Stellate microneedle

Abstract

Provided herein is technology relating to obtaining and testing biological samples for disease biomarkers and particularly, but not exclusively, to hydrogel-coated microneedles, arrays of hydrogel-coated microneedles, and related methods of making stellate microcolumns and hydrogel-coated microneedles and using the hydrogel-coated microneedles for capturing exosomes from subject tissues for diagnosing diseases such as cancer.

Description

[0001] STELLATE MICRONEEDLE

[0002] STATEMENT OF RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 713,802, filed October 30, 2024, the entire contents of which are incorporated herein by reference for all purposes.

[0004] FIELD

[0005] Provided herein is technology relating to obtaining and testing biological samples for disease biomarkers and particularly, but not exclusively, to hydrogel-coated microneedles, arrays of hydrogel-coated microneedles, and related methods of using hydrogel-coated microneedles for capturing exosomes from subject tissues for diagnosing diseases such as cancer.

[0006] BACKGROUND

[0007] Microneedles (MN) have been used to isolate disease biomarkers from bodily fluids (e.g., blood, interstitial fluid) and thereby provide a minimally invasive method for diagnosing disease. See, e.g., Smith (2023) “Deep reactive ion etched microneedle array for in-vivo melanoma cancer monitoring via cancer exosome isolation” Cancer Res (2023) 83 (7_Supplement): 3297 (Abstract 3297); and Park (2023) “Hydrogel Microneedles

[0008] Extracting Exosomes for Early Detection of Colorectal Cancer” Biomacromolecules 24(3): 1445-52, incorporated herein by reference. However, diagnosis of diseases using microneedles to collect samples has been hindered by previous microneedle designs and by inefficient sample collection and biomarker isolation. Accordingly, new technologies are needed.

[0009] SUMMARY

[0010] For example, in some embodiments, the technology disclosed herein provides a method for isolating exosomes. In some embodiments, the method comprises pressing a microneedle patch comprising a plurality of microneedles coated with a functionalized hydrogel onto a tissue! and recovering exosomes from the microneedle patch. In some embodiments, the plurality of microneedles is arranged in an array comprising a spacing of at least 250 pm to 1000 pm (e.g., 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 pm) between microneedles. In some embodiments, each microneedle of the plurality of microneedles comprises a shaft and a tip, wherein the shaft has a length of at least 450 gm to 650 gm (e.g., 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, or 650 pm), at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm (e.g., 50, 60, 70, 80, 90, or 100 nm). In some embodiments, the plurality of microneedles comprises 100 to 1000 microneedles (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 microneedles).

[0011] In some embodiments, recovering exosomes comprises dissolving the functionalized hydrogel to produce a dissolved hydrogel. In some embodiments, recovering exosomes comprises dissolving the functionalized hydrogel to produce a dissolved hydrogel and centrifuging the dissolved hydrogel.

[0012] In some embodiments, the tissue is skin. In some embodiments, a living organism comprises the tissue. In some embodiments, a human comprises the tissue. In some embodiments, a human having a cancer comprises the tissue. In some embodiments, the tissue comprises skin and pressing the microneedle patch onto the skin causes the microneedle patch to pierce the skin to a depth of approximately 300 pm to 350 pm (e.g., 300, 310, 320, 330, 340, or 350 pm). In some embodiments, the tissue comprises skin and pressing the microneedle patch onto the skin causes the functionalized hydrogel to contact interstitial fluid and / or blood. In some embodiments, the tissue comprises skin and pressing the microneedle patch onto the skin causes the functionalized hydrogel to contact an exosome. In some embodiments, the exosome or exosomes comprise / s an exosome marker and a cancer biomarker. In some embodiments, the exosome or exosome / s comprise MCAM and / or MCSP.

[0013] In some embodiments, the technology disclosed herein provides a method of diagnosing a disease. In some embodiments, methods comprise pressing a microneedle patch comprising a plurality of microneedles coated with a functionalized hydrogel onto a tissue; recovering exosomes from the microneedle patch; and detecting a disease biomarker present in the exosomes using an immunoassay. In some embodiments, the plurality of microneedles is arranged in an array comprising a spacing of at least 250 pm to 1000 pm (e.g., 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 pm) between microneedles. In some embodiments, each microneedle of the plurality of microneedles comprises a shaft and a tip, wherein the shaft has a length of at least 450 pm to 650 pm (e.g., 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, or 650 pm), at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm (e.g., 50, 60, 70, 80, 90, or 100 nm). In some embodiments, the plurality of microneedles comprises 100 to 1000 microneedles (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 microneedles). In some embodiments, recovering exosomes comprises dissolving the functionalized hydrogel to produce a dissolved hydrogel. In some embodiments, recovering exosomes comprises dissolving the functionalized hydrogel to produce a dissolved hydrogel and centrifuging the dissolved hydrogel.

[0014] In some embodiments, the tissue is skin. In some embodiments, a human comprises the tissue. In some embodiments, a human having a cancer or suspected of having a cancer comprises the tissue. In some embodiments, the cancer is a melanoma. In some embodiments, the tissue comprises skin and pressing the microneedle patch onto the skin causes the microneedle patch to pierce the skin to a depth of approximately 300 pm to 350 pm (e.g., 300, 310, 320, 330, 340, or 350 pm). In some embodiments, the tissue comprises skin and pressing the microneedle patch onto the skin causes the functionalized hydrogel to contact interstitial fluid and / or blood. In some embodiments, the tissue comprises skin and pressing the microneedle patch onto the skin causes the functionalized hydrogel to contact an exosome. In some embodiments, the exosome or exosomes comprise / s an exosome marker and a cancer biomarker. In some embodiments, the exosome or exosomes comprise / s MCAM and / or MCSP.

[0015] In some embodiments, the immunoassay is a lateral flow immunoassay test strip comprising an antigen-binding agent specific for the biomarker. In some embodiments, the immunoassay is a lateral flow immunoassay test strip comprising a test line comprising an anti’MCAM and / or anti-MCSP antibody.

[0016] Further embodiments relate to a microneedle patch comprising a substrate comprising a plurality of microneedles coated with a functionalized hydrogel. In some embodiments, the substrate comprises silicon. In some embodiments, the substrate comprises glass or quartz. In some embodiments, the substrate comprises a metal (e.g., titanium, stainless steel) and / or a polymer.

[0017] In some embodiments, the plurality of microneedles is arranged in an array comprising a spacing of at least 250 pm to 1000 pm (e.g., 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 pm) between microneedles. In some embodiments, each microneedle of the plurality of microneedles extends from the substrate substantially normal to a planar surface of the substrate. In some embodiments, each microneedle of the plurality of microneedles comprises a shaft and a tip, wherein the shaft has a length of at least 450 pm to 650 pm (e.g., 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, or 650 pm), at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm (e.g., 50, 60, 70, 80, 90, or 100 nm). In some embodiments, the stellate shape is a six-pointed stellate shape, In some embodiments, the plurality of microneedles comprises 100 to 1000 microneedles (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 microneedles).

[0018] In some embodiments, the functionalized hydrogel comprises polyvinyl alcohol (PVA), alginate, and an exosome-binding agent. In some embodiments, the exosome- binding agent comprises a phosphatidylserine binding agent. In some embodiments, the exosome-binding agent comprises an annexin. In some embodiments, the exosome- binding agent comprises an annexin V.

[0019] The technology disclosed herein further provides a method of producing a microneedle patch. In some embodiments, the method comprises etching a substrate using a photolithograpy mask and a deep reactive ion etching to produce an array of micropillars! and sharpening the array of micropillars by exposing the micropillars to acid to produce an array of microneedles. In some embodiments, deep reactive ion etching is performed at a bias power of less than 40 W (e.g., 15 W, 20 W, 25 W, 30 W, or 35 W). In some embodiments, the acid comprises a mixture of hydrofluoric acid and nitric acid. In some embodiments, the substrate comprises silicon. In some embodiments, the plurality of microneedles is arranged in an array comprising a spacing of at least 250 pm to 1000 pm (e.g., 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 pm) between microneedles. In some embodiments, each microneedle of the plurality of microneedles extends from the substrate substantially normal to a planar surface of the substrate. In some embodiments, each microneedle of the plurality of microneedles comprises a shaft and a tip, wherein the shaft has a length of at least 450 pm to 650 pm (e.g., 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, or 650 pm), at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm (e.g., 50, 60, 70, 80, 90, or 100 nm). In some embodiments, the stellate shape is a six-pointed stellate shape. In some embodiments, the plurality of microneedles comprises 100 to 1000 microneedles (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 microneedles). In some embodiments, methods further comprise coating the plurality of microneedles with a functionalized hydrogel In some embodiments, the functionalized hydrogel comprises polyvinyl alcohol (PVA), alginate, and an exosome-binding agent. In some embodiments, the exosome-binding agent comprises a phosphatidylserine-bindmg agent. In some embodiments, the exosome- binding agent comprises an annexin. In some embodiments, the exosome -binding agent comprises an annexin V. In some embodiments, methods comprise producing a photolithography mask comprising an array of shapes; coating the substrate with a photoresist; exposing the photoresist using the photolithography mask; and developing the photoresist.

[0020] The technology described herein finds use in isolating exosomes and diagnosing disease. For example, embodiments provide use of a microneedle patch comprising a plurality of microneedles and a functionalized hydrogel to isolate exosomes from a subject. In some embodiments, the plurality of microneedles is arranged in an array comprising a spacing of at least 250 gm to 1000 gm (e.g., 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 gm) between microneedles. In some embodiments, each microneedle of the plurality of microneedles comprises a shaft and a tip, wherein the shaft has a length of at least 450 gm to 650 gm (e.g., 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, or 650 gm), at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm (e.g., 50, 60, 70, 80, 90, or 100 nm). In some embodiments, the plurality of microneedles comprises 100 to 1000 microneedles (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 microneedles). In some embodiments, the exosomes comprise an exosome marker and a cancer biomarker. In some embodiments, the exosomes comprise MCAM and / or MCSP. Some embodiments provide use of a microneedle patch comprising a plurality of microneedles and a functionalized hydrogel to detect a cancer biomarker in a subject. In some embodiments, the cancer biomarker is a melanoma biomarker.

[0021] The technology disclosed herein provides kits. In some embodiments, kits comprise a microneedle patch comprising a substrate comprising a plurality of microneedles; a functionalized hydrogel solution; and a lateral flow immunoassay device. In some embodiments, kits comprise a microneedle patch comprising a substrate comprising a plurality of microneedles coated with a functionalized hydrogel; and a lateral flow immunoassay device.

[0022] The technology described herein provides systems. In some embodiments, systems comprise a microneedle patch comprising a substrate comprising a plurality of microneedles; a functionalized hydrogel solution; and a lateral flow immunoassay device. In some embodiments, a system comprises a microneedle patch comprising a substrate comprising a plurality of microneedles coated with a functionalized hydrogel; and a lateral flow immunoassay device. In some embodiments, systems further comprise a reader device configured to quantify a test line on the immunoassay device. Reader devices are known in the art and are reviewed in, e.g., Park “Lateral Flow Immunoassay Reader Technologies for Quantitative Point-of Care Testing” Sensors (Basel) 22(19) ^7398 (2022), which is incorporated herein by reference.

[0023] Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0026] These and other features, aspects, and advantages of the present technology will become better understood with regard to the following drawings.

[0027] FIG. 1A is a schematic showing the steps of a method for producing a plurality of stellate microcolumns (steps 1 and 2) and a plurality of micro needles (step 3).

[0028] FIG. IB is a schematic drawing of a microneedle patch 100 comprising a substrate 110 and a microneedle 120 as provided herein. FIG. IB further shows a coordinate system used to describe microneedle patches herein.

[0029] FIG. 1C is a schematic drawing showing a microneedle patch comprising a plurality of microneedles in an array (e.g., a rectangular array). Dotted lines indicate measurements of spacing between adjacent microneedles.

[0030] FIG. 2A is a drawing of a stellate microcolumn. In some embodiments, the drawing of the stellate microcolumn finds use in producing a microneedle as described herein. Measurements are provided in units of micrometers. In some embodiments, the drawing finds use in preparing a photoresist mask for producing a stellate microcolumn and microneedle as described herein.

[0031] FIG. 2B is a drawing of an array of stellate microcolumns. In some embodiments, the drawing of the stellate microcolumns finds use in producing an array of microneedles as described herein. Measurements are in micrometers. In some embodiments, the drawing finds use in preparing a photoresist mask for producing an array of stellate microcolumns and microneedles as described herein.

[0032] FIG. 2C is an electron micrograph of a stellate microcolumn as described herein. FIG. 2D is an electron micrograph of a microneedle as described herein. FIG. 2E is an electron micrograph showing the tip of a microneedle as described herein.

[0033] FIG. 2F is an electron micrograph showing an array of microneedles as described herein.

[0034] FIG. 2G is a photograph showing an array of microneedles as described herein.

[0035] FIG. 2H is an electron micrograph showing a microneedle produced by DRIE using a 40-W bias power.

[0036] FIG. 21 is an electron micrograph showing a microneedle produced by DRIE using a 20-W bias power.

[0037] FIG. 3 is an image from lightsheet microscopy showing the puncture of skin by microneedles to a depth of approximately 300 to 350 gm.

[0038] FIG. 4A is a photograph showing contact of a microneedle patch comprising a functionalized hydrogel on a melanoma mouse patient- derived xenograft sample.

[0039] FIG. 4B is a photograph showing the melanoma mouse patient derived xenograft sample after removing the microneedle patch comprising a functionalized hydrogel.

[0040] FIG. 5 is a bar plot showing the total protein concentration in melanoma exosomes and healthy exosomes isolated from melanoma mouse patient derived xenograft (PDX) samples and healthy mouse skin samples, respectively.

[0041] FIG. 6 is a photograph of a western blot used to detect calnexin (a cell marker), TSG101 (an exosome marker), flotillin 1 (an extracellular vesicle marker), and MCSP (a melanoma marker) in melanoma exosomes and healthy exosomes isolated from melanoma mouse patient-derived xenograft (PDX) samples and healthy mouse skin samples, respectively.

[0042] FIG. 7 is a plot showing data from an ELISA used to quantify CD63 in melanoma exosomes and healthy exosomes isolated from melanoma mouse patient derived xenograft (PDX) samples and healthy mouse skin samples, respectively. Data were collected using a microneedle patch comprising a functionalized hydrogel that was dipped into EVs in solution (x), a microneedle patch comprising a functionalized hydrogel that was pressed into melanoma skin (o), and a microneedle patch comprising a functionalized hydrogel that was pressed into healthy skin (•).

[0043] FIG. 8A is a SEM image showing exosomes isolated using a microneedle patch comprising a functionalized hydrogel pressed into melanoma skin.

[0044] FIG. 8B is a SEM image showing exosomes isolated using a microneedle patch comprising a functionalized hydrogel pressed into healthy skin. FIG. 8C is a SEM image showing that the exosomes isolated from the melanoma skin had diameters in the expected range of 30 to 200 nm.

[0045] FIG. 8D is a SEM image showing that the exosomes isolated from the melanoma skin had diameters in the expected range of 30 to 200 nm.

[0046] FIG. 9 is a bar plot showing results of validating a LFIA test strip for detecting melanoma biomarkers associated with exosomes isolated using a microneedle patch comprising a functionalized hydrogel that was pressed into melanoma skin.

[0047] FIG. 10A is a photograph showing LFIA test strips used to assay melanoma and healthy samples prepared using a microneedle patch comprising a functionalized hydrogel.

[0048] FIG. 10B is a bar plot showing quantification of the test line intensities for the melanoma sample and for the healthy sample shown in FIG. 10A.

[0049] It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.

[0050] DETAILED DESCRIPTION

[0051] Provided herein is technology relating to obtaining and testing biological samples for disease biomarkers and particularly, but not exclusively, to hydrogel-coated microneedles, arrays of hydrogel-coated microneedles, and related methods of using hydrogel-coated microneedles for capturing extracellular vesicles from subject tissues for diagnosing diseases such as cancer. In some embodiments, the microneedles have a stellate shape and are produced by a method comprising producing a stellate microcolumn and producing the microneedle from the stellate microcolumn.

[0052] In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that the sequences can be varied and still remain within the spirit and scope of the various embodiments disclosed herein.

[0053] All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belongs. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

[0054] Definitions

[0055] To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0056] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment’' as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0057] In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, and “the” include plural references. The meaning of “in” includes “in” and “on.”

[0058] As used herein, the terms “about”, “approximately”, “substantially”, and “significantly” are understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms that are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” mean plus or minus less than or equal to 10% of the particular term and “substantially” and “significantly” mean plus or minus greater than 10% of the particular term.

[0059] As used herein, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges. As used herein, the disclosure of numeric ranges includes the endpoints and each intervening number therebetween with the same degree of precision. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0060] As used herein, the suffix “-free” refers to an embodiment of the technology that omits the feature of the base root of the word to which “-free” is appended. That is, the term “X-free” as used herein means “without X”, where X is a feature of the technology omitted in the “X-free” technology. For example, a “calcium-free” composition does not comprise calcium, a “mixing-free” method does not comprise a mixing step, etc.

[0061] Although the terms “first”, “second”, “third”, etc. may be used herein to describe various steps, elements, compositions, components, regions, layers, and / or sections, these steps, elements, compositions, components, regions, layers, and / or sections should not be limited by these terms, unless otherwise indicated. These terms are used to distinguish one step, element, composition, component, region, layer, and / or section from another step, element, composition, component, region, layer, and / or section. Terms such as “first”, “second”, and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first step, element, composition, component, region, layer, or section discussed herein could be termed a second step, element, composition, component, region, layer, or section without departing from technology.

[0062] As used herein, the word “presence” or “absence” (or, alternatively, “present” or “absent”) is used in a relative sense to describe the amount or level of a particular entity (e.g., an analyte (e.g., a biomarker)). For example, when an analyte is said to be “present” in a test sample, it means the level or amount of this analyte is above a predetermined threshold; conversely, when an analyte is said to be “absent” in a test sample, it means the level or amount of this analyte is below a pre- determined threshold. The pre determined threshold may be the threshold for detectability associated with the particular test used to detect the analyte or any other threshold. When an analyte is “detected” in a sample it is “present” in the sample; when an analyte is “not detected” it is “absent” from the sample. Further, a sample in which an analyte is “detected” or in which the analyte is “present” is a sample that is “positive” for the analyte. A sample in which an analyte is “not detected” or in which the analyte is “absent” is a sample that is “negative” for the analyte.

[0063] As used herein, an “increase” or a “decrease” refers to a detectable (e.g., measured) positive or negative change, respectively, in the value of a variable relative to a previously measured value of the variable, relative to a pre-established value, and / or relative to a value of a standard control. An increase is a positive change preferably at least 10%, more preferably 50%, still more preferably 2 fold, even more preferably at least 5-fold, and most preferably at least 10-fold relative to the previously measured value of the variable, the pre-established value, and / or the value of a standard control. Similarly, a decrease is a negative change preferably at least 10%, more preferably 50%, still more preferably at least 80%, and most preferably at least 90% of the previously measured value of the variable, the pre-established value, and / or the value of a standard control. Other terms indicating quantitative changes or differences, such as “more” or “less,” are used herein in the same fashion as described above.

[0064] As used herein, the term “number” shall mean one or an integer greater than one (e.g., a plurality). As used herein, the term “plurality” shall mean more than one.

[0065] As used herein, the word “unitary” means a component is created as a single piece or unit. That is, a component that includes pieces that are created separately and then coupled together as a unit is not a “unitary” component or body.

[0066] As used herein, a “system” refers to a plurality of real and / or abstract components operating together for a common purpose. In some embodiments, a “system” is an integrated assemblage of hardware and / or software components. In some embodiments, each component of the system interacts with one or more other components and / or is related to one or more other components. In some embodiments, a system refers to a combination of components and software for controlling and directing methods. For example, a “system” or “subsystem” may comprise one or more of, or any combination of, the following: mechanical devices, hardware, components of hardware, circuits, circuitry, logic design, logical components, software, software modules, components of software or software modules, software procedures, software instructions, software routines, software objects, software functions, software classes, software programs, files containing software, etc., to perform a function of the system or subsystem. Thus, the methods and apparatus of the embodiments, or certain aspects or portions thereof, may take the form of program code (e.g., instructions) embodied in tangible media, such as floppy diskettes, CD-ROMs, hard drives, flash memory, or any other machine -readable storage medium wherein, when the program code is loaded into and executed by a machine, such as a computer, the machine becomes an apparatus for practicing the embodiments. In the case of program code execution on programmable computers, the computing device generally includes a processor, a storage medium readable by the processor (e.g., volatile and non-volatile memory and / or storage elements), at least one input device, and at least one output device. One or more programs may implement or utilize the processes described in connection with the embodiments, e.g., through the use of an application programming interface (API), reusable controls, or the like. Such programs are preferably implemented in a high-level procedural or object-oriented programming language to communicate with a computer system. However, the program(s) can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language, and combined with hardware implementations.

[0067] The terms “protein’ and “polypeptide’’ refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably. A “protein” or “polypeptide” encoded by a gene is not limited to the amino acid sequence encoded by the gene but includes post- translational modifications of the protein. Where the term “amino acid sequence” is used herein to refer to an amino acid sequence of a protein molecule, “amino acid sequence” and like terms, such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. Furthermore, an “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein.

[0068] The term “domain” when used in reference to a polypeptide refers to a subsection of the polypeptide which possesses a unique structural and / or functional characteristic! typically, this characteristic is similar across diverse polypeptides. The subsection typically comprises contiguous amino acids, although it may also comprise amino acids that act together or that are in close proximity due to folding or other configurations.

[0069] The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or “nucleic acid” refer to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.

[0070] As used herein, the term “antibody” is used in its broadest sense to refer to whole antibodies, monoclonal antibodies (including human, humanized, or chimeric antibodies), polyclonal antibodies, and antibody fragments that can bind antigen (e.g., Fab', F' (ab)2, Fv, single chain antibodies), comprising complementarity determining regions (CDRs) of the foregoing as long as they exhibit the desired biological activity.

[0071] As used herein, “antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments! diabodies! linear antibodies; single -chain antibody molecules! and multispecific antibodies formed from antibody fragments.

[0072] As used herein the term, “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments may include, but are not limited to, test tubes and cell cultures. The term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0073] As used herein the term “disease” refers to a deviation from the condition regarded as normal or average for members of a species, and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species (e.g., diarrhea, nausea, fever, pain, inflammation, etc.).

[0074] As used herein, the term “administration” refers to the act of giving a drug, prodrug, antibody, or other agent, or therapeutic treatment to a physiological system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplary routes of administration to the human body can be through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like. “Coadministration” refers to administration of more than one chemical agent or therapeutic treatment (e.g., radiation therapy) to a physiological system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). As used herein, administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. “Coadministration” of therapeutic treatments may be concurrent, or in any temporal order or physical combination.

[0075] As used herein, the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a disease or disorder through introducing in any way a therapeutic composition of the present technology into or onto the body of a subject, “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

[0076] As used herein, “therapeutically effective dose” refers to an amount of a therapeutic agent sufficient to bring about a beneficial or desired clinical effect. Said dose can be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired (e.g., aggressive vs. conventional treatment).

[0077] As used herein, the terms “detect”, “detecting”, or “detection” may describe either the general act of discovering or discerning or the specific observation of a detectably labeled composition.

[0078] As used herein, the term “stage of cancer” refers to a qualitative or quantitative assessment of the level of advancement of a cancer. Criteria used to determine the stage of a cancer include, but are not limited to, the size of the tumor and the extent of metastases (e.g., localized or distant).

[0079] As defined herein, “a tumor” is a neoplasm that may either be malignant or non- malignant. Tumors of the same tissue type originate in the same tissue and may be divided into different subtypes based on their biological characteristics.

[0080] As used herein, the term “cancer” refers to a malignant disease caused or characterized by the proliferation of cells that have lost susceptibility to normal growth control. “Malignant disease” refers to a disease caused by cells that have gained the ability to invade either the tissue of origin or to travel to sites removed from the tissue of origin. Particular cancers related to the technology provided herein include, but are not limited to, lung cancer and pancreatic cancer.

[0081] As used herein, the term “stellate” refers to a star shape, e.g., a shape comprising a center (e.g., a polyhedral or circular center) and a number of features (e.g., points, rays) radiating from the center. In some embodiments, a stellate shape is a star polygon. A star polygon may be described by a Schlafli symbol {n / m}, in which n is the number of vertices, m is the step used in sequencing the edges around it, and m and n have no common factor. In some embodiments, a stellate shape is an isotoxal star simple polygon (i.e. , having alternating vertices at two different radii). An isotoxal star simple polygon that matches the outline of a regular star polygon described by Schlafli symbol {n / m} may be described using the notation | n / m | or more generally by {nQ} to describe an isotoxal concave or convex simple 2n-gon with outer internal angle a. For an isotoxal star simple polygon described by I n / m | , the outer internal angle a = 180(1 - 2m / n) degrees and the inner vertices have an external angle Bext = 180(1 - 2(m - l) / n) degrees. While some embodiments of the technology are described in which a six-pointed stellate shape (e.g., an isotoxal star simple polygon described as {Gao0} and in which a is 30° and Bext is 90°) is used to construct a micropillar (see, e.g., FIG. 2A and 20), the technology is not limited to any particular stellate shape and / or any particular number of features radiating from the center. In some embodiments, a stellate shape may be constructed by extending edges or faces of a polygon until they meet to form a new polygon. One may draw a stellate shape in MAPLE (Maple 2024, Maplesoft, a division of Waterloo Maple Inc., Waterloo, Ontario) using the command stellate(gon, core, n), wherein gon is the name of the stellated polyhedron to be created, core is the core polyhedron, and n is a non negative integer. See, e.g., Grunbaum and Shephard, Tilings and Patterns (Dover Publications Inc.i 2nd ed., 2016), incorporated herein by reference.

[0082] As used herein, the term “exosome” refers to a membranous particle having a diameter (or a largest dimension of a non-spheroid shape) of between approximately 10 nm to approximately 5000 nm, more typically between approximately 30 nm and approximately 1000 nm, and most typically between approximately 50 nm and approximately 200 nm, wherein at least part of the membrane of the exosomes is directly obtained from a cell membrane.

[0083] As used herein, the terms “patient” or “subject” refer to organisms that are subjected to various tests provided by the technology. The term “subject” includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human.

[0084] As used herein, the term “sample” refers to any sample suitable for the methods described herein. The sample may be any sample that includes exosomes suitable for detection or isolation. Sources of samples include blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, sweat, tears, joint fluid, interstitial fluid (e.g., plasmatic, lymphatic, intraocular, synovial, pericardial, intrapleural, peritoneal, and digestive fluids), and bronchial washes. In particular assays described herein, the sample is a blood sample, including, for example, whole blood or any fraction or component thereof including serum and plasma. A blood sample suitable for use with the present disclosure may be extracted from any source known that includes blood cells or components thereof, such as venous, arterial, peripheral, tissue, cord, and the like. For example, a sample may be obtained and processed using well-known and routine clinical methods (e.g., procedures for drawing and processing whole blood). In one aspect, an exemplary sample may be peripheral blood drawn from a subject with cancer. In another embodiment, the sample may be platelet-free plasma.

[0085] As used herein, a “system” refers to a plurality of real and / or abstract components operating together for a common purpose. In some embodiments, a “system” is an integrated assemblage of hardware and / or software components. In some embodiments, each component of the system interacts with one or more other components and / or is related to one or more other components. In some embodiments, a system refers to a combination of components and software for controlling and directing methods. For example, a “system” or “subsystem” may comprise one or more of, or any combination of, the following: mechanical devices, hardware, components of hardware, circuits, circuitry, logic design, logical components, software, software modules, components of software or software modules, software procedures, software instructions, software routines, software objects, software functions, software classes, software programs, files containing software, etc., to perform a function of the system or subsystem. Thus, the methods and / or steps of methods described herein, or certain aspects or portions thereof, may take the form of program code (e.g., instructions) embodied in tangible media, such as floppy diskettes, CD-ROMs, hard drives, flash memory, or any other machine -readable storage medium wherein, when the program code is loaded into and executed by a machine, such as a computer, the machine becomes an apparatus for practicing the embodiments. In the case of program code execution on programmable computers, the computing device generally includes a processor, a storage medium readable by the processor (e.g., volatile and non-volatile memory and / or storage elements), at least one input device, and at least one output device. One or more programs may implement or utilize the processes described in connection with the embodiments, e.g., through the use of an application programming interface (API), reusable controls, or the like. Such programs are preferably implemented in a high-level procedural or object-oriented programming language to communicate with a computer system. However, the program (s) can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language and combined with hardware implementations. Description

[0086] Cancer is one of the leading causes of death. Accordingly, the technology provided herein relates to the early diagnosis and treatment of cancer, e.g., to improve patient outcomes. Successful treatment of cancer relies on diagnosis, staging the disease, selecting an effective therapy, and / or monitoring disease state after therapy to prevent or detect relapse. Conventional cancer diagnosis typically comprises histological evaluation of tumor material obtained from tissue biopsy. However, biopsy based sampling is invasive. Thus, provided herein is an improved and non-invasive or minimally invasive sampling technology for obtaining and testing biological samples for disease biomarkers and particularly, but not exclusively, to hydrogel-coated microneedles, arrays of hydrogebcoated microneedles, and related methods of using hydrogel-coated microneedles for capturing exosomes from subject tissues for diagnosing diseases such as cancer. The technology provided herein has the advantage of being non-invasive or minimally invasive.

[0087] Exosomes

[0088] Extracellular vesicles (EV) are released by all cells in both normal and abnormal functioning. Release of EV occurs in both prokaryotes and eukaryotes and is important in a broad range of physiological and pathological processes. Exosomes are a type of EV that originate from cellular endosomes, which sort and transport proteins and lipids within a cell. Exosomes comprise a membrane bilayer that separates the internal lumen space from the external milieu. Exosomes typically have a size of approximately 100 nm (e.g., ranging from approximately 40 nm to 160 nm). See, e.g., Yanez-Md (2015) “Biological properties of extracellular vesicles and their physiological functions” Journal of Extracellular Vesicles. 4: 27066, incorporated herein by reference.

[0089] After production by cells, exosomes interact with other cellular components. Thus, exosomes comprise molecular biomarkers produced by the cell of origin and the complement of biomarkers present in exosomes is associated with the state (e.g., healthy, diseased) and type of the cell producing the exosomes. Further, exosomes have been detected in all biological fluids, thus providing an easily accessible, noninvasive sample for obtaining and assaying exosomal biomarkers. Thus, exosome collection and analysis of exosome biomarkers provide a means for diagnosing disease in a subject. See, e.g., Nonaka (2022) “Saliva Diagnostics” Annual Review of Analytical Chemistry 15: 107-21; and van der Pol (2012) “Classification, functions, and clinical relevance of extracellular vesicles” Pharmacological Reviews 64: 676-705. each of which is incorporated herein by reference.

[0090] Exosomes comprise biomarkers in both the lumen and associated with the exosome membrane bilayer. Exemplary types of biomolecules that are carried by exosomes include nucleic acids (e.g., DNA (cell-free DNA (cfDNA)), RNA (e.g., mRNA, tRNA, microRNA (e.g., exosomal microRNA), hcRNA, circRNA. etc.)), lipids (e.g., phospholipids, cholesterol, lipid rafts, ceramides), metabolites, ribonucleoproteins, and proteins (e.g., adhesion molecules, cytoskeleton components, cytokines, ribosomal proteins, growth factors, metabolic enzymes).

[0091] While exosomes comprise biomarkers that vary according to the cell of origin and its status, exosomes also comprise an evolutionarily-conserved common set of protein molecules, e.g., CD9, CH63, CD81, ALIX, HSP70, TSG101, duramycin, and heparin.

[0092] Cancer biomarkers

[0093] Exosomes are useful in disease diagnosis because they are involved in a number of physiological functions. For example, exosomes carry biomolecules between cells - in particular, exosomes deliver biomolecules that influence apoptosis, metastasis, angiogenesis, tumor progression, thrombosis and immunity by directing T cells towards immune activation or immune suppression.

[0094] As noted above, exosomes comprise cytosolic and membrane components that reflect the properties of the cell from which they are produced. Accordingly, the type of originating cell can be used to describe the exosomes, e.g., the term “tumor-derived exosomes” or “tumor exosomes” or “cancer exosomes” refers to exosomes secreted by, derived from, and indicative of tumor, cancer, and / or malignant cells. Similarly, the term “normal exosomes” refers to exosomes secreted by, derived from, and indicative of normal cells. As used herein, “normal cells” refers to substantially healthy, nondiseased, non-apoptotic, and non-stressed cells (e.g., non-tumorigenic cells).

[0095] Embodiments of the technology relate to diagnosing, staging and classifying all cancers including lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, brain cancer, thyroid cancer, liver cancer, cervical cancer, ovarian cancer, gastrointestinal cancer, lymphomas, pre -neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer. Some cancer biomarkers include prostate -specific antigen (PSA) as a biomarker of prostate cancer, HER2 as a biomarker of certain types of breast cancer, CA125 as a biomarker of ovarian cancer, CEA as a biomarker of colorectal cancer, BRCA1 / BRCA2 as a biomarker of ovarian and breast cancer, MCAM and / or MCSP as a biomarker of melanoma, and EGFR as a biomarker of certain types of non small cell lung cancer.

[0096] Importantly, tumor cell-derived exosomes comprise phosphatidylserine (PS) in the outer leaflet of the bilayer membrane and thus PS provides an important biomarker of tumor exosomes. Accordingly, PS binding agents may be used to isolate tumor exosomes. One particular exemplary class of PS binding agents are annexins e.g., annexin V. Other PS binding agents include, e.g., anti-PS antibodies (e.g., monoclonal antibodies, antibody fragments, single chain antibodies, single chain variable fragments). See, e.g., U.S. Pat. No. 8,956,616, incorporated herein by reference. Additional PS binding agents include Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Factor VIII, Factor IX, Factor IX, Factor X, Mer (a PS-binding scavenger receptor), a5B3 integrin, CR3 complement receptor, the CR4 complement receptor, PSr (a phosphatidylserine receptor), Beta 2 -glycoprotein, protein kinase C (PKG), PLC8, synaptotagmin (e.g., comprising Ca2+-dependent 02 domains), Gas6, MFG- E8, Akapl2, Akap81, pinin, serum response factor binding protein 1 (Srfbp), Vtilb, Fibrillarin, Mylk, Prpf40a, C2cd21, Collla2, annexin Al, lactadheren, and apolipoprotein H (Apo H).

[0097] Subjects

[0098] Subjects include, e.g., an animal (e.g., a mammal (e.g., a human)) having a disease. The disease may be a neoplasm (e.g., a cancer). In some embodiments, the subject has a lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, brain cancer, thyroid cancer, liver cancer, cervical cancer, ovarian cancer, gastrointestinal cancer, lymphomas, pre -neoplastic lesions in the lung, colon cancer, melanoma, skin cancer (e.g., melanoma), or bladder cancer.

[0099] T reatment

[0100] In some embodiments, the technology relates to treating hyperplastic, dysplastic, or neoplastic diseases and conditions (e.g., cancer). In particular, the technology provides methods of diagnosing and treating lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, thyroid cancer, brain cancer, renal cancer, bone cancer, liver cancer, skin cancers (e.g., melanoma), testicular cancer, cervical cancer, ovarian cancer, gastrointestinal cancer, lymphomas, colon cancer, bladder cancer, and any other neoplastic disease. As used herein, the term “treatment of cancer” refers to include killing cancer cells, inhibiting cancer cell growth, inhibiting metastasis, decreasing tumor or tissue size, decreasing tumor cell burden, or otherwise reversing or reducing the malignant phenotype of tumor cells.

[0101] In some embodiments, methods comprise treating a subject having a cancer. In some embodiments, methods of treating a subject having a cancer comprise administering a pharmaceutical composition to the subject. In some embodiments, methods of treating a subject having a cancer comprise administering an effective amount of a pharmaceutical composition to the subject.

[0102] In some embodiments, treating cancer includes reducing or stabilizing the size of a tumor (e.g., a benign or malignant tumor), slowing or preventing an increase in the size of a tumor, reducing or stabilizing the number of tumor cells, increasing the disease-free survival time between the disappearance of a tumor and its reappearance, preventing an initial or subsequent occurrence of a tumor, or reducing or stabilizing an adverse symptom associated with a tumor. In some embodiments, the number of cancerous cells surviving the treatment is at least 10, 20, 40, 60, 80, or 100% lower than the initial number of cancerous cells, as measured using any standard assay. In some embodiments, the decrease in the number of cancerous cells induced by administration of a therapy is at least 2, 5, 10, 20, or 50-fold greater than the decrease in the number of non-cancerous cells. In some embodiments, the number of cancerous cells present after administration of a therapy is at least 2, 5, 10, 20, or 50-fold lower than the number of cancerous cells present after administration of a control (such as administration of saline or a buffer). In some embodiments, the methods of the present invention result in a decrease of 10, 20, 40, 60, 80, or 100% in the size of a tumor as determined using standard methods. In some embodiments, at least 10, 20, 40, 60, 80, 90, or 95% of the treated subjects have a complete remission in which there are no detectable cancerous cells. In some embodiments, the cancer does not reappear, or reappears after at least 2, 5, 10, 15, or 20 years. In some embodiments, the length of time a subject survives after being diagnosed with cancer and treated with a therapy of the invention is at least 10, 20, 40, 60, 80, 100, 200, or even 500% greater than (i) the average amount of time an untreated subject survives or (ii) the average amount of time a subject treated with another therapy survives.

[0103] In some embodiments, the technology provides methods for stratifying or classifying subjects involved in a clinical trial for the treatment of a disease or disorder in a mammal.

[0104] In various embodiments, routes of administration for administering a pharmaceutical composition to a subject vary according to the condition of the subject, type of cancer, location and nature of the lesion, and drug, and may include, e.g., intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration and formulation.

[0105] In some embodiments, the technology relates to methods, compositions, and kits for monitoring therapeutic efficacy of an anti-cancer treatment. In some embodiments, methods comprise quantifying tumor exosomes in a first sample from a subject obtained at a first time-point, the first time-point occurring before the cancer treatment, quantifying tumor exosomes in at least a second sample from the subject obtained at a second time-point, the second time-point occurring after the cancer treatment! and comparing the amount of tumor exosomes in the first and second samples. An increased amount of tumor exosomes in the second sample relative to the first sample is indicative of lack of therapeutic efficacy, and a decreased amount of tumor exosomes in the second sample relative to the first sample is indicative of therapeutic efficacy.

[0106] Devices

[0107] In some embodiments, the technology provided herein relates to devices comprising a number of microneedles (a “microneedle patch”). In some embodiments, a microneedle patch finds use in methods for isolating exosomes from a subject.

[0108] In some embodiments, a microneedle patch comprises a number of microneedles. In some embodiments, e.g., as shown in FIG. IB, the microneedle patch 100 comprises a substrate 110 comprising a number of microneedles 120. In some embodiments, the substrate is planar or substantially planar and the microneedles project from the substrate in a direction that is normal or substantially normal to the plane of the substrate. In some embodiments, the microneedle patch comprising the substrate and the microneedles is a unitary piece. In some embodiments, the microneedle patch comprising the substrate and the microneedles is a unitary piece comprising silicon (e.g., etched silicon).

[0109] In some embodiments, e.g., as shown in FIG. 1C, the microneedle patch comprises 10 to 1,000,000 microneedles. In some embodiments, the microneedle patch comprises at least 10! 100; 1000; 10,000; 100,000; or 1,000,000 microneedles. In some embodiments, the microneedle patch comprises 100 to 1000 microneedles (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 microneedles). In some embodiments, e.g., as shown in FIG. 1C, the microneedle patch comprises a plurality of microneedles arranged in an array. In some embodiments, the array has a spacing (i.e., a microneedle density) and an arrangement. In some embodiments, the array of microneedles comprises a spacing of at least 250 pm to 1000 pm (e.g., 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,

[0110] 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,

[0111] 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760,

[0112] 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,

[0113] 950, 960, 970, 980, 990, or 1000 pm) between adjacent microneedles. In some embodiments the microneedles are arranged in a rectangular array (e.g., having a number of rows and columns). In some embodiments, the microneedles are arranged in a hexagonal array. In some embodiments, the microneedles are arranged in a number of concentric circles. In some embodiments, the microneedles are arranged in a number of concentric geometric shapes.

[0114] As used herein, the term “width” refers to a dimension measured in the X Y plane of the microneedle patch substrate (or measured in a plane parallel or substantially parallel to the microneedle patch substrate), and the term “length” refers to a dimension measured in a direction normal to the X-Y plane of the microneedle substrate (e.g., in the Z direction).

[0115] In some embodiments, each microneedle comprises a shaft and a tip. In some embodiments, the shaft has a length of approximately 450 pm to 650 pm and the tip has a width of approximately 100 nm or less (e.g., 50, 60, 70, 80, 90, or 100 nm). In some embodiments, the shaft has a width of approximately 50 to 200 pm (e.g. , 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 pm). In some embodiments, each microneedle has a stellate shape. In some embodiments, the microneedle shaft has a stellate shape. In some embodiments, each microneedle has a six-pointed stellate shape, e.g., an isotoxal star simple polygon described as {6so0} and in which a is 30° and Bext is 90°).

[0116] During the development of embodiments of the microneedle technology described herein, experiments were conducted with microneedle patches, and data were collected indicating that the microneedles have a verticality, length, sharpness, and stability appropriate for piercing skin and isolating exosomes.

[0117] In some embodiments, the microneedle patch is coated with a functionalized hydrogel. In some embodiments, the functionalized hydrogel comprises polyvinyl alcohol (PVA), alginate, and an exosome-binding (e.g., PS-binding) agent (e.g., an annexin (e.g., annexin V)). In some embodiments, the functionalized hydrogel is gelated using a calcium chloride solution (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaC12).

[0118] Methods

[0119] In some embodiments, the technology provides methods. In some embodiments, the technology provides methods of producing a microneedle patch. In some embodiments, the technology provides methods of using a microneedle patch to isolate exosomes.

[0120] For instance, embodiments of methods for producing a microneedle patch comprise producing a photolithography mask, e.g., by producing a graphical image file comprising an outline of a mask shape and printing the image on a transparent medium. In some embodiments, methods comprise producing a photolithography mask comprising an array of outlined shapes (e.g., an array of outlined stellate shapes). In some embodiments, the photolithography mask comprises at least 10; 100; 1000; 10,000; 100,000; or 1,000,000 outlined shapes (e.g., outlined stellate shapes). In some embodiments, the photolithography mask comprises 100 to 1000 outlined shapes (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 outlined shapes (e.g., outlined stellate shapes)).

[0121] Next, methods comprise coating a substrate (e.g., a silicon wafer) with a layer of photoresist. In some embodiments, the substrate is a silicon wafer having a diameter of approximately 4 inches (e.g., approximately 100 mm) and a thickness of approximately 1 mm. Accordingly, embodiments of methods comprise providing a substrate (e.g., a silicon wafer) and photoresist. In some embodiments, the layer of photoresist is approximately 3 pm to 5 pm thick. Methods then comprise exposing the photoresist using the photolithography mask and developing the photoresist. After developing the photoresist, the substrate is etched (e.g., using wet etching, RIE, and / or DRIE) to produce a substrate comprising an array of micropillars, e.g., an array of micropillars having a stellate shape. In some embodiments, methods comprise etching using DRIE at a low bias power (e.g., less than 40 W, e.g., 20 W) until approximately 800 to 900 pm of the substrate is etched, e.g., to produce micropillars (e.g., stellate micropillars) having a length of approximately 800 to 900 pm. In some embodiments, methods comprise incubating the substrate comprising the micropillars (e.g., stellate micropillars) in an O2 plasma, e.g., to remove sidewall passivation. Further, embodiments comprise sharpening the micropillars to produce microneedles. For example, in some embodiments, methods comprise incubating the substrate comprising the micropillars in a wet etch solution comprising a mixture of one or more acids (e.g., a mixture of hydrofluoric acid and nitric acid (e.g., 1:20 v / v hydrofluoric acid to nitric acid)) for 35 to 40 minutes (e.g., 35, 36, 37, 38, 39, or 40 minutes) to produce a substrate comprising microneedles (e.g., a microneedle patch). Experiments conducted during the development of embodiments of the technology described herein produced data indicating that each microneedle had a length of approximately 450 to 650 pm from top to bottom and a tip approximately 100 nm in width. SEM indicated that the base of each microneedle had a stellate shape, which improved the stability of the microneedle.

[0122] In some embodiments, methods comprise coating microneedles with a functionalized hydrogel. In some embodiments, the functionalized hydrogel comprises polyvinyl alcohol (PVA), alginate, and an exosome-binding (e.g., PS-binding) agent (e.g., an annexin (e.g., annexin V)). In some embodiments, methods comprise producing a functionalized hydrogel. In some embodiments, producing a functionalized hydrogel comprises dissolving PVA in water to produce a heated PVA solution, dissolving alginate in water to produce a heated alginate solution, and mixing the heated PVA solution and the heated alginate solution to produce a hydrogel solution. Next, producing a functionalized hydrogel comprises conjugating the hydrogel solution with the exosome- binding (e.g., PS-binding) agent (e.g., an annexin (e.g., annexin V)) to produce a functionalized hydrogel solution. Finally, methods comprise gelating the functionalized hydrogel solution using a calcium chloride solution (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCh) to gelate (e.g., solidify) the functionalized hydrogel solution and thereby produce a functionalized hydrogel coating on the microneedles.

[0123] In some embodiments, a microneedle patch comprising a functionalized hydrogel finds use in isolating exosomes from a subject. Accordingly, embodiments of methods for isolating exosomes from a subject comprise providing a microneedle patch comprising a functionalized hydrogel, e.g., according to a method described herein. In some embodiments, methods comprise contacting (e.g., pressing) the microneedle patch onto the skin of a subject. In some embodiments, methods comprise contacting (e.g., pressing) the microneedle patch onto the skin of a subject for 5 to 30 minutes (e.g., 5, 10, 15, 20, 25, or 30 minutes). In some embodiments, contacting (e.g., pressing) the microneedle patch onto the skin of a subject provides that a number of microneedles pierce the skin of the subject. In some embodiments, contacting (e.g., pressing) the microneedle patch onto the skin of a subject provides that a number of microneedles penetrate the skin to a depth of approximately 300 to 350 pm (e.g., 300, 310, 320, 330, 340, or 350 pm). In some embodiments, contacting (e.g., pressing) the microneedle patch onto the skin of a subject provides that a number of microneedles (e.g., comprising a functionalized hydrogel) contact interstitial fluid of the subject. In some embodiments, contacting (e.g., pressing) the microneedle patch onto the skin of a subject provides that a number of microneedles (e.g., comprising a functionalized hydrogel) contact blood of the subject. In some embodiments, contacting (e.g., pressing) the microneedle patch onto the skin of a subject provides that a number of microneedles (e.g., comprising a functionalized hydrogel) contact exosomes of the subject (e.g., of the blood and / or interstitial fluid of the subject). In some embodiments, the exosomes comprise one or more cancer biomarkers. In some embodiments, the exosomes comprise one or more melanoma biomarkers, e.g., MCAM, and / or MCSP.

[0124] Next, methods comprise collecting exosomes from the microneedle patch comprising a functionalized hydrogel, e.g., by dissolving the functionalized hydrogel to produce a dissolved hydrogel and unbinding the exosomes (e.g., by adding EDTA). In some embodiments, exosomes are collected from the dissolved hydrogel by centrifugation.

[0125] In some embodiments, methods comprise assaying the exosomes and / or assaying one or more biomarkers isolated with the exosomes. In some embodiments, assaying biomarkers comprises performing an immunoassay, e.g., to detect and / or quantify a biomarker. The technology is not limited in the type of immunoassay that is used to detect and / or quantify a biomarker and may include, e.g., an ELISA, a lateral flow immunoassay, a fluoroimmunoassay, a chemiluminescence immunoassay, or a western blot. In some embodiments, assays detect and / or quantify one or more of CD9, CH63, CD81, ALIX, HSP70, TSG101, duramycin, or heparin. In some embodiments, assays detect and / or quantify MCAM and / or MCSP. In some embodiments, assay methods comprise contacting an exosome with an antigen-binding agent (e.g., an antibody or antibody fragment) specific for CD9, CH63, CD81, ALIX, HSP70, TSG101, duramycin, heparin, MCAM, and / or MCSP.

[0126] In some embodiments, any of the methods include generating a report (such as a written or electronic report) disclosing a result of the method (such as the presence, absence, or quantity of a biomarker). In some embodiments, any of the methods include taking a clinical action based on a result of a method and / or assay.

[0127] Kits

[0128] In some embodiments, the technology provides a kit comprising a microneedle patch comprising a functionalized hydrogel and an immunoassay (e.g., a lateral flow immunoassay test strip). In some embodiments, the functionalized hydrogel comprises Av and the LFIA comprises an antigen- binding agent specific for MCAM and / or MCSP.

[0129] Uses

[0130] In some embodiments, the technology provides use of a microneedle patch comprising a plurality of microneedles and a functionalized hydrogel to isolate exosomes from a subject. Further, embodiments relate to use of a microneedle patch comprising a plurality of microneedles and a functionalized hydrogel to detect a cancer biomarker in a subject. In some embodiments, the technology provides use of a microneedle patch comprising a plurality of micro needles to deliver a pharmaceutical composition (e.g., a drug, a vaccine) to a subject, e.g., by a transdermal, intraocular, vaginal, transungual, cardiac, vascular, gastrointestinal, or intracochlear route of administration. In some embodiments, the microneedles are solid, hollow, coated, and / or dissolvable. See, e.g., McConville (2018) “Mini-Review: Assessing the Potential Impact of Microneedle Technologies on Home Healthcare Applications” Medicines (Basel) 5(2): 50; Kim (2012) “Microneedles for drug and vaccine delivery” Adv Drug Deliv Rev 64(14): 1547-68; and Avcil (2021) “Microneedles in Drug Delivery: Progress and Challenges” Micromachines (Basel) 12(11): 1321, each of which is incorporated herein by reference.

[0131] Product-by-process

[0132] In some embodiments, the technology provides a microneedle patch comprising a substrate comprising a plurality of micro needles (e.g., a device as described herein), wherein said microneedle patch is produced by a method (e.g., a method as described herein), e.g., a method comprising etching a substrate using a photolithograpy mask and a deep reactive ion etching to produce an array of micropillars; and sharpening the array of micropillars by exposing the micropillars to acid to produce an array of microneedles. Although the disclosure herein refers to certain dlustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.

[0133] Examples

[0134] Several challenges are related to designing and producing microneedles to sample exosomes by contacting a microneedle patch to human skin. The microneedles need to have a sufficiently long length to penetrate to an appropriate depth and location in human tissue at which exosomes may be obtained for subsequent collection and assay. The microneedles also need to be substantially vertical with respect to the substrate (i.e., substantially normal to the substrate surface) and have a sufficiently narrow width to facilitate easy penetration of skin with a minimized force and minimized pain upon contact to skin. However, designing a microneedle with an aspect ratio having a long length and a narrow width can negatively affect the verticality of the microneedle with respect to the substrate surface (e.g., making the microneedle slanted or crooked) and / or negatively affect the stability of the microneedle such that it may bend or break upon insertion into skin.

[0135] During the development of embodiments of the technology provided herein, microneedle patches were produced and tested by pressing against models of human skin and obtaining exosomes from the skin. Data produced during the experiments indicated that the microneedles had a sufficient length and width to penetrate into regions of skin to contact and collect exosomes without suffering from structural instability. Further, data indicated that the microneedles collected exosomes from skin that were successfully tested for biomarkers of cancer and for markers of exosomes.

[0136] Example 1 - Fabrication of a microneedle array

[0137] During the development of embodiments of the technology described herein, experiments were conducted to produce an array of microneedles using photolithography. A general outline of the process is shown in FIG. 1A. A photolithography mask was drawn and produced having an array of stellate shapes. See, e.g., FIG. 2A and 2B. A silicon wafer (100 mm diameter x 1 mm thick) was spin coated with a layer (e.g., 3 pm to 5 pm thick) of photoresist (e.g., MEGAPOSIT SPR220, Dow Chemical), and the photoresist was exposed using the photolithography mask for approximately 15 seconds. After developing the photoresist for approximately 100 seconds, the spin-coated silicon wafer was lithographically patterned and etched by deep reactive ion etching (DRIE, e.g., using the standard Bosch processes). The DRIE used a low bias power (e.g., less than approximately 40 W, e.g., 20 W) until approximately 800 pm to 900 pm of the silicon wafer was etched to produce a number of stellate micropillars (e.g., in an array). The array of stellate micropillars on the silicon wafer was incubated at 250 °C in an O2 plasma for 5 minutes to remove sidewall passivation from the DRIE process. The Bosch processes are described in, e.g., U.S. Pat. Nos. 5501893! 6531068; and 6284148, each of which is incorporated herein by reference.

[0138] Next, the stellate micropillars were sharpened by placing the silicon wafer comprising the array of stellate micropillars in a wet etch solution for 35 to 40 minutes (e.g., 38 minutes). The wet etch solution comprised a mixture of hydrofluoric acid and nitric acid (e.g., F20 v / v hydrofluoric acid to nitric acid). After sharpening, the silicon wafer comprised an array of microneedles. Microneedle patches were cleaved from the wafer using a diamond scribe. The solution of hydrofluoric acid and nitric acid isotropically etches and sharpens the microcolumns to produce microneedles.

[0139] The stellate microcolumns and microneedles were evaluated using scanning electron microscopy (SEM). First, each microneedle patch was submerged in a solution of 2% v / v glutaraldehyde in calcium chloride solution (0. 1 M HEPES, 1.4 M NaCl, and 25 mM CaCh) overnight at 4 °C. Next, each patch was submerged in a solution of 50% v / v ethanol and 50% v / v calcium chloride solution (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCls) for 10 minutes and titrated at ethanol concentrations of 70% v / v, 90% v / v, 100%, and 100% for 10 minutes each. After a final wash in a solution of 50% v / v ethanol and 50% v / v hexamethyldisilazane (HMDS) for 10 minutes, the microneedle patches were incubated in 100% HMDS overnight and left to evaporate. Electron micrographs of the microneedle patches were obtained using a Hitachi SU8000 Field Emission SEM. FIG. 20 shows a stellate microcolumn prior to sharpening. Each stellate micropillar measured approximately 750 to 850 pm from top to bottom and had a diameter of approximately 100 pm. FIG. 2D shows a microneedle after sharpening. After sharpening, each microneedle measured approximately 450 to 650 pm from top to bottom. FIG. 2E shows a the tip of a microneedle, which is less than 100 nm in width. FIG. 2F shows an array of microneedles on a microneedle patch. FIG. 2G shows a photograph of a microneedle patch.

[0140] Experiments were conducted to test the effect of bias power on stellate micropillar geometry. Spin-coated silicon wafers were lithographically patterned and etched by DRIE (e.g., using the standard Bosch processes) using the photolithography mask and a 40-W bias power (FIG. 2H) and 20-W bias power (FIG. 21). The 20-W bias power produced stellate micropillars having an improved sidewall verticality. (Compare FIG. 2H with FIG. 21).

[0141] Example 2 - Penetration of microneedles into skin

[0142] During the development of embodiments of the technology described herein, experiments were conducted to evaluate the penetration of the microneedles into skin. In these experiments, pig skin was used because it has similar mechanical properties as human skin. A microneedle patch was inserted into a piece of pig skin and the patch and skin were fixed in 4% v / v paraformaldehyde overnight at 4 °C to crosslink and preserve the samples for microscopy. The skin was cleared by titrating the patch and skin in a solution of 50% v / v THF in distilled water, then in solutions of 70% v / v THF in distilled water, 80% v / v THF in distilled water, and 100% THF. Each incubation was approximately one hour long. The patch and skin was incubated in 100% THF overnight at 4 °C, then for an hour in fresh 100% THF for one hour, and in 100% methanol for one hour. The patch and skin were placed in dibenzyl ether and stored at 4 °C until imaging. The microneedle patch and skin were removed and imaged using lightsheet microscopy to evaluate the puncture of the skin by the microneedles. FIG. 3 shows the penetration of a microneedle in the pig skin to a depth of approximately 300 to 350 pm.

[0143] Example 3 - Coating microneedle array with a functionalized hydrogel

[0144] During the development of embodiments of the technology described herein, microneedles were coated with a hydrogel (e.g., comprising polyvinyl alcohol (PVA) and alginate) conjugated to annexin V. Annexin V specifically binds to phosphatidylserine on exosomes and thus provides a capture protein that is specific for cancer exosomes. (see, e.g., Kang (2019) “Isolation and Profiling of Circulating Tumor-Associated Exosomes Using Extracellular Vesicular Lip id-Protein Binding Affinity Based Microfluidic Device” Small 15(47): el903600, incorporated herein by reference

[0145] First, PVA and alginate were separately dissolved in heated water to produce a heated PVA solution and a heated alginate solution. The heated PVA solution and the heated alginate solution were mixed to provide a hydrogel solution. After centrifuging and filtering the hydrogel solution, the hydrogel solution was conjugated with annexin V (Av) protein (at a ratio of L 25 to L20 Av to hydrogel) to produce a hydrogel solution having an affinity for exosomes.

[0146] Microneedles were exposed briefly to an Os plasma to make the microneedles hydrophilic. Approximately 20 pl of the functionalized hydrogel solution (i.e., comprising the hydrogel'. \v protein complex) was layered on the microneedle array patches using a pipette. After removing the excess functionalized hydrogel solution, the functionalized hydrogel solution was gelated by pipetting a calcium chloride solution (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCh) onto the functionalized hydrogel solution to gelate (e.g., solidify) the functionalized hydrogel solution and thereby produce a functionalized hydrogel. The functionalized hydrogel solution was incubated with calcium chloride solution (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCls) for a minimum of 5 minutes to produce the gelated functionalized hydrogel. Example 4 - Isolation of exosomes from melanoma skin

[0147] During the development of embodiments of the technology described herein, a microneedle patch comprising a functionalized hydrogel was used to isolate exosomes from melanoma skin samples. Microneedle patches were produced and coated with a functionalized hydrogel as described above. The microneedle patches were pressed into melanoma mouse patient-derived xenograft (PDX) samples and healthy mouse skin samples for 15 minutes. FIG. 4A. The microneedle patches were removed from the skin samples. Microneedle puncture marks were observed on the skin. FIG. 4B. The microneedle patches were washed twice in 15 id of calcium chloride solution (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCh). Then, the microneedle patches were incubated in EDTA to dissolve the hydrogel and unbind the exosomes from the Av. The exosomes were isolated from the dissolved hydrogel using centrifugation (e.g., ultracentrifugation), and the exosomes isolated from melanoma skin (“melanoma exosomes’) and exosomes isolated from healthy skin (“healthy exosomes”) were assayed.

[0148] First, the total protein concentration in the melanoma exosomes and healthy exosomes was measuring using a bicinchoninic acid (BOA) assay. FIG. 5. Microneedle patches were submerged in 200 pl of radioimmunoprecipitation assay buffer (RIPA buffer) with 1% v / v protease inhibitor cocktail. As shown in FIG. 5, the melanoma exosomes (“Experimental”) comprised much more total protein than the healthy exosomes (“Control”). In particular, the melanoma exosomes (“Experimental”) comprised approximately 500 to 1000 pg / ml protein and the healthy exosomes (“Control”) comprised less than 100 pg / ml protein.

[0149] Next, immunoblot (“western blot”) assays were used to detect calnexin (a cell marker), TSG101 (an exosome marker), flotillinl (an extracellular vesicle marker), and MCSP (a melanoma marker) in the exosome samples prepared from the melanoma exosomes and healthy exosomes. FIG. 6. Homogenized melanoma mouse PDX tissue was used as a control. As shown in FIG. 6, the homogenized melanoma mouse PDX tissue was positive for all of MCSP (melanoma marker), calnexin (cell marker), TSG101 (exosome marker), and flotillin- 1 (extracellular vesicle marker). The exosomes isolated from the melanoma mouse PDX tissue were positive for MCSP (melanoma marker), TSG101 (exosome marker), and flotillin- 1 (extracellular vesicle marker); and the exosomes isolated from the melanoma mouse PDX tissue were negative for calnexin (cell marker). FIG. 6. The exosomes isolated from healthy mouse skin were negative for MCSP (melanoma marker), calnexin (cell marker), and flotillin- 1 (extracellular vesicle marker); the exosomes isolated from healthy mouse skin were positive for TSG101 (exosome marker). FIG. 6.

[0150] In addition, enzyme-linked immunosorbent assay (ELISA) was used to quantify CD63 in the exosome samples. FIG. 7. CD63 is an exosomal marker that is associated with the pathogenesis of cancer. FIG. 7 shows the ELISA calibration curve and CD63 concentrations measured for a melanoma skin sample, for cell line derived extracellular vesicles on microneedles, and from a healthy skin sample.

[0151] SEM was used to visualize the melanoma exosomes and healthy exosomes isolated using the microneedle patches. Briefly, microneedle patches were pressed into melanoma mouse patient-derived xenograft (PDX) samples and healthy mouse skin samples for 15 minutes and removed from the tissue. After removal, exosomes captured on the microneedles were washed twice in a calcium chloride solution (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCh) and then fixed overnight at 4°C in a solution of 2% v / v glutaraldehyde in calcium chloride solution (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCh solution). The microneedle patches were titrated in calcium chloride solution (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCL) and ethanol and then incubated in HMDS overnight to evaporate. FIG. 8A shows a microneedle patch pressed into melanoma skin and FIG. 8B shows a microneedle patch pressed into healthy skin. As shown in FIG. 8A and 8B, more exosomes were isolated from melanoma skin than from healthy skin. Further, the exosomes isolated from the melanoma skin had diameters in the expected range of 30 to 200 nm. FIG. 8C and FIG. 8D.

[0152] The data collected from the protein concentration assay (BCA), immunoassay (western blot), and electron microscopy indicated that significantly more exosomes were isolated from the mouse PDX models than from the healthy control.

[0153] Example 5 - Lateral flow immunoassay

[0154] During the development of embodiments of the technology described herein, experiments were conducted to prepare a lateral flow immunoassay (LFIA) strip for detecting cancer-associated exosomes. See, e.g., Koczula “Lateral flow assays” (2016) Essays in Biochemistry 60: 111-20; and Posthuma-Trumpie (2009) “Lateral flow (immuno)assays:its strengths, weaknesses, opportunities and threats. A literature survey” Anal Bioanal Chem 393:569-82, each of which is incorporated herein by reference, for a description of lateral flow immunoassay technologies and techniques.

[0155] First, anti-MCAM (melanoma cell adhesion molecule) antibodies and anti MCSP (melanoma marker) antibodies (each at 1 mg / ml) were placed at 0.1 pl / rnni on 5-mm wide nitrocellulose membranes (HF75 membrane cards) to produce a test line. Antimouse antibodies at 1 mg / ml and were placed at 0. 1 pl / mm on the membrane to produce a control line. Sample and absorbent pads were placed on the membrane.

[0156] A 1.5-ml volume of gold nanoparticles (AuNP, BB International) were mixed with 14 pg of anti-CD63 antibodies at a ratio of Pl and incubated with shaking for one hour to produce AuNP conjugated with anti-CD63 (anti-CD63 AuNP). The anti-CD63 AuNP were incubated with 100 pl of 1% w / v bovine serum albumin (BSA) for 20 minutes to block, the antrCD63 AuNP were collected by centrifugation at 6.8 k RCF for 20 minutes, and the collected anti-CD63 AuNP were resuspended in 1 mb of 2 mM borate solution in distilled water with 10% w / v sucrose and 1% w / v BSA. A running buffer was made having a concentration of 10 mM 2-[4-(2-hydroxyethyl)piperazin-l- yl] ethane sulfonic acid (HEPES) in phosphate buffered saline (PBS) and with 2% w / v BSA and 0.05% v / v TWEEN- 20.

[0157] Microneedle patches were produced and coated with a functionalized hydrogel as described above. The microneedle patches were pressed into melanoma mouse patient- derived xenograft (PDX) samples and healthy mouse skin samples for 15 minutes. A volume of 15 pl of 20 mM EDTA was pipetted onto the patches for 5 minutes, then collected; a second volume of 15 pl of 20 mM EDTA was pipetted onto the patches for 5 minutes, then collected. The collected solutions were mixed with 10 pl of anti-CD63 AuNP. 90 pl of the running buffer was added to the collected solutions and incubated for 5 minutes to provide a test sample. The test samples were pipetted onto the LFIA strip sample pad, developed in a conical tube containing running buffer, and then imaged. Dilutions of exosome preparations were prepared, mixed with anti’CD63 AuNP, and tested on the LFIA strip to determine an appropriate volume of sample to apply to the LFIA strip for visualization. FIG. 9. As shown in FIG. 9, the data collected indicated that a visual test line appeared on the LFIA strip when a volume of 8 pl of exosomes were added to the LFIA strip.

[0158] FIG. 10A shows the LFIA results from using microneedle patches to collect exosomes from melanoma mouse patient-derived xenograft (PDX) samples and healthy mouse skin samples. As shown in FIG. 10A, a visible test line appeared for the melanoma sample but not for the healthy skin sample. A control line was visible for both samples, indicating that the assay performed correctly. Quantification of the test line intensities for the melanoma sample and for the healthy sample is shown in FIG. 10B.

[0159] In summary, the data collected during the experiments described herein indicated that exosomes were isolated and quantified from melanoma PDX samples. Further, the isolated exosomes are detectable using LFIA. Accordingly, the microneedle patch and detection assays provide an easily implemented assay suitable for use at a point of care or in a home setting for rapid detection of melanoma in a patient.

[0160] All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

CLAIMSWE CLAIM:

1. A method of diagnosing a disease, said method comprising: pressing a microneedle patch comprising a plurality of microneedles coated with a functionalized hydrogel onto a tissue; recovering exosomes from the microneedle patch; and detecting a disease biomarker present in the exosomes using an immunoassay.

2. The method of claim 1, wherein the plurality of microneedles is arranged in an array comprising a spacing of at least 250 pm to 1000 pm between microneedles.

3. The method of claim 1, wherein each micronecdle of the plurality of micronecdles comprises a shaft and a tip, wherein the shaft has a length of at least 450 pm to 650 pm, at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm.

4. The method of claim 1. wherein the plurality of microneedles comprises 100 to 1000 microneedles.

5. The method of claim 1, wherein recovering exosomes comprises dissolving the functionalized hydrogel to produce a dissolved hydrogel.

6. The method of claim 5, further comprising centrifuging the dissolved hydrogel.

7. The method of claim 1, wherein the tissue is skin.

8. The method of claim 1, wherein a human comprises the tissue.

9. The method of claim 1, wherein a human having a cancer or suspected of having a cancer comprises the tissue.

10. The method of claim 9, wherein the cancer is a melanoma.

11. The method of claim 1, wherein the tissue comprises skin and pressing the microneedle patch onto the skin causes the microneedle patch to pierce the skin to a depth of approximately 300 pm to 350 pm.

12. The method of claim 1, wherein the tissue comprises skin and pressing the microneedle patch onto the skin causes the functionalized hydrogel to contact interstitial fluid and / or blood.

13. The method of claim 1, wherein the tissue comprises skin and pressing the microneedle patch onto the skin causes the functionalized hydrogel to contact an exosome.

14. The method of claim 1, wherein the exosomes comprise an exosome marker and a cancer biomarker.

15. The method of claim 1, wherein the exosomes comprise MCAM and / or MCSP.

16. The method of claim 1, wherein the immunoassay is a lateral flow immunoassay test strip comprising an antigen-binding agent specific for the biomarker.

17. The method of claim 1, wherein the immunoassay is a lateral flow immunoassay test strip comprising a test line comprising an anti MCAM and / or anti’MCSP antibody.

18. A kit comprising a microneedle patch comprising a substrate comprising a plurality of microneedles coated with a functionalized hydrogel; and a lateral flow immunoassay device.

19. A method for isolating exosomes, said method comprising: pressing a microneedle patch comprising a plurality of microneedles coated with a functionalized hydrogel onto a tissue! and recovering exosomes from the microneedle patch.

20. The method of claim 19. wherein the plurality of microneedles is arranged in an array comprising a spacing of at least 250 pm to 1000 pm between microneedles.

21. The method of claim 19. wherein each microneedle of the plurality of microneedles comprises a shaft and a tip, wherein the shaft has a length of at least 450 pm to 650 pm, at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm.

22. The method of claim 19, wherein the plurality of microneedles comprises 100 to 1000 microneedles.

23. The method of claim 19. wherein recovering exosomes comprises dissolving the functionalized hydrogel to produce a dissolved hydrogel.

24. The method of claim 23. further comprising centrifuging the dissolved hydrogel.

25. The method of claim 19, wherein the tissue is skin.

26. The method of claim 19, wherein a living organism comprises the tissue.

27. The method of claim 19. wherein a human comprises the tissue.

28. The method of claim 19, wherein a human having a cancer comprises the tissue.

29. The method of claim 19, wherein the tissue comprises skin and pressing the microneedle patch onto the skin causes the microneedle patch to pierce the skin to a depth of approximately 300 pm to 350 pm.

30. The method of claim 19, wherein the tissue comprises skin and pressing the microneedle patch onto the skin causes the functionalized hydrogel to contact interstitial fluid and / or blood.

31. The method of claim 19, wherein the tissue comprises skin and pressing the microneedle patch onto the skin causes the functionalized hydrogel to contact an exosome.

32. The method of claim 19, wherein the exosomes comprise an exosome marker and a cancer biomarker.

33. The method of claim 19. wherein the exosomes comprise MCAM and / or MCSP.

34. A microneedle patch comprising a substrate comprising a plurality of microneedles coated with a functionalized hydrogel.

35. The microneedle patch of claim 34. wherein the substrate comprises silicon.

36. The microneedle patch of claim 34, wherein the plurality of microneedles is arranged in an array comprising a spacing of at least 250 pm to 1000 pm between microneedles.

37. The microneedle patch of claim 34. wherein each microneedle of the plurality of microneedles comprises a shaft and a tip, wherein the shaft has a length of at least 450 pm to 650 pm, at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm.

38. The microneedlc patch of claim 37, wherein the stellate shape is a six-pointed stellate shape.

39. The microneedle patch of claim 34. wherein the plurality of microneedles comprises 100 to 1000 microneedles.

40. The microneedle patch of claim 34, wherein the functionalized hydrogel comprises poly-vinyl alcohol (PVA), alginate, and an exosome-binding agent.

41. The microneedle patch of claim 40, wherein the exosome-binding agent comprises a phosphatidylserine -binding agent.

42. The microneedle patch of claim 40, wherein the exosome-binding agent comprises an annexin.

43. The microneedle patch of claim 40. wherein the exosome-binding agent comprises an annexin V.

44. The microneedle patch of claim 34, wherein each microneedle of the plurality of microneedles extends from the substrate substantially normal to a planar surface of the substrate.

45. A kit comprising a microneedle patch according to any one of claims 34—44; and a lateral flow immunoassay device.

46. A method of producing a microneedle patch, the method comprising: etching a substrate using a photolithography mask and a deep reactive ion etching to produce an array of micropillars; and sharpening the array of micropillars by exposing the micropillars to acid to produce an array of microneedles.

47. The method of claim 46, wherein deep reactive ion etching is performed at a bias power of less than 40 W.

48. The method of claim 46. wherein the acid comprises a mixture of hydrofluoric acid and nitric acid.

49. The method of claim 46, wherein the substrate comprises silicon.

50. The method of claim 46. wherein the plurality of microneedles is arranged in an array comprising a spacing of at least 250 pm to 1000 pm between microneedles.

51. The method of claim 46. wherein each microneedle of the plurality of microneedles comprises a shaft and a tip, wherein the shaft has a length of at least 450 pm to 650 pm. at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm.

52. The method of claim 46. wherein the stellate shape is a six-pointed stellate shape.

53. The method of claim 46, wherein the plurality of microneedles comprises 100 to 1000 microneedles.

54. The method of claim 46. further comprising coating the plurality of microneedles with a functionalized hydrogel.

55. The method of claim 54, wherein the functionalized hydrogel comprises polyvinyl alcohol (PVA), alginate, and an exosome-binding agent.

56. The method of claim 55. wherein the exosome-binding agent comprises a phosphatidylserine-binding agent.

57. The method of claim 55. wherein the exosome-binding agent comprises an annexin.

58. The method of claim 55, wherein the exosome-binding agent comprises an annexin V.

59. The method of claim 46. wherein each microneedle of the plurality of microncedles extends from the substrate substantially normal to a planar surface of the substrate.

60. The method of claim 46, further comprising: producing a photolithography mask comprising an array of shapes; coating the substrate with a photoresist: exposing the photoresist using the photolithography mask; and developing the photoresist.

61. A kit comprising a microneedle patch comprising a substrate comprising a plurality of microneedles; a functionalized hydrogel solution: and a lateral flow immunoassay device.

62. A system comprising a microneedle patch comprising a substrate comprising a plurality of microneedles; a functionalized hydrogel solution; a lateral flowimmunoassay device; and a reader device configured to quantify a test line on the immunoassay device.

63. A system comprising a microneedle patch comprising a substrate comprising a plurality of microneedles coated with a functionalized hydrogel; a lateral flow immunoassay device; and a reader device configured to quantify a test line on the immunoassay device.

64. Use of a microneedle patch comprising a plurality of microneedles and a functionalized hydrogel to isolate exosomes from a subject.

65. The use of claim 64, wherein the plurality of microneedles is arranged in an array comprising a spacing of at least 250 pm to 1000 pm between microneedles.

66. The use of claim 64, wherein each microneedle of the plurality of microneedles comprises a shaft and a tip. wherein the shaft has a length of at least 450 pm to 650 pm, at least a portion of the shaft has a stellate shape, and the tip has a width of approximately 50 nm to 100 nm.

67. The use of claim 64, wherein the plurality of microneedlcs comprises 100 to 1000 microneedles.

68. The use of claim 64. wherein the exosomes comprise an exosome marker and a cancer biomarker.

69. The use of claim 64, wherein the exosomes comprise MCAM and / or MCSP.

70. Use of a microneedle patch comprising a plurality of microneedles and a functionalized hydrogel to detect a cancer biomarker in a subject.

71. The use of claim 70. wherein the cancer biomarker is a melanoma biomarker.

72. A microneedle patch comprising a substrate comprising a plurality of microneedles, wherein said microneedle patch is produced by a method comprising:etching a substrate using a photolithography mask and a deep reactive ion etching to produce an array of micropillars; and sharpening the array of micropillars by exposing the micropillars to acid to produce an array of microneedles.

73. A microneedle patch comprising a substrate comprising a plurality of microneedles, wherein said microneedle patch is produced by a method according to any one of claims 46-60.

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