Highly bright fluorescent dyes for extracellular vesicle quantification and phenotyping

Abstract

The compound includes a nanomaterial carrier, a first linker having a first end connected to the nanomaterial carrier, a second linker having a first end connected to the nanomaterial carrier, a fluorescent entity connected to the second end of the first linker, and a biomolecule connected to the second end of the second linker. The biomolecule is configured to connect to a cluster of differentiation (CD) of an extracellular vesicle (EV). A method thereof is also disclosed.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 62 / 889,691, filed August 21, 2019, International Application No. PCT / US2020 / 035568, filed June 1, 2020, and International Application No. PCT / US2020 / 035574, filed June 1, 2020. U.S. Provisional Patent Application No. 62 / 889,691, International Application No. PCT / US2020 / 035568, and International Application No. PCT / US2020 / 035574 are incorporated herein by reference in their entireties.

[0002] (Statement of Government Support) The invention described herein was made with government support under Grants #1261910, #1521057 and #1738466 awarded by the National Science Foundation. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Fluorophores are compounds with fluorescent properties that have biomedical applications. For example, fluorescent dyes can be used as tracers or dyes to stain specific molecules or structures. More specifically, fluorescent dyes are used to stain tissues, cells, or biological materials in various analytical methods, such as fluorescence imaging and spectroscopy.

[0004] Extracellular vesicles (EVs) are biological particles encapsulated in a phospholipid bilayer. EV diameters range from approximately 20 nanometers (nm) to several microns, but most are smaller than approximately 350 nm. Detecting EVs may be useful for certain clinical applications, such as early disease detection and treatment with diagnostic biomarkers and therapeutics. However, due to their small diameter, EVs have only a few biological markers. Therefore, accurately counting and phenotyping small EVs is challenging. High-resolution imaging flow cytometry is the most promising method for counting EVs by laser light scattering and phenotyping them by the fluorescent signals of fluorochromes tagged to them. However, due to their small size, low refractive index (n ~ 1.3-1.4), and swarming tendency, accurately quantifying EVs by light scattering remains challenging, and phenotyping remains difficult due to the weak fluorescent signals from the few fluorochromes in each EV. These issues hinder the effectiveness of EVs as diagnostic biomarkers and therapeutics. Summary of the Invention [Means for solving the problem]

[0005] A compound according to an exemplary embodiment of the present invention includes, inter alia, a nanomaterial carrier, a first linker having a first end connected to the nanomaterial carrier, a second linker having a first end connected to the nanomaterial carrier, a fluorescent entity connected to the second end of the first linker, and a biomolecule connected to the second end of the second linker, wherein the biomolecule is configured to connect to a cluster of differentiation (CD) of an extracellular vesicle (EV).

[0006] In further examples of the foregoing, the nanomaterial support is a boron nitride nanotube (BNNT) or a carbon nanotube (CNT).

[0007] In a further example of any of the foregoing, the nanomaterial is a nanodot.

[0008] In a further example of any of the foregoing, a first end of at least one of the first and second linkers is covalently attached to the nanomaterial support.

[0009] In a further example of any of the foregoing, a first end of at least one of the first and second linkers includes a functional group, which covalently bonds the linker to the nanomaterial support.

[0010] In a further example of any of the foregoing, the second end of at least one of the first and second linkers is covalently attached to a fluorescent entity or biomolecule via a functional group.

[0011] In a further example of any of the foregoing, a first end of at least one of the first and second linkers is non-covalently attached to the nanomaterial support.

[0012] In a further example of any of the foregoing, at least one of the first and second linkers is amphiphilic and includes a hydrophobic region and a hydrophilic region, the hydrophobic region non-covalently binding to the nanomaterial support.

[0013] In a further example of any of the foregoing, the linker has a molecular weight of about 1,000 to 10,000 Da.

[0014] In a further example of any of the foregoing, the nanomaterial support is a boron nitride nanotube.

[0015] In further examples of any of the foregoing, at least one of the first and second linkers is DSPE-PEG. n (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol) n ]), where n is the number of polyethylene glycol (PEG) molecules in the polyethylene glycol (PEG) chain.

[0016] A method according to an exemplary embodiment of the present invention includes, inter alia, linking at least one fluorescent entity and at least one biomolecule to a nanomaterial carrier. The biomolecule is configured to connect to clusters of differentiation (CDs) of extracellular vesicles (EVs) to form a compound. The method also includes applying the compound to the extracellular vesicles (EVs) so that the compound connects to the EVs via the biomolecule to form marked extracellular vesicles (EVs), and detecting at least one of light scattering and fluorescence of the marked EVs.

[0017] In further examples of the foregoing, the support is a boron nitride nanotube (BNNT) support, a carbon nanotube (CNT) support, or a nanodot.

[0018] In a further example of any of the foregoing, the attachment of at least one of the fluorescent entity and the biomolecule is via a linker, and the attachment of the linker to the nanomaterial support is via a covalent bond.

[0019] In a further example of any of the foregoing, a first end of the linker comprises a first functional group and a second end of the linker comprises a second functional group, the first functional group covalently attached to the nanomaterial support and the second functional group covalently attached to the fluorescent entity.

[0020] In a further example of any of the foregoing, the attachment of at least one of the fluorescent entity and the biomolecule is via a linker, and the attachment of the linker to the nanomaterial support is via a non-covalent bond.

[0021] In a further example of any of the foregoing, the linker is amphiphilic and comprises a hydrophobic region and a hydrophilic region, the hydrophobic region non-covalently binding to the nanomaterial carrier.

[0022] In a further example of any of the foregoing, the linker has a molecular weight of about 1,000 to 10,000 Da.

[0023] In a further example of any of the foregoing, the nanomaterial support is a boron nitride nanotube.

[0024] In further examples of any of the foregoing, the linker may be DSPE-PEG n (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol) n ]), where n is the number of polyethylene glycol (PEG) molecules in the polyethylene glycol (PEG) chain. [Brief explanation of the drawings]

[0025] [Figure 1A] FIG. 1 is a schematic diagram of a bright fluorescent dye structure. [Figure 1B] FIG. 1 is a schematic diagram of a dye-linker structure. [Figure 1C] FIG. 1 is a schematic diagram of an antibody-linker structure. [Figure 2] (A) shows the light scattering signal from PBS buffer containing EVs. (B) shows the fluorescence signal from the PBS buffer of Figure 2(A). [Figure 3] (A) Light scattering signal from short BNNTs. (B) Light scattering signal from short BNNTs at 4x BNNT concentration. (C) Length distribution of short BNNTs. [Figure 4] (A) shows the light scattering signal from a short BNNT labeled with a dye linker, and (B) shows the fluorescence signal from a short BNNT labeled with a dye linker. [Figure 5] (A) Light scattering signals from long BNNTs. (B) Length distribution of long BNNTs. DETAILED DESCRIPTION OF THE INVENTION

[0026] Most commonly, a bright fluorescent dye comprises a carrier element, a fluorescent element, and a linker that connects the carrier element to the fluorescent element. For biomedical applications, each of the carrier element, linker, and fluorescent element must be biocompatible (although biocompatibility requirements vary depending on the particular application).

[0027] Exemplary carrier elements are nanomaterials such as carbon nanotubes (CNTs) and boron nitride nanotubes (BNNTs), both of which are recognized as biocompatible nanomaterials for biomedical applications such as cellular drug delivery and spectroscopy applications. However, fluorescent elements bound to nanotubes have been shown to exhibit quenching, or a decrease in fluorescence brightness.

[0028] As described in U.S. Patent Application No. 15 / 953200, filed April 13, 2018, and published as U.S. Patent Application Publication No. 2018 / 0296705, and International Application No. PCT / US2020 / 035568 and International Application No. PCT / US2020 / 035574, filed June 1, 2020, certain fluorescent dyes with nanomaterial carriers have been found to not only exhibit no quenching effect, but also exhibit brightness several orders of magnitude higher than other known fluorescent dyes. U.S. Patent Application No. 15 / 953200, International Application No. PCT / US2020 / 035568, and International Application No. PCT / US2020 / 035574 are incorporated herein by reference in their entireties.

[0029] Extracellular vesicles (EVs) are biological particles encapsulated in a phospholipid bilayer. EVs are biological particles naturally released from cells but cannot replicate on their own. EV diameters range from approximately 20 nanometers (nm) to several microns, but most are smaller than approximately 350 nm. Various EV subtypes, defined by their size, cellular origin, and function, have been proposed, including exosomes (approximately 20–150 nm), ectosomes (approximately 150–1000 nm), and apoptotic bodies (approximately 1–5 μm). EVs are found in bodily fluids such as blood, urine, and cerebrospinal fluid. They are also released into the growth medium of cultured cells. They carry various proteins, nucleic acids, metabolites, and even organelles from their parent cells. In particular, EVs possess several biomarkers / biological molecules, so-called clusters of differentiation (CDs), on their surface. Biomarkers derived from their parent cells have specialized functions in physiological processes such as immune system regulation, inflammatory responses, and tissue regeneration, as well as intercellular communication processes. Therefore, the detection of those CDs may be useful for certain clinical applications, such as early disease detection and treatment with diagnostic biomarkers and therapeutics.

[0030] Referring now to FIG. 1A, a fluorescent dye 20 is shown schematically. The fluorescent dye 20 generally includes an inorganic nanoscale carrier 22, a linker 24, a fluorescent entity 26, and one or more biomolecules 28 (e.g., antibodies). The biomolecules 28 can be selected to interact with biomarkers on EVs. Examples of biomarkers include surface markers such as MHC, CD9, CD63, and CD81. This interaction connects the fluorescent dye 20 to the EVs through an interaction between the biomarker 28 on the fluorescent dye and the CD. In this way, EVs can be detected (and counted, identified, etc.) by detection of the fluorescent dye 20, as described in more detail below.

[0031] In one example, the support 22 is a BNNT or CNT support. The support 22 can be manufactured by any known method.

[0032] In a specific example, the support 22 is a multi-walled BNNT or CNT support, each having multiple coaxial shells of hexagonal boron nitride (h-BN in the case of BNNTs) or graphene (in the case of CNTs), with a typical outer diameter greater than about 1 nm and less than about 80 nm. The length of these BNNTs and CNTs is about 1-5000 nm. In another example, the support 22 may be another nanoscale inorganic material, such as boron nitride (h-BN) nanosheets / nanoparticles and graphene / graphite nanosheets / nanoparticles. Boron nitride nanodots and carbon nanodots are also contemplated. In one example, as described more fully in International Application No. PCT / US2020 / 035574, the nanodots are treated with mechanical agitation to promote the formation of defects in the nanostructure of the dots, which defects promote / enable binding to the linkers 24, thereby allowing more linkers 24, and therefore more fluorescent entities 26, to bind to the nanodots, improving the fluorescence of the resulting fluorescent dyes 20.

[0033] 1B-C, linker 24 is an amphiphilic polymer linker. That is, linker 24 includes a hydrophobic region 25 and a hydrophilic region 27. Hydrophobic region 25 non-covalently binds to nanotube support 22, while hydrophilic region 27 covalently binds to fluorescent entity 26 (or another entity, described below). One example linker 24 is DSPE-PEG. n (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol) n ]), where n is the number of polyethylene glycol (PEG) molecules in the PEG chain. Other linkers 24 can similarly include PEG chains of varying lengths (or different chains).

[0034] The hydrophilic region 27 is covalently attached to a biomolecule 28 (antibody, nucleic acid, etc.). An example linker 24 is DSPE-PEG n (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol) n]), where n is the number of polyethylene glycol (PEG) molecules in the PEG chain. Other linkers 24 can similarly include PEG chains of varying lengths (or different chains).

[0035] In one example, as more fully described in U.S. Patent Application No. 15 / 953,200, linker 24 has a molecular weight greater than about 1,000 Da (corresponding to an extended linker length of about 5-10 nm for linker 24 with PEG chains) and less than about 10,000 Da, which can improve the fluorescence of the resulting fluorescent dye 20 compared to prior art fluorescent dyes. In a further example, linker 24 has a molecular weight greater than about 2,400 Da and less than about 10,000 Da.

[0036] In addition to the DSPE-PEG linker 24 discussed above, many other potential linkers are known in the art. For example, linker 24 can contain one or more groups selected from -CH2-, -CH=, -C≡, -NH-, -N=, O-, -NH2-, -N3-, -S-, -C(O)-, -C(O)2-, -C(S)-, -S(O)-, -S(O)2-, or any combination thereof. It will be understood that linkers containing two or more of the above groups are selected so that linker 24 is stable (e.g., not susceptible to degradation) and biologically relevant, e.g., so that linker 24 does not contain two adjacent -O- groups that would create a labile peroxide bond. Linker 24 can be linear, branched, or contain one or more ring systems. Non-limiting exemplary linkers include fatty acids, phospholipids, sphingolipids, sphingophospholipids (such as, but not limited to, DSPE, 1-O-hexadecanyl-2-O-(9Z-octadecenyl)-sn-glycero-3-phospho-(1′-rac-glycerol) (ammonium salt), N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol) 5000, D-erythro sphingosylphosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine, 3-sn-phosphatidyl-L-serine (PS), glycosylphosphatidylinositol, 1,2-dioleoyl-sn-glycero-3-phosphoethanoamine) hydrophobic regions. The hydrophobic units are used to conjugate with water-soluble polymer chains, such as PEG (or PEO, polyethylene oxide), PMO (polymethyloxazoline), PEI (polyethyleneimine), polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polypeptides, carbohydrate anchors, etc. The water-soluble polymer chains are attached to a linker at one end and to a fluorescent entity (or another moiety, as described below) at the second end. These hydrophobic and hydrophilic units must have reactive groups, as described above, so that the groups can be attached together to the amphiphilic linker.

[0037] The fluorescent entity 26 may be any known fluorescent dye, including, but not limited to, coumarin, benzoxadiazole, acridone, acridine, bisbenzimide, indole, benzoisoquinoline, naphthalene, anthracene, xanthene, pyrene, porphyrin, fluorescein, rhodamine, boron-dipyrromethene (BODIPY), and cyanine derivatives. Many such fluorescent dyes are commercially available. As known in the art, the fluorescent entity 26 may be attached to the linker 24 by any suitable method, including, for example, by inducing a chemical reaction between the linker 24 and the fluorescent entity 26.

[0038] In another example, as described more fully in International Application No. PCT / US2020 / 035574, fluorescent dye 20 can be created by covalent functionalization of linker 24 to support 22. In this example, linker 24 includes a functional group "R" that interacts with support 22 and a functional group "R" that interacts with other moieties attached to support 22, such as fluorophores 26 and antibodies 28. An exemplary functional group is a hydroxyl group, although any well-known functional group is contemplated. In a further example described in International Application No. PCT / US2020 / 035574, support 22 is treated, such as by mechanical agitation in a polar liquid, to create defects in the nanostructure of support 22, which defects facilitate / enable binding to linker 24 via functional group R.

[0039] It has been confirmed that only the BNNT carrier 22 (before binding with the linker 24 and fluorescent entity 26) can cause sufficient light scattering for detection. Therefore, in another example, the BNNT carrier 22 can be used as a carrier for the fluorescent dye 26 without the linker 24 for the detection of EVs by flow cytometry.

[0040] Figure 2A shows the forward scatter (FSC) and side scatter (SSC) of laser light for a phosphate-buffered saline (PBS) solution containing EVs. As shown, 10 3 FSCs are induced by a 405 nm wavelength laser and 2The lower SSC is caused by the longer wavelength laser at 488 nm. These levels of scattering signal are considered "noise" in prior art methods of EV detection. Therefore, there is a detection window R1 where the scattering intensity of nanoparticles is high, which includes EVs and carriers of fluorescent dyes. The flow rate for all measurements described here is 0.25 μl / s, and the laser power is 100 mW at both 405 nm and 488 nm.

[0041] Figure 2B shows the fluorescence signal collected by the FITC channel (centered at 52 nm) of the PBS solution. As shown, there is no fluorescent dye 20 in the sample, so only noise is detected.

[0042] 3A shows the FSC and SSC of short BNNT support 22. In this example, the "short" BNNTs have lengths less than about 500 nm, with an average length of about 330 nm. The particle concentration of these BNNTs is 2.5×10 8 / ml. As shown, a higher scattering signal is detected within the R1 window defined in Figure 2A. The average signal intensity within the window is (2,698, 1,150). This means that BNNTs are detectable and therefore quantifiable by laser light scattering in a flow cytometer. This is useful for quantification of EVs when stained with fluorescent dyes 20 bearing BNNT carriers 22 as described here.

[0043] Figure 3B shows the FSC and SSC of BNNT support 22 when concentrated four-fold. As shown, the signal is detected within the R1 window, with an average signal intensity of (1,043, 934). This means that the detectable scattering signal depends on the concentration of BNNTs. In this particular case, some of the strong scattering is due to the higher particle concentration (1.0 × 10 9 The length distribution of these short BNNTs is shown in Figure 3C.

[0044] Figure 4A shows the FSC and SSC of a short BNNT carrier, a linker 24, and a fluorescent dye 20 with a fluorescent entity 26 (in this example, a FITC dye). As shown, a strong scattering signal is detected within the R1 window, with an average intensity of (2,627, 1,455).

[0045] FIG. 4B shows the fluorescent signal of a short BNNT carrier, a linker 24, and a fluorescent dye 20 with a FITC entity 26. As shown, 10 3 A strong signal centered on the BNNT is detected. This means that both the scattering and fluorescence signals of the fluorescent dye 20 with the short BNNT carrier, linker 24, and fluorescent entity 26 are detectable even under conventional settings / situations for light scattering and fluorescence detection, and are therefore suitable for use in EV labeling and detection.

[0046] In another example, the BNNT support 22 may be a "long" BNNT. The length of a "long" BNNT is approximately 500-5000 nm. More specifically, a long BNNT has a length between approximately 500-2000 nm. FIG. 5B shows the length distribution of an exemplary "long" BNNT sample, which has an average length of 900 nm, with many BNNTs longer than 1000 nm. FIG. 5A shows the FSC and SSC of the long BNNT support. The particle concentration of this BNNT sample is 6×10 10 / ml. As shown, the scattering signal intensity is strong, with an average intensity within the R1 window of (3,221,739).

[0047] The above results indicate that both short and long BNNTs can be used as carriers of fluorescent dyes, which cause stronger laser scattering signals in flow cytometry and enable the detection and counting of EVs.

[0048] The foregoing description is exemplary rather than limiting in nature. Variations and modifications to the disclosed examples will become apparent to those skilled in the art that do not necessarily depart from the essence of this invention. The scope of legal protection given to this invention can only be determined by studying the following claims.

Claims

1. a nanomaterial carrier; a first linker having a first end connected to the nanomaterial support; a second linker having a first end connected to the nanomaterial support; a fluorescent entity attached to the second end of the first linker; a biomolecule connected to the second end of the second linker; Equipped with The biomolecule is configured to connect to a cluster of differentiation (CD) of an extracellular vesicle (EV), and the cluster of differentiation (CD) of the extracellular vesicle (EV) is a biomolecule or biomarker present on the surface of the extracellular vesicle (EV); the nanomaterial support is either a short boron nitride nanotube (BNNT) or a long boron nitride nanotube (BNNT); The short boron nitride nanotubes (BNNTs) have a length of less than 500 nm, with an average length of 330 nm, and the long boron nitride nanotubes (BNNTs) have a length of 500-2000 nm, with an average length of 900 nm.

2. 2. The compound of claim 1, wherein the first end of at least one of the first and second linkers is covalently bonded to the nanomaterial support.

3. 3. The compound of claim 2, wherein the first end of at least one of the first and second linkers comprises a functional group, the functional group covalently bonding the linker to the nanomaterial support.

4. 3. The compound of claim 2, wherein the second end of at least one of the first and second linkers is covalently attached to the fluorescent entity or the biomolecule via a functional group.

5. 2. The compound of claim 1, wherein the first end of at least one of the first and second linkers is non-covalently attached to the nanomaterial carrier.

6. The compound of claim 5, wherein at least one of the first and second linkers is amphipathic and comprises a hydrophobic region and a hydrophilic region, and the hydrophobic region is non-covalently bound to the nanomaterial carrier.

7. The compound according to claim 5, wherein the linker has a molecular weight of 1,000 to 10,000 Da.

8. At least one of the first and second linkers is DSPE-PEG ｎ (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol) ｎ 2. The compound of claim 1, wherein n is the number of polyethylene glycol (PEG) molecules in the polyethylene glycol (PEG) chain.

9. At least one fluorescent entity and at least one biomolecule configured to connect to clusters of differentiation (CDs) of extracellular vesicles (EVs) to form a compound are linked to a nanomaterial carrier; applying the compound to the extracellular vesicles (EVs) such that the compound connects to the EVs via the biomolecule to form marked extracellular vesicles; detecting at least one of light scattering and fluorescence of the marked extracellular vesicles (EVs); Prepared for this, The cluster of differentiation (CD) of the extracellular vesicle (EV) is a biological molecule or biomarker present on the surface of the extracellular vesicle (EV), the nanomaterial support is either a short boron nitride nanotube (BNNT) or a long boron nitride nanotube (BNNT); The method for detecting extracellular vesicles, wherein the short boron nitride nanotubes (BNNTs) have a length of less than 500 nm and an average length of 330 nm, and the long boron nitride nanotubes (BNNTs) have a length of 500-2000 nm and an average length of 900 nm.

10. The detection method according to claim 9, characterized in that at least one of the fluorescent entity and the biomolecule is linked via a linker, and the linker is linked to the nanomaterial support via a covalent bond.

11. 11. The detection method of claim 10, wherein a first end of the linker comprises a first functional group, a second end of the linker comprises a second functional group, the first functional group covalently bonded to the nanomaterial carrier, and the second functional group covalently bonded to the fluorescent entity.

12. The detection method according to claim 9, characterized in that at least one of the fluorescent entity and the biomolecule is linked via a linker, and the linker is linked to the nanomaterial carrier via a non-covalent bond.

13. The detection method of claim 12, wherein the linker is amphipathic and comprises a hydrophobic region and a hydrophilic region, and the hydrophobic region is non-covalently bound to the nanomaterial carrier.

14. 13. The detection method according to claim 12, wherein the linker has a molecular weight of 1,000 to 10,000 Da.

15. The linker is DSPE-PEG ｎ (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol) ｎ 13. The method of claim 12, wherein n is the number of polyethylene glycol (PEG) molecules in the polyethylene glycol (PEG) chain.

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