Calibration set containing nylon 6 and dye-containing nanoparticles

Abstract

The calibration set includes a plurality of first nanoparticles comprising nylon 6 covalently bonded to a first dye, the first nanoparticles having a first average diameter and a first polydispersity index between 1.15 and 1.19. The calibration set also includes second nanoparticles comprising nylon 6 covalently bonded to a second dye different from the first dye, the second nanoparticles having a second average diameter and a second polydispersity index between 1.15 and 1.19. The first and second average diameters are different. The first and second average diameters are each independently between 30 nm and 3000 nm. The first average diameter is at least twice the second average diameter.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application No. 62 / 980,653, filed February 24, 2020, the entire disclosure of which is incorporated herein by reference. [Technical field]

[0002] This specification relates generally to calibration sets, and more particularly to calibration sets having properties similar to extracellular vesicles (EVs). [Background technology]

[0003] Research into extracellular vesicles (EVs) is expanding, both in terms of methods to isolate EVs from serum and cells and to enrich them for therapeutic applications. The term "extracellular vesicles" generally refers to lipid bilayer-enclosed particles that are naturally released from cells and cannot self-replicate. These particles are further classified according to size and function, with the smallest being exosomes with a size of about 30 nm and the largest being microvesicles with a size of about 2 μm.

[0004] However, characterization methods have been hampered by the lack of calibration standards for EV quantification and sizing techniques. Polystyrene beads have traditionally been used as standards in sizing instruments such as flow cytometers. However, the properties of polystyrene beads, such as density, light scattering effect, size, and binding affinity with fluorescent labels, match those of mammalian cells. However, although EVs are products of mammalian cells, the intrinsic properties of EVs are different from those of the cells from which they are derived and therefore also different from those of polystyrene beads. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need for alternative calibration sets, especially calibration sets that have similar properties to EV. [Means for solving the problem]

[0006] Various embodiments described herein provide a calibration set of nanoparticles having similar properties to EVs. Specifically, the calibration set includes a plurality of first nanoparticles of nylon 6 covalently bonded to a first dye and a plurality of second nanoparticles of nylon 6 covalently bonded to a second dye. The first nanoparticles have a first average diameter and the second nanoparticles have a second average diameter different from the first average diameter. The nanoparticles have a size range, density, and light scattering effect similar to EVs, as described in further detail below, and can be used as calibration standards for size measurement and quantification techniques.

[0007] A calibration set according to a first embodiment disclosed herein includes a plurality of first nanoparticles and a plurality of second nanoparticles. The first nanoparticles include nylon 6 covalently bonded to a first dye and have a first average diameter and a first polydispersity index between 1.15 and 1.19. The second nanoparticles include nylon 6 covalently bonded to a second dye different from the first dye and have a second average diameter and a second polydispersity index between 1.15 and 1.19. The first average diameter and the second average diameter are different. The first average diameter and the second average diameter are each independently between 30 nm and 3000 nm. The first average diameter is at least twice the second average diameter.

[0008] A calibration set according to a second embodiment disclosed in the present specification includes the calibration set according to the first embodiment, and both the first dye and the second dye include lipophilic fluorescent dyes.

[0009] A calibration set according to a third embodiment disclosed in the present specification includes the calibration set according to the first or second embodiment, and the first dye has a first excitation maximum wavelength λEx1. The second dye has a second excitation maximum wavelength λEx2. λEx1 and λEx2 are different. Both λEx1 and λEx2 are in the range of 350 nm to 750 nm.

[0010] A calibration set according to a fourth aspect disclosed in the present specification includes a calibration set according to any one of the first to third aspects, and the calibration set comprises a container containing a mixture including both the first nanoparticles and the second nanoparticles.

[0011] A calibration set according to a fifth aspect disclosed in the present specification includes a calibration set according to any one of the first to fourth aspects, and at least one of the first average diameter and the second average diameter is in the range of 30 nm to 250 nm.

[0012] A calibration set according to a sixth aspect disclosed in the present specification includes a calibration set according to any one of the first to fifth aspects, and both the first nanoparticles and the second nanoparticles have a density of 1.15 g / mL to 1.19 g / mL.

[0013] A calibration set according to a seventh aspect disclosed in the present specification includes a calibration set according to any one of the first to sixth aspects, and both the first nanoparticles and the second nanoparticles contain RNA.

[0014] A method according to an eighth aspect of the present disclosure includes the steps of: uniformly dispersing a plurality of first nanoparticles comprising nylon 6 covalently bonded to a first dye and a plurality of second nanoparticles comprising nylon 6 covalently bonded to a second dye different from the first dye in a calibration suspension; adding a fixed amount of the calibration suspension in which the first nanoparticles and the second nanoparticles are uniformly dispersed to a sample buffer to generate a sample containing the first nanoparticles and the second nanoparticles at a predetermined concentration in the sample buffer; analyzing the sample with a size measurement device to obtain at least one data output related to the fluorescence emission of the first dye and the fluorescence emission of the second dye; and adjusting one or more settings of the size measurement device based on the at least one data output.

[0015] A method according to a ninth aspect disclosed herein includes the method according to the eighth aspect, wherein the first plurality of nanoparticles have a first average diameter and a first polydispersity index greater than or equal to 1.15 and less than or equal to 1.19, and the second plurality of nanoparticles have a second average diameter and a second polydispersity index greater than or equal to 1.15 and less than or equal to 1.19.

[0016] A method according to a tenth aspect disclosed herein includes the method according to the eighth or ninth aspect, wherein the size measuring device is a flow cytometer.

[0017] A method according to an eleventh aspect disclosed herein includes any of the methods according to the eighth to tenth aspects, wherein adjusting one or more settings includes maximizing the signal, minimizing the coefficient of variation, or a combination thereof.

[0018] A method according to a twelfth aspect disclosed herein includes any of the methods according to the eighth to eleventh aspects, wherein the step of adjusting one or more settings includes adjusting settings related to the resolution limit of the particle size measurement, the range of the particle size measurement, the sensitivity of the scattering photomultiplier tube, the baseline noise of the instrument, laser alignment, optical system alignment, stability of the flow cytometer fluidics system, cell sorter drop delay, cell sorter efficiency, or a combination thereof.

[0019] A method according to a thirteenth aspect disclosed in the present specification includes the method according to any one of the eighth to twelfth aspects, in which the first dye and the second dye both include a fat-soluble fluorescent dye.

[0020] A method according to a fourteenth aspect disclosed in the present specification includes the methods according to any of the eighth to thirteenth aspects, and at least one of the first average diameter and the second average diameter is in the range of 30 nm to 250 nm.

[0021] A method according to a fifteenth aspect disclosed in the present specification includes any of the methods according to the eighth to fourteenth aspects, wherein the first nanoparticles and the second nanoparticles both have a density of 1.15 g / mL to 1.19 g / mL.

[0022] A calibration set according to a sixteenth embodiment disclosed herein includes a plurality of nanoparticle groups, each of which comprises a plurality of nanoparticles comprising nylon 6 and a dye. The dye contained in the nanoparticles of each nanoparticle group is different from the corresponding dye of the nanoparticles of the different nanoparticle groups. Each nanoparticle group has a corresponding average diameter and a polydispersity index of 1.15 to 1.19. The corresponding average diameter of each nanoparticle group is at least two times different from the corresponding average diameter of each of the other nanoparticle groups in the plurality of nanoparticle groups. The corresponding average diameters are 30 nm to 3000 nm.

[0023] A calibration set according to a seventeenth aspect disclosed in the present specification includes the calibration set according to the sixteenth aspect, and all of the dyes include lipophilic fluorescent dyes.

[0024] A calibration set according to an eighteenth aspect disclosed herein includes a calibration set according to either the sixteenth or seventeenth aspect, wherein the calibration set comprises a container containing a mixture including a plurality of groups of nanoparticles.

[0025] A calibration set according to a 19th aspect disclosed in the present specification includes a calibration set according to any one of the 16th to 18th aspects, and the multiple nanoparticle groups have a density of 1.15 g / mL to 1.19 g / mL.

[0026] A calibration set according to a twentieth aspect disclosed in the present specification includes a calibration set according to any one of the sixteenth to nineteenth aspects, and at least one of the multiple nanoparticle groups contains RNA.

[0027] Additional features and advantages are set forth in the detailed description that follows, and will become apparent to those skilled in the art in part from the description, or may be learned by practice of the embodiments described herein, including the following detailed description, claims, and accompanying drawings.

[0028] It should be understood that both the foregoing general description and the following detailed description are intended to describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments and are incorporated in and constitute a part of this specification. The drawings illustrate by way of example the various embodiments described herein and, together with the following detailed description, serve to explain the principles and operation of the claimed subject matter. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 is a schematic diagram illustrating nanoparticles having different average sizes compared to various extracellular vesicles according to one or more embodiments shown and described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] In various embodiments, the calibration set includes a plurality of first nanoparticles comprising nylon 6 covalently bonded to a first dye and a plurality of second nanoparticles comprising nylon 6 covalently bonded to a second dye different from the first dye. The first nanoparticles have a first average diameter and a first polydispersity index between 1.15 and 1.19, and the second nanoparticles have a second average diameter and a second polydispersity index between 1.15 and 1.19. In various embodiments, the first average diameter and the second average diameter are different, both of the first average diameter and the second average diameter are between 30 nm and 3000 nm, and the first average diameter is at least twice the second average diameter.

[0031] Reference will now be made in detail to various embodiments, as illustrated in the accompanying drawings, and wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0032] In the present specification, ranges may be expressed in the form of "about" a certain value or more, "about" a certain value to "about" another certain value, or "about" another certain value or less. When a range is expressed in this manner, there are other embodiments including the range or more, the range to the certain value, or the range or less. Similarly, when a value is expressed as an approximation by using "about", it will be understood that there are other embodiments that consist of the specific value. It will also be understood that the endpoints of each range are both relative to each other and independent of each other.

[0033] As used herein, directional terms (e.g., up, down, right, left, front, back, top, bottom, etc.) are merely used with reference to the drawings and are not intended to imply absolute orientations.

[0034] Unless expressly stated otherwise, no method described herein is intended to be construed as requiring that steps be performed in a particular order, nor is any method described herein intended to require a particular orientation for any apparatus. Thus, except where a method claim or embodiment actually describes the order of its steps, and an apparatus claim or embodiment actually describes the order or orientation of individual components, or where a claim or description is otherwise expressly made to the effect that the steps are limited to a particular order, and except where a particular order or orientation of apparatus components is described, no order or orientation is intended to be inferred in any respect. This applies to all non-express matters that may be the basis of interpretation, such as questions of reasoning about the order of steps, the flow of operations, the order of components, or the orientation of components, common meanings derived from grammatical constructions or punctuation, the number or type of embodiments described herein, and the like.

[0035] As used herein, the singular forms "a," "an," and "the" are intended to include reference to the corresponding plurals unless the context clearly indicates that they do not include the plurals. Thus, for example, an expression introducing a certain component with the article "a" also includes embodiments having two or more of that component unless the context clearly indicates otherwise.

[0036] Additionally, the term "formed from" can mean one or more of "comprises," "consists essentially of," or "consists of." For example, when a component is "formed from" a particular material, the component may comprise, consist essentially of, or consist of the particular material.

[0037] Nanoparticles containing nylon 6 and lipophilic fluorescent dye FIGURE 1 is a schematic diagram illustrating one embodiment of a calibration set 100 including multiple nanoparticle groups 102. In FIGURE 1, the individual groups are identified as 102a-102f, with reference numeral 102 referring collectively to any one or more of the multiple groups. Additionally, the individual groups 102a-102f are illustrated as nanoparticle strings each including a number of individual nanoparticles, with one nanoparticle in each group identified as 102a1-102f1, respectively. Although the embodiment illustrated in FIGURE 1 includes six nanoparticle groups, it is contemplated that any number of nanoparticle groups may be included in the calibration set 100.

[0038] Each group 102 includes a plurality of nanoparticles that include nylon 6 covalently bonded to a corresponding dye. Such dyes can include organic dyes (e.g., fluorescein, rhodamine, aminomethylcoumarin (AMCA)), biofluorescent molecules (e.g., green fluorescent protein, phycoerythrin, allophycocyanin), quantum dots, or lipophilic fluorescent dyes. Thus, in FIG. 1, a first group of nanoparticles 102a includes nylon 6 covalently bonded to a first dye, a second group of nanoparticles 102b includes nylon 6 covalently bonded to a second dye, a third group of nanoparticles 102c includes nylon 6 covalently bonded to a third dye, a fourth group of nanoparticles 102d includes nylon 6 covalently bonded to a fourth dye, a fifth group of nanoparticles 102e includes nylon 6 covalently bonded to a fifth dye, and a sixth group of nanoparticles 102f includes nylon 6 covalently bonded to a sixth dye. The dye of each nanoparticle population is different from the dyes of other nanoparticle populations and is therefore a dye uniquely assigned to that particular nanoparticle population, labeling it as such.

[0039] In various embodiments, each dye is a lipophilic fluorescent dye, such as a lipophilic carbocyanine dye. Non-limiting examples of suitable commercially available lipophilic carbocyanine dyes include the lipophilic fluorescent tracer dyes sold as DiB, DiA, DiO, DiI, DiD, and DiR, all available from Biotium, Inc. (Hayward, Calif.). The color, excitation maximum wavelength (λEx), and emission maximum wavelength (λEm) of each of these example dyes are shown in Table 1 below.

[0040] [Table 1]

[0041] As shown in Table 1, these lipophilic fluorescent dyes have excitation maximum wavelengths (e.g., peak wavelengths of the excitation spectrum showing absorption maximum) measured by a monochromator of 353 nm to 750 nm, and therefore the lipophilic fluorescent dyes shown in Table 1 are suitable for use in common size measurement techniques. Such size measurement techniques include, but are not limited to, flow cytometers incorporating optics such as lamps and lasers that can generate optical signals of the excitation maximum wavelengths to excite these dyes. Table 1 also shows that the emission maximum wavelengths measured by a monochromator (corresponding colors that can be observed in response to excitation) are different for each dye. Therefore, the sensitivity and resolution of the measurement can be improved by using multiple fluorescent dyes and associating each fluorescent dye with nanoparticles having a corresponding average diameter.

[0042] In various embodiments, the dye is covalently attached to a polymer, including nylon 6. In some embodiments, as described in more detail below, the dye is attached to the polymer prior to the formation of the nanoparticles, resulting in the dye being uniformly distributed throughout each nanoparticle. Nylon 6 has the following structure:

[0043] [ka]

[0044] The carbonyl group of the nylon 6 structure allows for covalent binding with the dye, which not only allows tracking of the nylon 6 nanoparticles, but also reduces or prevents leakage of the dye from the particles or dissociation of the dye. For example, in conventional standards in which the dye is encapsulated or incubated in polymer particles, it is believed that no direct chemical bond is formed between the dye and the particles. Therefore, in conventional standards, the dye may leak from the particles, causing errors in the measurement. In contrast, in various embodiments, the fluorescent dye described herein is covalently bound to nylon 6 and incorporated into the nanoparticles, and is believed not to release the fluorescent dye unless the polymer is decomposed, thereby reducing the possibility of dissociation of the dye from the nanoparticles.

[0045] In various embodiments, the nanoparticles have a density of 1.15 g / mL to 1.19 g / mL, as measured according to ASTM standard D 792. For example, the density of the nanoparticles can be 1.15 g / mL to 1.19 g / mL, 1.15 g / mL to 1.18 g / mL, 1.15 g / mL to 1.17 g / mL, 1.15 g / mL to 1.16 g / mL, 1.16 g / mL to 1.19 g / mL, 1.16 g / mL to 1.18 g / mL, 1.16 g / mL to 1.17 g / mL, 1.17 g / mL to 1.19 g / mL, 1.17 g / mL to 1.18 g / mL, or 1.18 g / mL to 1.19 g / mL. Thus, the problem of differences in material density between EVs and reference nanoparticles is eliminated, as the density of the nanoparticles is similar to that of EVs (i.e., 1.15-1.19 g / mL) and measurably higher than that of mammalian cells (i.e., 1.04-1.05 g / mL).

[0046] Each nanoparticle group has a mean hydrodynamic diameter (referred to herein as the "mean diameter") measured according to ISO 22412, collectively referred to as d n The average hydrodynamic diameter of each nanoparticle group is measured by light scattering measurement techniques such as dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), and visually confirmed by microscopy such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In Figure 1, d a is the average diameter of the first nanoparticle group 102a, d b is the average diameter of the second nanoparticle group 102b, d c is the average diameter of the third nanoparticle group 102c, d d is the average diameter of the fourth nanoparticle group 102d, d e is the average diameter of the fifth nanoparticle group 102e, d fis the average diameter of the sixth nanoparticle group 102f.

[0047] In various embodiments, the standard deviation of particle size of each nanoparticle group (e.g., the standard deviation of the maximum diameter of each particle in each group) is statistically determined by software calculation and is 15% or less. For example, the standard deviation of particle size of each nanoparticle group can be 15% or less, 12.5% ​​or less, 10% or less, 8% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less. In some embodiments, the standard deviation of the average diameter of each nanoparticle group is 0 to 15%, 0 to 12.5%, 0 to 10%, 0 to 8%, 0 to 6%, 0 to 5%, 0 to 4%, 0 to 3%, 0 to 2%, 0 to 1%, 0 to 0.5%, 0.5% to 15%, 0.5% to 12.5%, 0.5% to 10%, 0.5% to 8%, 0.5% to 6%. , 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2%, 0.5% to 1%, 1% to 15%, 1% to 12.5%, 1% to 10%, 1% to 8%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, or 1% to 2%, including all ranges and subranges within these ranges. In some embodiments, it is contemplated that each standard deviation can be a number within this range and may be the same or different than the standard deviation of one or more other nanoparticle populations.

[0048] In various embodiments, each nanoparticle population can be further characterized by a polydispersity index of 1.15 to 1.19. The polydispersity index (also referred to as "PDI" or D-stroke) is a measure of the spread of particle size distribution determined by cumulant analysis of a nanoparticle population and is measured using dynamic light scattering (DLS) techniques in accordance with ISO 22412 and ISO 13321. In some embodiments, the PDI of each nanoparticle population can be 1.15 to 1.18, 1.15 to 1.17, 1.15 to 1.16, 1.16 to 1.19, 1.16 to 1.18, 1.16 to 1.17, 1.17 to 1.19, 1.17 to 1.17, or 1.18 to 1.19 (including all ranges and subranges within these ranges). It is contemplated that, in some embodiments, each PDI can be a value within this range and can be the same or different from the PDI of one or more other groups of nanoparticles.

[0049] The mean diameter of each group, d n is different from the average diameter of each of the other nanoparticle groups. Therefore, in Figure 1, d a d b , d c , d d , d e , d f Unlike d b d c , d d , d e , d f Unlike d c d d , d e , d f Unlike d d d e , d f Unlike d e d f In various embodiments, each average diameter is at least twice the average diameter of the next largest. For example, in FIG. b (100 nm) is d a (50 nm) x 2, d c (250 nm) is db (100 nm) at least twice, d d (500nm) is d c (250 nm), d e (1000nm) is d d (500 nm), d f (2000nm) is d e (1000 nm).

[0050] In various embodiments, each average diameter d n are independently 30 nm or more and 3000 nm or less. For example, each average diameter d n can be 30 nm to 3000 nm, 30 nm to 2500 nm, 30 nm to 2000 nm, 30 nm to 1500 nm, 30 nm to 1000 nm, 30 nm to 750 nm, 30 nm to 500 nm, 30 nm to 300 nm, 30 nm to 250 nm, 30 nm to 100 nm, 50 nm to 3000 nm, 50 nm to 2500 nm, 50 nm to 2000 nm, 50 nm to 1500 nm, 50 nm to 1000 nm, 50 nm to 750 nm, 50 nm to 500 nm, 50 nm to 300 nm, or 50 nm to 250 nm (including all any ranges and subranges within these ranges). In some embodiments, the average diameter of at least one population of nanoparticles is 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 75 nm or less, or 50 nm or less.

[0051] It should be understood that while FIG. 1 illustrates groups 102a-102f having particular average diameters, the average diameters of the calibration set 100 may vary depending on the specific embodiment. For example, one or more groups of nanoparticles may be selected such that the average diameter of the nanoparticles is in a relatively close range (within 50 nm, within 100 nm, or within 250 nm) to the type of EV being investigated. In addition to the calibration set 100, FIG. 1 also shows a schematic representation of various types of EVs 104 and their approximate average diameters. Specifically, FIG. 1 includes exosomes 106 having an average diameter of about 100 nm or less, a first set of microvesicles 108 having an average diameter of about 100 nm to about 1000 nm, a second set of microvesicles 110 having an average diameter of about 1000 nm to about 2000 nm, and oncosomes 112 having an average diameter of more than about 2000 nm. Thus, as shown in FIG. 1, the nanoparticles described herein may be used to approximate the size of various types of EVs.

[0052] In some embodiments, one or more of the nanoparticle populations may comprise RNA. In such embodiments, the RNA is attached to the polymer via the amide groups of the polymer prior to nanoparticle formation, as described in further detail below. Thus, the RNA is uniformly distributed throughout each nanoparticle. As used herein, the term "RNA molecule" or "RNA" refers to ribonucleic acid, i.e., a polymer of nucleotides. The nucleotides are typically the monomers adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate, which are linked together along a so-called backbone formed by phosphodiester bonds between the sugar (i.e., ribose) of a first monomer and the phosphate moiety of an adjacent second monomer. In embodiments in which the nanoparticles comprise RNA, the RNA may have any predefined RNA sequence. This RNA sequence may, for example, be a general RNA sequence or may be a specific sequence of interest. Thus, as described in further detail below, the results of the analysis may be verified by adding nanoparticles containing RNA to a biological sample and observing the effect of each technological step in the overall experimental protocol.

[0053] Method for producing nanoparticles Nanoparticles of various embodiments can be produced according to any one of a number of nanoprecipitation techniques, including, but not limited to, pipette drop nanoprecipitation, flow-controlled T-mixer nanoprecipitation, and flow-controlled microfluidic nanoprecipitation. Generally, nanoparticles are prepared by dissolving nylon 6 pellets in a protic polar solvent (e.g., acetic acid, formic acid, ethanol, etc.) or aprotic polar solvent (e.g., tetrahydrofuran, acetone, acetonitrile, etc.) until homogeneous (e.g., nylon 6 is uniformly dispersed in the solvent). Dyes are either dissolved in the nylon 6 and solvent mixture or introduced by emulsion with a miscible solvent. Aqueous mixtures of water and surfactants (e.g., anti-agglomerating agents such as polyvinyl alcohol and Pluronic®) are prepared as the mixing and recovery solutions.

[0054] In various embodiments, the size of the nanoparticles is controlled by controlling the polymer concentration in the organic phase, the selection of the organic solvent, the surfactant concentration in the aqueous phase, the selection of the surfactant, the mixing ratio of the organic phase to the aqueous phase, the flow rates of the organic and aqueous phases during mixing, and the device used for nanoprecipitation. For example, increasing the polymer concentration in the organic phase or the mixing ratio of the organic phase to the aqueous phase increases the particle size. For example, increasing the surfactant concentration in the aqueous phase or the ratio of the aqueous phase to the organic phase decreases the particle size. In addition, the selection of the organic solvent or surfactant can also have various effects on the particle size. Such effects vary depending on the chemical properties of the solvent or surfactant itself, such as chemical density, charge, and miscibility, and can be identified experimentally. In addition, an increase in the flow rate during mixing is also thought to affect the particle size (generally, the particle size decreases with an increase in the flow rate). However, apart from such effects, the flow rates of the organic phase and the aqueous phase can be changed independently, which may result in an effect similar to that of the change in the mixing ratio.

[0055] Furthermore, the specific nanoparticle formation method used will affect particle size variation. The more precisely the device is controlled, the more accurate and precise the target particle size can be achieved. For example, a flow-controlled microfluidic method will produce particles with a lower PDI than a T-mixer method, which in turn will produce particles with a lower PDI than a pipette method.

[0056] In an embodiment for preparing nanoparticles by the pipette drop method, a small amount of polymer (e.g., 0.5% to 2.0% w / v of nylon 6 polymer) is dissolved in an organic solvent along with a lipophilic fluorescent dye. In some embodiments, the fluorescent dye is added in an amount that results in a dye / polymer ratio of 0.25% to 1.5% by weight, and the polymer to dye ratio is 50:1 to 250:1. In certain embodiments, the polymer to dye ratio is approximately 100:1. In embodiments where the dye is not soluble in the organic solvent, the emulsion is made by dissolving the dye in a miscible solvent (e.g., dimethylsulfoxide (DMSO)) and adding it to the organic phase while vortexing or sonicating until a homogenous solution is obtained. The organic phase containing the polymer and dye is then added dropwise to the aqueous phase while stirring. In some embodiments, for example, the aqueous phase contains 0.5% to 5.0% w / v of a surfactant in water. The organic solvent is then removed by evaporation. In some embodiments, evaporation can be performed by stirring (eg, for at least 3 hours) or using a rotary evaporator (eg, for about 15 minutes).

[0057] The nanoparticles are then recovered from the aqueous medium by centrifugation. In some embodiments, the aqueous medium can be centrifuged at about 16,000×g for about 15 minutes at room temperature. The aqueous supernatant is decanted, and the nanoparticle pellet is washed by resuspending the nanoparticles in 10 mL of water and centrifuging at about 16,000×g for about 15 minutes at room temperature. In some embodiments, this washing process can be performed two or more times. Finally, the washed dispersion is lyophilized for at least 48 hours to obtain lyophilized nanoparticles. The nanoparticles can be stored at a temperature of −20° C. until needed.

[0058] In an embodiment of preparing nanoparticles using a flow-controlled T-mixer method or a flow-controlled microfluidic method, nylon 6 at a concentration of about 50 mg / mL and a lipophilic fluorescent dye at a concentration of 0.5% by weight relative to the polymer are dissolved in an organic solvent. An aqueous phase is prepared by adding a surfactant of 0.5% to 5.0% by weight / volume to water. Each phase is then placed in a reservoir. In various embodiments, the reservoir is a syringe. In an embodiment using a T-mixer method, the syringes are attached to a syringe pump with the outlet lines from each syringe communicating with the respective inlet ports of the T-mixer. A collection vessel is then placed at the outlet port of the T-mixer. The syringe pump is programmed based on experimentally determined flow rates to simultaneously deliver the organic and aqueous phases to the T-mixer at the appropriate flow rates and total amounts. In an embodiment using a flow-controlled microfluidic method, the organic and aqueous phases are delivered to the microfluidic chip at experimentally determined flow rates. The nanoparticles are then collected and washed in the same manner as described above.

[0059] It is contemplated that the methods for preparing nanoparticles described herein may be adjusted or varied depending on the particular embodiment, e.g., amounts of raw materials, centrifugation times, etc., may be adjusted or varied based on the desired size of the nanoparticles to be formed.

[0060] In embodiments in which RNA is incorporated into nanoparticles, the RNA molecules can be added to the aqueous phase of the above mixture. In such embodiments, the aqueous phase contains a weakly acidic buffer (e.g., sodium acetate or Tris-EDTA) to promote interaction of the RNA with the amide groups in nylon 6. In some embodiments, the pH of the aqueous phase is 4.5 to 7.

[0061] In various embodiments, the nanoparticles can be filtered using size selection methods, such as size exclusion centrifugal filtration, to achieve a desired size distribution of the nanoparticles.

[0062] Although various methods have been described above for forming nanoparticles containing nylon 6 and a dye, it should be understood that other methods known to those skilled in the art can be used to produce the nanoparticles, including, but not limited to, emulsion methods, electrospray methods, and plasma deposition methods.

[0063] In various embodiments, two or more groups of nanoparticles are added in equal amounts to a single container, such as a vial, to form a standard set. In some embodiments, water or other solvents can be added to resuspend the nanoparticles before use. It is believed that storing the nanoparticles in a concentrated state can reduce hydrolysis of the polymer that can occur when the nanoparticles are suspended in water, thereby extending the shelf life of the nanoparticles.

[0064] How nanoparticles are used The nanoparticles according to various embodiments can be used in a variety of ways. For example, the nanoparticles according to various embodiments can be used to characterize small biological vesicles such as various EVs. For example, in some embodiments, the nanoparticles are used as standards spiked into biological samples to evaluate isolation efficiency and standardize techniques. In some embodiments, nanoparticles carrying specific RNA sequences may allow independent validation of results. Such validation may include, for example, demonstrating that EVs retain their functionality (e.g., ability to transcribe RNA) after isolation using a particular isolation method. Also, for example, the nanoparticles are used as calibration standards for EV quantification and sizing techniques. Such techniques include, but are not limited to, flow cytometry.

[0065] In some embodiments, the calibration set can be used to calibrate a size measuring device, such as a flow cytometer. Thus, a method for calibrating a size measuring device includes uniformly dispersing first nanoparticles and second nanoparticles in a calibration suspension. The first nanoparticles include a first dye, and the second nanoparticles include a second dye different from the first dye. Then, a certain amount of the calibration suspension in which the first nanoparticles and the second nanoparticles are uniformly dispersed is added to a sample buffer to generate a sample containing a predetermined concentration of the first nanoparticles and the second nanoparticles in the sample buffer.

[0066] The sample is analyzed by a size measuring device (e.g., a Luminex Guava® easyCyte™ flow cytometer or a Thermo Fisher Attune™ NxT flow cytometer) to obtain at least one data output related to the fluorescence emission of the first dye and the fluorescence emission of the second dye. One or more settings of the size measuring device are then adjusted based on the at least one data output. In some embodiments, adjusting one or more settings can include maximizing the signal, minimizing the coefficient of variation, or a combination thereof. In some embodiments, the settings to be adjusted relate to the resolution limit of the particle size measurement, the range of the particle size measurement, the sensitivity of the scatter photomultiplier tube, the baseline noise of the device, the laser alignment, the optical alignment, the stability of the flow cytometer fluidics system, the drop delay of the cell sorter, the efficiency of the cell sorter, or a combination thereof. It is contemplated that other adjustments can be made. Such other adjustments will vary depending on the particular embodiment and will be based at least in part on the specific size measuring device being calibrated.

[0067] In embodiments in which the nanoparticles comprise RNA, the nanoparticles can be used as an artificial EV spike-in standard to standardize the recovery of EVs and associated RNA from biological fluids and facilitate independent validation of results. In such embodiments, the artificial EV standard can be used to ensure that the RNA is not damaged by the experimental protocol. As will be appreciated by those skilled in the art, the experimental protocol can include, for example, a quantitative assay such as the RiboGreen® assay or a functional assay.

[0068] In various embodiments described herein, the calibration set includes multiple nanoparticle populations of nylon 6 and corresponding dye nanoparticles, each with a corresponding mean diameter. In this case, these nanoparticles exhibit a density, light scattering effect, and size range similar to EVs, making them particularly suitable for use as standards for EV studies. Furthermore, the nanoparticles of various embodiments can contain RNA molecules, which allows the nanoparticles to be used as an artificial EV spike in some experimental protocols to standardize the recovery of EVs and their associated RNA and facilitate independent validation of results.

[0069] It will be apparent to those skilled in the art that various modifications and variations may be made to the embodiments described herein without departing from the spirit and scope of the claimed subject matter. Accordingly, to the extent that modifications and variations of the various embodiments described herein do not depart from the scope of the appended claims and their equivalents, such modifications and variations are intended to be included within the scope of this specification.

[0070] Preferred embodiments of the present invention will be described below in detail.

[0071] EMBODIMENT 1 a plurality of first nanoparticles comprising nylon 6 covalently bonded to a first dye, the first nanoparticles having a first average diameter and a first polydispersity index greater than or equal to 1.15 and less than or equal to 1.19; second nanoparticles comprising nylon 6 covalently bonded to a second dye different from the first dye, the second nanoparticles having a second average diameter and a second polydispersity index greater than or equal to 1.15 and less than or equal to 1.19; A calibration set comprising: the first average diameter and the second average diameter are different; the first average diameter and the second average diameter are each independently 30 nm or more and 3000 nm or less; The first average diameter is at least twice the second average diameter of a calibration set.

[0072] EMBODIMENT 2 2. The calibration set of embodiment 1, wherein the first dye and the second dye both comprise lipophilic fluorescent dyes.

[0073] EMBODIMENT 3 the first dye has a first excitation maximum wavelength λEx1; the second dye has a second excitation maximum wavelength λEx2; λEx1 and λEx2 are different, 2. The calibration set according to embodiment 1, wherein λEx1 and λEx2 are both in the range of 350 nm to 750 nm.

[0074] EMBODIMENT 4 2. The calibration set of embodiment 1, wherein the calibration set comprises a container containing a mixture including both the first nanoparticles and the second nanoparticles.

[0075] EMBODIMENT 5 2. The calibration set according to embodiment 1, wherein at least one of the first average diameter and the second average diameter is in the range of 30 nm to 250 nm.

[0076] EMBODIMENT 6 2. The calibration set according to embodiment 1, wherein the first nanoparticles and the second nanoparticles both have a density of 1.15 g / mL to 1.19 g / mL.

[0077] EMBODIMENT 7 2. The calibration set of embodiment 1, wherein the first nanoparticles and the second nanoparticles both comprise RNA.

[0078] EMBODIMENT 8 1. A method of using a calibration set, the method comprising: uniformly dispersing in a calibration suspension a plurality of first nanoparticles comprising nylon 6 covalently bonded to a first dye and a plurality of second nanoparticles comprising nylon 6 covalently bonded to a second dye different from the first dye; adding a fixed amount of the calibration suspension in which the first nanoparticles and the second nanoparticles are uniformly dispersed to a sample buffer to generate a sample containing the first nanoparticles and the second nanoparticles at a predetermined concentration in the sample buffer; analyzing the sample with a size measurement device to obtain at least one data output relating to the fluorescence emission of a first dye and the fluorescence emission of a second dye; adjusting one or more settings of the size measuring device based on the at least one data output; The method includes:

[0079] EMBODIMENT 9 the plurality of first nanoparticles having a first average diameter and a first polydispersity index greater than or equal to 1.15 and less than or equal to 1.19; 9. The method of embodiment 8, wherein the plurality of second nanoparticles has a second average diameter and a second polydispersity index greater than or equal to 1.15 and less than or equal to 1.19.

[0080] EMBODIMENT 10 9. The method of embodiment 8, wherein the size measuring device is a flow cytometer.

[0081] EMBODIMENT 11 11. The method of embodiment 10, wherein adjusting the one or more settings comprises maximizing the signal, minimizing the coefficient of variation, or a combination thereof.

[0082] EMBODIMENT 12 11. The method of embodiment 10, wherein adjusting one or more settings comprises adjusting settings related to a resolution limit of particle size measurement, a range of particle size measurement, a scattering photomultiplier sensitivity, an instrument baseline noise, a laser alignment, an optical system alignment, a flow cytometer fluidics stability, a cell sorter drop delay, a cell sorter efficiency, or a combination thereof.

[0083] EMBODIMENT 13 9. The method of embodiment 8, wherein the first dye and the second dye both comprise lipophilic fluorescent dyes.

[0084] EMBODIMENT 14 9. The method according to embodiment 8, wherein at least one of the first average diameter and the second average diameter is in the range of 30 nm to 250 nm.

[0085] EMBODIMENT 15 9. The method of embodiment 8, wherein the first nanoparticles and the second nanoparticles both have a density between 1.15 g / mL and 1.19 g / mL.

[0086] EMBODIMENT 16 A calibration set including a plurality of nanoparticle groups each including a plurality of nanoparticles including nylon 6 and a dye, the dye contained in the nanoparticles of each nanoparticle group is different from the corresponding dye contained in the nanoparticles of a different nanoparticle group; each of the nanoparticle populations has a corresponding mean diameter and polydispersity index of 1.15 or greater and 1.19 or less; the corresponding average diameter of each nanoparticle group differs by at least a factor of two from the corresponding average diameter of each of the other nanoparticle groups in the plurality of nanoparticle groups; A calibration set, wherein the corresponding mean diameter is 30 nm or more and 3000 nm or less.

[0087] EMBODIMENT 17 17. The calibration set of embodiment 16, wherein the dyes all comprise lipophilic fluorescent dyes.

[0088] EMBODIMENT 18 17. The calibration set of embodiment 16, wherein the calibration set comprises a container containing a mixture comprising the plurality of nanoparticles.

[0089] EMBODIMENT 19 17. The calibration set of embodiment 16, wherein the plurality of nanoparticle groups have a density of 1.15 g / mL to 1.19 g / mL.

[0090] EMBODIMENT 20 17. The calibration set of embodiment 16, wherein at least one of the plurality of nanoparticles comprises RNA. [Explanation of symbols]

[0091] 100 Calibration Sets 102 Nanoparticles 104 Extracellular Vesicles (EVs) 106 Exosomes 108 Microvesicles 110 Microvesicles 112 Oncosome

Claims

1. a plurality of first nanoparticles comprising nylon 6 covalently bonded to a first dye, the first nanoparticles having a first average diameter and a first polydispersity index greater than or equal to 1.15 and less than or equal to 1.19; second nanoparticles comprising nylon 6 covalently bonded to a second dye different from the first dye, the second nanoparticles having a second average diameter and a second polydispersity index greater than or equal to 1.15 and less than or equal to 1.19; A calibration set comprising: the first average diameter and the second average diameter are different; the first average diameter and the second average diameter are each independently 30 nm or more and 3000 nm or less; The first average diameter is at least twice the second average diameter.

2. The calibration set of claim 1 , wherein the first dye and the second dye both comprise lipophilic fluorescent dyes.

3. the first dye has a first excitation maximum wavelength λEx1; the second dye has a second excitation maximum wavelength λEx2; λEx1 and λEx2 are different, 2. The calibration set according to claim 1, wherein λEx1 and λEx2 are both in the range of 350 nm to 750 nm.

4. The calibration set of claim 1 , wherein the calibration set comprises a container containing a mixture including both the first nanoparticles and the second nanoparticles.

5. 2. The calibration set of claim 1, wherein at least one of the first average diameter and the second average diameter is in the range of 30 nm to 250 nm.

6. 2. The calibration set of claim 1, wherein the first nanoparticles and the second nanoparticles both have a density between 1.15 g / mL and 1.19 g / mL.

7. The calibration set of claim 1 , wherein the first nanoparticles and the second nanoparticles both comprise RNA.

8. A method of using a calibration set, the method comprising: uniformly dispersing in a calibration suspension a plurality of first nanoparticles comprising nylon 6 covalently bonded to a first dye and a plurality of second nanoparticles comprising nylon 6 covalently bonded to a second dye different from the first dye; adding a fixed amount of the calibration suspension, in which the first nanoparticles and the second nanoparticles are uniformly dispersed, to a sample buffer to generate a sample containing the first nanoparticles and the second nanoparticles at a predetermined concentration in the sample buffer; analyzing the sample with a size measurement device to obtain at least one data output relating to the fluorescence emission of a first dye and the fluorescence emission of a second dye; adjusting one or more settings of the size measuring device based on the at least one data output; The method includes:

9. the plurality of first nanoparticles having a first average diameter and a first polydispersity index greater than or equal to 1.15 and less than or equal to 1.19; The method of claim 8 , wherein the plurality of second nanoparticles has a second average diameter and a second polydispersity index greater than or equal to 1.15 and less than or equal to 1.

19.

10. The method of claim 8 , wherein the size measuring device is a flow cytometer.

11. The method of claim 10 , wherein adjusting the one or more settings comprises maximizing a signal, minimizing a coefficient of variation, or a combination thereof.

12. 11. The method of claim 10, wherein adjusting the one or more settings comprises adjusting settings related to a resolution limit of particle size measurement, a range of particle size measurement, a scattering photomultiplier sensitivity, an instrument baseline noise, a laser alignment, an optical system alignment, a stability of a flow cytometer fluidics system, a cell sorter drop delay, a cell sorter efficiency, or a combination thereof.

13. The method of claim 8 , wherein the first dye and the second dye both comprise lipophilic fluorescent dyes.

14. The method of claim 8, wherein at least one of the first average diameter and the second average diameter is in the range of 30 nm to 250 nm.

15. The method of claim 8, wherein the first nanoparticles and the second nanoparticles both have a density between 1.15 g / mL and 1.19 g / mL.

16. A calibration set including a plurality of nanoparticle populations each including a plurality of nanoparticles including nylon 6 and a dye, the dye contained in the nanoparticles of each nanoparticle group is different from the corresponding dye contained in the nanoparticles of a different nanoparticle group; each of the nanoparticle populations having a corresponding mean diameter and polydispersity index of 1.15 or greater and 1.19 or less; the corresponding average diameter of each nanoparticle group differs from the corresponding average diameter of each of the other nanoparticle groups in the plurality of nanoparticle groups by a factor of at least two; A calibration set, wherein the corresponding average diameter is 30 nm or more and 3000 nm or less.

17. The calibration set of claim 16 , wherein the dyes all comprise lipophilic fluorescent dyes.

18. The calibration set of claim 16 , wherein the calibration set comprises a container containing a mixture including the plurality of nanoparticles.

19. 17. The calibration set of claim 16, wherein the plurality of nanoparticles have a density between 1.15 g / mL and 1.19 g / mL.

20. The calibration set of claim 16 , wherein at least one of the plurality of nanoparticles comprises RNA.

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