Methods for quantifying extracellular vesicle surface markers and compositions for performing the same

Abstract

Methods for quantifying surface markers on extracellular vesicles are provided. Aspects of the methods include comparing the mean fluorescence intensity of a surface marker of interest (surface marker MFI) of a labeled extracellular vesicle (EV) sample to a calibration curve obtained from a liposome calibration composition to quantify the surface marker on the extracellular vesicles of the EV sample. Compositions for practicing embodiments of the invention are also provided.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to the filing date of U.S. Provisional Patent Application Serial No. 62 / 951968, filed December 20, 2019, pursuant to 35 USC §119(e); the disclosure of which is incorporated herein by reference.

[0003] introduction

[0004] Extracellular vesicles (also known in the art as EV particles) are vesicles released from cells and enclosed by a lipid membrane in body fluids. EVs vary in composition and size, ranging from 50 nm to 1000 nm in diameter (Nat. Rev. Drug Discov. 12(5)(2013) 347-357; Curr. Opin. Cell Biol. 29C(2014) 116-125). EVs originate from their donor cells, resulting from outward budding of the plasma membrane. Multiple reports have shown that EVs play important roles in pathophysiological processes such as immune responses and tumor growth (Nat. Cell Biol. 10(12)(2008) 1470-1476; Nat. Cell Biol. 10(5)(2008) 619-624). Therefore, EVs present in body fluids have been used as biomarkers for the diagnosis and monitoring of diseases (J. Biol. Chem. 277(6)2012) 3842-3849). Because tumor-derived EVs are released in easily accessible bodily fluids such as blood or urine (Nat. CellBiol. 10(12)(2008) 1470-1476; Nat. Med. 18(12)(2012) 1835-1840), analysis of these EVs for disease surveillance may avoid biopsies.

[0005] Flow cytometry is widely used to analyze EV particles stained with fluorescent antibodies targeting known cell surface markers. Commercial flow cytometers only measure the relative fluorescence of surface markers in the form of mean (or median) fluorescence intensity (MFI). Variations in instrument setup, maintenance, or replacement, as well as variations in fluorescent antibodies from different suppliers, can affect the relative MFI value of the same sample. This effect limits the use of MFI values ​​to compare data acquired over time by different laboratories and instruments.

[0006] Despite the increasing recognition of EVs as important biological and therapeutic entities, standardized methodologies for their analysis remain lacking. Currently used EV analytical strategies are outlined below:

[0007] • Quantification of EV particles rather than their surface markers according to the count or enumeration of EV particles, for example, by counting using nanoparticle tracking analysis (NTA), high resolution flow cytometry and Trucount beads (J. Control. Release 200 (2015) 87-96);

[0008] • using fluorescent dyes that are either incorporated into the lipid bilayer or are non-specifically staining any phospholipid membrane, like Di-8-ANEPPS, PKH67 or Annexin (Clin. Chem. 64 (4) (2018) 680-689), wherein such non-specific markers cannot provide an estimate of EV surface markers stained with fluorescent antibodies; and

[0009] • staining of EVs with fluorescent antibodies to identify EV surface markers (e.g. CD61-APC, EpCAM-APC), but not to quantify them.

[0010] However, quantification of surface markers according to the number of molecules or the number of antibodies bound per specific marker remains one of the biggest challenges in the field of EV research. SUMMARY

[0011] Methods for quantifying surface markers of extracellular vesicles are provided. Aspects of the methods include comparing the mean fluorescence intensity of a surface marker of interest (surface marker MFI) of a labeled extracellular vesicle (EV) sample to a calibration curve obtained from a liposome calibration composition to quantify the surface marker on the extracellular vesicles of the EV sample. Compositions for practicing embodiments of the invention are also provided.

[0012] In one embodiment, a liposome calibration composition consisting of a series of fluorescently labeled liposomes with a known number of fluorophores attached to their surface is provided. If desired, the known number of fluorophores can be expressed in units called equivalent soluble fluorescent molecules (MESF). A curve of the known number of fluorophores (or MESF) versus the fluorescence intensity of the liposomes provides a standard curve (i.e., a calibration curve) from which the fluorescence intensity (MFI value) of a stained EV sample can be converted to the number of fluorophores (or MESF value) bound to the surface of the EV particles. By this method, the MFI value is converted to a standardized MESF value (or MESF value) of the number of fluorophores that is independent of the instrument differences in different laboratories, further improving the inter-laboratory standardization. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figures 1A to 1C PE-labeled liposomes as calibrators are shown. Figure 1A is a schematic representation of liposomes labeled with different numbers of PE fluorescent dyes.Figure 1B is a schematic of PE liposome with PE fluorescence analysis in SSC. Figure 1C A standard curve is provided generated by plotting the MESF values of PE Quantibrite beads against their PEMFI values of the MESF assigned to the same color of labeled liposomes.

[0014] Figures 2A to 2C FITC labeled liposomes analyzed by DLS and flow cytometry are shown. Figure 2A Liposome size analyzed by dynamic light scattering (DLS) is provided. Figure 2B Flow cytometry analysis of FITC labeled liposomes on a BD FACS Aria Fusion flow cytometer (Becton, Dickinson and Company) is provided. Figure 2C FITC labeled liposome analysis after sorting in a FACS Aria Fusion flow cytometer is provided.

[0015] Figures 3A to 3B FITC labeled liposome calibrators are provided. Figure 3A Flow cytometry analysis of FITC labeled liposomes on a BD FACSAria Fusion before sorting is provided. Figure 3B FITC labeled liposome calibrators generated after sorting in a FACS Aria Fusion flow cytometer are provided. The data demonstrate that liposome calibrators labeled with fluorophores can be generated. DETAILED DESCRIPTION

[0016] Methods for quantifying extracellular vesicle surface markers are provided. Aspects of the methods include comparing the mean fluorescence intensity of a surface marker of interest (surface marker MFI) of a labeled extracellular vesicle (EV) sample to a calibration curve obtained from a liposome calibration composition to quantify the surface marker on the extracellular vesicles of the EV sample. Compositions for practicing embodiments of the application are also provided.

[0017] Before the present application is described in detail, it is to be understood that this application is not limited to the particular embodiments described herein because such embodiments can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present application will be limited only by the appended claims.

[0018] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the application.

[0019] Certain ranges are presented herein with numerical values being presented as approximations. The numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, can inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, representative illustrative methods and materials are now described.

[0021] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior application. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

[0022] Notably, the use of the indefinite article "a" or "an" preceding an element, as used in the specification herein and the appended claims, does not exclude the presence of more than one of that element unless the context clearly dictates otherwise. It should also be noted that claims can be drafted to exclude any optional element. Thus, such claims should be understood to have been drafted in view of the statutory requirement that the claims be "clear" and "concrete" and that the claims be "supported" by the examples.

[0023] It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and illustrated herein has independent components and features that can be readily separated from the features of any of the other several embodiments without departing from the scope or spirit of the present application. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0024] Although the apparatus and method have been or will be described for the sake of grammatical fluency and functional interpretation, it should be clearly understood that, unless expressly stated in 35 USC §11, the claims should not be construed as necessarily being limited in any way to “apparatus” or “step,” but should conform to the meaning of the definitions provided by the claims under the principle of equivalence and the full scope of equivalents, and where the claims are expressly stated in 35 USC §112, a full legal equivalent will be given in 35 U.S.C §112.

[0025] Methods for quantifying extracellular vesicle surface markers

[0026] As described above, aspects of this disclosure include methods for quantifying one or more extracellular vesicle surface markers in a sample. Therefore, aspects of the invention include methods for quantifying one or more extracellular vesicle surface markers (i.e., EV surface markers) in a sample containing extracellular vesicles (i.e., EV samples). In some cases, the method is a method for quantifying a single EV surface marker. In other cases, the method is a method for quantifying two or more different EV surface markers (wherein the different EV surface markers are molecules with different molecular formulas, such as different proteins with different amino acid sequences). Although the number of different EV surface markers quantified in this embodiment may vary, in some cases the number of different EV surface markers is 2 to 10, for example 2 to 8, for example 3 to 5.

[0027] Because this method quantifies one or more EV surface markers, it provides an estimate of at least the amount of a given marker on the EV surface in an EV sample, for example, in the form of an estimate of at least the number of molecules of a given marker on the EV surface in the EV sample. The quantification obtained using embodiments of this method can be an average of many (including all) EVs in the EV sample. Therefore, the quantification of a given EV surface marker obtained by the method of this invention differs from the average fluorescence intensity (MFI) obtained for that marker, because the average fluorescence intensity is not a quantitative representation of the marker, but rather a relative representation. The quantification can be provided in any convenient form, for example, based on the number of molecules of the given marker, based on the number of fluorophores bound to the surface of EV particles (e.g., as molecules of an equal amount of soluble fluorescent molecules or MESF), etc.

[0028] An EV sample from which one or more surface markers are quantified according to the present application can vary, wherein the EV sample is a sample comprising EVs of interest. Examples of EV samples include, but are not limited to, biological samples, such as biological fluids, such as, but not limited to: urine, blood, plasma, serum, saliva, semen, fecal matter, sputum, cerebrospinal fluid, tears, mucus, sperm, amniotic fluid, and the like.

[0029] In embodiments in which the method is practiced, the mean fluorescence intensity of a surface marker of interest obtained from an EV sample is compared to a calibration curve obtained from a liposome calibration composition, e.g., as described in more detail below. The mean fluorescence intensity of a surface marker used in the methods of the present application (i.e., surface marker MFI) is obtained by flow cytometric analysis of a labeled EV sample. The labeled EV sample is an EV sample labeled with a surface marker label, wherein the surface marker label comprises a specific binding member of a surface marker to be quantified and a fluorophore. The labeled EV sample can be subjected to flow cytometric analysis to obtain the surface marker MFI using any convenient protocol. In some cases, the surface marker MFI is a median or geometric mean, wherein the particular MFI used can be selected based on the properties of the histogram from which it is derived. In some cases, the MFI is obtained using a commercially available program, such as FlowJo® TM Software flow data analysis software (Becton, Dickinson and Company).

[0030] In some cases, the method comprises preparing a labeled EV sample and obtaining the MFI of a surface marker thereof. The labeled EV sample can be prepared using any convenient protocol. In some cases, an EV sample is combined with an appropriate amount of a surface marker label that specifically binds to a surface marker to be quantified under conditions sufficient for the surface marker label to bind to the surface of interest, thereby producing a labeled EV sample. As described above, the surface marker label comprises a specific binding member of a surface marker of interest and a fluorescent label.

[0031] The specific binding member of the surface marker label specifically binds to a surface marker such that the specific binding member and the surface marker have affinity for each other. The affinity between the specific binding member and the surface marker can vary, with in some cases they can specifically bind to each other in a binding complex characterized by a KD (dissociation constant) of 10-5M or less than 10-5M, 10-6M or less than 10-6M, 10-7M or less than 10-7M, 10-8M or less than 10-8M, 10-9M or less than 10-9M, 10-10M or less than 10-10M, 10-11M or less than 10-11M, 10-12M or less than 10-12M, 10-13M or less than 10-13M, 10-14M or less than 10-14M, or 10-15M or less than 10-15M. Any suitable surface marker binding moiety can be used as the specific binding member, such as a protein binding moiety, an antibody or fragment thereof, an aptamer, a small molecule, a ligand, a peptide, an oligonucleotide, etc., or any combination thereof. For example, the specific binding member can include an antibody, such as an antibody specific for a surface marker (e.g., a receptor) on an extracellular vesicle. The antibody can be a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene segment recombination processes) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule, such as an antibody fragment. The antibody fragment can be, for example, a portion of an antibody, such as a F(ab’)2, Fab’, Fab, Fv, sFv, etc. In some embodiments, the antibody fragment can bind to the same antigen recognized by the full-length antibody. The antibody fragment can include an isolated fragment consisting of the variable regions of an antibody, such as a “Fv” fragment consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which the light and heavy variable regions are connected by a peptide linker (“scFv proteins”).

[0032] In addition to specific binding members, surface marker labels also include fluorophores. Fluorophores of interest can include, but are not limited to, dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.). A large number of dyes are commercially available from a variety of sources. For example, the fluorophore can be 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid; acridine and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-l-naphthyl)maleimide; anthranilamide; auramine; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine and derivatives such as oxirane B, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7; 4',6-diamidino-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonphthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriaminepentaacetic acid; 4,4'-dihydroxy-2,2'-disulfonic acid stilbene-2,2'-disulfonic acid; 4,4'-dihydroxystilbene-2,2-disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives, such as eosin and eosin isothiocyanate; erythrosin and derivatives, such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives, such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazine, naphthofluorescein, and QFITC (XRITC); fluorescamine; IR144; IR1446; green fluorescent protein (GFP); red fluorescent protein (RCFP); lissamine TM ; lissamine rhodamine, Lucifer Yellow; malachite green isothiocyanate; 4-methylumbelliferone; o-tolualidine; nitrotyrosine; pararosaniline base; Nile blue; Oregon Green; phenol red; B-phycoerythrin (PE); PE-Cy7, o-phthaldehyde; pyrene and derivatives, such as pyrene, pyrene butyrate, and succinimidyl 1-pyrene butyrate; reactive red 4 (Cibacron TMBrilliant Red 3B-A); Rhodamine and its derivatives, such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine, Rhodamine B, sulfonyl chloride, Rhodamine (Rhod), Rhodamine B, Rhodamine 123, Rhodamine X isothiocyanate, sulfonylrhodamine B, sulfonylrhodamine 101, sulfonyl chloride derivatives of sulfonylrhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosinic acid and terbium chelate derivatives; xanthones; carotenoid-protein complexes, such as polydinophyll-chlorophyll protein (PerCP); allophycocyanin (APC); or combinations thereof.

[0033] In some cases, the fluorophores labeled by surface markers are polymeric dyes. Polymeric dyes of interest include, but are not limited to, those published by Gaylord et al. in U.S. Publications Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20110257374, 20120028828, 20120252986, 20130190193, 20160264737, 20160266131, 20180231530, 20180009990, 20180009989, and 201... The dyes described in 80163054, the disclosure of which is incorporated herein by reference in its entirety; and the dyes described in Gaylord et al., J. Am. Chem. Soc., 2001, 123(26), pp. 6417-6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133(32), pp. 12600-12607, the disclosure of which is incorporated herein by reference in its entirety.

[0034] In some embodiments, the polymeric dye comprises a conjugated polymer including a plurality of first optically active units forming a conjugated system, having a first absorption wavelength (e.g., as described herein) at which the first optically active units absorb light to form an excited state. The conjugated polymer (CP) may be a polycationic, polyanionic, and / or charge-neutral conjugated polymer.

[0035] The CP can be water soluble for use in biological samples. Any convenient substituent can be included in the polymeric dye to provide increased water solubility, such as a hydrophilic substituent, such as a hydrophilic polymer, or a charged substituent, such as a group that is positively or negatively charged in aqueous solution, such as under physiological conditions. Any convenient water soluble group (WSG) can be used in the light harvesting multichromophores of the subject matter. The term “water soluble group” refers to a functional group that is well solvated in an aqueous environment and imparts improved water solubility to the molecule to which it is attached. In some embodiments, the WSG increases the solubility of the multichromophore in a predominantly aqueous solution (e.g., as described herein) compared to a multichromophore lacking the WSG. The water soluble group can be any convenient hydrophilic group that is well solvated in an aqueous environment. In some cases, the hydrophilic water soluble group is charged, such as positively or negatively charged. In certain cases, the hydrophilic water soluble group is a neutral hydrophilic group. In some embodiments, the WSG is a hydrophilic polymer, such as polyethylene glycol, cellulose, chitosan, or a derivative thereof.

[0036] As described herein, the terms “polyethylene oxide,” “PEO,” “polyethylene glycol,” and “PEG” are used interchangeably and refer to polymers comprising a chain described by the formula -(CH2-CH2-O-) n - described chain, or a derivative thereof. In some embodiments, “n” is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 5 to 15, or 10 to 15. It will be appreciated that the PEG polymer can be of any convenient length and can include various end groups, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and terminal amino groups. Functionalized PEGs suitable for use in the subject multichromophores include those described in S. Zalipsky, “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates,” Bioconjugate Chemistry 1995, 6(2), 150-165. Water soluble groups of interest include, but are not limited to, carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate, ester, polyethylene glycol (PEG) and modified PEG, hydroxyl, amine, ammonium, guanidinyl, polyamine and sulfonium, polyol, straight or cyclic sugar, primary, secondary, tertiary, or quaternary and polyamine, phosphonate ester group, phosphinate ester group, ascorbate group, diol, including polyether, -COOM', -SO3M', -PO3M', -NR3 + , Y', (CH2CH2O)p R and mixtures thereof, wherein Y' can be any halogen, sulfate, sulfonate, or oxygen-containing anion, p can be 1 to 500, each R can independently be H or an alkyl group (such as methyl), M' can be a cationic counterion or hydrogen, -(CH2CH20)p- yy CH2CH2XR yy CH2CH2X- yy CH2CH2X-, -X(CH2CH20)p- yy CH2CH2-, diols, and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S, and NR ZZ , and R ZZ , and R YY are independently selected from H and C 1-3 alkyl.

[0037] The polymeric dye can be of any convenient length. In some cases, the polymeric dye can have a specific number of monomeric repeat units or segments ranging from 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000, or 2 to 1,000 units or segments, or such as 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments.

[0038] The polymeric dye can be of any convenient molecular weight (MW). In some cases, the MW of the polymeric dye can be expressed as an average molecular weight. In some cases, the average molecular weight of the polymeric dye is 500 to 500,000, such as 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000, or even 50,000 to 100,000 average molecular weight. In certain embodiments, the average molecular weight of the polymeric dye is 70,000.

[0039] In certain cases, the polymeric dye comprises the following structure:

[0040]

[0041] wherein CP1, CP2, CP3, and CP4 are independently a conjugated polymer segment or oligomeric structure, wherein one or more of CP1, CP2, CP3, and CP4 is a band gap-reduced n-conjugated repeat unit, and each n and each m is independently 0 or an integer from 1 to 10,000, and p is an integer from 1 to 100,000.

[0042] In some cases, the polymeric dye comprises the following structure:

[0043]

[0044] wherein each R 1independently a solubilizing group or a linker dye; L 1 and L 2 is optionally a linker; each R 2 independently is H or an aryl substituent; each A 1 and A 2 independently is H, an aryl substituent, or a fluorophore; G 1 and G 2 each independently is selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member; each n and each m independently is 0 or an integer from 1 to 10,000; and p is an integer from 1 to 100,000. Solubilizing groups of interest include alkyl, aryl, and heterocyclic groups further substituted with hydrophilic groups such as polyethylene glycol (e.g., 2 to 20 units of PEG), ammonium, sulfonium, phosphonium, and the like.

[0045] In some cases, the polymeric dye includes a conjugated segment having one of the following structures as part of the polymer backbone:

[0046]

[0047] where each R 3 independently is an optionally substituted alkyl or aryl group; Ar is an optionally substituted aryl or heteroaryl group; and each n is an integer from 1 to 10,000. In certain embodiments, R 3 is an optionally substituted alkyl group. In certain embodiments, R 3 is an optionally substituted aryl group. In certain cases, R 3 is substituted with a polyethylene glycol, a dye, a chemoselective functional group, or a specific binding moiety. In some cases, Ar is substituted with a polyethylene glycol, a dye, a chemoselective functional group, or a specific binding moiety.

[0048] In some cases, the polymeric dye includes the following structure:

[0049]

[0050] where each R 1 independently is a solubilizing group or a linker-dye group; each R 2 independently is H or an aryl substituent; each L 1 and L 3 independently is an optional linker; each A 1 and A 3 independently is H, a fluorophore, a functional group, or a specific binding moiety (e.g., an antibody); and n and m are each independently 0 or an integer from 1 to 10,000, where n+m > 1.

[0051] The polymeric dye can have one or more desired spectral properties, such as a particular maximum absorption wavelength, a particular maximum emission wavelength, an extinction coefficient, a quantum yield, and the like (see, e.g., Chattopadhyay et al., “Brilliant violet fluorophores: A new class of ultrabright fluorescent compounds for immunofluorescence experiments.” Cytometry Part A, 81A(6), 456-466, 2012).

[0052] In some embodiments, the polymeric dye has an absorption curve from 280 nm to 475 nm. In certain embodiments, the polymeric dye has a maximum absorption (maximum excitation) from 280 nm to 475 nm. In some embodiments, the polymeric dye absorbs incident light having a wavelength from 280 nm to 475 nm.

[0053] In some embodiments, the maximum emission wavelength of the polymeric dye is from 400 nm to 850 nm, for example, from 415 nm to 800 nm. Specific examples of the maximum emission wavelength of interest include, but are not limited to, 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm. In some cases, the maximum emission wavelength of the polymeric dye is selected from 410 nm to 430 nm, 500 nm to 520 nm, 560 nm to 580 nm, 590 nm to 610 nm, 640 nm to 660 nm, 700 nm to 720 nm, and 775 nm to 795 nm. In some embodiments, the maximum emission wavelength of the polymeric dye is 421 nm. In some cases, the maximum emission wavelength of the polymeric dye is 510 nm. In some cases, the maximum emission wavelength of the polymeric dye is 570 nm. In some embodiments, the maximum emission wavelength of the polymeric dye is 602 nm. In some cases, the maximum emission wavelength of the polymeric dye is 650 nm. In some cases, the maximum emission wavelength of the polymeric dye is 711 nm. In some embodiments, the maximum emission wavelength of the polymeric dye is 786 nm. In some cases, the maximum emission wavelength of the polymeric dye is 421 nm ± 5 nm. In some embodiments, the maximum emission wavelength of the polymeric dye is 510 nm ± 5 nm. In some cases, the maximum emission wavelength of the polymeric dye is 570 nm ± 5 nm. In some cases, the maximum emission wavelength of the polymeric dye is 602 nm ± 5 nm. In some embodiments, the maximum emission wavelength of the polymeric dye is 650 nm ± 5 nm. In some cases, the maximum emission wavelength of the polymeric dye is 711 nm ± 5 nm. In some cases, the maximum emission wavelength of the polymeric dye is 786 nm ± 5 nm. In some embodiments, the maximum emission wavelength of the polymeric dye is selected from 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm.

[0054] In some cases, the extinction coefficient of polymeric dyes is 1×10⁻⁶. 6 cm -1 M -1 or greater than 1×10 6 cm -1 For example, 2×10 6 cm -1 M -1 or greater than 2×10 6 cm -1 M -1 2.5×10 6 cm -1 M -1 or greater than 2.5 × 10 6 cm -1 M -1 3×106 cm -1 M -1 or greater than 3×10 6 cm -1 M -1 4×10 6 cm -1 M -1 or greater than 4×10 6 cm -1 M -1 5×10 6 cm -1 M -1 or greater than 5×10 6 cm -1 M -1 6×10 6 cm -1 M -1 or greater than 6×10 6 cm -1 M -1 7×10 6 cm -1 M -1 or greater than 7×10 6 cm -1 M -1 Or 8×10 6 cm -1 M -1 or greater than 8×10 6 cm -1 M -1 In some embodiments, the quantum yield of the polymeric dye is 0.05 or greater than 0.05, for example, 0.1 or greater than 0.1, 0.15 or greater than 0.15, 0.2 or greater than 0.2, 0.25 or greater than 0.25, 0.3 or greater than 0.3, 0.35 or greater than 0.35, 0.4 or greater than 0.4, 0.45 or greater than 0.45, 0.5 or greater than 0.5, or even greater. In some cases, the quantum yield of the polymeric dye is 0.1 or greater than 0.1. In some cases, the quantum yield of the polymeric dye is 0.3 or greater than 0.3. In some cases, the quantum yield of the polymeric dye is 0.5 or greater than 0.5. In some embodiments, the extinction coefficient of the polymeric dye is 1 × 10⁻⁶. 6 cm -1 M -1 or greater than 1×10 6 cm -1 M -1 The quantum yield is 0.3 or greater than 0.3. In some embodiments, the extinction coefficient of the polymeric dye is 2 × 10⁻⁶. 6 cm -1 M -1 or greater than 2×10 6cm -1 M -1 , a quantum yield of 0.5 or greater than 0.5.

[0055] Specific polymeric dyes that can be used include, but are not limited to, BD Horizon Brilliant TM dyes, such as BD Horizon Brilliant TM Violet dyes (e.g., BV421, BV510, BV605, BV650, BV711, BV786); BD Horizon Brilliant TM UV dyes (e.g., BUV395, BUV496, BUV737, BUV805); and BD Horizon Brilliant TM Blue dyes (e.g., BB515).

[0056] As described above, the MFI of the surface marker of interest is compared to a calibration curve obtained from the liposome calibration composition to quantify the target EV surface marker. The calibration curve is a standard curve of known quantities of fluorophores (e.g., relative to the average fluorescence intensity of the liposomes in the liposome calibration composition). More specifically, the calibration curve is a standard curve prepared by plotting the quantity of fluorophores expressed in units called equivalent soluble fluorescent molecules (MESF) versus the MFI of two or more different liposome subpopulations of the liposome calibration composition, wherein each different liposome subpopulation comprises a known quantity of fluorophores that is different from the quantity of any other subpopulation in the liposome calibration composition. As described in more detail below, a given liposome calibration composition can include two or more different liposome subpopulations, such as three or more, four or more, five or more, including ten or more different liposome subpopulations. Thus, the calibration curve can be a line derived from the values of 2 or more liposome subpopulations in the calibration composition, such as 3 or more, 4 or more, 5 or more, including 10 or more, and up to all of the different liposome subpopulations in the calibration composition from which it is derived.

[0057] In some cases, the method can include obtaining a calibration curve for comparison to the surface marker MFI. The calibration curve can be obtained by flow cytometric analysis of a liposome calibration composition having the same fluorophore as the surface marker label to obtain the MFI of different liposome subpopulations of the calibration composition, and then plotting the observed MFI values for each subpopulation versus the known quantity of fluorophores (e.g., MESF).

[0058] Flow cytometry systems and methods for analyzing samples that can be used in the methods of the present application include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rded., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan; 49(pt 1): 17-28; Linden et al., Semin Throm Hemost. 2004 Oct 30(5):502-11; Alison et al., J Pathol, 2010 Dec; 222(4):335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosures of which are incorporated herein by reference. In certain instances, flow cytometry systems of interest include BD Biosciences FACSCanto TM II Flow Cytometer, BD Accuri TM Flow Cytometer, BD Biosciences FACSCelesta TM Flow Cytometer, BD Biosciences FACSLyric TM Flow Cytometer, BD Biosciences FACSVerse TM Flow Cytometer, BD Biosciences FACSymphony TM Flow Cytometer, BD Biosciences LSR Fortessa TM Flow Cytometer, BD Biosciences LSR Fortess TM X-20 Flow Cytometer and BD Biosciences FACSCalibur TM Cell Sorter, BD Biosciences FACSCount TM Cell Sorter, BD Biosciences FACSLyric TM Cell Sorter and BD Biosciences Via TMCell Sorter, BD Biosciences Influx TM Cell Sorter, BD Biosciences Jazz TM Cell Sorter, BD Biosciences Aria TM Cell Sorter, and BD Biosciences FACSMelody TM Cell Sorter, etc. In some embodiments, the subject particle sorting system is a flow cytometry system, such as those described in U.S. Patent Nos. 9952076; 9933341; 9726527; 9453789; 9200334; 9097640; 9095494; 9092034; 8975595; 8753573; 8233146; 8140300; 7544326; 7201875; 7129505; 6821740; 6813017; 6809804; 6372506; 5700692; 5643796; 5627040; 5620842; 5602039; the disclosures of which are incorporated by reference herein in their entireties.

[0059] MFI values can be obtained as described above, e.g., using FlowJo TM software to process data obtained from a flow cytometer, e.g., data obtained using a flow cytometer as described above. In embodiments, flow cytometer- obtained data for a liposome calibration composition is obtained using the same flow cytometer settings (e.g., fluorescence and compensation settings), e.g., same voltage parameters for photomultiplier tubes of the flow cytometer, etc., as used to obtain EV surface marker MFIs.

[0060] As described above, surface marker MFIs for a labeled EV sample are compared to a calibration curve to obtain the number of fluorophores bound to the EV vesicle surface of the EV sample, thereby quantifying the EV surface marker of interest. Any convenient protocol can be used in comparing surface marker MFIs to a calibration curve. For example, a surface marker MFI can be matched to a corresponding MFI of a calibration curve, and from this the corresponding number of fluorophores (e.g., MESF) is determined. An equation for the slope of the curve can be obtained, and the surface marker MFI used in the equation to solve for the number of fluorophores (e.g., MESF). The obtained number of fluorophores (e.g., MESF value) for a surface marker can be used alone as a quantitative value for the surface marker, or further used to determine the bound surface marker label per EV (e.g., by calculating the ratio of fluorophores to specific binding members in a given surface marker label), and thus the surface marker per EV.

[0061] The methods described herein can be used to quantify surface markers of a variety of different types of EVs. EVs whose surface markers can be quantified using the methods of the present application include, but are not limited to, vesicles, exosomes, and the like. In some cases, the extracellular vesicles have a diameter of 5 μ or less, e.g., 1 μ or less, where in some cases the extracellular vesicles have a diameter of 30 nm to 2500 nm, e.g., 30 nm to 1000 nm. In some cases, the EVs are exosomes (e.g., having a diameter of 30 nm to 150 nm).

[0062] The EV surface markers that can be quantified using the methods of the present application can vary. Surface markers of interest include, but are not limited to: ALCAM; CD166; ASGR1; BCAM; BSG; CD147; CD14; CD19; CD2; CD200; CD127; CD25; CD161; CD45RA; CD15S; CD4; CD127; CD15S; CD3; EpCAM; CD44; Her2 / Neu; ACVR1B; ALK4; ACVR2A; ACVR2B; BMPR1A; BMPR2; CSF1R; MCSFR; CSF2RB; EGFR; EPHA2; EPHA4; EPHB2; EPHB4; ERBB2; androgen receptor; CAR; ERa; ERb; ESRRA; ESRRB; ESRRG; FXR; glucocorticoid receptor; LXR-a; LXR-b; PPARA; PPARD; PPARG; PXR; SXR; estrogen receptor beta; progesterone receptor; RARA; RARB; RARG; RORA; RXRA; RXRB; THRA; THRB; vitamin D3 receptor; AGER; APP; CLEC12A; MICL; CTLA4; FOLR1; FZD1; FRIZZLED-1; KLRB1A; LRPAP1; NCR3; NKP30; OLR1; PROCR; PTPN1; SOX9; SCARB2; TACSTD2; TREM1; TREM2; TREML1; and VDR.

[0063] In some cases, the method can also include using a liposome calibration composition as a size calibrant for the EV particles. Because both liposomes and EVs are made from phospholipids, the refractive index (RI) of the liposomes is essentially the same as that of the EVs. Also, because the diameter of the labeled liposomes of the liposome calibration composition is 100 nm to 500 nm (which is the same as the EV particles), the labeled liposomes can be used as a size estimate for the diameter of the EV particles. Currently, polystyrene and silica beads are used as size calibrants, which are significantly different from the RI of biological samples such as EVs. This difference in RI values introduces error in the size estimate of the EV particles. Liposomes are composed of phospholipids, cholesterol, and fatty acid derivatives, such as l-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine ((DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). These components are the same as the chemical components that make up the membrane of the EV particles. Therefore, the refractive index of the liposomes is the same as that of the EV particles. In one embodiment, a liposome with an average diameter of 210 nm (e.g., as shown in FIG. 1) is used for all different liposome intensities, such that the size of the particles will remain constant, and the average size of the labeled liposomes provides a size estimate reference for the EV particles. Figure 2A

[0064] Liposome calibration composition

[0065] As described above, the methods of the present application use a liposome calibration composition. The liposome calibration composition is a composition comprising two or more different liposome subpopulations, each subpopulation comprising a known amount of a fluorophore that is different in amount from any other subpopulation in the liposome calibration composition. Thus, a given liposome calibration composition includes at least a first liposome subpopulation and a second liposome subpopulation, the first liposome subpopulation including a first known amount of a fluorophore, e.g., expressed in MESF, and the second liposome subpopulation including a second known amount of the same fluorophore, e.g., expressed in MESF, wherein the number of fluorophore molecules of the second subpopulation is different from the number of fluorophores of the first subpopulation. A given liposome calibration composition can include two or more different liposome subpopulations, e.g., three or more, four or more, five or more, including ten or more different liposome subpopulations.

[0066] ​The liposome size of the liposome calibration composition can vary. In some cases, the average size (e.g., average diameter) of the liposomes is 1000 nm or less, e.g., 900 nm or less, or 800 nm or less, or 700 nm or less, or 600 nm or less, or 500 nm or less, or 400 nm or less, or 300 nm or less, or 250 nm or less, or 200 nm or less, or 150 nm or less, or 100 nm or less, or 75 nm or less, or 50 nm or less, or 25 nm or less, or 20 nm or less, or 15 nm or less, or 10 nm or less, or 5 nm or less, or 1 nm or less, where in some cases the average size is 1 nm or greater, e.g., 5 nm or greater. In certain cases, the average size of the liposomes is 1000 nm or less. In certain cases, the average size of the liposomes is 800 nm or less. In certain cases, the average size of the liposomes is 500 nm or less. In certain cases, the average size of the liposomes is 400 nm or less. In certain cases, the average size of the liposomes is 300 nm or less. In certain cases, the average size of the liposomes is 250 nm or less. In certain cases, the average size of the liposomes is 200 nm or less. In certain cases, the average size of the liposomes is 100 nm or less. In certain cases, the average size of the liposomes is 50 nm or less. For example, the liposomes can include small unilamellar vesicles (SUVs), e.g., unilamellar vesicles having an average size of 100 nm or less, e.g., 10 nm to 100 nm. In some cases, the size of the liposomes is 50 nm to 1000 nm, e.g., 100 nm to 500 nm.

[0067] In certain embodiments, the liposomes are uniform in size, and thus they exhibit low polydispersity. "Dispersity" or "polydispersity" is a measure of the heterogeneity of particle size in a mixture. In the context of liposomes, polydispersity can range from 0 to 1, where a polydispersity of 0 indicates a monodisperse population of liposomes (e.g., liposomes of the same average size), and where a polydispersity of 1 indicates a heterogeneous mixture of liposomes. In some cases, the size (and polydispersity) of the liposomes can be determined by dynamic light scattering (DLS). In some cases, the polydispersity of the liposomes can be 0.1 or less than 0.1. In some cases, the polydispersity of the liposomes can be 0.05 or less than 0.05. In some cases, the polydispersity of the liposomes can be 0.01 or less than 0.01. In certain cases, the polydispersity of the liposomes is 0.01 to 0.5, e.g., 0.01 to 0.4, or 0.01 to 0.3, or 0.01 to 0.2, or 0.01 to 0.1. In other embodiments, the polydispersity of the liposomes is 0.01 to 0.5, e.g., 0.01 to 0.5, or 0.01 to 0.4, or 0.01 to 0.3, or 0.01 to 0.2. In other embodiments, the polydispersity of the liposomes is 0.01 to 0.5, e.g., 0.05 to 0.5, or 0.1 to 0.5, or 0.1 to 0.4, or 0.1 to 0.3. In other embodiments, the polydispersity of the liposomes is 0.01 to 0.5, e.g., 0.05 to 0.5, or 0.1 to 0.5, or 0.2 to 0.5, or 0.2 to 0.4.

[0068] In embodiments of the disclosure, the liposomes useful in the present disclosure are composed of lipids. In certain embodiments, the lipids are amphiphilic. Amphiphilic lipids can include a hydrophilic group and one or more than one lipophilic group covalently bonded to the hydrophilic group. In some cases, the hydrophilic group is a charged group, e.g., an anionic group or a cationic group. In some cases, the hydrophilic group is an uncharged polar group. In some embodiments, the hydrophilic group includes both a charged group and a polar group. Examples of hydrophilic groups include, but are not limited to, phosphates, phosphocholines, phosphoglycerols, phosphoethanolamines, phosphoserines, phosphoinositols, ethylphosphosphorylcholine, polyethylene glycols, polyglycerols, sphingosines, phosphoshingosines, trinitroligacetic acid, melamine, glucosamine, trimethylamine, spermine, spermidine, and conjugate carboxylic acid salts, sulfate salts, borate, sulfonic acid salts, sulfate salts, sugars, amino acids, and the like. In some cases, the hydrophilic group includes a phosphocholine.

[0069] In certain embodiments, the lipophilic group includes an aliphatic chain, such as a saturated or unsaturated, straight or branched, substituted or unsubstituted aliphatic chain. For example, the lipophilic group can include an aliphatic chain of 2 to 40 carbon atoms in length, and can be saturated or unsaturated, straight or branched, substituted or unsubstituted. For example, the lipophilic group can include a saturated or unsaturated, straight or branched, substituted or unsubstituted hydrocarbon chain having 2 to 40 carbon atoms, such as 4 to 30 carbon atoms, or 4 to 25 carbon atoms, or 6 to 24 carbon atoms, or 10 to 20 carbon atoms. In certain cases, the lipophilic group includes a saturated or unsaturated, straight or branched hydrocarbon chain having 18 carbon atoms. In certain cases, the lipophilic group includes a saturated or unsaturated, straight or branched hydrocarbon chain having 16 carbon atoms.

[0070] Embodiments of liposomes include liposomes having a known amount of a fluorophore stably associated therewith. The fluorophore of the liposomes of the calibration composition is the same as the fluorophore of the surface marker label, examples of which are provided above. By "stably associated" is meant that one moiety is bound or otherwise associated with another moiety or structure under standard conditions. Bonds can include covalent bonds and non-covalent interactions, such as but not limited to ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, and the like. In certain embodiments, the fluorescence is covalently bound to the liposome. For example, as described above, the lipids that make up the liposome can include a hydrophilic group, which in some cases can include an activated functional group that provides for covalent linkage to a fluorophore, e.g., such that the fluorophore is conjugated to the liposome. Any convenient activated functional group useful in chemical synthesis can be used to covalently bind a detectable label to the hydrophilic group of a lipid, such as but not limited to an amine, carboxyl, amide, hydroxyl, azide, maleimide, bromoacetyl, 2-pyridyldithiol, haloalkyl, alkene, or alkyne group, and the like.

[0071] The average number of fluorophores stably associated with the liposomes of a given subpopulation of liposomes (e.g., expressed in MESF) can be different. The average number of fluorophores is different in any two subpopulations of liposomes in a given calibration composition.

[0072] The liposome calibration composition used in embodiments of the present invention can be prepared using any convenient protocol. In some cases, the protocol includes preparing two or more different subpopulations of liposomes, each subpopulation comprising a known amount of a fluorophore; and combining the two or more different subpopulations of liposomes to produce the liposome calibration composition, wherein the known amount of fluorophore of each subpopulation is different from the amount of any other subpopulation in the liposome calibration composition.

[0073] The fluorophores comprising the liposomes of each subpopulation can be prepared using any convenient protocol. In some cases, the protocol comprises preparing an initial population of fluorophores comprising liposomes, and then generating liposome subpopulations from the initial population using a liposome extrusion device, e.g., as described in U.S. Patent Application Serial No. 15 / 472053 (Publication No. US 2017-0341049 Al), the disclosure of which is incorporated herein by reference. After preparing a given fluorophore comprising subpopulation of liposomes, the average number of fluorophores per liposome of the subpopulation, For example, represented by MESF, can be determined using any convenient protocol, e.g., using the protocol of Quantibrite TM beads (Becton Dickinson and Company), where further details regarding such beads and protocols for using the beads can be found in U.S. Patent Nos. 6350619; 7738094; and 8248597, the disclosure of each of which is incorporated herein by reference in its entirety.

[0074] After preparation, the different fluorophore comprising liposome subpopulations are combined to generate a liposome calibration composition. The number of liposomes in the different subpopulations can be the same or different. The different liposome subpopulations can be combined to generate a liposome calibration composition using any convenient protocol, e.g., by combining the different subpopulations into a single container and mixing.

[0075] Kit

[0076] Aspects of the present disclosure also include a liposome calibration composition, e.g., as described above, where the liposome calibration composition is present in a suitable container, e.g., a test tube. The liposome calibration composition can be provided as a liquid composition or a dry composition, including a lyophilized composition, as desired. The kit can also include a surface marker label, e.g., as described above, where the fluorophore of the surface marker label is the same as the fluorophore of the liposome calibration composition.

[0077] The kit can also include a liquid. For example, the kit can include a buffer, e.g., a sample buffer, a wash buffer, an assay buffer, etc. In some cases, the kit can include a liquid suitable for a liposome suspension. The kit can also include additional reagents, such as, but not limited to, detectable labels (e.g., fluorescent labels, colorimetric labels, chemiluminescent labels, multicolor reagents, avidin-streptavidin related detection reagents, radioactive labels, gold particles, magnetic labels, etc.), etc.

[0078] In certain embodiments, the kit can also include a calibration standard. For example, the kit can include a set of labeled beads, such as a set of standard fluorescent labeled beads. The calibration standard can be used to determine the accuracy of the assay instrument and ensure consistency between subsequent assays. For example, the calibration standard can be used to determine the accuracy of a flow cytometer. In some cases, the calibration standard includes labeled beads, such as fluorescently labeled beads. The fluorescently labeled beads can be standard fluorescently labeled beads that are commonly used as calibration standards. Examples of standard fluorescently labeled beads include, but are not limited to, fluorescently labeled microparticles or nanoparticulates. In some cases, the fluorescently labeled beads are configured such that they remain suspended in the assay mixture and do not substantially settle or aggregate. In some embodiments, the fluorescently labeled beads include, but are not limited to, fluorescently labeled polystyrene beads, fluorescein beads, rhodamine beads, and other beads labeled with a fluorescent dye. Additional examples of fluorescently labeled beads are described in U.S. Patent Nos. 6350619; 7738094; and 8248597, the disclosure of each of which is incorporated herein by reference in its entirety.

[0079] Additionally, the kit can include packaging configured to house the various components. The packaging can be a sealed package, such as a water vapor resistant container, optionally under airtight and / or vacuum seal. In certain cases, the packaging is a sterile package configured to maintain the devices enclosed in the packaging in a sterile environment. “Sterile” means substantially free of microorganisms (such as fungi, bacteria, viruses, spore forms, and the like). The kit can also include a liquid container, such as described above.

[0080] In addition to the above components, the subject kits can also include instructions for practicing the subject methods. These instructions can be present in the subject kits in a variety of forms, one or more of which can be present in the kit. One form in which these instructions can be present is as printed information on a suitable medium or substrate, such as a paper or paperboard sheet, in the packaging of the kit, in a package insert, and the like. Another means by which such instructions can be present is as the information recorded or stored on a computer readable medium, such as a CD, DVD, Blu-ray disc, computer readable memory (such as flash memory), and the like, which can be read by a computer system, either as a whole or as on a chip. Another means by which such instructions can be present is as a website address which can be accessed via the internet or other distributed network as known to one of skill in the computer and computer software arts. Any of these forms of instructions which are present in the kits described herein are optionally accompanied by packaging materials, such as a box, a tray, a tube, and the like, for handling and storing the instruments and materials in the kit.

[0081] Applications

[0082] The subject methods, devices, and systems can be used in applications requiring quantification of EV surface markers. These applications include research and diagnostic / therapeutic applications. For example, the methods can be used in various research applications, e.g., to identify new and useful EV markers. In other cases, the methods can be used in diagnostic / therapeutic applications, e.g., to obtain a labeled EV sample from a live subject and use it to obtain a diagnosis of a condition, e.g., a disease condition of the subject. In such cases, the methods can further include treating the disease condition of the subject based on the diagnosis obtained.

[0083] From the disclosure provided above, it will be appreciated that the embodiments of the disclosure have broad applications. Accordingly, the examples shown herein are provided for illustrative purposes and are not intended to be construed as limiting the embodiments of the disclosure in any way. One of ordinary skill in the art will readily recognize various noncritical parameters that can be changed or modified to yield essentially similar results. Accordingly, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the disclosure, and are not intended to limit the scope of what the inventors regard as their application nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric pressure.

[0084] Examples

[0085] A. Preparation of Liposome Compositions

[0086] (1) Fluorescent protein-modified liposome

[0087] Primary amine functional groups were incorporated into the liposome membrane using DSPE-PEG(2000) amine (l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000-amine) as described in U.S. Patent Application Serial No. 15 / 472,053, the disclosure of which is incorporated herein by reference. The resulting liposomes containing primary amine functional groups on their surface were reacted with SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate). The SPDP-liposomes were then treated with DTT to generate free thiol groups (-SH) on the surface of the liposomes. The resulting thiol-liposome preparation was reacted with maleimide derivatized green fluorescent protein (GFP), phycoerythrin (PE), allophycocyanin (APC), PerCp and their tandem conjugates (PE-CF594, PE-Cy5, PE-Cy7, PE-H7, APC-R700, Percp-Cy5.5, etc.) to generate liposomes bearing various fluorescent proteins. The labeled liposomes were further purified by size exclusion chromatography to provide purified preparations of labeled liposomes.

[0088] (2) Organic fluorescent dye-modified liposome

[0089] Primary amine functional groups are incorporated into the liposome membrane as described in Example A(l) above. The liposome preparation bearing primary amine groups on the surface is then reacted with various active fluorescent dyes containing NHS esters, isothiocyanates, or sulfonyl chlorides. Typical examples of fluorescent dyes with active groups include various Alexa-NHS ester dyes, fluorescein, rhodamine isothiocyanate, and Texas Red sulfonyl chloride. The labeled liposomes are purified from unreacted dyes by size exclusion chromatography to provide a pure fraction of labeled liposomes. Alternatively, liposomes can be labeled with fluorophores by incorporating various commercially available fluorescent lipids or cholesterols directly into the liposome membrane.

[0090] (3) Deposition of functional polymer on the surface of liposome to enhance fluorescent signal

[0091] During the liposome preparation as described in Examples A(l) and A(2) above, carboxyl functional groups are incorporated into the liposome membrane using DSPE-PEG(2000) carboxylic acid (l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxyl(polyethylene glycol)-2000]), as described in U.S. Patent Application Serial No. 15 / 472,053, the disclosure of which is incorporated herein by reference. In the presence of a carbodiimide, the carboxylated liposomes are reacted with various aminated polymers such as polyallylamine, polyvinylamine, polyethyleneimine, polylysine, aminated dextran, or chitosan to create amide bonds between the liposome carboxyl groups and the aminated polymers. The resulting polymeric amine-liposome structures are then directly reacted with various fluorescent dyes as described in Examples A(l) and A(2) above. Alternatively, the aminated polymers described above are labeled with fluorescent dyes prior to reaction with the liposomes to covalently attach to the liposome surface via the chemistry described above. The deposition of functional polymers on the liposome surface increases the number of fluorescent dyes attached to the liposome due to the increased number of amine functional groups, thus enhancing the fluorescence intensity of the liposome particles.

[0092] (4) Click chemistry-modified liposome

[0093] Liposomes are labeled with fluorescent dyes designed for azide-alkyne cycloaddition or click chemistry. As previously described, the liposome membrane is modified with various alkyne or azide functional groups such as alkyne phospholipids, alkyne cholesterols, azide phosphocholine, azide distearoyl-glycero-phosphoethanolamine, etc. The resulting functionalized liposomes are then reacted with fluorescent dyes designed for click chemistry bearing alkyne or azide functional groups. The fluorescent liposomes are further purified using size exclusion chromatography.

[0094] (5) Dye wrapped in liposome

[0095] A variety of fluorescent proteins such as GFP, PE, APC, PercP and their corresponding tandem fluorophores (PE-CF594, PE-Cy5, PE-Cy7, PE-H7, APC-R700, Percp-Cy5.5, etc.) are encapsulated within the liposomes during their preparation. Similarly, various polymers labeled with fluorescent dyes such as those described in Example (3) and fluorescent conjugated polymers (or polymeric dyes) are directly encapsulated within the liposomes.

[0096] (6) Preparation of fluorescent liposome by combining the above chemical methods

[0097] Various fluorescent liposomes are constructed using a combination of surface labeling and encapsulation of fluorophores within the liposomes to provide MESF liposome calibrators.

[0098] B. Number of fluorophores assigned to liposomes characterized:

[0099] As described in Examples A1-4 above, liposomes with different fluorescent intensities are produced using different concentrations of active fluorescent dyes such as FITC-NHS ester or phycoerythrin-maleimide with a fixed number of liposome particles. Figure 1A Examples of liposomes incorporating different numbers of fluorophores are shown. As Figure 1B Each labeled liposome is analyzed in a flow cytometer as shown. The plot of SSC (side scatter) versus PE fluorescence signal provides the mean fluorescence intensity (MFI) values for each liposome population at different labeling levels.

[0100] To assign the number of fluorophores to the liposomes with different MFI values, a set of fluorescent calibration beads with known numbers of fluorophore molecules, such as PE-QuantiBRITE beads, are analyzed in a flow cytometer under the same PMT voltage and fluorescence channel compensation settings as used for the liposomes. The standard curve (plot of MFI values versus their corresponding fluorophore numbers) from these calibration beads is used to calculate the number of fluorophore molecules for each liposome intensity. Figure 1C PE calibrator beads and their corresponding curve are shown. Once the number of fluorophores is assigned to the liposomes of different intensities, the liposomes are used as a calibrator set for determining the number of fluorophores for unknown EV samples.

[0101] C. Characterization of liposomes by dynamic light scattering and flow cytometry:

[0102] Once labeled liposomes with defined fluorescent intensities are prepared, the quality and fluorescent intensities of the liposomes are analyzed by two independent methods. Dynamic light scattering (DLS) is used to provide information about the size distribution of the population of liposomes. Figure 2AThe size distribution of the FITC labeled liposome preparation analyzed in a DLS instrument is shown. The liposome population has a diameter of 150 nm to 400 nm with an average diameter of 210 nm. Figure 2B The same liposome preparation analyzed in a flow cytometer is shown in dot plot (SSC vs. FITC intensity) and histogram (counts vs. FITC intensity). The dot plot shows the gated liposome population (P1) and the histogram shows the fluorescence intensity of the gated P1 liposomes. As shown by the data, the %CV of the histogram of the entire labeled liposomes shown in gate P3 is about 67.3% and the MFI value is 27834 (the circled values in the table, respectively). This indicates that the MFI distribution of the liposome population is fairly broad as indicated by its large %CV value (67%). In order to obtain a liposome population with a smaller %CV of its MFI values, it is necessary to sort a narrower portion of the liposome population in the flow cytometer. Figure 2B Gate P2 of the middle histogram shows the labeled portion of the liposome population for sorting. Figure 2C The data analysis of the sorted liposome population is shown. As shown by the circled values in the table, the %CV of the sorted liposomes is significantly improved from 67% to 25.8% or 2.6 fold, while the MFI value of the sorted population remains essentially the same (30061 FITC MFI) with no loss of FITC signal intensity. Thus, by sorting labeled liposomes of different intensities, it is possible to produce a series of liposomes with MFI values having a sufficiently narrow %CV that they can be mixed in a single vial and have well separated MFI histograms in the fluorescence channel. These well separated labeled liposomes have a defined number of fluorophore molecules assigned to each intensity and can be used as calibrators for quantifying EV surface markers. Figures 3A to 3B Results of the FITC labeled liposome calibrators are provided. Figure 3A Flow cytometry analysis of the FITC labeled liposomes on the BD FACS Aria Fusion before sorting is provided. Figure 3B FITC labeled liposome calibrators produced after sorting in the FACS Aria Fusion flow cytometer are provided. The data demonstrate that it is possible to produce liposome calibrators labeled with fluorophores.

[0103] The disclosure is also defined by the following clauses, notwithstanding the appended claims:

[0104] 1. A method of quantifying a surface marker on extracellular vesicles of a sample, the method comprising:

[0105] Comparing:

[0106] (i) the mean fluorescence intensity of a surface marker (surface marker MFI) of a labelled extracellular vesicle (EV) sample, wherein the labelled EV sample has been labelled with a surface marker label comprising a specific binding member for the surface marker and a fluorophore; and

[0107] (ii) a calibration curve obtained from a liposome calibration composition comprising two or more different liposome subpopulations, each subpopulation comprising a known amount of fluorophore that is different from the amount of any other subpopulation in the liposome calibration composition;

[0108] to obtain the number of fluorophores bound to the surface of the extracellular vesicles of the EV sample and to quantify the surface marker on the extracellular vesicles of the EV sample.

[0109] 2. The method of clause 1, wherein the calibration curve comprises a curve of the known amount of fluorophore per different liposome subpopulation against the fluorescence intensity of the calibration liposome composition.

[0110] 3. The method of any one of clauses 1 and 2, wherein the method further comprises obtaining the surface marker MFI of the labelled EV sample.

[0111] 4. The method of clause 3, wherein the surface marker MFI of the labelled EV sample is obtained by a flow cytometry assay of the labelled EV sample.

[0112] 5. The method of clause 4, wherein the method further comprises preparing the labelled EV sample.

[0113] 6. The method of clause 5, wherein the specific binding member of the surface marker label comprises an antibody or binding fragment thereof.

[0114] 7. The method of any one of the preceding clauses, wherein the method further comprises obtaining the calibration curve.

[0115] 8. The method of clause 7, wherein the calibration curve is obtained by a flow cytometry assay of the liposome calibration composition.

[0116] 9. The method of clause 8, wherein the liposome calibration composition is flow cytometry assayed under the same settings as used for the flow cytometry assay of the labelled EV sample using the same flow cytometer.

[0117] 10. The method of clause 9, wherein the same settings comprise the same voltage parameters of the flow cytometer photomultiplier tubes.

[0118] 11. The method of any of the preceding clauses, wherein the fluorophore is selected from PE, PE-Cy7, APC, BV421, BV510, and BV605.

[0119] 12. The method of clause 11, wherein the fluorophore is PE.

[0120] 13. The method of any of the preceding clauses, wherein the liposome calibration composition comprises three or more than three different subpopulations.

[0121] 14. The method of clause 13, wherein the liposome calibration composition comprises four different subpopulations.

[0122] 15. The method of any of the preceding clauses, wherein the liposomes of the different subpopulations are uniform in size.

[0123] 16. The method of clause 15, wherein the uniform size is 100 nm to 150 nm.

[0124] 17. The method of any of the preceding clauses, wherein the method further comprises using the liposome calibration composition as a size calibrator for a labeled EV sample.

[0125] 18. The method of any of the preceding clauses, wherein the labeled EV sample is prepared from an initial sample obtained from a living subject.

[0126] 19. The method of clause 18, wherein the method further comprises obtaining a diagnosis of a disease condition of the subject based on the obtained quantification.

[0127] 20. The method of clause 19, wherein the method further comprises obtaining a treatment of the disease condition of the subject based on the obtained diagnosis.

[0128] 21. A liposome calibration composition comprising two or more than two different liposome subpopulations, each subpopulation comprising a known amount of a fluorophore that is different in amount from any other subpopulation in the liposome calibration composition.

[0129] 22. The liposome calibration composition of clause 21, wherein the liposome calibration composition comprises three or more than three different subpopulations.

[0130] 23. The liposome calibration composition of clause 22, wherein the liposome calibration composition comprises four different subpopulations.

[0131] 24. The liposome calibration composition of any of clauses 21 to 23, wherein the liposomes of the different subpopulations are uniform in size.

[0132] 25. The liposome calibration composition of clause 24, wherein the uniform size is 100 nm to 150 nm.

[0133] 26. The liposome calibration composition of any one of clauses 21 to 25, wherein the fluorophore is selected from PE, PE-Cy7, APC, BV421, BV510, and BV605.

[0134] 27. The liposome calibration composition of clause 26, wherein the fluorophore is PE.

[0135] 28. The liposome calibration composition of any one of clauses 21 to 27, wherein the fluorophore is conjugated to the liposome.

[0136] 29. A method of making a liposome calibration composition, the method comprising:

[0137] (a) making two or more different subpopulations of liposomes, each subpopulation of liposomes comprising a known amount of a fluorophore; and

[0138] (b) combining the two or more different subpopulations of liposomes to produce the liposome calibration composition, wherein the known amount of fluorophore of each subpopulation is different from the amount of any other subpopulation in the liposome calibration composition.

[0139] 30. The liposome calibration composition of clause 29, wherein the liposome calibration composition comprises three or more different subpopulations.

[0140] 31. The method of clause 30, wherein the liposome calibration composition comprises four different subpopulations.

[0141] 32. The method of any one of clauses 29 to 31, wherein the liposomes of the different subpopulations are uniform in size.

[0142] 33. The method of clause 32, wherein the uniform size is 100 nm to 500 nm.

[0143] 34. The method of any one of clauses 29 to 33, wherein the fluorophore is selected from PE, PE-Cy7, APC, BV421, BV510, and BV605.

[0144] 35. The method of clause 34, wherein the fluorophore is PE.

[0145] 36. The method of any one of clauses 29 to 35, wherein the fluorophore is conjugated to the liposome.

[0146] 37. A kit comprising:

[0147] a liposome calibration composition comprising two or more different liposome subpopulations, each subpopulation comprising a known amount of a fluorophore in a different amount than any other subpopulation in the liposome calibration composition; and

[0148] a container for the liposome calibration composition.

[0149] 38. The kit of clause 37, wherein the liposome calibration composition comprises three or more different subpopulations.

[0150] 39. The kit of clause 38, wherein the liposome calibration composition comprises four different subpopulations.

[0151] 40. The kit of any one of clauses 37 to 39, wherein the liposomes of the different subpopulations are uniform in size.

[0152] 41. The kit of clause 40, wherein the uniform size is 100 nm to 500 nm.

[0153] 42. The kit of any one of clauses 37 to 41, wherein the fluorophore is selected from the group consisting of PE, PE-Cy7, APC, BV421, BV510, and BV605.

[0154] 43. The kit of any one of clauses 37 to 42, wherein the kit further comprises a surface marker label comprising a specific binding member for a surface marker and a fluorophore.

[0155] 44. The kit of clause 43, wherein the specific binding member comprises an antibody or binding fragment thereof.

[0156] In at least some of the previously described embodiments, one or more elements used in an embodiment can be used interchangeably in another embodiment, unless such substitution is not technically feasible. Those skilled in the art will appreciate that various other omissions, additions and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be included herein within the scope of the subject matter defined by the claims.

[0157] Those skilled in the art will appreciate that, in general, terminology used herein, and especially in the appended claims (for example, in the body of the appended claims), is generally intended to be open and permissive, for example, the term "comprising" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "including" should be interpreted as "including but not limited to", etc. Those skilled in the art will further appreciate that, if it is intended that a particular number of introduced claim recitations be limited, then such limitation will be explicitly recited in the claims, and no inference in that regard will be drawn from the absence of such explicit recitation. For example, to help understand, the following appended claims can contain the use of introductory phrases such as "at least one" and "one or more" to introduce claim recitations. However, the use of these phrases is not to be construed as implying that through the use of unnumbered introduced claim recitations that any particular claim limitation is limited to only one such recitation, even though such claim limitation is included in the same claim that includes the introductory phrases "one or more" or "at least one" and unnumbered (for example, should be interpreted to mean "at least one" or "one or more"); this applies the same to the use of the definite article "the" to introduce claim recitations. Moreover, even if a particular number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, "two statements" without further modification means at least two statements, or two or more statements). Also, in instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in open and permissive sense (for example, a system having at least one of A, B, and C would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general such a construction is intended in open and permissive sense (for example, a system having at least one of A, B, or C would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further recognize that virtually any disjunctive word and / or phrase presenting two or more alternative terms is intended to cover the possibility of including one of the terms, either of the terms, or both terms, unless otherwise indicated. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B".

[0158] Further, where a feature or aspect of the disclosure is described according to a Markush group, it is understood that the disclosure also encompasses any individual member or subgroup of members of the Markush group.

[0159] As will be understood by those skilled in the art, all ranges disclosed herein include any and all possible subranges and combinations thereof, for any and all purposes. Any listed range can be easily reduced to at least one half, third, fourth, fifth, tenth, etc. As a non-limiting example, each range discussed herein can be easily halved, one thirded, one fourthed, one fifthed, one tenthed, etc. Those skilled in the art will further appreciate that any language expressing a possibility or a potential for some aspect to be augmented is used in accordance with the principle that every aspect is possible and

[0160] While the foregoing application has been described in some detail for purposes of clarity and the specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be practiced that are

[0161] Therefore, the foregoing merely illustrates the principles of the application. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the application and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to be only for pedagogical purposes to aid the reader in understanding the principles of the application and the concepts contributed by the inventor to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the application, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that any herein- disclosed integrations of the aspects and embodiments of the application with other aspects and embodiments thereof are to be construed as being within the scope of the present application. Furthermore, those skilled in the art will recognize that bonds, including single and double bonds, can be saturated or unsaturated, and that any bond can be a carbon-heteroatom bond. It is therefore clear to those skilled in the art that the present application can be practiced with alterations and modifications and falls within the spirit and scope of the appended claims. Accordingly, the application is not limited to that precisely as shown and described.

[0162] Accordingly, the scope of the present application is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope of the present application is intended to be limited only by the appended claims. In the claims, the recitation of a 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) limitation is expressly defined as being invoked only when the exact phrase "means for" or the exact phrase "steps for" is recited in the claim; if the claim does not recite the exact phrase "means for" or the exact phrase "steps for", then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

Claims

1. A method for quantifying surface markers on extracellular vesicles of a sample, the method comprising: Compare: (i) The mean fluorescence intensity (MFI) of surface markers in labeled extracellular vesicle (EV) samples, wherein the labeled EV samples have been labeled with surface markers comprising a specific binding member and a fluorophore of the surface marker; (ii) Calibration curves obtained from a liposome calibration composition comprising two or more different liposome subpopulations, each subpopulation containing a known amount of fluorophore that differs from the amount of any other subpopulation in the liposome calibration composition, wherein the fluorophore is covalently bound to the surface of the liposome or encapsulated within the liposome. The aim was to obtain the number of fluorophores bound to the surface of extracellular vesicles of EV samples and to quantify the surface markers on extracellular vesicles of EV samples.

2. The method of claim 1, wherein the calibration curve comprises a curve of the known amount of fluorophores of each different liposome subpopulation relative to the fluorescence intensity of the calibration liposome composition.

3. The method according to any one of claims 1 and 2, wherein the method further comprises obtaining a surface marker MFI of the labeled EV sample.

4. The method of claim 3, wherein the surface marker MFI of the labeled EV sample is obtained by flow cytometry determination of the labeled EV sample.

5. The method of claim 4, wherein the method further comprises preparing a labeled EV sample.

6. The method of claim 5, wherein the specific binding member of the surface marker comprises an antibody or a binding fragment thereof.

7. The method of claim 1, wherein the method further comprises obtaining a calibration curve.

8. The method of claim 7, wherein the calibration curve is obtained by flow cytometry determination of the liposome calibration composition.

9. The method of claim 8, wherein the liposome calibration composition is used for flow cytometry assays with the same flow cytometer in the same settings as those used for flow cytometry assays of labeled EV samples.

10. The method of claim 1, wherein the liposome calibration composition comprises three or more different subgroups.

11. The method of claim 1, wherein the liposomes of different subgroups are of uniform size.

12. The method of claim 1, wherein the method further comprises using a liposome calibration composition as a size calibrator for the labeled EV sample.

13. A liposome calibration composition comprising two or more different liposome subpopulations, each subpopulation comprising a known amount of a fluorophore that differs from the amount of any other subpopulation in the liposome calibration composition, and wherein the fluorophore is covalently bound to the surface of the liposome or encapsulated within the liposome.

14. The liposome calibration composition according to claim 13, wherein the liposomes of different subgroups are of uniform size.

15. A reagent kit comprising: a liposome calibration composition comprising two or more different liposome subpopulations, each subpopulation comprising a known amount of a fluorophore in a different amount than any other subpopulation in the liposome calibration composition, wherein the fluorophore is covalently bound to the surface of the liposome or encapsulated within the liposome; and a container for the liposome calibration composition.

16. The method of claim 1, wherein the fluorophore is covalently bound to the surface of the liposome via a hydrophilic group of a lipid of the liposome.

17. The method of claim 16, wherein the fluorophore is covalently bound to the hydrophilic group via an amine group, a carboxyl group, an amide group, a hydroxyl group, an azido group, a maleimide group, a bromoacetyl group, a 2-pyridyldithiol group, a haloalkyl group, an alkenyl group, or an alkyne propyl group.

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