fig. 4, panel B, sEVs demonstrated the closest fit (Spearman’s coefficient, p = 0.9998 for sEVs and p = 0.9943 for lEVs, p < 0.0001). Finally, for the Gaussian distribution shown in Fig. 4, panel C, both sEVs and lEVs had similar fits, with sEVs yielding a slightly better fit (Spearman’s coefficient, p = 0.9942 for sEVs and p = 0.9926 for lEVs, p < 0.0001). Interestingly, at lower points of the gradients, the lEVs consistently demonstrated greater adsorption. Although the EVs could not fully reproduce the patterns, with the edges of the pattern demonstrating decreases in fluorescence, the EVs followed the desired trend and demonstrated spatial actuation not currently achieved in any reported techniques. Quantification of the relative mean fluorescence intensity for the U-87 MG lEVs and sEVs compared to the desired template are reported in Fig. 4, panel D, indicating that in the future, the templates can be tuned for specific types of EVs in order to produce the intended pattern more precisely.

[0081] U-87 sEVs were co-labeled with anti-CD63-AF488 and hsa-miR-21-5p molecular beacons and patterned in gradients to determine whether EV digital titration could be used in future colocalization studies. Unlike conventional ELISA assays, wherein antibodies or other ligands are used to first capture EVs of interest and fluorescent probes are then added to label the EVs while blocking nonspecific binding, EV digital titration requires pre-labeling EVs in solution followed by a cleanup step to avoid deposition of unbound probes that are indistinguishable from the EVs. Both ultrafiltration with repeated PBS washing steps and size exclusion chromatography (SEC) were tested, with SEC resulting in less background fluorescent contamination from unbound antibodies and molecular beacons. An example of successful pre-labeled and cleaned-up U-87 sEVs in the Gaussian distribution is shown in Fig. 4, panel E, with a magnified region showing colocalization of both labels shown in Fig. 4, panel F.

[0082] Migrasome-mimetic EV Trails. Given the abundance of lEVs produced from the U- 87 MG bioreactor cultures, the study attempted to micropattern migrasome-mimetic trails in accordance with those seen in several recent publications[28, 54, 55], The U-87 MG lEVs used for this study were isolated using an established migrasome isolation protocol involving density gradients

[0031] . For purposes of this study, the geometry and concentration of the deposited EV trails were kept simple in order to ascertain whether LEVA could be used to produce platforms for studying the effects of surface-bound EVs on cell migration generally and to determine whether the surface-bound EVs themselves could influence cell migration behavior compared to a similarly engineered surface treated with FBS rather than U-87 MG lEVs. Following patterning, U-87 MG cells were recorded using timelapse microscopy. As shown in Fig. 5, single U-87 MG cells not only attached more frequently on the engineered surface that was treated with lEVs but also exhibited ID migration behavior along the patterned regions much more than the similarly engineered but FBS-treated surface shown in Fig. 7.

[0083] OMV-mediated Neutrophil Swarming. To display the dynamic, highly tunable, nanoparticle patterning capabilities of LEVA, bacterial OMVs were used as a stimulant for human peripheral blood neutrophils, which induced the neutrophil swarming cascade. For validation of E. coli OMV size distribution and conformation, NTA and negative stain TEM were utilized and can be seen in Fig. 6, panel A. Then, to identify whether OMVs produced morphological differences among neutrophils, identified as a state of activation, OMVs and neutrophils were allowed to incubate together for 30 minutes before imaging using inverted epifluorescence microscopy. In Fig. 6, panel B phase contrast images of native neutrophils (1), neutrophils with a WGA dye-only control (2), and neutrophils with WGA stained OMVs (3) demonstrate WGA-labeled neutrophils underwent significantly more morphological changes. This morphological change is shown in the bar graph, where nearly 80% of neutrophils in this condition are activated. As neutrophils are activated with OMVs, it was of great interest to see if surface-bound OMVs could initiate the neutrophil swarming cascade for the first time. Fig. 6, panels C-D displays the initial template utilized by LEVA, where an 8-point star is displayed in Fig. 6, panel C and a square in Fig. 6, panel D (surface area = 2830 / / m2), the resulting OMV patterns imaged through TIRFM and a 15, 30, and 60-minute time-point images of the OMV-mediated neutrophil swarm.

[0084] LEVA’S capabilities include the ability to micropattern EVs across large surface areas, and this is displayed by the large arrays of OMV-mediated neutrophil swarms in Fig. 6, panel E, where 2500 «m x 2500 «m fields of view (FOV) of an FBS-only control 17 x 17 30 «m diameter 150 «m center-to-center spacing pattern (far left), 17 x 17 array of the same pattern OMV induced neutrophil swarms, and 6 x 6 60 «m diameter 400 «m center-to-center spacing OMV induced neutrophil swarms. In these images, there are roughly 15 more neutrophils per swarm present in the 60 «m diameter pattern compared to the 30 «m diameter pattern after 90 minutes. A closer view of the 30 «m OMV pattern can be seen in Fig. 7, where the OMVs have been stained with WGA.

[0085] Further demonstrating the versatility of LEVA, multiple different geometric templates were utilized within the same microdomain. This can be seen in Fig. 6, panel F, where 15, 30, and 60-minute time-point images of a circle on the left and an 8-point star on the right, which are 400 «m apart, both patterns with an equal area of 2830 / / m2. Interestingly, despite the same surface area, it was observed that the cells’ migration differed when these two patterns were in close proximity, as observed through the cells' migratory tracks in Fig. 6, panel G, where the red spots represent cells that are on the star half of the FOV and the blue spots represent cells which are on the circle half.

[0086] Interestingly, within the blue tracks, the neutrophils appear to turn around shortly after swarming begins. This change in migratory behavior can be measured through the chemotactic index. The chemotactic index is a measurement of the neutrophils' directionality, where a positive value indicates the cell is traversing toward the described OMV pattern, and a negative value indicates the cell is traversing away. The chemotactic index towards the circular OMV pattern (blue on the heatmap) demonstrates that after 600 seconds, there is a clear change in the directionality from the circular pattern to the star OMV pattern, whereas the chemotactic index towards the star (red on the heatmap) increases from 0-600 seconds, and has substantially larger amounts of cells migrating towards the OMV pattern. When the chemotactic index of a star-circle swarm is compared to a circle-circle swarm, it can be seen in the circle-circle swarm that there is no noticeable shift in directionality over time, which is apparent in the circle-star swarms’ chemotactic index. Additionally, an image can be seen of the neutrophil tracks after 20 minutes in Fig. 7. These tracks further display the difference in migration between the circle-circle and circle-star swarm, as there is no noticeable shift between the tracks at any time-points.

[0087] Discussion. The present example illustrates the basic principles of the LEVA technique and demonstrates the range of applications to which it can be applied. The potential value that this platform technology could provide to the EV field is limitless, particularly as EV researchers have recently begun to appreciate the role of ECM and surface-bound EVs in human health and disease. Single-EV characterization by digital titration and fluorescence colocalization, single-cell migration on migrasome or migrasome-mimetic trails, and OMV- mediated neutrophil swarming are only a few of the many EV and nanoparticle-related applications, which are enabled by LEVA.

[0088] While the COMSOL simulation results rely on several assumptions to approximate the EV properties and underlying LEVA kinetics, the results are promising. This initial study used lEVs and sEVs from a U87-MG CELLine adherent bioreactor culture, but the similarities in trends from the simulations and time-lapse TIRFM imaging indicate that this model could be useful for other types of particles. It is noted that several other factors can also be included in this approach to kinetics characterization, such as lipoprotein contamination, changes in EV corona due to processing or storage conditions, or the use of different buffers and surface materials[56-59]. For example, applications enabled by LEVA are the mimicking of migrasomes or other matrix and surface-bound mammalian EVs followed by cell culture could be valuable. The present study focused on the micropatterning of GBM lEVs and subsequent attachment and migration of GBM cells, however, optimization and tuning of the engineered surface for specific microenvironments can move towards ideal biomimicry of ECM-bound EVs, particularly migrasomes. For example, in the case of migrasomes, a study of the EV-bound retraction fibers, often found attached to the migrasomes’ pomegranate-like structures can be utilized. It is noted that the production and isolation of migrasomes, as well as their deposition, can be modified to ensure that their morphology reflects their native state as closely as possible. These surface-bound EVs can then be compared to other engineered microenvironmental stimuli to compare their relative influence on cell behavior and function compared to other stimuli. Finally, in addition to engineering scalable, biomimetic EV-containing microenvironments, it is noted that LEVA can improve the fundamental understanding of migrasomes and ECM- bound EVs, encouraging the development of relevant therapies therapies from applications ranging from cancer to wound healing based on these findings. OMVs have been shown to elicit a stronger response from antigen-presenting cells

[0060] , but little is known about the change in neutrophil response when presented OMVs as compared to live bacteria

[0045] or antigen-coated microspheres

[0061] . Furthermore, this platform permits the multifaceted study of other immune cell-OMV interactions, which are yet to be explored. For example, there have been studies aiming to understand the neutrophilbiofilm coexistence

[0062] and biofilms are understood to be continually releasing OMVs

[0063] , therefore LEVA permits this relationship between biofilms, OMVs, and neutrophils (or other immune cells) to be better understood. Additionally, super-resolution microscopy of neutrophil swarms has been shown to identify the changes of organelles within the neutrophil crowd

[0064] , and the LEVA platform also enables these studies.

[0089] LEVA is not only versatile in the type of EVs that can be used with the reported version of the surface patterning technique but also the underlying surfaces themselves. For instance, during the initial LEVA steps when the surface is coated with PLL, ECM proteins of interest could be used independently, or in combination with PLL to mimic different microenvironments. While the exact location of photocleavage produced by the UV illumination is still unclear, it is likely that some, if not all of this initial layer remains on the surface and could be changed to mimic different microenvironments or modify the interactions with adsorbed EVs. In fact, any initial layer, which allows subsequent functionalization with PEG by targeting amines should be possible with slight modifications to the exemplified approach. It is also possible to cleave any residual PEG following micropatterning, in a manner similar to sacrificial layer removal in photolithography by using flood exposure of the entire surface with UV illumination. However, this optional step requires optimization and careful consideration of the effects that UV exposure could have on the captured EVs. Furthermore, in this study, it was largely performed patterning on glass cover slips to enable simple TIRFM imaging as required for continual validation in this type of study. However, as long as the general surface chemistry approach is the same and similar UV illumination is possible, LEVA could be applied to many other types of surfaces with similar resolution, tunability, and throughput. While this may affect TIRFM or other single EV-resolution imaging techniques, once the EV micropatterning is validated, a wide range of other functional surfaces could be patterned using LEVA. These could include engineered topographical cues and other 2D and 3D micro and nanostructures, polymer thin films, and other surfaces to create multifunctional platforms and devices. In these cases, electron microscopy can be used to validate LEVA, but once developed, these more advanced surfaces could also be incorporated into microfluidic and other bioMEMS devices to incorporate dynamic fluidic conditions for both the EVs and cells of interest.

[0090] Conclusion. In this study, a powerful new platform technology was introduced called Light-induced Extracellular Vesicle Adsorption (LEVA), enabling the efficient patterning of EVs onto surfaces in a scalable, versatile, and high-resolution manner. LEVA can rapidly advance the study of matrix- and surface-bound EVs from diverse sources and provide a clearer understanding of their role in cell-to-cell communication. The development and optimization of LEVA were enabled by the production of vast amounts of EVs from bioreactors and validated with commercially available GFP-EV standards and E. coli OMVs. Expanding on the reported simulation and time-lapse high-resolution microscopy studies will enable more efficient patterning, and has already clearly established LEVA’S dependence on EV size as well as charge. This study focused on validating the LEVA process and demonstrating its use in three applications; however, one of ordinary skill in the art would appreciate that LEVA can provide a platform for many other applications.

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[0158] The materials and methods of the appended claims are not limited in scope by the specific materials and methods described herein, which are intended as illustrations of a few aspects of the claims and any materials and methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the materials and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative materials, methods, and aspects of these materials and methods are specifically described, other materials and methods and combinations of various features of the materials and methods are intended to fall within the scope of the appended claims, even if not specifically recited. Thus a combination of steps, elements, components, or constituents can be explicitly mentioned herein; however, all other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

CLAIMSWhat is claimed is:

1. A device (e.g., a microfluidic device) comprising: a micropatterned substrate defining a binding region and a passivation region, wherein the binding region comprises a photoetched charged surface to non-specifically adsorb a population of extracellular particles (EPs) (e.g., extracellular vesicles (EVs), lipoproteins, viruses, non-vesicular particles, etc.) from a sample.

2. The device of claim 1, wherein the charged surface comprises a surface functionalized with a cationic polymer (e.g., a cationic protein).

3. The device of claim 2, wherein the cationic polymer comprises a polyamine.

4. The device of any one of claims 2-3, wherein the cationic polymer comprises an aminosilane.

5. The device of any one of claims 2-4, wherein the cationic polymer comprises poly- L-lysine (PLL).

6. The device of any one of claims 1-5, wherein the passivation region comprises a photocleavable antifouling coating.

7. The device of claim 6, wherein the photocleavable antifouling coating comprises methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA), polyether, polyethylene glycol, and / or poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG).

8. The device of any one of claims 1-7, wherein the binding region of the micropatterned substrate is monolithically etched into the passivation region.

9. The device of any one of claims 1-8, wherein at least a portion of the binding region is arranged in a gradient pattern.

10. The device of claim 9, wherein the gradient pattern comprises a linear gradient, an exponential decay gradient, and / or a Gaussian distribution.

11. The device of any one of claims 1-10, wherein the binding region is arranged in an array (e.g., an addressable array).

12. The device of any one of claims 1-11, wherein the binding region comprises a plurality of distinct geometric features.

13. The device of any one of claims 1-12, wherein the binding region is patterned in a migrasome-mimetic or matrix-bound EP trail.

14. The device of any one of claims 1-13, wherein the population of EPs comprises large EVs (IE Vs).

15. The device of any one of claims 1-14, wherein the population of EPs comprises small EVs (sEVs).

16. The device of any one of claims 1-15, wherein the population of EPs comprises migrasomes.

17. The device of any one of claims 1-16, wherein the population of EPs comprises lipoproteins.

18. The device of any one of claims 1-17, wherein the population of EPs comprises viruses.

19. The device of any one of claims 1-18, wherein the population of EPs comprises non- vesicular particles.

20. The device of any one of claims 1-19, wherein the population of EPs comprises a labeling group.

21. The device of any one of claims 1-20, wherein the population of EPs comprises a plurality of labeling groups.

22. The device of any one of claims 1-21, wherein the population of EPs comprises fluorescently labeled EPs.

23. A system comprising: the device according to any one of claims 1-22; and an imaging element positionable about the device to collect images of adsorbed EP.

24. A method for analyzing and / or characterizing extracellular particles (EPs) (e.g., extracellular vesicles (EVs), lipoproteins, viruses, non-vesicular particles, etc.), comprising: contacting a sample comprising a population of EPs (e.g., isolated EPs) with the device according to any one of claims 1-22; and obtaining images via an imaging element positionable about the device.