Calibration device comprising nanoparticles with nylon-6 and a dye

Abstract

A calibration device comprising first nanoparticles comprising nylon-6 covalently bound 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. The calibration device further comprises second nanoparticles comprising nylon-6 covalently bound to a second dye, the second dye being 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. The first average diameter is different from the second average diameter, the first average diameter and the second average diameter are each independently greater than or equal to 30 nm and less than or equal to 3000 nm, and the first average diameter is at least twice the second average diameter.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 980,653, filed February 24, 2020, pursuant to 35 U.SC §119, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This specification generally relates to calibration devices, and more specifically, to calibration devices having properties similar to extracellular vesicles (EVs). Background Technology

[0004] Extracellular vesicle (EV) research has expanded to methods for isolating and concentrating EVs from serum and cells for therapeutic applications. The term "extracellular vesicle" generally refers to particles that are naturally released from cells, defined by a liposome bilayer, and cannot replicate. These particles can be subdivided by size and function, with the smallest being exosomes as small as 30 nm and the largest being microvesicles as large as 2 μm.

[0005] However, characterization methods lack calibration standards suitable for quantification and sizing techniques of EVs. While polystyrene bead standards are routinely used in sizing instruments such as flow cytometers, polystyrene beads possess properties that match mammalian cells, such as density, light scattering effects, size, and fluorescent label binding affinity. Although EVs are products that can be derived from mammalian progenitor cells, their inherent properties differ from those of their progenitor cells, and therefore also differ from those of polystyrene beads.

[0006] Therefore, alternative calibration devices are needed, especially those with properties similar to EVs. Summary of the Invention

[0007] The various embodiments described herein provide calibration devices for nanoparticles with properties similar to EV. Specifically, the calibration device includes first nanoparticles of nylon-6 covalently bonded to a first dye and 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, which is different from the first average diameter. The nanoparticles have a size range, density, and light scattering effect similar to EV and can be used as calibration standards for sizing and quantifying techniques, as will be described in more detail below.

[0008] According to a first aspect disclosed herein, a calibration device includes a first nanoparticle and a second nanoparticle, the first nanoparticle comprising nylon-6 covalently bonded to a first dye, the first nanoparticle 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 second nanoparticle comprising nylon-6 covalently bonded to a second dye, the second dye being different from the first dye, the second nanoparticle 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, wherein: the first average diameter and the second average diameter are different; the first average diameter and the second average diameter are each independently greater than or equal to 30 nm and less than or equal to 3000 nm; and the first average diameter is at least twice the second average diameter.

[0009] According to a second aspect disclosed herein, a calibration apparatus includes the calibration apparatus according to the first aspect, wherein the first dye and the second dye each comprise a lipophilic fluorescent dye.

[0010] According to a third aspect disclosed herein, a calibration apparatus includes a calibration apparatus according to any of the preceding aspects, wherein: a first dye includes a first maximum excitation wavelength λEx1; a second dye includes a second maximum excitation wavelength λEx2; λEx1 and λEx2 are not the same; and λEx1 and λEx2 are each from 350 nm to 750 nm.

[0011] According to a fourth aspect disclosed herein, a calibration apparatus includes the calibration apparatus according to any of the preceding aspects, wherein the calibration apparatus includes a container comprising a mixture containing first nanoparticles and second nanoparticles.

[0012] According to the fifth aspect disclosed herein, a calibration apparatus includes the calibration apparatus according to any of the preceding aspects, wherein at least one of the first average diameter and the second average diameter is 30 nm to 250 nm.

[0013] According to the sixth aspect disclosed herein, a calibration device includes the calibration device according to any of the preceding aspects, wherein the first nanoparticle and the second nanoparticle each have a density of 1.15 g / mL to 1.19 g / mL.

[0014] According to the seventh aspect disclosed herein, a calibration device includes the calibration device according to any of the preceding aspects, wherein the first nanoparticle and the second nanoparticle each comprise RNA.

[0015] According to the eighth aspect disclosed herein, a method includes: uniformly dispersing first nanoparticles and second nanoparticles in a calibration suspension, the first nanoparticles comprising nylon-6 covalently bound to a first dye, the second nanoparticles comprising nylon-6 covalently bound to a second dye, the second dye being different from the first dye; adding a predetermined volume of the calibration suspension containing the uniformly dispersed first nanoparticles and second nanoparticles to a sample buffer to produce a sample containing a predetermined concentration of the first nanoparticles and second nanoparticles in the sample buffer; analyzing the sample with a sizing instrument to obtain at least one data output relating to the fluorescence output of the first dye and the fluorescence output of the second dye; and adjusting one or more settings of the sizing instrument based on said at least one data output.

[0016] According to the ninth aspect disclosed herein, a method includes the method according to the eighth aspect, wherein: a first nanoparticle has 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 a second nanoparticle 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.

[0017] According to the 10th aspect disclosed herein, a method includes the method according to the 8th or 9th aspect, wherein the sizing instrument is a flow cytometer.

[0018] According to the 11th aspect disclosed herein, a method includes the method according to any one of the 8th to 10th aspects, wherein adjusting the one or more settings includes maximizing the signal, minimizing the coefficient of variation, or a combination thereof.

[0019] According to the 12th aspect disclosed herein, a method includes the method according to any one of aspects 8 to 11, wherein adjusting the one or more settings includes adjusting settings regarding: the resolution limit of particle size measurement, the range of particle size measurement, the sensitivity of the scattering photomultiplier tube, baseline instrument noise, laser alignment, optical alignment, stability of the flow cytometer's fluid system, drop delay of the cell sorter, cell sorter efficiency, or a combination thereof.

[0020] According to aspect 13 disclosed herein, a method includes the method according to any one of aspects 8 to 12, wherein the first dye and the second dye each comprise a lipophilic fluorescent dye.

[0021] According to aspect 14 disclosed herein, a method includes the method according to any one of aspects 8 to 13, wherein at least one of the first average diameter and the second average diameter is 30 nm to 250 nm.

[0022] According to aspect 15 disclosed herein, a method includes the method according to any one of aspects 8 to 14, wherein the first nanoparticle and the second nanoparticle each have a density of 1.15 g / mL to 1.19 g / mL.

[0023] According to the 16th aspect disclosed herein, a calibration device includes: multiple groups of nanoparticles comprising nylon-6, wherein each nanoparticle in one group further includes a dye different from the corresponding dye in different groups of nanoparticles, wherein: each group of nanoparticles has a corresponding average diameter and a polydispersity index greater than or equal to 1.15 and less than or equal to 1.19; the corresponding average diameter of each group of nanoparticles differs from the corresponding average diameter of each of the other groups of nanoparticles by at least two times; and each corresponding average diameter is greater than or equal to 30 nm and less than or equal to 3000 nm.

[0024] According to the 17th aspect disclosed herein, a calibration apparatus includes the calibration apparatus according to the 16th aspect, wherein each of the dyes comprises a lipophilic fluorescent dye.

[0025] According to aspect 18 disclosed herein, a calibration apparatus includes the calibration apparatus according to aspect 16 or 17, wherein the calibration apparatus includes a container comprising a mixture containing multiple groups of nanoparticles.

[0026] According to aspect 19 disclosed herein, a calibration device includes the calibration device according to any one of aspects 16 to 18, wherein the plurality of nanoparticles have a density of 1.15 g / mL to 1.19 g / mL.

[0027] According to aspect 20 disclosed herein, a calibration device includes the calibration device according to any one of aspects 16 to 19, wherein at least one of the plurality of nanoparticles comprises RNA.

[0028] Further features and advantages are set forth in the following detailed description, some of which will be apparent to those skilled in the art from the description, or will be recognized by practicing the embodiments described herein, including the following detailed description, the claims, and the accompanying drawings.

[0029] It should be understood that the foregoing general description and the following detailed description both depict various embodiments and are intended to provide an overall assessment or framework for understanding the nature and characteristics of the claimed subject matter. The included drawings provide a further understanding of the various embodiments and are incorporated in and form a part of this specification. The drawings illustrate the various embodiments described herein and, together with the description, serve to explain the principles and operation of the claimed subject matter. Attached Figure Description

[0030] Figure 1 The diagram illustrates a group of nanoparticles according to one or more embodiments shown and described herein, the nanoparticles having a different average diameter compared to various extracellular vesicles. Detailed Implementation

[0031] In various embodiments, a calibration device includes a first nanoparticle and a second nanoparticle, the first nanoparticle comprising nylon-6 covalently bonded to a first dye, and the second nanoparticle comprising nylon-6 covalently bonded to a second dye, the second dye being different from the first dye. The first nanoparticle has 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 nanoparticle 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. In various embodiments, the first average diameter and the second average diameter are different, and each of the first average diameter and the second average diameter is greater than or equal to 30 nm and less than or equal to 3000 nm, wherein the first average diameter is at least twice the second average diameter.

[0032] The various embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. The same reference numerals will be used as much as possible in the drawings to denote the same or similar parts.

[0033] In this document, a range may be expressed as beginning "about" a specific value and / or ending "about" another specific value. Another implementation of expressing such a range includes beginning with said specific value and / or ending with said other specific value. Similarly, when a numerical value is expressed as an approximation using the antecedent "about," it should be understood that the specific value constitutes another implementation. It should also be understood that the endpoints of each range are important both in relation to and independent of the other endpoint.

[0034] The directional terms used in this article, such as up, down, left, right, front, back, top, and bottom, are only for reference to the accompanying drawings and are not intended to imply absolute orientation.

[0035] Unless otherwise expressly stated, no method described herein should be construed as requiring its steps to be performed in a specific order or requiring any device to have a particular orientation. Therefore, if a method claim or implementation does not actually describe the order in which its steps are to be followed, or if any device claim or implementation does not actually describe the order or orientation of the components, or if the claims or description do not otherwise specifically state that the steps are limited to a specific order, or do not describe a specific order or orientation of the device components, then in no way should the order or orientation be inferred. This applies to any possible non-expressive basis of interpretation, including: logical questions concerning the arrangement of steps, operational flow, the order of components, or the orientation of components; questions of obvious meaning derived from grammatical organization or punctuation; and questions of the number or type of embodiments described in the specification.

[0036] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the / said” used herein include plural references. Thus, for example, a reference to “an” component includes aspects having two or more such components, unless otherwise explicitly stated in the text.

[0037] The term "formed from" can mean one or more of the following: containing, substantially composed of, or consisting of. For example, a component formed from a particular material can contain that particular material, consist substantially of that particular material, or consist of that particular material.

[0038] Nanoparticles containing nylon-6 and lipophilic fluorescent dyes

[0039] Figure 1 An exemplary embodiment of the calibration device 100 is illustrated, which includes multiple groups of nanoparticles 102, wherein each individual group is designated 102a-102f, and reference numeral 102 generally refers to any one or more groups. Individual groups 102a-102f are shown as rows of individual nanoparticles, and individual nanoparticles from each group are designated 102a1-102f1 respectively. Although Figure 1 The illustrated embodiment includes six groups of nanoparticles, but it is believed that any number of groups of nanoparticles may be included in the calibration device 100.

[0040] Each group 102 includes nanoparticles containing nylon-6, which are covalently bound to a corresponding dye, such as an organic dye [e.g., fluorescein, rhodamine, aminomethylcoumarin (AMCA)], a biofluorescent material [e.g., green fluorescent protein, phycoerythrin, allophycocyanin], quantum dots, or a lipophilic fluorescent dye. Therefore, in Figure 1In this structure, the first group of nanoparticles 102a comprises nylon-6 covalently bonded to a first dye, the second group of nanoparticles 102b comprises nylon-6 covalently bonded to a second dye, the third group of nanoparticles 102c comprises nylon-6 covalently bonded to a third dye, the fourth group of nanoparticles 102d comprises nylon-6 covalently bonded to a fourth dye, the fifth group of nanoparticles 102e comprises nylon-6 covalently bonded to a fifth dye, and the sixth group of nanoparticles 102f comprises nylon-6 covalently bonded to a sixth dye. The dye in each group of nanoparticles is different from each of the other dyes in the other groups of nanoparticles; therefore, the dye is unique to a particular group of nanoparticles and indicates the specific group of nanoparticles.

[0041] In each embodiment, each dye is a lipophilic fluorescent dye comprising a lipophilic carbocyanine dye. Suitable commercially available new lipophilic carbocyanines include, for example, but not limited to, lipophilic fluorescent tracer dyes sold as DiB, DiA, DiO, DiI, DiD, and DiR, all of which are available from Biotium, Inc. (Hayward, California). The color, maximum excitation wavelength (λEx), and maximum emission wavelength (λEm) of each of these exemplary dyes are shown in Table 1 below.

[0042] Table 1:

[0043]

[0044] As shown in Table 1, lipophilic fluorescent dyes have maximum excitation wavelengths ranging from 353 nm to 750 nm (e.g., the peak wavelength at which maximum absorption occurs in the excitation spectrum), measured using a monochromator. Therefore, the lipophilic fluorescent dyes in Table 1 are suitable for common size-controlled techniques, including but not limited to flow cytometry, which includes optical systems such as lamps and lasers that generate light signals at the maximum excitation wavelengths, thus exciting the dyes. Additionally, Table 1 shows that each dye exhibits a different maximum emission wavelength (measured using a monochromator), corresponding to the observable color in response to excitation. Therefore, the use of multiple fluorescent dyes, when each dye is associated with nanoparticles having a corresponding average diameter, can achieve increased sensitivity and resolution.

[0045] In various embodiments, the dye is covalently bound to a polymer, said polymer comprising nylon-6. In embodiments, as described in more detail below, the dye is first bound to the polymer and then formed into nanoparticles, such that the dye is uniformly distributed within each nanoparticle. Nylon-6 has the following structure:

[0046]

[0047] The carbonyl group in the nylon-6 structure enables covalent bonding with the dye, which not only tracks nylon-6 nanoparticles but also reduces or eliminates dye leakage or dissociation from the particles. For example, conventional standards that encapsulate or incubate polymer particles with dye may not form direct chemical bonds between the dye and the particles. Therefore, dye leakage from the particles can introduce errors into measurements using conventional standards. In contrast, in various embodiments, the fluorescent dye described herein is covalently bound to nylon-6 and incorporated into the nanoparticles; the polymer will need to degrade to release the fluorescent dye, thereby reducing the likelihood of dye dissociation from the nanoparticles.

[0048] In various embodiments, the nanoparticles have a density greater than or equal to 1.15 g / mL and less than or equal to 1.19 g / mL, as measured according to ASTM D792. For example, the nanoparticles have the following densities: greater than or equal to 1.15 g / mL and less than or equal to 1.19 g / mL, greater than or equal to 1.15 g / mL and less than or equal to 1.18 g / mL, greater than or equal to 1.15 g / mL and less than or equal to 1.17 g / mL, greater than or equal to 1.15 g / mL and less than or equal to 1.16 g / mL, greater than or equal to 1.16 g / mL and less than or equal to 1.19 g / mL, greater than or equal to 1.16 g / mL and less than or equal to 1.18 g / mL, greater than or equal to 1.16 g / mL and less than or equal to 1.17 g / mL, greater than or equal to 1.17 g / mL and less than or equal to 1.19 g / mL, greater than or equal to 1.17 g / mL and less than or equal to 1.18 g / mL, or greater than or equal to 1.18 g / mL and less than or equal to 1.19 g / mL. Therefore, the nanoparticles have a density similar to that of EVs (i.e., 1.15-1.19 g / mL) and are appropriately higher than the density of mammalian cells (i.e., 1.04-1.05 g / mL), thereby eliminating the problem of different material densities between EVs and standard nanoparticles.

[0049] Each group of nanoparticles has an average hydraulic diameter (referred to as "average diameter" in this document) as measured according to ISO 22412, which is generally referred to as d. n The average hydraulic diameter of each group of nanoparticles was determined by light scattering measurements and visual confirmation via microscopy. Light scattering measurements included dynamic light scattering (DLS) or nanoparticle tracking analysis (NAT), while microscopy methods included scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Figure 1 In the middle, d a It is the average diameter of the first group of nanoparticles, 102a, d b It is the average diameter of the second group of nanoparticles, 102b, d cIt is the average diameter of the third group of nanoparticles, 102c, d d It is the average diameter of the fourth group of nanoparticles, 10²d. e It is the average diameter of the fifth group of nanoparticles, 10²e, and d f It is the average diameter of the sixth group of nanoparticles, 102f.

[0050] In various embodiments, each group of nanoparticles has a size standard deviation of less than or equal to 15% (e.g., the standard deviation of the maximum diameter of each particle in the group), which is statistically determined by software calculation. For example, the standard deviation of each group of nanoparticles may be less than or equal to 15%, less than or equal to 12.5%, less than or equal to 10%, less than or equal to 8%, less than or equal to 6%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%. In the implementation, the standard deviation of the average diameter of each group of nanoparticles can be greater than or equal to 0 and less than or equal to 15%, greater than or equal to 0 and less than or equal to 12.5%, greater than or equal to 0 and less than or equal to 10%, greater than or equal to 0 and less than or equal to 8%, greater than or equal to 0 and less than or equal to 6%, greater than or equal to 0 and less than or equal to 5%, greater than or equal to 0 and less than or equal to 4%, greater than or equal to 0 and less than or equal to 3%, greater than or equal to 0 and less than or equal to 2%, greater than or equal to 0 and less than or equal to 1%, greater than or equal to 0 and less than or equal to 0.5%, greater than or equal to 0.5% and less than or equal to 15%, greater than or equal to 0.5% and less than or equal to 12.5%, greater than or equal to 0.5% and less than or equal to 10%, greater than or equal to 0.5% and less than or equal to 8%, greater than or equal to 0.5% and less than or equal to 10%, and less than or equal to 8%. The ranges are 6% or higher, greater than or equal to 0.5% and less than or equal to 5%, greater than or equal to 0.5% and less than or equal to 4%, greater than or equal to 0.5% and less than or equal to 3%, greater than or equal to 0.5% and less than or equal to 2%, greater than or equal to 0.5% and less than or equal to 1%, greater than or equal to 0% and less than or equal to 0.5%, greater than or equal to 1% and less than or equal to 15%, greater than or equal to 1% and less than or equal to 12.5%, greater than or equal to 1% and less than or equal to 10%, greater than or equal to 1% and less than or equal to 8%, greater than or equal to 1% and less than or equal to 6%, greater than or equal to 1% and less than or equal to 5%, greater than or equal to 1% and less than or equal to 4%, greater than or equal to 1% and less than or equal to 3%, or greater than or equal to 1% and less than or equal to 2%, including any and all ranges and subranges within these ranges. It is conceivable that, in embodiments, each standard deviation can be a number within that range and can be the same as or different from the standard deviations of one or more other groups of nanoparticles.

[0051] In various embodiments, each group of nanoparticles can be further characterized by a polydispersity index greater than or equal to 1.15 and less than or equal to 1.19. The polydispersity index (sometimes referred to as "PDI" or "D") is a measure of the width of the size distribution calculated by cumulative analysis of the nanoparticle group, and uses standards conforming to ISO 22412 and ISO 14419. The PDI is determined by dynamic light scattering (DLS) of 13321. In embodiments, the PDI of each group of nanoparticles can be greater than or equal to 1.15 and less than or equal to 1.18, greater than or equal to 1.15 and less than or equal to 1.17, greater than or equal to 1.15 and less than or equal to 1.16, greater than or equal to 1.16 and less than or equal to 1.19, greater than or equal to 1.16 and less than or equal to 1.18, greater than or equal to 1.16 and less than or equal to 1.17, greater than or equal to 1.17 and less than or equal to 1.19, greater than or equal to 1.17 and less than or equal to 1.17, or greater than or equal to 1.18 and less than or equal to 1.19, including any and all ranges and subranges within these ranges. It is conceivable that, in embodiments, each PDI can be a number within this range and can be the same as or different from the PDI of one or more other groups of nanoparticles.

[0052] The average diameter d of each group n The average diameter is different from that of each group in the other nanoparticle groups. Therefore, in Figure 1 In the middle, d a With d b d c d d d e and d f Different, d b With d c d d d e and d f Different, d c With d d d e and d f Different, d d With d e and d f They are not the same, and d e With d f They are not the same. In each implementation, each average diameter is at least twice the next maximum average diameter. For example, in Figure 1 In the middle, d b (100nm) is d a (50nm) twice as much, d c (250nm) is d b At least twice that of (100nm), d d (500nm) is d c(250nm) twice as much, d e (1000nm) is d d (500nm) twice as much, and d f (2000nm) is d e Twice that of (1000nm).

[0053] In each embodiment, each average diameter d n Independently greater than or equal to 30 nm and less than or equal to 3000 nm. For example, each average diameter d n The following wavelengths are possible: greater than or equal to 30nm and less than or equal to 3000nm; greater than or equal to 30nm and less than or equal to 2500nm; greater than or equal to 30nm and less than or equal to 2000nm; greater than or equal to 30nm and less than or equal to 1500nm; greater than or equal to 30nm and less than or equal to 1000nm; greater than or equal to 30nm and less than or equal to 750nm; greater than or equal to 30nm and less than or equal to 500nm; greater than or equal to 30nm and less than or equal to 300nm; greater than or equal to 30nm and less than or equal to 250nm; and greater than or equal to 30nm and less than or equal to 100nm. The ranges are: greater than or equal to 50 nm and less than or equal to 3000 nm; greater than or equal to 50 nm and less than or equal to 2500 nm; greater than or equal to 50 nm and less than or equal to 2000 nm; greater than or equal to 50 nm and less than or equal to 1500 nm; greater than or equal to 50 nm and less than or equal to 1000 nm; greater than or equal to 50 nm and less than or equal to 750 nm; greater than or equal to 50 nm and less than or equal to 500 nm; greater than or equal to 50 nm and less than or equal to 300 nm; or greater than or equal to 50 nm and less than or equal to 250 nm, including any and all ranges and sub-ranges within these ranges. In an embodiment, at least one group of nanoparticles has an average diameter less than or equal to 300 nm, less than or equal to 250 nm, less than or equal to 200 nm, less than or equal to 150 nm, less than or equal to 100 nm, less than or equal to 75 nm, or less than or equal to 50 nm.

[0054] It should be understood that, although Figure 1 The groups 102a-102f shown have specific average diameters, but the average diameter in calibration device 100 can vary depending on the specific implementation. For example, one or more groups of nanoparticles can be selected such that the average diameter is relatively close to the EV type of interest in a range (within 50 nm, within 100 nm, within 250 nm). In addition to the calibration device 100 shown, Figure 1 The various types of EV 104 and their approximate average diameters are also illustrated. Specifically, Figure 1This includes exosomes 106 having an average diameter less than or equal to about 100 nm; microvesicles 108 in a first set with an average diameter greater than or equal to about 100 nm and less than or equal to about 1000 nm; microcavities 110 in a second set with an average diameter greater than or equal to about 1000 nm and less than or equal to about 2000 nm; and oncosomes 112 with an average diameter greater than about 2000 nm. Therefore, as... Figure 1 As shown, the nanoparticles described in this paper can be used to approximate the size of various different EVs.

[0055] In some embodiments, one or more groups of nanoparticles may include RNA. In such embodiments, as will be described in more detail below, the RNA is first bound to the polymer via the amide groups of the polymer before the nanoparticles are formed. Thus, the RNA is uniformly distributed in each nanoparticle. As used herein, the phrase “RNA molecule” or “RNA” refers to ribonucleic acid, i.e., a polymer composed of nucleotides. Nucleotides are typically adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate monomers linked together along a so-called backbone formed by phosphodiester bonds between the sugar (i.e., ribose) of the first monomer and the phosphate ester of the adjacent second monomer. In embodiments where the nanoparticles include RNA, the RNA can possess any predetermined RNA sequence. The RNA sequence can be, for example, a common RNA sequence or a specific sequence of interest. Therefore, analytical results can be validated by adding RNA-containing nanoparticles to a biological sample and observing the effects of technical steps throughout the experimental protocol, as will be described in more detail below.

[0056] Methods for manufacturing nanoparticles

[0057] Nanoparticles for each embodiment can be fabricated using any of a variety of nanoprecipitation methods, including but not limited to pipette droplet nanoprecipitation, flow-controlled T-mixer nanoprecipitation, and flow-controlled microfluidic nanoprecipitation. Generally, to formulate nanoparticles, nylon-6 groups are dissolved in a polar protic solvent (e.g., acetic acid, formic acid, ethanol, etc.) or a polar aprotic solvent (e.g., tetrahydrofuran, acetone, acetonitrile, etc.) until homogenized (e.g., nylon-6 is uniformly distributed throughout the solvent). The dye is dissolved in a mixture of nylon-6 and the solvent or introduced via an emulsion through a miscible solvent. An aqueous mixture of water and a surfactant (e.g., polyvinyl alcohol, Pluronic acid, or another aggregation protectant) is prepared for mixing and collecting the solution.

[0058] In various embodiments, the size of the nanoparticles is controlled by controlling the polymer concentration in the organic phase, the choice of the organic solvent, the surfactant concentration in the aqueous phase, the surfactant selection, the ratio of the organic phase to the aqueous phase in the mixture, the flow rates of the organic and aqueous phases during mixing, and the apparatus for nanoprecipitation. For example, increasing the polymer concentration in the organic phase or increasing the ratio of the organic phase to the aqueous phase in the mixture results in an increase in particle diameter. Conversely, increasing the surfactant concentration in the aqueous phase or increasing the ratio of the aqueous phase to the organic phase results in a decrease in particle diameter. The choice of organic solvent and surfactant can have various effects on particle size, which can be empirically determined based on the chemical density, charge, miscibility, and other chemical properties of the solvent or surfactant itself. Increasing the flow rate during mixing can also affect the particle diameter (generally, higher flow rates result in a decrease in particle diameter). However, the flow rates of the organic and aqueous phases can vary independently, which may have different effects, similar to the effects described regarding changes in the ratios in the mixture.

[0059] Furthermore, the specific method used to form nanoparticles can affect the variability of particle size. The more control the device has, the more precise and accurate the target particle size can be achieved. For example, flow-controlled microfluidic methods can produce particles with smaller particle density (PDI) compared to T-mixers, while T-mixers can produce particles with smaller particle size (PDF) compared to droplet methods.

[0060] In embodiments of nanoparticle production using the pipette droplet method, a small amount of polymer (e.g., greater than or equal to 0.5% and less than or equal to 2.0% nylon-6 by weight / volume (w / v)) along with a lipophilic fluorescent dye is dissolved in an organic solvent. In embodiments, a fluorescent agent is added at a dye / polymer ratio of greater than or equal to 0.25% (by weight, w / w) and less than or equal to 1.5%, and the polymer to dye ratio is greater than or equal to 50:1 and less than or equal to 250:1. In specific embodiments, the polymer to dye ratio is approximately 100:1. In embodiments where the dye is insoluble in the organic solvent, an emulsion is prepared by dissolving the dye in a miscible solvent (e.g., dimethyl sulfoxide (DMSO)) and added to the organic phase, while vortexing or sonicating until a homogenized solution is achieved. The organic phase containing the polymer and dye is then added dropwise to the aqueous phase while stirring. In this embodiment, the aqueous phase is a surfactant present in water at a concentration of 0.5% or more and 5.0% (w / v) or less. The organic solvent is then removed by evaporation. In this embodiment, evaporation can be carried out by stirring (e.g., for at least 3 hours) or by using a rotary evaporator (e.g., for about 15 minutes).

[0061] Nanoparticles are recovered from an aqueous medium by centrifugation. In one embodiment, the aqueous medium is centrifuged at about 6,000 x g for about 15 minutes at room temperature. The aqueous supernatant is discarded, and the nanoparticles are washed by resuspending them in 10 mL of water and centrifuging at about 16,000 x g for about 15 minutes at room temperature. In one embodiment, the washing procedure may 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 -20°C until needed.

[0062] In embodiments of nanoparticle preparation using a flow-controlled T-mixer method or a flow-controlled microfluidic method, nylon and a lipophilic fluorescent dye are dissolved in an organic solvent at a concentration of approximately 50 mg / mL of nylon-6 and 0.5% (w / w) of dye / polymer. An aqueous phase is also prepared by adding greater than or equal to 0.5% and less than or equal to 5.0% (w / v) of surfactant to water. Each phase is placed in a reservoir, which in various embodiments is a syringe. In embodiments using the T-mixer method, each syringe is mounted on a syringe pump, and the syringe output line is directed to an inlet port of the T-mixer. A collection container is positioned at the outlet port of the T-mixer. Using an empirically determined flow rate, the syringes deliver each phase simultaneously to the T-mixer at appropriate flow rates and volumes. In embodiments using the flow-controlled microfluidic method, the organic and aqueous phases are supplied to the microfluidic chip at an empirically determined rate. The nanoparticles are recovered and washed as described above.

[0063] It is conceivable that the method for preparing nanoparticles described herein can be adjusted and modified, for example, including the amount of components, the number of centrifugations, etc., depending on the specific implementation and, for example, based on the desired size of the nanoparticles to be formed.

[0064] In embodiments where RNA is incorporated into nanoparticles, RNA molecules may be added to the aqueous phase of the mixture described above. In such embodiments, the aqueous phase comprises a slightly acidic buffer (e.g., sodium acetate or tri-EDTA) to promote the interaction between the amide groups in nylon-6 and the RNA. In embodiments, the pH of the aqueous phase is greater than or equal to 4.5 and less than or equal to 7.

[0065] In various embodiments, size exclusion centrifuge filters or other size-selective methods can be used to filter nanoparticle groups to achieve the desired size distribution.

[0066] While various methods for forming nanoparticles containing nylon-6 and dyes have been described, it should be understood that other methods known to those skilled in the art can also be used to manufacture nanoparticles, including but not limited to emulsification techniques, electrospraying, and plasma deposition.

[0067] In various embodiments, two or more sets of nanoparticles are added in equal volumes to a single container (e.g., a vial) to form a standard set. In some embodiments, the nanoparticles can be resuspended by adding water or another solvent before use. It is believed that storing the nanoparticles in a concentrated state can extend their shelf life by limiting the hydrolytic degradation of the polymer, which can occur when the nanoparticles are suspended in water.

[0068] How to use nanoparticles

[0069] Nanoparticles from various embodiments can be used in a variety of ways; for example, they can be used to characterize small biological vesicles, such as various EVs. For instance, in some embodiments, nanoparticles are used as standards incorporated into biological samples to assess separation efficiency and provide technical standardization. In some embodiments, nanoparticles loaded with specific RNA sequences can independently validate results, for example, by demonstrating that a specific separation method preserves the functionality (e.g., RNA transport capacity) of the separated EVs. As another example, nanoparticles are used as calibration standards for EV quantification and sizing techniques—including, but not limited to, flow cytometry.

[0070] In one embodiment, the calibration device can be used to calibrate a dimensional instrument, such as a flow cytometer. Therefore, a method for calibrating a dimensional instrument includes: uniformly dispersing a first nanoparticle and a second nanoparticle in a calibration suspension. The first nanoparticle comprises a first dye, and the second nanoparticle comprises a second dye different from the first dye. Next, a predetermined volume of the calibration suspension containing the uniformly dispersed first and second nanoparticles is added to a sample buffer to generate a sample, the sample containing a predetermined concentration of the first and second nanoparticles in the sample buffer.

[0071] Use a dimensional measuring instrument (e.g., purchased from Luminex). easyCyte TM Flow cytometer or Attune from Thermo Fisher Scientific TMAn NxT flow cytometer analyzes a sample to obtain at least one data output regarding the fluorescence output of a first dye and the fluorescence output of a second dye. Then, one or more settings of the calibrated instrument are adjusted based on this at least one data output. In embodiments, adjusting the one or more settings may include maximizing the signal, minimizing the coefficient of variation, or a combination thereof. In some embodiments, the adjusted settings may relate to the resolution limit of particle size measurement, the range of particle size measurement, the sensitivity of the scattering photomultiplier tube, baseline instrument noise, laser alignment, optical alignment, the stability of the flow cytometer's fluid system, cell sorter droplet delay, cell sorter efficiency, or a combination thereof. Depending on the specific implementation, other adjustments are contemplated, and these will be at least in part based on the specific calibrated instrument.

[0072] In embodiments where the nanoparticles include RNA, the nanoparticles can be used as artificial EV incorporation standards to standardize the recovery of EVs and associated RNA from biological fluids and facilitate independent validation of results. In such embodiments, artificial EV standards can be used to ensure that RNA is not damaged by the experimental protocol. As those skilled in the art will understand, the experimental protocol may include, for example, a RIBOGREEN assay or other quantitative assay, or functional analysis.

[0073] In the various embodiments described herein, the calibration apparatus comprises groups of nylon-6 and corresponding dye nanoparticles, each group of nanoparticles having a corresponding average diameter. Therefore, the nanoparticles exhibit density, light scattering effects, and size range comparable to EVs, making them particularly suitable as standards for EV research. Additionally, the nanoparticles in each embodiment may include RNA molecules, enabling the nanoparticles to be used as artificial EV incorporations in experimental protocols to facilitate independent validation of results and standardized recovery of EVs and associated RNA.

[0074] It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments described herein without departing from the spirit and scope of the claimed subject matter. Therefore, this specification is intended to cover modifications and variations to the various embodiments described herein, provided that such modifications and variations are within the scope of the appended claims and their equivalents.

Claims

1. A calibration device comprising: The first nanoparticle comprises nylon-6 covalently bonded to a first dye, the first nanoparticle 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; and The second nanoparticle comprises nylon-6 covalently bound to a second dye, which is different from the first dye. The second nanoparticle 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, wherein: The first average diameter is different from the second average diameter; The first average diameter and the second average diameter are each independently greater than or equal to 30 nm and less than or equal to 3000 nm; and The first average diameter is at least twice the second average diameter.

2. The calibration apparatus according to claim 1, wherein, The first dye and the second dye each comprise a lipophilic fluorescent dye.

3. The calibration apparatus according to claim 1, wherein: The first dye includes a first maximum excitation wavelength λEx1; The second dye includes a second maximum excitation wavelength λEx2; λEx1 and λEx2 are not the same; and λEx1 and λEx2 are each between 350 nm and 750 nm.

4. The calibration apparatus according to claim 1, wherein, The calibration device includes a container containing a mixture of first nanoparticles and second nanoparticles.

5. The calibration apparatus according to claim 1, wherein, At least one of the first average diameter and the second average diameter is between 30 nm and 250 nm.

6. The calibration apparatus according to claim 1, wherein, The first nanoparticle and the second nanoparticle each have a density of 1.15 g / mL to 1.19 g / mL.

7. The calibration apparatus according to claim 1, wherein, The first and second nanoparticles each contain RNA.

8. A method of using a calibration device, the method comprising: The first nanoparticle and the second nanoparticle are uniformly dispersed in a calibration suspension. The first nanoparticle includes nylon-6 covalently bound to a first dye, and the second nanoparticle includes nylon-6 covalently bound to a second dye. The second dye is different from the first dye. A calibration suspension containing uniformly dispersed first and second nanoparticles in volume is added to a sample buffer to generate a sample, wherein the sample contains a predetermined concentration of first and second nanoparticles in the sample buffer. The sample is analyzed using a fixed-size instrument to obtain at least one data output regarding the fluorescence output of the first dye and the fluorescence output of the second dye; as well as Adjust one or more settings of the dimensional measuring instrument based on the at least one data output. in: The first nanoparticle has 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 nanoparticle 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.

9. The method according to claim 8, wherein, The instrument used for sizing is a flow cytometer.

10. The method according to claim 9, wherein, Adjusting one or more of the settings includes maximizing the signal, minimizing the coefficient of variation, or a combination thereof.

11. The method according to claim 9, wherein, Adjusting one or more of the settings includes adjusting settings related to: the resolution limit of particle size measurement, the range of particle size measurement, the sensitivity of the scattering photomultiplier tube, baseline instrument noise, laser alignment, optical alignment, stability of the flow cytometer's fluid system, droplet delay of the cell sorter, cell sorter efficiency, or a combination thereof.

12. The method according to claim 8, wherein, The first dye and the second dye each comprise a lipophilic fluorescent dye.

13. The method according to claim 8, wherein, At least one of the first average diameter and the second average diameter is between 30 nm and 250 nm.

14. The method according to claim 8, wherein, The first nanoparticle and the second nanoparticle each have a density of 1.15 g / mL to 1.19 g / mL.

15. A calibration apparatus comprising: Multiple groups of nylon-6 nanoparticles, wherein each nanoparticle in one group also includes a dye different from the corresponding dye in different groups of nanoparticles, wherein: Each group of nanoparticles has a corresponding average diameter and a polydispersity index greater than or equal to 1.15 and less than or equal to 1.19; The average diameter of each group of nanoparticles differs from the average diameter of the corresponding nanoparticles in each of the multiple groups by at least two times; and Each corresponding average diameter is greater than or equal to 30 nm and less than or equal to 3000 nm.

16. The calibration apparatus according to claim 15, wherein, Each of the dyes includes a lipophilic fluorescent dye.

17. The calibration apparatus according to claim 15, wherein, The calibration device includes a container containing a mixture of multiple groups of nanoparticles.

18. The calibration apparatus according to claim 15, wherein, Multiple groups of nanoparticles have densities ranging from 1.15 g / mL to 1.19 g / mL.

19. The calibration apparatus according to claim 15, wherein, At least one of the multiple groups of nanoparticles includes RNA.

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