Patent Description:
Extracellular vesicle (EV) research has expanded for both separation and concentration methods from serum and cells for therapeutic applications. The phrase "extracellular vesicle" generally refers to particles naturally released from a cell that are delimited by a lipid bilayer and cannot replicate. These particles can be subcategorized by size and function, with the smallest being exosomes as small as <NUM> and the largest being microvesicles as large as <NUM>.

However, characterization methods lack appropriate calibration standards for EV quantitation and sizing technologies. Although polystyrene bead standards are conventionally used in sizing instruments, such as flow cytometers, the polystyrene beads have properties that match those of mammalian cells, such as density, light scattering effects, size, and fluorescent marker binding affinity. Though EVs are products that may be of mammalian cell origin, their inherent properties differ from the properties of their origin cells and, thus, from the polystyrene beads. <CIT> describes a kit of uniform size microbead standards for flow cytometer alignment, compensation, and/or calibration.

Accordingly, a need exists for alternative calibration sets, and in particular, for calibration sets with properties similar to EVs.

According to appended claim <NUM> there is provided a calibration set for use with extracellular vesicle quantitation and/or sizing instruments. According to appended claim <NUM> there is provided a method of using said calibration set.

It is to be understood that the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter.

<FIG> schematically illustrates groups of nanoparticles having different average diameters as compared to various extracellular vesicles according to one or more embodiments shown and described herein.

In various embodiments, a calibration set comprises first nanoparticles comprising nylon-<NUM> covalently bound to a first dye and second nanoparticles comprising nylon-<NUM> covalently bound to a second dye that is different from the first dye. The first nanoparticles have a first average diameter and a first polydispersity index of greater than or equal to <NUM> and less than or equal to <NUM>, and the second nanoparticles have a second average diameter and a second polydispersity index of greater than or equal to <NUM> and less than or equal to <NUM>. In various embodiments, the first average diameter is different from the second average diameter, and each of the first average diameter and the second average diameter are greater than or equal to <NUM> and less than or equal to <NUM>, with the first average diameter being at least two times the second average diameter.

Reference will now be made in detail to various embodiments, examples of which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout the drawings to refer to the same or like parts.

Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

Directional terms as used herein - for example up, down, right, left, front, back, top, bottom - are made only with reference to the figures as drawn and are not intended to imply absolute orientation.

Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order, nor that with any apparatus specific orientations be required. Accordingly, where a method claim or embodiment does not actually recite an order to be followed by its steps, or that any apparatus claim or embodiment does not actually recite an order or orientation to individual components, or it is not otherwise specifically stated in the claims or description that the steps are to be limited to a specific order, or that a specific order or orientation to components of an apparatus is not recited, it is in no way intended that an order or orientation be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps, operational flow, order of components, or orientation of components; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a" component includes aspects having two or more such components, unless the context clearly indicates otherwise.

The term "formed from" can mean one or more of comprises, consists essentially of, or consists of. For example, a component that is formed from a particular material can comprise the particular material, consist essentially of the particular material, or consist of the particular material.

<FIG> schematically illustrates an embodiment of a calibration set <NUM> including a plurality of groups of nanoparticles <NUM>, with each individual group being identified as 102a - 102f, and reference numeral <NUM> referring generally to any one or more of the groups. The individual groups 102a-102f are shown as rows of individual nanoparticles, with a single nanoparticle from each group identified as 102a1-102f1, respectively. While the embodiment shown in <FIG> includes six groups of nanoparticles, it is contemplated that any number of groups of nanoparticles can be included in the calibration set <NUM>.

Each group <NUM> comprises nanoparticles comprising nylon-<NUM> covalently bound to a corresponding dye, such as an organic dye (e.g., fluorescein, rhodamine, aminomethylcoumarin (AMCA)), a biological fluorophore (e.g., green fluorescent protein, phycoerythrin, allophycocyanain), quantum dots, or a fluorescent lipophilic dye. Accordingly, in <FIG>, the first group of nanoparticles 102a comprises nylon-<NUM> covalently bound to a first dye, the second group of nanoparticles 102b comprises nylon-<NUM> covalently bound to a second dye, the third group of nanoparticles 102c comprises nylon-<NUM> covalently bound to a third dye, the fourth group of nanoparticles 102d comprises nylon-<NUM> covalently bound to a fourth dye, the fifth group of nanoparticles 102e comprises nylon-<NUM> covalently bound to a fifth dye, and the sixth group of nanoparticles 102f comprises nylon-<NUM> covalently bound 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 such that the dye is unique to and indicative of the particular group of nanoparticles.

In various embodiments, each dye is a fluorescent lipophilic dye comprising a lipophilic carbocyanine dye. Suitable commercially available lipophilic carbocyanine dyes include, by way of example and not limitation, fluorescent lipophilic tracer dyes sold as DiB, DiA, DiO, DiI, DiD, and DiR, all available from Biotium, Inc. (Hayward, CA). The color, excitation maximum wavelength (λEx), and emission maximum wavelength (λEm) for each of these example dyes is presented in Table <NUM> below.

As shown in Table <NUM>, the fluorescent lipophilic dyes have an excitation maximum wavelength (e.g., the peak wavelength in the excitation spectrum where the maximum absorption occurs) of from <NUM> to <NUM> as measured using a monochromator and, as such, the fluorescent lipophilic dyes in Table <NUM> are suitable for use in common sizing technologies, including but not limited to, flow cytometers, which incorporate optical systems such as lamps and lasers that produce light signals at the excitation maximum wavelength and can, therefore, excite the dyes. Moreover, Table <NUM> shows that each of the dyes exhibits a different emission maximum wavelength measured using a monochromator, which corresponds to the color that is observable in response to excitation. Accordingly, the use of multiple fluorescent dyes can enable increased sensitivity and resolution when each fluorescent dye is associated with nanoparticles having a corresponding average diameter.

In various embodiments, the dye is covalently bound to a polymer comprising nylon-<NUM>. In embodiments, the dye is bound to the polymer prior to the formation of the nanoparticles, as will be described in greater detail below, such that the dye is homogeneously distributed throughout the nanoparticle. Nylon-<NUM> has the following structure:
<CHM>
The carbonyl group of the nylon-<NUM> structure enables covalent binding with the dye which, in addition to enabling the tracing of the nylon-<NUM> nanoparticles, can also reduce or eliminate dye leakage or disassociation of the dye from the particles. For example, conventional standards which encapsulate or incubate polymer particles with the dye may not form direct chemical bonds between the dye and the particles. Accordingly, dye leaking from the particles can introduce error into measurements made using conventional standards. In contrast, in various embodiments, the fluorescent dyes described herein are covalently bound to the nylon-<NUM> and incorporated into the nanoparticles and the polymer would have to degrade to release the fluorescent dye, thereby reducing the likelihood of disassociation of the dye from the nanoparticles.

In various embodiments, the nanoparticles have a density of greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, as measured in accordance with ASTM D792. For example, the nanoparticles have a density of greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL, or greater than or equal to <NUM>/mL and less than or equal to <NUM>/mL. Accordingly, the nanoparticles have a density that is similar to the density of EVs (i.e., <NUM>-<NUM>/mL) and measurably higher than the density of mammalian cells (i.e., <NUM>-<NUM>/mL), thereby eliminating the issue of different material density between the EVs and the standard nanoparticles.

Each group of nanoparticles has an average hydrodynamic diameter (referred to herein as "average diameter"), determined in accordance with ISO <NUM>, which is generally referred to as dn. The average hydrodynamic diameter for each group of nanoparticles is determined by light scattering measurement techniques, such as dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA) and visually confirmed via microscopy, such as scanning electron microscopy (SEM) or transmission electron microscopy (TEM). In <FIG>, da is the average diameter of the first group of nanoparticles 102a, db is the average diameter of the second group of nanoparticles 102b, dc is the average diameter of the third group of nanoparticles 102c, dd is the average diameter of the fourth group of nanoparticles 102d, de is the average diameter of the fifth group of nanoparticles 102e, and df is the average diameter of the sixth group of nanoparticles 102f.

In various embodiments, each group of nanoparticles has a standard deviation of size (e.g., a standard deviation of the maximum diameter of each of the particles in the group) of <NUM>% or less, which is determined statistically via software calculations. For example, each group of nanoparticles may have a standard deviation of less than or equal to <NUM>%, less than or equal to <NUM>%, less than or equal to <NUM>%, less than or equal to <NUM>%, less than or equal to <NUM>%, less than or equal to <NUM>%, less than or equal to <NUM>%, less than or equal to <NUM>%, less than or equal to <NUM>%, less than or equal to <NUM>%, or less than or equal to <NUM>%. In embodiments, the average diameter of each group of nanoparticles may have a standard deviation of greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM> and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, greater than or equal to <NUM>% and less than or equal to <NUM>%, or greater than or equal to <NUM>% and less than or equal to <NUM>%, including any and all ranges and sub-ranges within these ranges. It is contemplated that, in embodiments, each standard deviation can be a number within this range, and may be the same as or different from the standard deviation of one or more other groups of nanoparticles.

In various embodiments, each group of nanoparticles can be further characterized by a polydispersity index of greater than or equal to <NUM> and less than or equal to <NUM>. The polydispersity index (sometimes referred to as the "PDI" or D) is a measure of the broadness of the size distribution calculated from the cumulants analysis of the group of nanoparticles and is determined using dynamic light scattering (DLS) in compliance with ISO <NUM> and ISO <NUM>. In embodiments, each group of nanoparticles may have a PDI of greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, or greater than or equal to <NUM> and less than or equal to <NUM>, including any and all ranges and sub-ranges within these ranges. It is contemplated that, in embodiments, each PDI can be a number within this range, and may be the same as or different from the PDI of one or more other groups of nanoparticles.

The average diameter dn of each group is different from the average diameter of each of the other groups of nanoparticles. Accordingly, in <FIG>, da is different from db, dc, dd, de, and df, db is different from de, dd, de, and df, dc is different from dd, de, and df, dd is different from de and df, and de is different from df. In various embodiments, each average diameter is at least two times the next largest average diameter. For example, in <FIG>, db (<NUM>) is two times da (<NUM>), dc (<NUM>) is at least two times db (<NUM>), dd (<NUM>) is two times dc (<NUM>), de (<NUM>) is two times dd (<NUM>), and df (<NUM>) is two times de (<NUM>).

In various embodiments, each average diameter dn is independently greater than or equal to <NUM> and less than or equal to <NUM>. For example, each average diameter dn can be greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, greater than or equal to <NUM> and less than or equal to <NUM>, or greater than or equal to <NUM> and less than or equal to <NUM>, including any and all ranges and sub-ranges within these ranges. In embodiments, at least one group of nanoparticles has an average diameter less than or equal to <NUM>, less than or equal to <NUM>, less than or equal to <NUM>, less than or equal to <NUM>, less than or equal to <NUM>, less than or equal to <NUM>, or less than or equal to <NUM>.

It should be appreciated that although the groups 102a-102f shown in <FIG> have particular average diameters, the average diameters in the calibration set <NUM> can vary depending on the particular embodiment. For example, one or more groups of nanoparticles can be selected to have an average diameter that is relatively close in range (within <NUM>, within <NUM>, or within <NUM>) to a type of EV of interest. In addition to showing the calibration set <NUM>, <FIG> also schematically illustrates various types of EVs <NUM> and their approximate average diameters. In particular, <FIG> includes exosomes <NUM>, which have an average diameter of less than or equal to about <NUM>, a first set of microvesicles <NUM> having an average diameter of greater than or equal to about <NUM> and less than or equal to about <NUM>, a second set of microvesicles <NUM> having an average diameter of greater than or equal to about <NUM> and less than or equal to about <NUM>, and oncosomes <NUM> having an average diameter of greater than about <NUM>. Accordingly, as shown in <FIG>, the nanoparticles described herein can be used to approximate the size of various different EVs.

In some embodiments, one or more groups of nanoparticles may include RNA. In such embodiments, as will be described in greater detail below, the RNA is bound to the polymer via the amide group of the polymer prior to formation of the nanoparticle. Accordingly, RNA is homogenously distributed throughout each nanoparticle. As used herein, the phrase "RNA molecule" or "RNA" refers to ribonucleic acid, i.e., a polymer consisting of nucleotides. The nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate, and cytidine-monophosphate monomers which are connected to each other along a so-called backbone formed by phosphodiester bonds between the sugar (i.e., ribose) of a first monomer and a phosphate moiety of a second, adjacent monomer. In embodiments in which nanoparticles include RNA, the RNA may possess any predetermined RNA sequence. The RNA sequence can be, for example a generic RNA sequence, or a specific sequence of interest. Accordingly, results of analysis could be validated by adding the nanoparticles including RNA into a biologic sample and observing the effects of technical steps throughout the experimental protocol, as will be described in greater detail below.

The nanoparticles of various embodiments can be made in accordance with any one of a number of methods of nanoprecipitation, including, but not limited to, pipette droplet nanoprecipitation, flow-controlled T-mixer nanoprecipitation, and flow-controlled microfluidics nanoprecipitation. In general, to formulate nanoparticles, nylon-<NUM> pellets 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 homogenous (e.g., the nylon-<NUM> is uniformly distributed throughout the solvent). The dye is dissolved into the nylon-<NUM> and solvent mixture or introduced via a miscible solvent via emulsion. An aqueous mixture of water and surfactant (e.g., polyvinyl alcohol, pluronic, or another aggregation protectant) is prepared for both mixing and collecting solutions.

In various embodiments, the size of the nanoparticles is controlled by controlling the polymer concentration in the organic phase, the organic solvent choice, the surfactant concentration in the aqueous phase, the surfactant choice, the ratio of the organic phase to the aqueous phase in mixing, the flow rates of the organic phase and aqueous phase during mixing, and the apparatus used for nanoprecipitation. For example, increasing the polymer concentration in the organic phase or an increase in the ratio of organic phase to the aqueous phase in mixing results in an increase in particle diameter. As another example, an increase in surfactant concentration in the aqueous phase or an increase in aqueous phase to organic phase results in a decrease in particle diameter. The organic solvent and surfactant choice can have various effects that could be determined empirically in particle size depending on the chemical densities, charges, miscibility, and other chemical properties of the solvent or surfactants themselves. An increase in flow rates during mixing can also impact the particle diameter (generally, higher flow rates results in a decrease in particle diameter). However, the flow rates for organic and aqueous phase can be changed independently which may have a different effect similar to that described with their changes associated with ratio in the mixture.

Moreover, the particular method for forming the nanoparticles can impact the variability of particle size. The more control in the apparatus, the more accurate and precise the target particle size can be achieved. For example, the flow-controlled microfluidic method will be able to produce particles with a smaller PDI than the T-mixer, and the T-mixer will be able to produce particles with a smaller PDF than the pipette method.

In embodiments in which the pipette droplet method is used to prepare the nanoparticles, a small amount of polymer (e.g., greater than or equal to <NUM>% and less than or equal to <NUM>% w/v of nylon-<NUM>) is dissolved into organic solvent along with the fluorescent lipophilic dye. In embodiments, the fluorescent is added in an amount of greater than or equal to <NUM>% and less than or equal to <NUM>% w/w dye/polymer, and the ratio of polymer to dye is greater than or equal to <NUM>:<NUM> and less than or equal to <NUM>:<NUM>. In particular embodiments, the ratio of polymer to dye is approximately <NUM>:<NUM>. In embodiments in which the dye is not soluble in the organic solvent, an emulsion is made 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 homogeneous solution is achieved. Next, the organic phase containing the polymer and dye is added dropwise to the aqueous phase while stirring. In embodiments, the aqueous phase is greater than or equal to <NUM>% and less than or equal to <NUM>% w/v surfactant in water. The organic solvent is then removed via evaporation. In embodiments, evaporation can be performed by stirring (e.g., for at least <NUM> hours) or through the use of a rotary evaporator (e.g., for about <NUM> minutes).

Nanoparticles are recovered from the aqueous medium by centrifugation. In embodiments, the aqueous medium can be centrifuged at about <NUM>,<NUM> x g for about <NUM> minutes at room temperature. The aqueous supernatant is decanted and the nanoparticle pellet is washed by resuspending the nanoparticles in <NUM> of water and centrifuging at about <NUM>,<NUM> x g for about <NUM> minutes at room temperature. In embodiments, the washing procedure can be performed two or more times. Finally, the washed dispersion is lyophilized for at least <NUM> hours to yield freeze-dried nanoparticles. The nanoparticles can be stored at a temperature of -<NUM> until needed.

In embodiments in which the flow-controlled T-mixer method or flow-controlled microfluidic method is used to prepare the nanoparticles, nylon-<NUM> and fluorescent lipophilic dye are dissolved into organic solvent at a concentration of about <NUM>/mL nylon-<NUM> and <NUM>% w/w dye to polymer. An aqueous phase is also prepared by adding greater than or equal to <NUM>% and less than or equal to <NUM>% w/v surfactant in water. Each phase is put into a reservoir which, in various embodiments, is a syringe. In embodiments in which a T-mixer method is used, each syringe is mounted on a syringe pump with an output line of the syringe leading to one entrance port of a T-mixer. A collection vessel is positioned at the exit port of the T-mixer. Using an empirically-determined flow rate, the syringe pumps are programmed to deliver the appropriate flow and volumes of each phase simultaneously to the T-mixer. In embodiments in which a flow-controlled microfluidic method is used, organic and aqueous phases are provided to a microfluidic chip at empirically-determined rates. Nanoparticles are recovered and washed as described above.

It is contemplated that adjustments and modifications may be made to the methods for preparing the nanoparticles described herein, including, for example, amounts of ingredients, times of centrifugation, and the like, depending on the particular embodiment, and, for example, based on the desired size of nanoparticles to be formed.

In embodiments in which RNA is incorporated into the nanoparticles, the RNA molecules can be added into the aqueous phase of the mixtures described above. In such embodiments, the aqueous phase includes a slightly acidic buffer (e.g., sodium acetate or tris-EDTA) to facilitate the interaction of the amide group in the nylon-<NUM> and the RNA. In embodiments, the pH of the aqueous phase is greater than or equal to <NUM> and less than or equal to <NUM>.

In various embodiments, groups of nanoparticles can be filtered using size exclusion centrifuge filters or other size selection methods in order to achieve desired size distributions.

Although various methods for forming nanoparticles comprising nylon-<NUM> and dye have been described, it should be understood that other methods known to those skilled in the art may be used to manufacture the nanoparticles, including, but not limited to, emulsion techniques, electrospraying, and plasma deposition.

In various embodiments, two or more groups of nanoparticles are added in equal volumes to a single vessel, such as a vial, to form a standard set. In embodiments, the nanoparticles may be resuspended by adding water or another solvent prior to use. It is believed that by storing the nanoparticles in a concentrated state may prolong the shelf life of the nanoparticles by limiting hydrolytic degradation of the polymer that may occur when the nanoparticles are suspended in water.

The nanoparticles of various embodiments can be used in a variety of ways and can, for example, be used to characterize small biologic vesicles, such as various EVs. For example, in some embodiments, the nanoparticles are used as standards that are spiked into biological samples to assess isolation efficiency and furnish technical normalization. In some embodiments, nanoparticles loaded with a particular RNA sequence can enable independent validation of results, such as by demonstrating that a particular isolation method retains function (e.g., RNA transfer capacity) of isolated EVs. As another example, the nanoparticles are used as calibration standards for EV quantitation and sizing technologies, including but not limited to, flow cytometry.

In embodiments, the calibration set can be used to calibrate a sizing instrument, such as a flow cytometer. Accordingly, a method for calibrating a sizing instrument 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 that is different from the first dye. Next, a volume of the calibration suspension comprising the uniformly dispersed first and second nanoparticles is added to a sample buffer to generate a sample comprising a predetermined concentration of the first nanoparticles and the second nanoparticles in the sample buffer.

The sample is analyzed with a sizing instrument (e.g., Guava® easyCyte™ Flow Cytometer from Luminex or Attune™ NxT Flow Cytometer from Thermo Fisher) to obtain at least one data output regarding a fluorescence output of the first dye and a fluorescence output of the second dye. Next, one or more settings of the sizing instrument are adjusted based on the at least one data output. In embodiments, adjusting the one or more settings can include maximizing a signal, minimizing a coefficient of variation, or a combination thereof. In some embodiments, the setting that is adjusted may be related to resolution limit of particle size measurement, range of particle size measurement, sensitivity of scatter photomultiplier tubes, baseline instrument noise, laser alignment, optical alignment, stability of a fluidics system of the flow cytometer, cell sorter drop delay, cell sorter efficiency, or combinations thereof. Other adjustments are contemplated, depending on the particular embodiment, and will be based, at least in part, on the particular sizing instrument being calibrated.

In embodiments in which the nanoparticles include RNA, the nanoparticles may be used as artificial EV spike-in standards to standardize recovery of EV and associated RNA from biofluids and to facilitate independent verification of results. In such embodiments, the artificial EV standard may be used to confirm that the RNA is not damaged by the experimental protocol. Experimental protocols can include, for example, ribogreen assays or other quantification assays, or functional assays, as will be appreciated by those skilled in the art.

In various embodiments described herein, the calibration set includes groups of nanoparticles of nylon-<NUM> and a corresponding dye, each of which has a correpsonding average diameter. Accordingly, the nanoparticles exhibit a density, light scattering effects, and a size range comparable to EVs, making them particularly well-suited for use as standards in EV research. Moreover, the nanoparticles of various embodiments may include RNA molecules to enable the nanoparticles to be used as an artificial EV spike in experimental protocols to facilitate independent verification of results and to standardize recovery of EVs and associated RNA.

Claim 1:
A calibration set for use with extracellular vesicle quantitation and/or sizing instruments, comprising:
first nanoparticles comprising nylon-<NUM> covalently bound to a first dye, the first nanoparticles having a first average diameter and a first polydispersity index of greater than or equal to <NUM> and less than or equal to <NUM>; and
second nanoparticles comprising nylon-<NUM> covalently bound to a second dye that is different from the first dye, the second nanoparticles having a second average diameter and a second polydispersity index of greater than or equal to <NUM> and less than or equal to <NUM>, 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 <NUM> and less than or equal to <NUM>; and
the first average diameter is at least two times the second average diameter.