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WO2025043144A1 - Caractérisation simultanée d'arn et de protéines dans des vésicules extracellulaires et des lipoprotéines - Google Patents

Caractérisation simultanée d'arn et de protéines dans des vésicules extracellulaires et des lipoprotéines Download PDF

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WO2025043144A1
WO2025043144A1 PCT/US2024/043552 US2024043552W WO2025043144A1 WO 2025043144 A1 WO2025043144 A1 WO 2025043144A1 US 2024043552 W US2024043552 W US 2024043552W WO 2025043144 A1 WO2025043144 A1 WO 2025043144A1
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mir
rna
molecular
capture antibodies
sievs
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Eduardo Reategui
Truc NGUYEN KIM
Xilal RIMA
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Ohio State Innovation Foundation
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Ohio State Innovation Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present disclosure relates to methods and systems for simultaneously detecting RNAs and proteins in situ, in Extracellular Vesicles (EVs) and/or Lipoproteins (LPs), via one or more capture antibodies.
  • EVs Extracellular Vesicles
  • LPs Lipoproteins
  • Extracellular vesicles are small membranous vesicles secreted by cells that are trafficked intercellularly and present in various biofluids. EVs are involved in various biological processes from immunomodulation to embryonic development. In cancer, EVs promote drug resistance, immunosuppression, the epithelial-to-mesenchymal transition, disruption of the blood-brain barrier, and organotropism. However, the biomolecular composition of EVs is highly heterogeneous, with proteins, RNAs, DNAs, lipids, and metabolites reflecting their tissue of origin.
  • siEVP single EV and particle
  • Nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), and microfluidic resistive pulse sensing (MRPS) are routinely used to measure the size and concentration of siEVs, with the minimum detectable size in the 50 - 100 nm range.
  • NTA, TRPS, and MRPS integrate siEVP signals non-specifically due to limitations in phenotyping.
  • Atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are often utilized to provide morphological and mechanical properties of siEVs while surpassing the optical limit of diffraction.
  • FAM Atomic force microscopy
  • SEM scanning electron microscopy
  • TEM transmission electron microscopy
  • Optical techniques can also be applied to tunable signal-enhancing surfaces, such as plasmonic and interferometric surfaces, to examine siEV surface protein composition via immunoselective immobilization.
  • antibody-DNA conjugates incorporating random-tag sequences in a proximity barcoding assay with NGS have been used to improve the simultaneous profiling of surface proteins in siEVs.
  • these promising technologies have demonstrated their ability to resolve subpopulations of siEVs from different tissues, the complex intraluminal cargo of siEVs, such as nucleic acids, still requires the same rigor and optimization.
  • evidence on the bioactivity of LP -transported miRNA and LP -bound proteins has inspired novel engineering approaches for their quantification.
  • RNAs and proteins in situ, in Extracellular Vesicles (EVs) and/or Lipoproteins (LPs).
  • EVs Extracellular Vesicles
  • LPs Lipoproteins
  • RNA and a protein in situ comprising tethering a plurality of an Extracellular Vesicle (EV) and/or a Lipoprotein (LP), isolated from a sample obtained from a subject, to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises of anti-CD63, anti-CD9, anti-ApoAl, anti-ApoB, or a combination thereof for EV and/or LP capture.
  • EV Extracellular Vesicle
  • LP Lipoprotein
  • the method comprises obtaining a sample from a subject; isolating an EV and/or an LP from the sample; tethering a plurality of the EV and/or the LP to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-CD63, anti-CD9, anti-ApoAl, anti- ApoB, or a combination thereof; binding a detection antibody and a molecular beacon to the EV and/or the LP tethered on the micropattern array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.
  • the method comprises obtaining a sample from a subject; isolating an EV and/or an LP from the sample; tethering a plurality of the EV and/or the LP to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-ApoAl, or anti-ApoB; binding a detection antibody and a molecular beacon to the EV and/or the LP tethered on the micropattem array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.
  • the method further comprises fluorescently imaging the micropattern array to capture an image data; and detecting an occurrence of a single EV and/or an LP expressing the first target type of the molecular cargo based on a fluorescent spot of a first color associated with the detection antibody in the image data captured; detecting an occurrence of a single EV and/or an LP expressing the second target type of the molecular cargo based on a fluorescent spot of a second color associated with the molecular beacon in the image data captured; and detecting an occurrence of the single EV and/or the LP expressing both the first target type of the molecular cargo, and the second target type of the molecular cargo based on a fluorescent spot of a third color in the image data captured.
  • the glass substrate is coated with poly-L-lysine (PLL) through physical adsorption prior to coating the glass substrate with a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG is covalently bound to the PLL through N-hydroxysuccinimide (NHS) chemistry.
  • the PEG is methoxy-poly(ethylene glycol)- succinimidyl valerate (mPEG-SVA).
  • the micropattern array is photoetched on the glass substrate in presence of a photoactivator, wherein the photoactivator is 4-benzoylbenzyl- trimethylammonium chloride (PLPP).
  • PLPP 4-benzoylbenzyl- trimethylammonium chloride
  • the micropattern array comprises a five-by-five array of circles, wherein each individual circle has a diameter ranging from about 2 pm to about 200 pm.
  • the one or more capture antibodies are biotinylated and attached to the micropattern array by binding to a physically adsorbed Neutravidin layer on the micropattern array. In some embodiments, the one or more capture antibodies bind to surface proteins expressed on the EV and/or the LP. In some embodiments, the one or more capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof. In some embodiments, the RNA is selected from a microRNA (miRNA), a messenger RNA (mRNA), or combinations thereof.
  • miRNA microRNA
  • mRNA messenger RNA
  • the detection antibody is conjugated with one or more fluorophores for fluorescent imaging.
  • the molecular beacon comprises one or more fluorescent dye sequences for fluorescent imaging. In some embodiments, the molecular beacon comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the molecular beacon is selected from any one of SEQ ID NO: 1-14.
  • LNA locked nucleic acid
  • fluorescently imaging the micropattem array to capture image data comprises total internal reflection fluorescence microscopy (TIRFM).
  • TIRFM produces an exponentially decaying electromagnetic wave that only excites fluorophores near a surface of the glass substrate to visualize signals from immobilized EV and/or an LP.
  • the subject is human.
  • FIG. 19C shows WB analyses on unprocessed serum, isolated very low-density LP / low-density LP (VLDL/LDL), isolated high-density LP (HDL), and the TFF retentate for EV biomarkers (annexin Al, ARF6, CD63, and CD9) and LP biomarkers (ApoB and ApoAl), indicate a co-isolation of the particles after TFF.
  • FIG. 19D shows WB analyses in cell and EV lysates demonstrate a lack of signal for LP biomarkers.
  • FIG. 19E shows TEM images of Gli36-derived EVs and serum-isolated LPs show different morphologies.
  • FIGS. 20A-20C show the detection of apolipoprotein corona on siEVs.
  • FIG. 20A shows a schematic of the preparation of siEVs with apolipoprotein corona, where TFF-purified EVs are incubated with EV-depleted plasma (EVDP) for 30 minutes at room temperature (RT) and subsequently purified with size-exclusion chromatography (SEC).
  • FIG. 20B shows representative TIRFM images for siEVs with apolipoprotein corona and the respective control demonstrate the detection of ApoAl and ApoB on siEVs.
  • FIG. 20A shows a schematic of the preparation of siEVs with apolipoprotein corona, where TFF-purified EVs are incubated with EV-depleted plasma (EVDP) for 30 minutes at room temperature (RT) and subsequently purified with size-exclusion chromatography (SEC).
  • FIG. 20B shows representative TIRFM images for siEVs with apolipoprotein cor
  • FIGS. 22A-22B show NCAN detection with the S1EVP PRA.
  • FIG. 22 A shows representative TIRFM images for FIG. 5C demonstrating the detection of NCAN in siEVs.
  • FIG. 22B shows distributions of fluorescence intensity for the expression of NCAN in siEVs across the six glioma cell lines as detected by the S1EVP PRA show homogeneous profiles. All micropatterns were functionalized with an anti-CD63/CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.
  • an “Fv” fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) has the ability to recognize and bind target, although at a lower affinity than the entire binding site.
  • Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods such as fluorescent imaging and spectroscopy.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see,
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
  • sequence comparisons typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
  • “increased” or “increase” as used herein generally means an increase by a statistically significant amount, for example “increased” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10- fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level so long as the increase is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur.
  • the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
  • a “protein”, “polypeptide”, or “peptide” each refer to a polymer of amino acids and does not imply a specific length of a polymer of amino acids.
  • the terms peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide.
  • This term also includes polypeptides with post-expression modification, such as glycosylation (e.g., the addition of a saccharide), acetylation, phosphorylation, and the like.
  • nucleic acid as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides.
  • nucleobase refers to the part of a nucleotide that bears the Watson/Crick base-pairing functionality.
  • the most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.
  • ribonucleic acid and “RNA” as used herein mean a polymer composed of ribonucleotides.
  • deoxyribonucleic acid and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.
  • a “subject” (or a “host”) is meant an individual.
  • the "subject” can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal.
  • the subject can be a mammal such as a primate or a human.
  • Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject.
  • Extracellular vesicles are particles that are released by cells into the extracellular space. They are lipid bilayer-delimited clusters of different sizes, cargo, and surface markers. EVs are carriers that can transport a variety of cargo, including proteins, lipids, nucleic acids, and metabolites.
  • Lipoproteins are spherical particles made of fat and protein that transport lipids (such as, for example, cholesterol) throughout a human body's bloodstream to the cells. They play a vital role in transporting lipids between organs, forming structural components in nervous tissue, and helping transmit electrical impulses.
  • lipids such as, for example, cholesterol
  • HDL high-density lipoprotein
  • LDL low-density lipoprotein
  • Extracellular vesicles and lipoproteins can interact to form functional complexes such as single EV and particle (siEVP) co-isolates.
  • siEVP single EV and particle
  • Such complexes have been observed in biofluids (such as, for example, saliva, serum, plasma, urine, sputum, nasal swab, fecal, tears, or cerebral spinal fluid) from healthy human donors and in various in vitro disease models (such as, for example, breast cancer and hepatitis C infection).
  • Apolipoprotein Al is a protein that is a major component of high-density lipoprotein (HDL) and plays a role in lipid metabolism and transport. ApoAl helps move cholesterol and phospholipids from inside cells to the cell's outer surface, converts cholesterol into a form that can be integrated into HDL and helps transform free cholesterol into cholesterol ester, which can then be transported to the liver for degradation.
  • Apolipoprotein B is a protein that carries lipids in the bloodstream, including cholesterol and fats. It's a building block of low-density lipoproteins (LDLs), intermediatedensity lipoproteins (IDLs), and very low-density lipoproteins (VLDLs), which are also known as "bad" cholesterol.
  • ApoB is encoded by the APOB gene and is produced in the liver and small intestine. There are two forms of ApoB that circulate in the body: ApoB48, which comes from the small intestine, and ApoB 100, which comes from the liver. ApoB48 serves in the absorption of dietary fats from the intestine. ApoB 100 is necessary for the assembly of VLDL in the liver and is the primary ligand for LDL receptor-mediated clearance of LDL particles from the blood.
  • a method of simultaneously detecting an RNA and a protein in situ comprising: obtaining a sample from a subject; isolating an Extracellular Vesicle (EV) and/or a Lipoprotein (LP) from the sample; tethering a plurality of the EV and/or the LP to a micropattem array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-CD63, anti-CD9, anti-ApoAl anti-ApoB, or a combination thereof; and binding a detection antibody and a molecular beacon to the EV and/or the LP tethered on the micropattern array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is
  • the method comprises obtaining a sample from a subject; isolating an EV and/or an LP from the sample; tethering a plurality of the EV and/or the LP to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-CD63, anti-CD9, anti-ApoAl, anti-ApoB, or a combination thereof; binding a detection antibody and a molecular beacon to the EV and/or the LP tethered on the micropattem array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.
  • the second target type of the molecular cargo is a DNA fragment.
  • the one or more capture antibodies comprises anti-ApoAl. In some embodiments, the one or more capture antibodies comprises anti-ApoB. In some embodiments, the one or more capture antibodies comprises anti-CD63. In some embodiments, the one or more capture antibodies comprises anti-CD9.
  • a method of simultaneously detecting an RNA and a protein in situ comprising: obtaining a sample from a subject; isolating an Extracellular Vesicle (EV) and/or a Lipoprotein (LP) from the sample; tethering a plurality of the EV and/or the LP to a micropattem array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-ApoAl or anti-ApoB; and binding a detection antibody and a molecular beacon to the EV and/or the LP tethered on the micropattern array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.
  • EV Extracellular Vesicle
  • LP Lipoprotein
  • the method comprises obtaining a sample from a subject; isolating an EV and/or an LP from the sample; tethering a plurality of the EV and/or the LP to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-ApoAl or anti-ApoB; binding a detection antibody and a molecular beacon to the EV and/or the LP tethered on the micropattern array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.
  • the second target type of the molecular cargo is a DNA fragment.
  • the micropattern array comprises a five-by-five array of circles, wherein each individual circle has a diameter ranging from about 2 pm to about 200 pm. In some embodiments, each individual circle has a diameter of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
  • each individual circle has the diameter of about 20 pm. In some embodiments, each individual circle has a center-to-center spacing of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
  • each individual circle has a center-to-center spacing of about 80 pm in relation to an adjacent circle.
  • the one or more capture antibodies are biotinylated and attached to the micropattern array by binding to a physically adsorbed Neutravidin layer on the micropattern array. In some embodiments, the one or more capture antibodies bind to surface proteins expressed on the EV and/or the LP. In some embodiments, the one or more capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof.
  • the RNA is selected from a microRNA (miRNA), a messenger RNA (mRNA), or combinations thereof.
  • RNA encodes AXL , AXL-2, AXL-3, NSF, NCAN, p53, GAPDH, hsa-miR-21-5p, hsa-miR-9-5p, hsa-miR-1246-5p, cel-miR- 39-3p, cel-miR-54-3p or cel-miR-238-3p.
  • DNA fragments such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and mitochondrial DNA (mtDNA)
  • the detection antibody is conjugated with one or more fluorophores, (such as, for example, Alexa Fluor 488, Alexa Fluor 546, Alexa Flour 647, Alexa Fluor 55, FITC or CoraLite 594) for fluorescent imaging.
  • the molecular beacon comprises one or more fluorescent dye sequences for fluorescent imaging. In some embodiments, the molecular beacon comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the molecular beacon is selected from any one of SEQ ID NO: 1-14. In some embodiments, the molecular beacon comprises SEQ ID NO: 1. In some embodiments, the molecular beacon comprises SEQ ID NO: 2. In some embodiments, the molecular beacon comprises SEQ ID NO: 3. In some embodiments, the molecular beacon comprises SEQ ID NO: 4. In some embodiments, the molecular beacon comprises SEQ ID NO: 5. In some embodiments, the molecular beacon comprises SEQ ID NO: 6.
  • LNA locked nucleic acid
  • the molecular beacon comprises SEQ ID NO: 7. In some embodiments, the molecular beacon comprises SEQ ID NO: 8. In some embodiments, the molecular beacon comprises SEQ ID NO: 9. In some embodiments, the molecular beacon comprises SEQ ID NO: 10. In some embodiments, the molecular beacon comprises SEQ ID NO: 11. In some embodiments, the molecular beacon comprises SEQ ID NO: 12. In some embodiments, the molecular beacon comprises SEQ ID NO: 13. In some embodiments, the molecular beacon comprises SEQ ID NO: 14.
  • fluorescently imaging the micropattem array to capture image data comprises total internal reflection fluorescence microscopy (TIRFM).
  • TIRFM produces an exponentially decaying electromagnetic wave that only excites fluorophores near a surface of the glass substrate to visualize signals from immobilized EV and/or an LP.
  • detection of the EV and/or LP comprises flowcytometry.
  • TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section (usually less than 200 nanometers) that is supported on a glass slide.
  • the technique is based on the principle that when excitation light is totally internally reflected in a transparent solid glass at its interface with a liquid medium, an electromagnetic field (also known as an evanescent wave) is generated at the solid-liquid interface with the same frequency as the excitation light.
  • the intensity of the evanescent wave exponentially decays with distance from the surface of the solid so that only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. Two-dimensional images of the fluorescence can then be obtained, although there are also mechanisms in which three- dimensional information on the location of vesicles or structures in cells can be obtained.
  • the sample is a biofluid.
  • the biofluid is saliva, serum, plasma, urine, sputum, nasal swab, fecal, tears, or cerebral spinal fluid.
  • the subject is a human.
  • a system for simultaneously detecting an RNA and a protein in situ comprising: one or more of capture antibodies attached to a micropattem array on a glass substrate, wherein the one or more of capture antibodies comprises anti-CD63, anti-CD9, anti- ApoAl, anti-ApoB or a combination thereof; a detection antibody and a molecular beacon, wherein the detection antibody is configured to bind to a first target type of molecular cargo, and wherein the molecular beacon is configured to bind to a second target type of molecular cargo wherein first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA; and a fluorescent imaging device to capture image data.
  • a method for simultaneously detecting an RNA and a protein in situ comprises a one or more of capture antibodies attached to a micropattern array on a glass substrate, a detection antibody and a molecular beacon and a fluorescent imaging device to capture image data.
  • the one or more of capture antibodies comprises either anti-CD63, anti-CD9, anti-ApoAl, anti-ApoB or a combination thereof.
  • the detection antibody is configured to bind to a first target type of molecular cargo.
  • the molecular beacon is configured to bind to a second target type of molecular cargo.
  • the first target type of molecular cargo is a protein
  • the second target type of molecular cargo is an RNA.
  • the second type of molecular cargo is a DNA fragment.
  • a system for simultaneously detecting an RNA and a protein in situ comprising: a plurality of capture antibodies attached to a micropattern array on a glass substrate, wherein the plurality of capture antibodies comprises anti-CD63, anti-CD9, anti- ApoAl, anti-ApoB or a combination thereof; a plurality of detection antibodies and a plurality of molecular beacons, wherein the plurality of detection antibodies is configured to bind to a first target type of molecular cargo, and wherein the molecular beacon is configured to bind to a second target type of molecular cargo wherein first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA; and a fluorescent imaging device to capture image data.
  • a method for simultaneously detecting an RNA and a protein in situ comprises a plurality of capture antibodies attached to a micropattern array on a glass substrate, a plurality of detection antibodies and a plurality of molecular beacons and a fluorescent imaging device to capture image data.
  • the plurality of capture antibodies comprises either anti-CD63, anti-CD9, anti- ApoAl, anti-ApoB or a combination thereof.
  • the plurality of detection antibodies is configured to bind to a first target type of molecular cargo.
  • the plurality of molecular beacons is configured to bind to a second target type of molecular cargo.
  • the first target type of molecular cargo is a protein
  • the second target type of molecular cargo is an RNA.
  • the second type of molecular cargo is a DNA fragment.
  • the one or more capture antibodies comprises anti-CD63. In some embodiments, the one or more capture antibodies comprises anti-CD9. In some embodiments, the one or more capture antibodies comprises anti-ApoAl. In some embodiments, the one or more capture antibodies comprises anti-ApoB.
  • a system for simultaneously detecting an RNA and a protein in situ comprising: one or more of capture antibodies attached to a micropattem array on a glass substrate, wherein the one or more of capture antibodies comprises anti-ApoAl or anti-ApoB; a detection antibody and a molecular beacon, wherein the detection antibody is configured to bind to a first target type of molecular cargo, and wherein the molecular beacon is configured to bind to a second target type of molecular cargo wherein first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA; and a fluorescent imaging device to capture image data.
  • a method for simultaneously detecting an RNA and a protein in situ comprises a one or more of capture antibodies attached to a micropattern array on a glass substrate, a detection antibody and a molecular beacon and a fluorescent imaging device to capture image data.
  • the one or more of capture antibodies comprises either anti-ApoAl or anti-ApoB.
  • the detection antibody is configured to bind to a first target type of molecular cargo.
  • the molecular beacon is configured to bind to a second target type of molecular cargo.
  • the first target type of molecular cargo is a protein
  • the second target type of molecular cargo is an RNA.
  • the second type of molecular cargo is a DNA fragment.
  • the one or more capture antibodies comprises anti-ApoAl . In some embodiments, the one or more capture antibodies comprises anti-ApoB.
  • a system for simultaneously detecting an RNA and a protein in situ comprising: a plurality of capture antibodies attached to a micropattern array on a glass substrate, wherein the plurality of capture antibodies comprises anti-ApoAl or anti-ApoB; a plurality of detection antibodies and a plurality of molecular beacons, wherein the plurality of detection antibodies is configured to bind to a first target type of molecular cargo, and wherein the molecular beacon is configured to bind to a second target type of molecular cargo wherein first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA; and a fluorescent imaging device to capture image data.
  • a method for simultaneously detecting an RNA and a protein in situ comprises a plurality of capture antibodies attached to a micropattern array on a glass substrate, a plurality of detection antibodies and a plurality of molecular beacons and a fluorescent imaging device to capture image data.
  • the plurality of capture antibodies comprises anti-ApoAl or anti-ApoB.
  • the plurality of detection antibodies is configured to bind to a first target type of molecular cargo.
  • the plurality of molecular beacons is configured to bind to a second target type of molecular cargo.
  • the first target type of molecular cargo is a protein
  • the second target type of molecular cargo is an RNA.
  • the second type of molecular cargo is a DNA fragment.
  • the one or more capture antibodies comprises anti-ApoAl . In some embodiments, the one or more capture antibodies comprises anti-ApoB.
  • the glass substrate is coated with poly-L-lysine (PLL) through physisorption prior to coating the glass substrate with a polyethylene glycol (PEG).
  • PLL poly-L-lysine
  • PEG polyethylene glycol
  • Some other coatings that can be used on glass surfaces include but are not limited to epoxy silane, 3-D Hydrogel, aminosilane, streptavidin, poly-L-Lysine, aldehydesilane, or 3-D Polymer.
  • the PEG is covalently bound to the PLL through N- hydroxysuccinimide (NHS) chemistry.
  • N- hydroxysuccinimide (NHS) chemistry Polyethylene glycol (PEG) is a synthetic polymer that can be categorized into different types based on molecular weight, synthesis geometry, specific functional groups, and applications. Some types of PEG molecules include branched PEG, star PEG, comb PEG, alkyne-PEG, PEG 200, PEG 300, PEG 400, PEG 600, PEG 4000, PEG 6000 and PEG 8000.
  • the PEG is methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA).
  • Physical adsorption or physisorption is the process wherein adsorbate molecules are held to the surface of an adsorbent by weak Van der Waals forces.
  • the micropattern array is photoetched on the glass substrate in presence of a photoactivator, wherein the photoactivator is 4-benzoylbenzyl- trimethylammonium chloride (PLPP).
  • PLPP 4-benzoylbenzyl- trimethylammonium chloride
  • the micropattem array comprises a five-by-five array of circles, wherein each individual circle has a diameter ranging from about 2 pm to about 200 pm. In some embodiments, each individual circle has a diameter of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • each individual circle has a center-to-center spacing of about 80 pm in relation to an adjacent circle.
  • the one or more capture antibodies is biotinylated and attached to the micropattern array by binding to a physically adsorbed Neutravidin layer on the micropattern array. In some embodiments, the one or more capture antibodies binds to surface proteins expressed on the EV and/or the LP. In some embodiments, the one or more capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof.
  • the plurality of capture antibodies is biotinylated and attached to the micropattern array by binding to a physically adsorbed Neutravidin layer on the micropattern array. In some embodiments, the plurality of capture antibodies binds to surface proteins expressed on the EV and/or the LP. In some embodiments, the plurality of capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof.
  • the system further comprises a first color associated with the detection antibody in a captured image data in a first channel. In some embodiments, the system further comprises a second color associated with the molecular beacon in the captured image data in a second channel. In some embodiments, the system comprises a third color in the captured image data in a third channel.
  • the system further comprises a first color associated with the plurality of detection antibodies in a captured image data in a first channel. In some embodiments, the system further comprises a second color associated with the plurality of molecular beacons in the captured image data in a second channel. In some embodiments, the system comprises a third color in the captured image data in a third channel.
  • the RNA is selected from a microRNA (miRNA), a messenger RNA (mRNA), or combinations thereof.
  • RNA encodes AXL- , AXL-2, AXL-3, NSF, NCAN, p53, GAPDH, hsa-miR-21-5p, hsa-miR-9-5p, hsa-miR-1246-5p, cel-miR- 39-3p, cel-miR-54-3p or cel-miR-238-3p.
  • DNA fragments such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and mitochondrial DNA (mtDNA)
  • the detection antibody is conjugated with one or more fluorophores (such as, for example, Alexa Fluor 488, Alexa Fluor 546, Alexa Flour 647, Alexa Fluor 55, FITC or CoraLite 594) for fluorescent imaging.
  • fluorophores such as, for example, Alexa Fluor 488, Alexa Fluor 546, Alexa Flour 647, Alexa Fluor 55, FITC or CoraLite 594.
  • the plurality of detection antibodies is conjugated with one or more fluorophores (such as, for example, Alexa Fluor 488, Alexa Fluor 546, Alexa Flour 647, Alexa Fluor 55, FITC or CoraLite 594) for fluorescent imaging.
  • fluorophores such as, for example, Alexa Fluor 488, Alexa Fluor 546, Alexa Flour 647, Alexa Fluor 55, FITC or CoraLite 594
  • the molecular beacon comprises one or more fluorescent dye sequences for fluorescent imaging. In some embodiments, the molecular beacon comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the molecular beacon is selected from any one of SEQ ID NOs: 1-14. In some embodiments, the molecular beacon comprises SEQ ID NO: 1. In some embodiments, the molecular beacon comprises SEQ ID NO: 2. In some embodiments, the molecular beacon comprises SEQ ID NO: 3. In some embodiments, the molecular beacon comprises SEQ ID NO: 4. In some embodiments, the molecular beacon comprises SEQ ID NO: 5. In some embodiments, the molecular beacon comprises SEQ ID NO: 6.
  • LNA locked nucleic acid
  • the molecular beacon comprises SEQ ID NO: 7. In some embodiments, the molecular beacon comprises SEQ ID NO: 8. In some embodiments, the molecular beacon comprises SEQ ID NO: 9. In some embodiments, the molecular beacon comprises SEQ ID NO: 10. In some embodiments, the molecular beacon comprises SEQ ID NO: 11. In some embodiments, the molecular beacon comprises SEQ ID NO: 12. In some embodiments, the molecular beacon comprises SEQ ID NO: 13. In some embodiments, the molecular beacon comprises SEQ ID NO: 14.
  • the plurality of molecular beacons comprises one or more fluorescent dye sequences for fluorescent imaging. In some embodiments, the plurality of molecular beacons comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the plurality of molecular beacons is selected from any one of SEQ ID NOs: 1-14. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 1. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 2. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 3. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 4.
  • LNA locked nucleic acid
  • the plurality of molecular beacons comprises SEQ ID NO: 5. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 6. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 7. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 8. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 9. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 10. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 11. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 12. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 13. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 14.
  • fluorescently imaging the micropattem array to capture image data comprises total internal reflection fluorescence microscopy (TIRFM).
  • detection of the EV and/or LP comprises flowcytometry.
  • Example 1 Engineering a Tunable Micropattern- Array Assay to Sort Single Extracellular Vesicles and Particles to Detect RNA and Protein In situ
  • a single-EV and particle (siEVP) protein and RNA assay S1EVP PRA
  • S1EVP PRA single-EV and particle protein and RNA assay
  • the S1EVP PRA immobilizes and sorts, particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ.
  • ELPs extracellular vesicles and particles
  • the S1EVP PRA outperformed the sensitivities of bulkanalysis benchmark assays for RNA and protein.
  • EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation.
  • GBM glioblastoma multiforme
  • the S1EVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs.
  • the S1EVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.
  • dSTORM Direct stochastic optical reconstruction microscopy
  • qSMLM quantitative single-molecule localization microscopy
  • TIRFM total internal reflection fluorescence microscopy
  • siEVP protein and RNA assay capable of multiplexing protein and RNA biomarker detection at a single-particle resolution.
  • the assay consists of an array of micropatterns surrounded by a non-biofouling polymer film that can be functionalized with various antibodies to sort and immobilize siEVPs.
  • ADP-ribosylation factor 6 (ARF6), annexin Al, CD63, and CD9 were targeted as EV-specific epitopes; epidermal growth factor receptor (EGFR) as a tumor-specific epitope; and apolipoprotein Al (ApoAl) and apolipoprotein B (ApoB) as LP-specific epitopes to immobilize and quantify siEVP subpopulations, revealing intervesicular and interparticle heterogeneity.
  • RNA-targeting molecular beacons (MBs) and fluorescently labeled antibodies generated signals for mRNA, miRNA, and protein on siEVPs, which were then visualized by TIRFM and quantified via automatic image acquisition.
  • the S1EVP PRA exceeded the detection limit for both qRT-PCR and ELISA by three orders of magnitude without tedious lysis and amplification steps.
  • single-LP-EV (siLP-EV) co-isolates were discovered expressing CD63 by subjecting serum-isolated siLPs to CD63/CD9-mediated capture on the S1EVP PRA, which were obscured by bulk-analysis methods.
  • the combinatorial multiplexing of various biomarkers across biomolecular species in siEVs allowed the investigation of siEV intravesicular heterogeneity.
  • RNA-seq small RNA sequencing
  • Capture and detection antibodies used in the study are provided in Table 3. Capture antibodies (except the select few pre-biotinylated) were biotinylated using an EZ-LinkTM micro Sulfo-NHS-biotinylation kit (ThermoFisher Scientific). All MBs used in the study are provided in Table 4.
  • Substrate fabrication Coverslips were cleaned with ethanol and then deionized (DI) water via sonication for 3 min. The surface of the coverslip was treated with oxygen plasma for 1 minute to activate the surface. A small drop of 0.01 % (w/v) PLL was placed onto parafilm on which the treated coverslip was then placed for an even distribution of the PLL. After incubating the coverslip for 30 minutes at room temperature, the PLL-coated coverslip was rinsed with DI water and dried with nitrogen flow. Following the same method, 100 mg/mL of mPEG-SVA diluted in 0.1 M HEPES was evenly distributed on the PLL-coated coverslip. The coverslip was incubated at room temperature for 1 hour before rinsing with DI water and drying with a nitrogen airflow.
  • DI deionized
  • the passivated coverslip was photoetched using the PRIMO optical module (Alveole) mounted on an automated inverted microscope (Nikon Eclipse Ti Inverted Microscope System, Melville, NY). Briefly, grayscale images were translated into UV light via a digital-micromirror device (DMD) that allows for a maskless illumination of different UV intensities correlating to the corresponding grayscale values.
  • DMD digital-micromirror device
  • PLPP gel was diluted in 96 % ethanol to distribute the gel evenly throughout the surface of the coverslip. After the ethanol evaporated, a silicone spacer (W x L 3.5 mm x 3.5 mm, 64 wells; Grace Bio Labs, Bend, OR) was placed on the PEG-coated coverslip.
  • a five-by-five array of 20-pm diameter circles spaced 80 pm center-to-center was exposed onto the coverslip with the PRIMO optical module.
  • micropatterns at different grayscale values, including 0, 25, 50, 75, 95, and 100 % with UV doses, including 10, 20, and 30 mJ/mm2 were examined (Table 1).
  • the photoetched coverslip was washed under a stream of DI water and dried by nitrogen flow.
  • a microscopy slide (ThermoFisher Scientific) was placed under the coverslip, and the 64-well ProPlate microarray system (Grace Bio Labs) was placed gently on the photoetched coverslip.
  • the assembled array was secured by self-cut Delrin snap clips (Grace Bio Labs) to avoid leakage or potential contamination.
  • the photoetched coverslip was rehydrated in phosphate-buffered saline (PBS) for 15 minutes before further functionalizing the micropatterns.
  • PBS phosphate-buffered saline
  • DMEM Dulbecco’s Modified Eagle Medium
  • SF268, SF295, SF539, SNB19, and SNB-75 glioma cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. All cell culture media was prepared with 10 % (v/v) fetal bovine serum (FBS) and 1 % (v/v) penicillin-streptomycin. Cell lines were cultured to 70 % confluence at 37 °C in a 5 % CO2 incubator. Before EV collection, cells were washed with PBS three times, after which the cells were incubated in serum-free media. After two days of cell culture, the EV-enriched cell culture media was collected and centrifuged at 2,000 x g for 10 minutes at room temperature to separate cell debris before further analysis.
  • RPMI Roswell Park Memorial Institute
  • GBM patient serum was obtained under Institutional Review Board (IRB)-approved protocols at MD Anderson Cancer Center (PA 19- 0661) following national guidelines. All patients signed informed consent forms during clinical visits before surgery and sample collection. Patients did not receive compensation in return for their participation in this study.
  • IRS Institutional Review Board
  • Healthy donor serum and plasma collection 10 mL of whole blood from healthy donors was collected into BD Serum Separation Tubes (SST; Thermo Fisher Scientific) and BD Plasma Preparation Tubes (PPT; Thermo Fisher Scientific) for serum and plasma collection, respectively.
  • SSTs were gently placed upright to coagulate for 60 minutes after being rocked 10 times.
  • PPTs were rocked 10 times. Both SSTs and PPTs were centrifuged at room temperature at 1,100 * g for 10 min. After The serum and plasma were stored in 1 mL aliquots at -80 °C. All blood samples were collected under an approved IRB at The Ohio State University (IRB #2018H0268).
  • LP isolation Healthy donor serum was subjected to the low-density LP / very-low- density LP (LDL/VLDL) and high-density LP (HDL) purification kit (Cell Biolabs, San Diego, CA). 1 mL of serum on ice, a dextran solution, and precipitation solution A was added and incubated on ice for 5 min. The sample was centrifuged at 6,000 * g for 10 minutes at 4°C. The supernatant was removed for further HDL processing, while the remaining pellet was subj ected to further LDL purification.
  • LDL/VLDL very-low- density LP
  • HDL high-density LP
  • the pellet was resuspended in 40 pL of a bicarbonate solution and centrifuged at 6,000 x g for 10 minutes at 4°C, whereby the supernatant was transferred to ImL of lx precipitation solution B and centrifuged at 6,000 x g for 10 minutes at 4°C.
  • the pellet was resuspended with 20 pL NaCl solution, added to ImL of lx precipitation solution C, and centrifuged at 6,000 x g for 10 minutes at 4 °C. The last process was repeated and after centrifugation, the pellet was resuspended in 20 pL of a NaCl solution.
  • the supernatant was added to 60 pL of a dextran solution and 150 pL of precipitation solution A and was then incubated for 2 hours at room temperature and centrifuged at 16,000 x g for 30 minutes at 4°C.
  • the pellet was resuspended in 500 pL of an HDL resuspension buffer and centrifuged at 6,000 x g for 10 minutes at 4°C.
  • the pellet was resuspended in 600 pL of a lx HDL wash solution, incubated on a rocker for 30 minutes at 4°C, and centrifuged at 6,000 x g for 10 minutes at 4°C.
  • the HDL supernatant was transferred to 90 pL of a dextran removal solution, while the LDL resuspension was added to 80 pL of the dextran removal solution.
  • the mixtures were incubated for 1 hour at 4°C and centrifuged at 6,000 x g for 10 minutes at 4°C by which the supernatants were recovered into a 20-kDa Slide- A-Lyzer® MINI Dialysis devices (Thermo Fisher Scientific) and incubated in PBS for 1 day.
  • Engineered-EV RNA model system Cell transfection was conducted via a cellular nanoporation (CNP) biochip. Briefly, a single layer of Gli36 cells ( ⁇ 8 x 10 6 ) was spread overnight on a 1 cm x 1 cm 3D CNP silicon chip surface. Individual CNP chips were transfected separately with cel-miR-39-3p, cel-miR-54-3p, and cel-miR-238-3p plasmids at 400 ng/pL concentration in PBS. For multi-plasmid transfection, a weight ratio of 1 : 1 : 1 was pre-mixed at a 400 ng/pL concentration each in PBS.
  • the plasmid solutions were injected into the cells via nanochannels using a 150 V electric field for 10 pulses, at 10 ms durations and 0.1 s intervals. EVs were collected from the cell supernatant 24 hours after cell transfection.
  • Tangential flow filtration (TFF) EVP purification The EV-enriched cell culture media and serum samples were introduced into a TFF system as described by previous techniques to purify EVPs. In brief, cell culture media or serum was circulated through a 500 kDa TFF hollow fiber filter cartridge, where EVPs were retained and enriched in the system ( ⁇ 5 mL), while free proteins and nucleic acids permeated through the filter.
  • Constant-volume diacycles of PBS were performed until pure EVPs were obtained (350 mL of PBS).
  • the EVPs were further enriched by centrifuging the sample within a 10 kDa centrifugal unit at 3,000 x g at 4 °C until a final volume of 100 pL was achieved.
  • Protein concentrations were measured using a Micro BCATM Protein Assay Kit (ThermoFisher Scientific), according to the manufacturer’s protocol.
  • Apolipoprotein corona EVs with apolipoprotein corona were prepared according to an established protocol. Briefly, plasma was diluted into PBS 1 : 1 and passed through a 2-pm filter then a 0.8-pm filter. The filtered plasma was ultracentrifuged at 20,000 x g at 16 °C for 40 min. The supernatant was collected and was ultracentrifuged at 100,000 x g at 4 °C for 16 hr. The supernatant was collected and referred to hereafter as EV-depleted plasma (EVDP). 60 pL of TFF-purified EVs harvested from Gli36 cells grown in serum-free conditions were incubated in 500 pL of EVDP for 30 minutes at room temperature.
  • EVDP EV-depleted plasma
  • the solution was purified via size-exclusion chromatography (SEC) with the qEV (Izon Sciences, Boston, MA), according to the manufacturer’s protocol.
  • SEC size-exclusion chromatography
  • the samples were concentrated to 10 9 particles/mL with a 3 kDa centrifugal unit at 3,000 x g at 4°C.
  • the purified EVs with apolipoprotein corona were immediately added to the S1EVP PRA.
  • TRPS The qNano Gold (Izon Sciences) was employed to quantify the size and concentration of EVPs via NP100 (50 - 330 nm) and NP600 (275 - 1570 nm) nanopore membranes. A pressure of 10 mbar and a voltage of 0.48 and 0.26 V was applied for the NP 100 and the NP600, respectively. Polystyrene nanoparticles (CPC 100 and CPC400) were used to calibrate the samples.
  • CPC 100 and CPC400 Polystyrene nanoparticles
  • MBs (listed 5'-3') targeting RNAs detected in this study are provided in Table 4.
  • the designed MBs were custom synthesized and purified using high-performance liquid chromatography (HPLC; Integrated DNA Technologies, Coralville, IA). Locked nucleic acid nucleotides (depicted as +) were incorporated into the oligonucleotide strands to improve the thermal stability and nuclease resistance of the MBs for incubation at 37 °C.
  • siEVP capture using the siEVP PRA 0.1 mg/mL of NA was added to the chip and allowed to adsorb onto the photoetched micropatterns for 30 min. The chip was washed with PBS thoroughly to remove excess NA.
  • a blocking solution of 3 % BSA and 100 mg/mL of mPEG-SVA was added to avoid unwanted non-specific binding.
  • biotinylated anti-CD63 and anti-CD9 were added at 20 pg/mL each and allowed to sit overnight at 4 °C.
  • anti-CD63, anti-CD9, anti-EGFR, anti-ARF6, anti-annexin Al, anti-ApoAl, anti-ApoB, and IgG were added separately at 20 pg/mL each and allowed to sit overnight at 4 °C.
  • 3 % BSA was added for 1 hour to further block after washing away the capture antibodies.
  • a concentration of 10 9 parti cles/mL (apart from dilution experiments, which employed 10 6 - 10 11 particles/mL) was then added and allowed to tether to the antibodies for 2 hours at room temperature. Unbounded EVPs were washed away with PBS and further blocked with 3 % BSA for 1 hour.
  • siEVP protein and RNA staining 10 pg/pL of MBs diluted in a lx TE buffer was added to the immobilized siEVPs for 1 hour at 37 °C.
  • As for protein detection 0.4 pg/mL of the fluorescently labeled antibodies were diluted into a solution of 1 % BSA was added to the EVP sample for 1 hour at room temperature.
  • Residual detection probes were washed away with PBS before imaging. For single biomarker analysis, sole detection probes were added. To analyze multiple proteins or RNAs, the probes were added sequentially, fluorescently labeled antibodies were added first, followed by MBs.
  • Image analysis Images of fluorescently labeled siEVPs were obtained by TIRFM (Nikon Eclipse Ti Inverted Microscope System, Melville, NY) with a lOOx oil immersion lens. An automatic algorithm was used to quantify the TIRFM images by detecting all bright signals determined via the defined outline of each bright signal by localizing the fluctuating fluorescence intensities throughout the image. The background noise was removed using a Wavelet de-noising method, and the net signal for all bright signals was obtained. The sum of all the bright signals within each micropattern was employed to calculate the TFI of the sample alongside distributions of fluorescence intensity of the siEVPs. The TFI of samples was normalized to the average TFI of the negative controls as the RFI.
  • ELISA EGFR protein expression levels in Gli36-derived EVs were quantified using an EGFR Human ELISA kit (ThermoFisher Scientific). EVs were spiked in healthy donor serum at concentrations ranging from 0 to 10 11 particles/mL while maintaining the serum- derived EVP concentration at 10 9 particles/mL. EGFR concentrations were quantified according to the manufacturer’s instructions.
  • qRT-PCR cel-miR-39-3p levels within the engineered EVs were quantified using qRT-PCR. EVs were spiked in healthy donor serum at concentrations ranging from 0 to 10 11 parti cles/mL while maintaining the serum-derived EVP concentration at 10 9 parti cles/mL.
  • RNA from the EVPs was isolated and purified using an RNeasy Mini Kit and an miRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany), respectively, according to the manufacturer’ s instructions.
  • cDNA was synthesized from the total RNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) on a thermal cycler (Veriti 96-Well Thermal Cycler; Applied Biosystems).
  • cel-miR-39-3p expression was quantified using a TaqMan Gene Expression assay (Assay Id: HsOl 125301 ml; ThermoFisher Scientific) on a Real-Time PCR System (Applied Biosystems).
  • Immunoblotting Gli36 cells, Gli36-derived EV, serum-isolated VLDL/LDL, serum- isolated HDL, unprocessed serum, and TFF-purified serum samples were lysed in radioimmunoprecipitation assay (RIP A) buffer (ThermoFisher Scientific, Waltham, MA) with the addition of Pierce protease and phosphatase inhibitor (ThermoFisher Scientific) for 15 minutes on ice. Protein concentrations were quantified using a Micro BCATM Protein Assay Kit (ThermoFisher Scientific), according to the manufacturer’s protocol.
  • RIP A radioimmunoprecipitation assay
  • SEM Scanning electron microscopy: Gli36-derived EVs were immobilized to the micropatterned coverslip overnight at 4°C.
  • the immobilized siEVs were fixed in a 2 % glutaraldehyde (Millipore Sigma) and 0.1 M sodium cacodylate solution (Electron Microscopy Sciences, Hatfield, PA) for 3 hours.
  • EVs were incubated in 1 % osmium tetraoxide (Electron Microscopy Sciences) and 0.1 M sodium cacodylate for 2 hours after washing with a 0.1 M sodium cacodylate solution. Subsequently, the sample was dehydrated with increasing ethanol concentrations (50, 70, 85, 95, and 100 %) for 30 minutes each.
  • the CO2 critical point dryer (Tousimis, Rockville, MD) was applied to dry the sample.
  • a ⁇ 2 nm layer of gold coating was deposited on the surface using a sputtering machine (Leica EM ACE 600, Buffalo Grove, IL) and was imaged using an SEM (Apreo 2, FEI, ThermoFisher Scientific).
  • TEM Two 20-pL DI water droplets and two 20-pL droplets of UranyLess EM contrast stain (Electron Microscopy Science) droplets were placed on parafilm.
  • TEM grids were plasma treated for 1 minute before 10 pL of Gli36-derived EVs and serum-isolated LPs were drop-cast onto the treated surface. The samples were incubated on the TEM surface for 1 minute and then blotted away with filter paper. The TEM grids were washed immediately by dipping into the DI water droplet, blotted with filter paper, and repeated with the other droplet. The same technique was repeated for the contrast stain with 22 s incubations. The TEM grid was kept in the grid box overnight to completely dry before imaging. TEM imaging was carried out with a Tecnai TF-20 operating at 200kV.
  • Cryogenic TEM Cryogenic TEM: 3-pL aliquots of EV samples with and without a lx TE buffer incubated at 37 °C for 2 hours were added to lacey 300-mesh copper specimen grids (Product #01883; Ted Pella Inc., Redding, CA). Excess liquid was blotted away for 4 s with WhatmanTM grade 1 filter papers (ThermoFisher Scientific), after which the grid was immediately plunged into liquid ethane with the Vitrobot Mark IV system (ThermoFisher Scientific) to rapidly form a thin layer of amorphous ice. The grid was then transferred under liquid nitrogen to a GlaciosTM Cryo-TEM (ThermoFisher Scientific). Lastly, images were collected with a FelconTM direct electron detector (ThermoFisher Scientific).
  • RNA sequencing was isolated from cells and the cell- derived EVs using the miRNeasy kit (Qiagen). The RNA was eluted with 50 pl of nuclease- free water and the quality was assessed using an RNA (Pico) chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). A sRNA-seq library construction method that utilizes adapters with four degenerated bases to reduce adapter-RNA ligation bias was used to characterize the miRNA. Size selection was performed using a Pippin HT automated size-selection instrument (Sage Science, Beverly, MA), and library concentrations were measured with the NEBNext Library Quant Kit (New England Biolabs, Ipswich, MA).
  • RNA from cells and EVs were analyzed using Agilent Human Whole Genome 8 x 60 microarrays with fluorescent probes prepared from isolated RNA samples using Agilent QuickAmp Labeling Kit according to the manufacturer’s instructions (Agilent). Gene expression information was obtained with Agilent’s Feature Extractor and processed with the in-house SLIM pipeline.
  • Colocalization efficiency An open-source plugin for Imaged called EzColocalization was employed to visualize and measure the colocalization of EV biomarkers from acquired TIRFM images.
  • S1EVP PRA was fabricated with the PRIMO optical module (FIG. 1A). Glass coverslips were coated with poly-L-lysine (PLL) through physisorption. Methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA) was covalently bound to the surface through N-hydroxysuccinimide (NHS) chemistry, creating a non-biofouling surface.
  • PLL poly-L-lysine
  • mPEG-SVA Methoxy-poly(ethylene glycol)-succinimidyl valerate
  • NHS N-hydroxysuccinimide
  • a five-by-five array of 20-pm diameter circles was photoetched from the mPEG monolayer via UV projections translated by a digital -micromirror device (DMD) in the presence of 4-benzoylbenzyl-trimethylammonium chloride (PLPP) as a photoactivator (FIG. 1 A-i).
  • DMD digital -micromirror device
  • PLPP 4-benzoylbenzyl-trimethylammonium chloride
  • FIG. 1 A-i 4-benzoylbenzyl-trimethylammonium chloride
  • the photoetching of the mPEG monolayer promotes the adsorption of proteins61, such as NeutrAvidin (NA), that can further be functionalized via biotin motifs. Therefore, biotinylated antibodies against surface proteins expressed on EVPs were immobilized strictly within the micropatterns to selectively sort siEVPs (FIG. lA-ii).
  • siEVP proteins were tagged with fluorescently labeled antibodies, while RNA species, including mRNAs and miRNAs, were tagged with MBs (FIG. 1 A-iii).
  • TIRFM was utilized to visualize the signals from immobilized siEVPs, as TIRFM produces an exponentially decaying electromagnetic wave that only excites fluorophores near the glass surface.
  • the micropattem-based design thus allows for a facile multiplexed analysis of siEVPs by immediately identifying and colocalizing signals in different regions of the glass surface.
  • the photoetching level correlates to the grayscale value of a digital template and the
  • EVs were harvested from Gli36 cells, a human glioma cell line, which were grown in serum-free media to minimize LP-EV interactions during EV collection.
  • the collected EVs purified by tangential flow filtration (TFF), alongside a negative control, phosphate-buffered saline (PBS), were tested on the various micropattern configurations, which were functionalized with antibodies targeting CD63 and CD9, common membrane proteins constitutively expressed in various subpopulations of EVs.
  • the captured EVs were then detected with a fluorescently labeled antibody against CD63.
  • a 50 % grayscale value and a 20 mJ/mm2 dose rendered the highest fluorescence intensity on siEVs relative to the control and minimized the non-specific binding of the fluorescently labeled antibody to the micropatterns (Table 1). Furthermore, the optimized grayscale value and dose demonstrated the homogenous adsorption of NA with specificity to the photoetched micropatterns (FIG. 7).
  • RNA within siEVs tethered to the micropatterns a fluorescently labeled antibody against CD63 and a MB targeting hsa-miR-21-5p, an abundant vesicular miRNA in GBM, were used as detection probes simultaneously, and visualized via TIRFM.
  • Each signal represented a siEV expressing CD63, while each signal representing a siEV carrying hsa-miR-21-5p in the acquired TIRFM images.
  • Each colocalized signal thus demonstrated the colocalization of both biomarkers.
  • fluorescence signals in the control were significantly lower, indicating the ability of S1EVP PRA to selectively multiplex different biomolecular species in siEVs (FIG. IB).
  • the TIRFM images could be quantified as distributions of fluorescence intensity of the siEVs to analyze the heterogenous expression of biomarkers on siEVs (FIG. 1C) or to quantify and statistically compare various samples utilizing relative fluorescence intensities (RFI; the total fluorescence intensity of signals detected in the sample divided by the average total fluorescence intensity of signals detected in PBS within the five-by-five array): where TFI is the total fluorescence intensity, s is the fluorescence intensity from the jth signal, and n is the number of signals within the ith micropattern.
  • RFI relative fluorescence intensities
  • SEM scanning electron microscopy
  • RNA detection in siEVs Although various methods are available to detect proteins on siEVPs, detecting RNA at a single-particle resolution without altering or damaging the integrity of the vesicles remains challenging. Therefore, it was aimed to optimize the specificity and sensitivity of RNA detection in siEVs from Gli36 cells with the S1EVP PRA.
  • TO detect vesicular RNA MBs were diluted in a tris-ethylenediaminetetraacetic acid (TE) buffer that is frequently used to solubilize and protect nucleic acids against degradation.
  • TE tris-ethylenediaminetetraacetic acid
  • TE contains ethylenediaminetetraacetic acid (EDTA), which electrostatically intercalates into the lipid bilayer causing its fluidization, and a tris buffer, which synergizes with EDTA. Therefore, it was hypothesized that TE buffer be used to stabilize the MBs and partially permeabilize the lipid bilayer of siEVs, allowing the MBs to reach the lumen of intact siEVs and hybridize with the desired RNA sequences. To test this, both the integrity of siEVs and the specificity of probes to intraluminal targets post-treatment with the TE buffer were quantified.
  • EDTA ethylenediaminetetraacetic acid
  • hsa-miR-21-5p a miRNA abundant in Homo sapiens
  • cel-miR-39-3p a non-human miRNA abundant in Caenorhabditis elegans
  • RNA specificity using S1EVP PRA Gli36 cells were transfected via electroporation to express the non-human miRNAs: cel-miR-39-3p, cel-miR- 54-3p, and cel-miR-238-3p (FIG. 10A).
  • siEVs harvested from the transfected cells were then detected with MBs targeting cel-miR-39-3p, cel-miR-54-3p, and cel-miR-238-3p.
  • the engineered siEVs enriched with non-human miRNAs were successfully detected as single fluorescent signals within the micropatterns with MBs targeting the corresponding miRNA, while control samples showed a negligible number of signals (FIG. 2A).
  • cel-miR-39-3p-enriched siEVs detected by MBs targeting cel-miR-39-3p exhibited a RFI of 9.10 ⁇ 2.07, while serum-derived siEVPs and the disparate MBs produced an average RFI of 1.08 ⁇ 0.11 (FIG.
  • the S1EVP PRA was compared against the benchmark method for bulk RNA detection, qRT-PCR. EVs harvested from Gli36 cells enriched with 400 ng/pL of the cel-miR-39-3p plasmid were diluted serially into EVPs isolated from healthy donor serum and were detected with the S1EVP PRA and qRT-PCR for cel- miR-39-3p.
  • qRT-PCR quantitative PCR
  • FIG. 2D concentration of 10 9 vesicles/mL
  • the sensitivity of the S1EVP PRA for protein detection in siEVs was compared to ELISA, the benchmark bulk-analysis method.
  • Gli36-derived EVs were diluted serially into EVPs isolated from healthy donor serum and were detected with both methods for a cytoplasmic epitope of EGFR, a transmembrane protein upregulated in GBM-associated EVs with external and intraluminal epitopes.
  • RNA detection (FIGS. 12A-12B) with similar observations to RNA detection (FIGS. 12A-12B).
  • the ability of the S1EVP PRA to focus intact siEVs and highlight biomarkers of interest at minimal concentrations affords a unique ability to colocalize biomolecular species on siEVs and explore intravesicular heterogeneity, which cannot be realized by bulk-analysis methods.
  • Simultaneous detection of various biomolecular species in siEVs To first determine the ability of the S1EVP PRA to multiplex various probes at a single-particle resolution, a tetraspanin analysis was performed on the siEVs, a commonplace procedure for in situ screening of siEVs.
  • Gli36-derived siEVs were screened for CD63, CD9, and CD81 with fluorescently labeled antibodies targeting the respective tetraspanins, whereby each antibody was chosen to excite at distinct wavelengths.
  • the fluorescence signals were pseudocolored as the primary colors of light such that the colocalization of two detection probes could be visualized, while white signals illustrated the colocalization of all detection probes (FIG. 3 A).
  • the fluorophores only emitted light when matched by their corresponding excitation wavelengths (Tukey’s HSD, p ⁇ 0.0001 for the matched channels with siEVs only), ensuring the validity of the colocalization as originating from the co-expression of the tetraspanins (FIG. 12).
  • 3B shows the colocalization efficiencies for CD63 and CD9 (20.08 ⁇ 2.09 %), CD81 and CD9 (19.31 ⁇ 1.59 %), CD63 and CD81 (20.84 ⁇ 2.52 %), and all three proteins (2.16 ⁇ 0.58 %).
  • various methods exist to simultaneously detect proteins on siEVs such as single-particle interferometric reflectance imaging sensing (SP- IRIS). Therefore, the S1EVP PRA was compared with a commercial SP-IRIS, the Exo View. Both the S1EVP PRA and the Exo View produced similar signals whereby the colocalization of the tetraspanins were illustrated (FIG. 14A).
  • the Exo View differentiated positive signals from their isotype control at lower signal-to-noise ratios due to high levels of non-specificity (FIG. 14B).
  • the colocalization profiles are higher on the Exo View, interestingly, the highest frequency of colocalization tends to be the complementary color to the antibody used to capture the siEVs; specifically, CD9+/CD81+ siEVs for CD63-mediated capture, CD63+/CD81+ siEVs for CD9-mediated capture, and CD9+/CD63+ siEVs for CD81- mediated capture (FIG. 14C).
  • an antibody cocktail as performed with the S1EVP PRA appears to normalize the bias to the capture antibody (FIG.
  • the S1EVP PRA provides higher signal-to-noise ratios for protein detection ( ⁇ 12 for the S1EVP PRA versus ⁇ 3 for the Exo View), an ability to colocalize with nucleic acid cargo, working concentrations one order of magnitude lower, and multiple technical replicates for a reliable colocalization analysis.
  • FIG. 15D shows the colocalization efficiencies for HAL- 1 and AXL-2 (26.89 ⁇ 2.61 %), AXL-2 andHAL-3 (28.57 ⁇ 3.24 %), AXL- 1 and AXL-3 (23.05 ⁇ 6.21 %), and all three regions (2.87 ⁇ 1.03 %).
  • siEVs harvested from Gli36 cells transfected with cel-miR-39-3p, cel-miR-54-3p, and cel-miR-238- 3p plasmids were detected by their respective MBs as was previously performed, but in conjunction, revealing the co-expression of multiple miRNAs within the same siEV (FIG. 16A).
  • FIG. 16A shows the ability to colocalize signals on the same RNA biomarker in siEVs with the S1EVP PRA.
  • 16B shows the colocalization efficiencies for cel-miR-39-3p and cel-miR-54-3p (32.94 ⁇ 1.47 %), cel-miR-54-3p and cel-miR-238-3p (31.10 ⁇ 1.03 %), cel-miR-238-3p and cel-miR- 39-3p (31.26 ⁇ 2.90 %), and all three miRNAs (5.51 ⁇ 0.51 %).
  • RNA species were screened in Gli36-derived siEVs.
  • AXL- ⁇ , hsa-miR-9-5p, and hsa-miR-21-5p were colocalized in various siEVs revealing the co-expression of mRNA and miRNA (FIG. 3C).
  • FIG. 3C shows the co-expression of mRNA and miRNA.
  • 3D shows the colocalization efficiencies for HAL- 1 and hsa-miR-9-5p (21.15 ⁇ 2.29 %), hsa-miR-21-5p and hsa-miR-9-5p (22.62 ⁇ 1.08 %), HAL-1 and hsa-miR-21-5p (20.67 ⁇ 2.58 %), and all three RNA biomarkers (2.95 ⁇ 0.18 %).
  • the two methods of detection, immunoaffinity and MB hybridization were tested together to test the ability of the S1EVP PRA to multiplex proteins and RNA simultaneously.
  • FIG. 3E shows the colocalization efficiencies for CD63 and hsa-miR-9-5p (19.30 ⁇ 1.05 %), hsa-miR- 21-5p and hsa-miR-9-5p (22.52 ⁇ 1.90 %), hsa-miR-21-5p and CD63 (20.71 ⁇ 2.23 %), and all three biomarkers (2.12 ⁇ 0.48 %).
  • FIG. 17B shows the colocalization efficiencies for CD63 and HAL-2 (21.49 ⁇ 5.78 %), hsa-miR-21-5p and AXL-2 (14.16 ⁇ 2.84 %), CD63 and hsa-miR-21-5p (13.68 ⁇ 2.72 %), and all three biomarkers (0.43 ⁇ 0.19 %). Therefore, the marriage of the two detection methods on intact siEVs with the S1EVP PRA broadens the horizon of current in situ methods, illustrating a rare display of siEV intravesicular heterogeneity with various biomolecular species.
  • Sorting siEVPs into subpopulations Tailoring the surface chemistry of the micropatterns enables the examination of intervesicular heterogeneity by first sorting siEVs into subpopulations based on membrane-protein composition.
  • CD63 and CD9 are expressed in higher quantities in small EVs and are considered “classical” exosomal biomarkers due to their enrichment and involvement in cargo loading despite being present in some ectosome subpopulations.
  • ARF6 and annexin Al are considered ectosomal biomarkers due to their enrichment and contribution towards the budding of vesicles from the plasma membrane.
  • Cetuximab a chimeric monoclonal antibody, was utilized to capture tumor-specific siEVs, which target the extracellular domain of EGFR and efficiently immobilize tumor-derived EVs from GBM patients.
  • WB analyses confirmed that tetraspanins CD63, CD9, and CD81 were enriched in the TFF-purified EVs from Gli36 cells when compared to their cellular concentrations.
  • EGFR, annexin Al, and ARF6 were upregulated in Gli36 cells, but were still present in the TFF-purified EVs (FIG. 4A).
  • micropattems were decorated with antibodies targeting CD63, CD9, annexin Al, ARF6, and EGFR with IgG as a negative isotype control for siEV capture.
  • Two miRNAs hsa-miR-21-5p and hsa-miR-9-5p
  • two mRNAs GPDH and AXL-2 four proteins
  • CD63, CD9, CD81, and EGFR a control for RNA detection
  • p53 a gene downregulated in GBM
  • the average RFI of the nine biomarkers was analyzed with respect to biogenesis pathways and the clustering from the linear discriminant analysis as “classical” exosomes (CD63+/CD9+ siEVs), ectosomes (ARF6+/annexin A1+ siEVs), tumor- derived siEVs (EGFR+ siEVs), and an isotype control (IgG), exhibiting an enrichment of most biomarkers in CD63+/CD9+ siEVs (FIG. 4C). Therefore, an anti-CD63/CD9 antibody cocktail was utilized for the remainder of the investigation to immobilize siEVPs.
  • high- density LPs HDL
  • VLDL very-low-density LPs
  • LDL low-density LPs
  • the isolated LPs demonstrated an absence of annexin Al, ARF6, CD63, and CD9, and an abundance of ApoAl in the HDL fraction and ApoB in the VLDL/LDL fraction (FIG. 19C), which are absent in Gli36 cells and their EV secretions (FIG. 19D).
  • the isolated LPs demonstrated dense morphologies as opposed to the classical “cup shapes” of EVs observed in TEM (FIG. 19E).
  • TFF on the healthy donor serum demonstrated enrichment of CD63, annexin Al, and ApoB and retention with a slight loss of ARF6, CD9, and ApoAl (FIG. 19C) indicating the co-isolation of LPs and EVs after TFF purification. Therefore, Gli36- derived EVs and a mixture of the two LP isolates were deposited on the S1EVP PRA functionalized with an anti-ApoB/ApoAl antibody cocktail and screened for ApoAl, ApoB, and CD63.
  • TFF-purified EVs harvested from Gli36 cells cultured in serum-free culture were incubated in EV-depleted plasma (EVDP) and subsequently purified to remove soluble proteins and enrich the EVs with apolipoprotein corona (FIG. 20A).
  • EVDP EV-depleted plasma
  • FIG. 20A The EVs incubated in EVDP were then introduced to the S1EVP PRA with CD63/CD9-mediated capture and detected for ApoAl and ApoB.
  • siLP-EV co-isolates that are simply siEVs disguised with apolipoprotein corona
  • the investigation was advanced to test the ability of the S1EVP PRA to detect vesicular RNA in a complex biofluid notwithstanding the observed complexity of biological samples.
  • RNA-seq and microarrays To demonstrate the pathological heterogeneity of gliomas, astrocytoma, gliosarcoma, and glioblastoma cell lines were included.
  • RNAs exhibited high concentrations in both cells and EVs with miRNA showing more differential expression levels (FIG. 5A).
  • four transcripts, two mRNAs (NSF and NCAN) and two miRNAs (hsa-miR-9-5p and hsa-miR- 1246-5p) were selected for further analysis, due to their previous association with GBM.
  • the heterogeneity of these transcripts in EVs was further explored with the S1EVP PRA at a single-particle resolution (FIG. 5C).
  • siEV fluorescence intensity demonstrated a more homogeneous expression for the mRNAs than the miRNAs across the six cell lines (FIGS. 21-24).
  • hsa-miR-9-5p cargo from SF268-, SF295-, SF539-, and SNB75-derived siEVs indicated more heterogeneous profiles with distribution maxima shifted to the right (FIG. 23).
  • hsa-miR-1246-5p cargo from SF268-, SNB75-, and SNB19- derived siEVs also demonstrated a heterogeneous expression with distribution maxima shifted to the right (FIG. 24).
  • the S1EVP PRA was used to characterize siEVPs from TFF-purified serum from GBM patients. For each individual, 20 pL of the purified serum was processed with the S1EVP PRA. A cohort of 10 GBM patients and 10 age-matched healthy individuals were chosen for the investigation (Table 2). Although the presence of siLP-EV co-isolates were demonstrated in serum, higher frequencies of positive signals for NSF, hsa-miR-9-5p, NCAN, and hsa-miR- 1246-5p were obtained from purified GBM patient serum in comparison to healthy donor serum (FIG. 6A).
  • siEVP PRA single-EV and particle protein and RNA assay
  • the S1EVP PRA is the first assay to demonstrate intravesicular heterogeneity across multiple biomolecular species with colocalization analyses, including protein and RNA, which was realized with a facile incubation with a TE buffer that stabilizes MBs, ensures the integrity of intact siEVs, preserves external epitopes, and delivers detection probes into the lumen of siEVs via partial permeabilization.
  • Intervesicular and interparticle heterogeneity were recognized by tuning the surface chemistry on the micropattern within the S1EVP PRA to capture siEVP subpopulations. Testing various capture antibodies and detection probes on Gli36-derived siEVs further elucidated intervesicular heterogeneity between subpopulations as a function of biomarker expression. Multivariate analyses on the grouped biomolecular species revealed similar profilometric trends for CD63+, CD9+, and EGFR+ siEV subpopulations, which corresponds with the upregulation of EGFR in Gli36-derived “classical” exosomes.
  • capturing siEVs by CD63 and CD9 yielded the most holistic signature across all biomolecules tested, which coincides with immunoselective immobilization strategies for siEVPs from non-small cell lung cancer patient serum.
  • a possibility for the enhanced capture and detection utilizing an anti- CD63/CD9 antibody cocktail to immobilize siEVs is that the cocktail does not discriminate cellular origin and rather captures siEVs from all cells. While originally considered a “classical” exosomal biomarker, CD9 is present in larger EVs albeit enriched in small EVs and is found on small ectosomes.
  • the linear discriminant analysis on the single biomarker expression demonstrated similarities between CD9+ and ARF6+ siEV subpopulations.
  • the anti-CD63/CD9 antibody cocktail captures exosomes and ectosomes, thus widening the breadth of capture.
  • the antibody cocktail was insufficient in sorting out LPs as CD63+/CD9+ siLP-EV co-isolates were uncovered, concurring with newer evidence on the complexity of LPs and EVs.
  • the co-expression of ApoAl and ApoB on the surface of EVs as protein corona with the S1EVP PRA was confirmed, which maintains bioactivity and has implications in disease progression.
  • Various studies utilize tetraspanins to immobilize siEVs and assume negligible interactions with LPs. This erroneous assumption may result from the dilution of the subpopulation in bulk-analysis methods, further motivating the necessity for siEVP methods in uncovering interparticle heterogeneity.
  • the S1EVP PRA is highly sensitive and capable of discerning heterogeneous subpopulations amongst siEVPs, colocalization is limited to the number of compatible fluorophores, indicating the possibility for siEVP subpopulations immobilized on the micropattern that downregulate all targets.
  • capturing all signals leads to a more comprehensive compositional analysis.
  • Advances in spectral microscopy, sequential labeling, and quantum-dot synthesis, to name a few, are strategic methods for overcoming spectral overlapping in bandpass filters, the applications of which further increase the scope of targeted siEVPs.
  • SP-IRIS demonstrated higher colocalization rates for the tetraspanin analyses, albeit with high levels of nonspecificity.
  • a complex subpopulation of siLP-EV co-isolates, which competitively interacts with the micropatterned surface was found. While their presence indicated minimal interference with the capability of the S1EVP PRA to measure GBM-associated vesicular RNA profiles in GBM patient serum, the possible contributions of siLPs as RNA carriers cannot be missed.
  • siEVP PRA The siEVP method circumvented the artifact of dilution often experienced in bulkanalysis methods. As such, disease-associated proteins and RNAs were preserved in intact siEVPs thus enhancing sensitivities and allowing detection at low concentrations and volumes.
  • the multivariate heterogeneity analysis afforded by the S1EVP PRA aids in uncovering differences in subpopulation-dependent packaging of biomolecules and illuminate biogenesis pathways.
  • Table 1 List of antibodies.
  • Table 2 List of MB designs.
  • Table 3 RFI optimization by controlling monolayer degradation.

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Abstract

La présente divulgation concerne des méthodes et des systèmes visant à détecter simultanément des ARN et des protéines in situ, dans des vésicules extracellulaires (VE) et/ou des lipoprotéines (LP), isolées à partir d'un échantillon obtenu auprès d'un sujet par l'intermédiaire d'un ou de plusieurs anticorps de capture.
PCT/US2024/043552 2023-08-24 2024-08-23 Caractérisation simultanée d'arn et de protéines dans des vésicules extracellulaires et des lipoprotéines Pending WO2025043144A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200018755A1 (en) * 2017-03-30 2020-01-16 Sysmex Corporation Method for measuring an ability of high-density lipoprotein to uptake cholesterol
WO2023114970A2 (fr) * 2021-12-16 2023-06-22 Ohio State Innovation Foundation Dosage d'arn et de protéine de vésicule extracellulaire unique par microscopie à fluorescence in situ dans un réseau de micromotifs uv
WO2024173717A2 (fr) * 2023-02-15 2024-08-22 Ohio State Innovation Foundation Compositions et procédés de caractérisation structurale et moléculaire de particules extracellulaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200018755A1 (en) * 2017-03-30 2020-01-16 Sysmex Corporation Method for measuring an ability of high-density lipoprotein to uptake cholesterol
WO2023114970A2 (fr) * 2021-12-16 2023-06-22 Ohio State Innovation Foundation Dosage d'arn et de protéine de vésicule extracellulaire unique par microscopie à fluorescence in situ dans un réseau de micromotifs uv
WO2024173717A2 (fr) * 2023-02-15 2024-08-22 Ohio State Innovation Foundation Compositions et procédés de caractérisation structurale et moléculaire de particules extracellulaires

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