Microscale nanoparticle tracking analysis systems and methods

Abstract

A nanoparticle tracking analysis (NTA) system (100) can include a tracking volume (102) to accommodate a microscale droplet at an NTA sample location, with at least one wall of the tracking volume having an unobstructed opening (108). The system (100) can also include an excitation light source (112) that introduces a light beam (114) to the NTA sample location through the unobstructed opening (108). An imaging device (116) can capture the particle light scattering from the microscale droplet. A method can include dispensing a microscale droplet into an NTA sample location, introducing a light beam to the NTA sample location through an unobstructed opening, capturing the particle light scattering from the illuminated particles, and collecting data and analyzing nanoparticles in the microscale droplet.

Description

[0001] MICROSCALE NANOPARTICLE TRACKING ANALYSIS SYSTEMS AND METHODS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No.

[0004] 63 / 746,429, filed January 17, 2025, which is incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] Not applicable.

[0007] BACKGROUND

[0008] Nanoparticle tracking analysis is a known process for correlating the Brownian motion of particles to particle size distributions. Typically, a laser excitation light source is passed through a sample volume. Light scattering patterns from that illumination of the sample are captured over several time frames. Motion of individual particles is mapped and tracked over these time frames and then correlated to particle sizes via the Stoke-Einstein equation. Commercial NTA systems typically require at least milliliters of sample, which is disadvantageous when working with precious or limited samples, such as biological fluids or expensive reagents. Additionally, these commercial systems generally lack integrated liquid-handling automation, thus requiring manual preparation of multiple dilutions to achieve optimal particle concentrations. Further, these systems use relatively high sample volumes due to enclosed flow cell designs with four walls that are fully filled to ensure consistent flow, avoid air pockets, and achieve uniform particle distribution for accurate measurements. The combination of high sample volume requirements and lack of automated dilution significantly limits practical applications, particularly for high-throughput screening and quantitative biological assays.

[0009] SUMMARY

[0010] A nanoparticle tracking analysis (NTA) system can include a tracking volume that has an NTA sample location. The volume can be configured to accommodate a microscaledroplet, with at least one wall of the volume having an unobstructed opening. Additionally, the NTA system can include an excitation light source configured to introduce a light beam to the NTA sample location through the unobstructed opening of the volume. The light beam can be sufficient to illuminate particles within the microscale droplet to produce particle light scattering. The NTA system can further include an imaging device configured to capture the particle light scattering from the illuminated particles within the microscale droplet to be used for NTA data collection and analysis. The NTA system can further include integrated automated fluid handling configured to automatically prepare and dispense microscale droplets into the NTA sample location, and regions configured for automated dilution and calibration of samples prior to analysis.

[0011] A method of tracking and analyzing nanoparticles in microscale volumes can include dispensing a microscale droplet into an NTA sample location. Dispensing can be performed using automated fluid handling integrated within the NTA system. The method can also include introducing a light beam to the NTA sample location through an unobstructed opening, with the light beam being sufficient to illuminate particles within the microscale droplet to produce particle light scattering. Furthermore, the method can include capturing the particle light scattering from the illuminated particles within the microscale droplet using an imaging device and collecting data and analyzing nanoparticles in the microscale droplet. The method can optionally further include culturing cells in microscale volumes at the NTA sample location, performing particle analysis of lipid nanoparticles (LNPs) prior to cell treatment and extracellular vesicles (EVs) secreted by cells treated with LNPs, and screening multiple LNP candidates using integrated datasets to evaluate potency. The method can further optionally include culturing cells in microscale volumes, performing particle analysis of LNPs prior to cell treatment and EVs secreted by cells treated with LNPs, and screening multiple LNP candidates using integrated orthogonal datasets including particle analysis and cell-based assays to produce a comprehensive potency metric for comparative evaluation among the LNP candidates.

[0012] There has thus been outlined, rather broadly, the more important features of the invention so that the detailed description thereof that follows may be better understood, and so that the present contribution to the art may be better appreciated. Other features of the present invention will become clearer from the following detailed description of theinvention, taken with the accompanying drawings and claims, or may be learned by the practice of the invention.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a schematic of a side view of a nanoparticle tracking analysis system in accordance with an example of the present technology.

[0014] FIG. IB is an exploded view of the sampling region 113 from FIG. 1A.

[0015] FIG. 1C is a schematic of a top view of the nanoparticle tracking analysis system of FIG. 1A.

[0016] FIG. 2A is a schematic of a digital microfluidics chip in accordance with an example of the present technology.

[0017] FIG. 2B is a schematic of a nanoparticle tracking analysis system coupled with a digital microfluidics platform in accordance with another example.

[0018] FIG. 2C is an expanded schematic illustration of the cell culture region of FIG. 2B. FIG. 2D is a side view of the cell culture region of FIG. 2C.

[0019] FIG. 2E is an expanded schematic illustration of the dilution region of FIG. 2B. FIG. 2F is an expanded schematic illustration of the particle analysis region of FIG.

[0020] 2B.

[0021] FIG. 2G is a schematic illustration of the sample interrogation region of FIG. 2F. FIG. 3 is a flowchart illustrating an example method of tracking and analyzing nanoparticles in microscale volumes in accordance with examples of the present disclosure.

[0022] FIG. 4 is a graph of relative frequency (density) as a function of diameter (nm) in accordance with one example.

[0023] FIG. 5 is a graph of relative frequency (density) as a function of diameter (nm) in accordance with another example.

[0024] FIG. 6A shows the results of NTA analysis of polymeric particles of various sizes in accordance with one example.

[0025] FIG. 6B shows the results of NTA analysis of extracellular vesicles (EVs) in accordance with one example.

[0026] FIG. 6C shows the results of NTA analysis of lipid nanoparticles encapsulating messenger RNA (LNP-mRNA) in accordance with one example.FIG. 7A shows transfection of retinal epithelial cells by commercially purchased and lipid-dyed (DiD) LNPs encapsulating GFP mRNA in accordance with one example.

[0027] FIG. 7B compares transfection potency of three different formulations of LNPs in accordance with one example.

[0028] FIGs. 7C and 7D quantify GFP protein expression and internalization of DiD-labeled LNPs in retinal epithelial cells treated or not treated with LNPs, in accordance with one example.

[0029] FIG. 7E is a graph which quantitatively compares GFP protein expression levels in retinal epithelial cells treated with three different LNP formulations and a negative control, in accordance with one example.

[0030] FIGs. 8A and 8B are graphs showing EV secretion by retinal epithelial cells following LNP treatment in accordance with one example. FIG. 8A shows increased EV secretion measured after transient LNP exposure followed by washing. FIG. 8B illustrates a tendency towards enhanced EV secretion in cells preconditioned with EVs derived from LNP-treated cells prior to LNP treatment, compared to cells receiving LNP treatment without EV preconditioning.

[0031] FIG. 8C compares transfection efficacy of retinal epithelial cells treated with LNPs with or without EV preconditioning in accordance with one example.

[0032] FIGs. 8D and 8E provide quantitative data on GFP protein expression and the internalization of DiD-labeled LNPs in retinal epithelial cells. These cells were treated with LNPs, both with and without EV preconditioning, in accordance with one example.

[0033] FIG. 9A illustrates EV secretion over 24 hours from activated and control Jurkat T cells cultured in microscale droplets, showing increased EV secretion upon activation, in accordance with one example.

[0034] FIG. 9B compares EV secretion levels at 24 hours between activated and control Jurkat T cells, indicating significantly elevated EV release from activated cells, in accordance with one example.

[0035] FIG. 9C compares EV size distributions from activated and control Jurkat T cells at 24 hours, demonstrating activation-dependent shifts toward larger EV sizes, in accordance with one example.These drawings are provided to illustrate various aspects of the invention and are not intended to be limiting of the scope in terms of dimensions, materials, configurations, arrangements or proportions unless otherwise limited by the claims.

[0036] DETAILED DESCRIPTION

[0037] While these exemplary embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, it should be understood that other embodiments may be realized and that various changes to the invention may be made without departing from the spirit and scope of the present invention. Thus, the following more detailed description of the embodiments of the present invention is not intended to limit the scope of the invention, as claimed, but is presented for purposes of illustration only and not limitation to describe the features and characteristics of the present invention, to set forth the best mode of operation of the invention, and to sufficiently enable one skilled in the art to practice the invention. Accordingly, the scope of the present invention is to be defined solely by the appended claims.

[0038] Definitions

[0039] In describing and claiming the present invention, the following terminology will be used.

[0040] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a collimating lens” includes reference to one or more of such lenses and reference to “the laser” refers to one or more of such devices.

[0041] As used herein with respect to an identified property or circumstance, “substantially” refers to a degree of deviation that is sufficiently small so as to not measurably detract from the identified property or circumstance. The exact degree of deviation allowable may in some cases depend on the specific context.

[0042] As used herein, “adjacent” refers to the proximity of two structures or elements. Particularly, elements that are identified as being “adjacent” may be either abutting or connected. Such elements may also be near or close to each other without necessarily contacting each other. The exact degree of proximity may in some cases depend on the specific context.As used herein, the term “about” is used to provide flexibility and imprecision associated with a given term, metric or value. The degree of flexibility for a particular variable can be readily determined by one skilled in the art. However, unless otherwise enunciated, the term “about” generally connotes flexibility of less than 2%, and most often less than 1%, and in some cases less than 0.01%.

[0043] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[0044] As used herein, the term “at least one of’ is intended to be synonymous with “one or more of.” For example, “at least one of A, B and C” explicitly includes only A, only B, only C, or combinations of each.

[0045] Numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also to include individual numerals such as 2, 3, 4, and subranges such as 1 to 3, 2 to 4, etc. The same principle applies to ranges reciting only one numerical value, such as “less than about 4.5,” which should be interpreted to include all of the above-recited values and ranges. Further, such an interpretation should apply regardless of the breadth of the range or the characteristic being described.

[0046] Any steps recited in any method or process claims may be executed in any order and are not limited to the order presented in the claims. Means-plus-function or step-plus- function limitations will only be employed where for a specific claim limitation all of the following conditions are present in that limitation: a) “means for” or “step for” is expressly recited; and b) a corresponding function is expressly recited. The structure, material or acts that support the means-plus function are expressly recited in the description herein.Accordingly, the scope of the invention should be determined solely by the appended claims and their legal equivalents, rather than by the descriptions and examples given herein. Example Embodiments

[0047] Systems and methods described herein can support microliter-scale volumes in NTA, representing 100 to 1000-fold reduction in volume requirements for samples. However, at the same time, these systems and methods can provide comparable or better capacity to determine the size and concentration of nanoparticles in the sample. Additionally, the systems and methods described in the present disclosure have the ability to integrate with fluid cells that are powered by digital microfluidics (DMF).

[0048] Turning now to FIG. 1 A and FIG. 1C, where FIG. 1A is a side view and FIG. 1C is a top view, a microscale nanoparticle tracking analysis (NTA) system 100 can include a tracking volume 102. In some examples, the tracking volume 102 can be formed using a plurality of walls. In one example, the tracking volume can be supported simply by a bottom wall 104. In other examples, the tracking volume 102 can be formed between two walls, with a bottom wall 104 and a top wall 106. In other examples, the tracking volume 102 can be defined by a bottom wall 104, a top wall 106, and side walls, enclosing the tracking volume 102. In examples where the tracking volume 102 is enclosed, at least one wall has an unobstructed opening 108. Alternatively, at least a part of the wall can have a transparent section. In one example, the system can include a single unobstructed opening, and in some cases a single unobstructed opening adjacent the NTA sample location. The unobstructed opening or transparent section can be various shapes, sizes, and locations on the wall. In one example, a transparent section can be made of glass, quartz, or other transparent material. In a further example, regardless of the number of walls, at least one wall of the tracking volume 102 includes an unobstructed opening 108.

[0049] As shown in FIG. IB, the volume 102 can include an NTA sample location 110. The NTA sample location 110 can be where a droplet 111 of sample liquid is placed in order to be analyzed. The NTA sample location 110 can be sized to fit a wide range of sample volumes. In some cases, the NTA sample location 110 can be sized to accommodate a microscale droplet. As a general guideline, the droplet can have a volume of 100 picoliter to less than 100 microliters, in some cases 1 nanoliter to 50 microliters, in other cases 500 nanoliter to 10 microliters. The NTA sample location 110 can be in any part of the trackingvolume 102. In some examples, there can be multiple NTA sample locations within the tracking volume. In some examples, the tracking volume can be a microscale volume, and in some cases can be a microscale channel.

[0050] In some cases, the tracking volume can be formed as a continuous channel through which the droplet can be moved, or as a dedicated well or reservoir. The channel can be formed in any shape including, but not limited to, a box, a concave depression, a slit, or the like. In one example, the volume 102 can be formed as a continuous channel between two walls, a top wall 106 and a bottom wall 104. In some examples, the walls can be plates. The plates can be made of glass, metal, plastic, or other suitable material that does not react with the liquid being analyzed. In some examples, the plates can be positioned to be a specific distance apart from one another. Depending on how far away the plates are from one another creates a certain gap size. In some cases, the gap size can be from about 30 to about 100 microns, from about 75 to about 200 microns, from about 100 to about 400 microns, or from about 100 to about 500 microns. In another example, the gap size can be from about 100 to about 300 microns. In further examples, at least one of the walls has a hydrophobic coating. In some examples, all walls have a hydrophobic coating. In this case, the NTA sample location does not have side walls (i.e. only top and bottom walls). In another example, the hydrophobic coating can be patterned to form hydrophilic islands where corresponding small droplets can be dispensed. In this manner, larger droplets can be moved across them while the small droplets remain behind due to differences in surface energy. Optionally, the NTA sample location can also be further defined or constrained by side walls as long as the unobstructed opening 108 remains unobstructed. In another example, the NTA sample location can be an open volume which is only physically bounded by a single floor plate, while surrounding space is unobstructed. Regardless, this system employs a distinct approach, combining a hydrophobic-coated surface to stabilize droplets, a cell design allowing unobstructed light excitation to the droplets, and specialized optics tailored to illuminate those droplets. This combination eliminates the need to fdl an entire chamber, allowing for more precise particle analysis with significantly reduced sample volumes.

[0051] In some examples, the NTA system 100 further includes an excitation light source 112. In some examples, the excitation light source 112 can be a laser, although other nonlimiting examples can include light-emitting diodes (LED), super-luminescent diodes (SLD),emission from one or more optical fibers, or waveguides coupled to one or more light sources. In one example, the excitation light source can be a multi-spatial mode laser. In this example, the laser can be coupled to an optical fiber 113 which is terminated with a fiber collimator 115. Regardless, the excitation light source 112 can be configured to introduce a light beam 114 to the NTA sample location 110. In one example, the light beam 114 can be a light sheet. This can be accomplished by the presence of a collimator to align the light and a slit aperture 117 which narrows the light beam 114 introduced from the light source 112. However, such light shaping can also be obtained by numerous configurations of lenses, apertures, amplitude and / or phase masks, and / or diffractive elements to shape a beam into a light sheet of dimensions suitable for the sample volume shape. As a general guideline, a light sheet can have a width to thickness ratio of 100 to 1, and most often 10 to 1 (where width and thickness refer to measures of the beam size, such as FWHM, along these respective dimensions). Similarly, a minimum thickness of the light sheet (FWHM) can range from about 5 pm to about 500 pm and in some cases 50 to 300 pm. The width (FWHM) of the light sheet can broadly range from about 50 to 10000 pm, and in some cases about 500 to 5000 pm. Alternatively, the light beam 114 can be a beam which is symmetric (e.g. circular cross-section) having a diameter (FWHM) from 5 to 500 pm and in some cases 50 to 300 pm. The cross-section of a beam can exhibit various shapes, including Gaussian, elliptical, Laguerre-Gaussian, Hermite-Gaussian, Ince-Gaussian, Super-Gaussian, Gaussian-like profiles, and top-hat (uniform intensity) distributions. Additional configurations such as scallop-shaped (characterized by higher intensity at the edges and a drop in the center) and ring or annular beam profiles are also considered. Furthermore, beams with extended depths of focus are contemplated, along with other potential cross-sectional shapes.

[0052] Additionally, the light beam 114 can be introduced to the NTA sample location 110 through the optically unobstructed opening 108 of the tracking volume 102. In some instances, the light beam 114 introduced can be sufficient to illuminate particles within the microscale droplet to produce particle light scattering. The light beam 114 can uniformly illuminate across the microscale droplet with negligible interaction with the wall. The illumination intensity across the droplet can vary from 100% to 5%, from 100% to 10%, from 100% to 33%, from 100% to 50%, from 100% to 70%, or from 100% to 90%. Uniform illumination, where intensity across the droplet remains within less than 20% of themaximum intensity, facilitates quantification of scatter intensity and particle concentration, but may not be required for size determination. Generally, the light beam 114 can be shaped to directly illuminate as much of the microscale droplet as possible without also interacting with walls of the tracking volume 102 (i.e. at least until after passing the sample location 110). As a general guideline, the NTA sample location can have an opening dimension where the light beam is shaped to have a maximum dimension relative to that opening. Accordingly, the light beam 114 can be shaped in a way that has a maximum dimension from about 20% to about 40% of the NTA sample location 110 size, from about 30% to about 60% of the NTA sample location 110 size, or from about 20% to less than 100% of the NTA sample location 110 size. In one example, the light beam can be shaped in a way that has a maximum dimension from about 30% to about 70% of the NTA sample location 110 size, and in some cases to about 90%. In some cases, the maximum dimension is a light beam width and the NTA sample location size is an opening width.

[0053] In one embodiment, the excitation light source 112 can be shaped into a light beam 114 to the NTA sample location 110 as a laser light sheet formed through a series of lenses and optical elements, including refractive and diffractive components. As an example, light from the source 112 can be directed through at least one spherical lens 118 and / or at least one cylindrical lens 120. Spherical and / or cylindrical lenses can be used to reduce beam divergence from the source, and prism pairs or other optical elements can be used to circularize a beam or make it elliptical. In order to form a light sheet, cylindrical lenses can be used to concentrate or expand the light beam along a common axis (i.e. perpendicular to the propagation direction). A slit aperture 117 can also be used to adjust the thickness of the light sheet in combination with subsequent optical elements that refocus the beam into a light sheet entering the opening 108. Additionally, the light beam can be directed through a fixed circular aperture 122 before reaching the NTA sample location 110 in order to remove diffractive artifacts created through the slit aperture 117. In this manner, the light beam can be shaped before it reaches the NTA sample location 110 such that the light sheet is formed within the sample chamber. One or more folding mirrors (124 and 128) can be inserted into the optical path in order to convert a long linear path into a more compact square or rectangular-shaped layout.In further examples, the NTA system 100 can include an imaging device 116. The imaging device 116 can be configured to capture light scattering from the illuminated particles within the microscale droplet. The captured particle light scattering can be used for NTA data collection and analysis. In some examples, the imaging device 116 can be a standard optical microscope or other suitable imaging system with or without optical magnification. The imaging system can contain, for example, a charged-coupled device (CCD) or complimentary metal-oxide-semiconductor (CMOS) array to detect the particle light scattering across the field of view. The imaging system 116 can be positioned in a way that can best capture particle light scattering. In some examples, the imaging system can be positioned above, below, or to the side of the NTA sample location 110. In examples where the imaging system is positioned above or below, if a top wall 106 or bottom wall 104 is present, the at least one wall of the tracking volume can further include a transparent window oriented between the illuminated particles and the imaging device 116. This transparent window allows for the imaging device 116 to capture at least a portion of the particle light scattering. In examples that include a transparent window, the at least one wall of the tracking volume can further include a second transparent window oriented opposite the transparent window. This second transparent window can reduce reflection of the particle light scattering. For example, when the imaging system is positioned above the NTA sample location, the transparent window can be oriented within the top wall while the second transparent window can be oriented within the bottom wall. Similarly, if the imaging system is positioned below the NTA sample location, the transparent window can be oriented within the bottom wall while the second transparent window can be oriented within the top wall. For example, a quartz window can be oriented within the bottom wall 104 while a second quartz window can be oriented within the top wall 106. In further examples, there can be multiple imaging systems or detection devices used, and multiple walls can be transparent.

[0054] In one embodiment of the NTA system 100, as seen in FIG. 2A, the tracking volume 200 can be formed between a top plate 202 and a bottom plate 204. The two plates can further form a microfluidic array. In some cases, the two plates can be made of glass. In some examples, the tracking volume 200 can be a digital microfluidics (DMF) chip. DMF, also described as electrowetting-on-dielectric (EWOD), is a method of droplet-based fluid manipulation as discrete droplets on a surface array of electrode via electric fields. In someexamples, there are no side walls present in defining the tracking volume. In such examples, the NTA sample location 208 can be oriented at a periphery of the DMF chip. In DMF, droplets 210 are controlled by applying electric fields between actuation electrodes 214a and 214b, which are on the bottom plate 204, and the ground electrode 212, which is on the top plate 202. The actuation electrodes 214a, 214b can be horizontally separated by a gap or dielectric so that each electrode pad can act as a digitized location for creation of a local electric field. In the illustrated example, electrode 214a is actuated so that a positive charge builds adjacent the volume surface which attracts a preferential negative charge within the droplet which encourages movement of the droplet toward that electrode. The electrode can be actuated using AC, DC, or a combination of electrical signals, which attract opposite charges or dipoles within the droplet. Thus, upon application of electric fields, charges accumulate, which serve as an electrostatic handle for droplet manipulation. In this manner, the degree of droplet control can depend on the number and spacing of individual electrodes within a digital array of electrodes (e.g. coplanar with electrodes 214). In some examples, typical droplet manipulation can include measuring precise volumes, mixing samples or reagents, and transferring droplets for analysis and retrieval. Additionally, the bottom plate 204 can further include a dielectric insulation layer 216, with a hydrophobic coating 218 on both the top plate 202 and the bottom plate 204.

[0055] When integrated with the NTA system 100, DMF can provide many new capabilities, such as more sophisticated fluid handling, further decreasing volume requirements for analysis, and the ability to rapidly switch samples by moving droplets in an automated manner. Additionally, DMF enables the retrieval of the complete droplet sample for additional downstream analysis. Thus, intricate, software-programmable sample preparation can be performed prior to analysis.

[0056] FIG. 2B illustrates a DMF-NTA platform 220 which integrates an NTA system 222 with a digital microfluidics (DMF) platform 224. The NTA system includes a light source 226 which produces a light beam directed into a sample location through an unobstructed opening. The NTA system can also include an imaging device 228 which, in this case, can be oriented perpendicular to the light beam and / or a plane of the DMF platform 224. The DMF platform 224 can include upper contact pads 230 and lower contact pads 232 which are individually connected to respective upper and lower electrodes within the DMFworkspace 234. The DMF workspace 234 can include an array of actuation electrodes which can be selectively activated to drive droplets of fluid around the array. For example, fluid can be directed from a cell culture media reservoir 242 and a dilution reservoir 244 through a cell culture region 236, a dilution region 238, and a particle analysis region 240.

[0057] More specifically, FIG. 2C illustrates the cell culture region 236 where a culture medium fluid 246 is directed from the culture reservoir 242 (FIG. 2B) to a cell culture pad 248. FIG. 2D shows a side view of the cell culture pad 248 which shows a droplet 250 of the culture medium fluid contacting attached cells 252. Hydrophobic layers 254 are coated on each interior surface to prevent fluid from adhering. On the upper plate, a dielectric layer 256 can be used to insulate the hydrophobic layer 254 from actuating electrodes 258. Glass layer 260 can form an outer transparent support layer. A conductive reference electrode layer 262 can be formed on the lower plate along with a corresponding glass layer 260. In this case, the cells are adhered to a bottom surface of the cell culture region 236. Alternatively, cells can be introduced separately or together with the culture media, or can be adhered to suspended carrier particles. These cells can be attached via any suitable mechanism (i.e. naturally adherent, surface functionalized, trapping, encapsulation, electrostatic, magnetic, or the like).

[0058] Referring back to FIG. 2C, upon completion of culturing and production of extracellular vesicles or other particles, the cultured fluid 250 is directed toward the dilution region 238 which is shown in more detail in FIG. 2E. FIG. 2E shows a dilution medium 264 being directed from the dilution reservoir 244 (FIG. 2A) to mix with the cultured fluid 250 to form a diluted sample 266.

[0059] The diluted sample 266 is then directed toward the particle analysis region 240 (FIG.

[0060] 2A) which is shown in further detail in FIG. 2F and 2G. The diluted sample 266 can then be subjected to interrogation using the NTA system as described previously. As more fully shown in FIG. 2G, the diluted sample 266 can be directed to a sample interrogation location 267. A light source 268 can direct a light beam 269 through an unobstructed opening 270 such that the light beam interacts with particles in the diluted sample 266. As described previously, an imaging device 272 can receive scattered light 273 through a transparent window or layer 274 from interaction of the light beam 269 with particles in the dilutedsample 266. In this case, a bottom layer 276 can include a transparent window 278 to minimize reflection back to the imaging device 272.

[0061] Consistent with the above description and as illustrated by FIG. 3, a method 300 of tracking and analyzing nanoparticles in microscale volumes can include dispensing a droplet into an NTA sample location 310. The NTA sample location can be sized to fit a wide range of sample volumes, with different sized droplets dispensed. In some cases, a microscale droplet can be dispensed into the NTA sample location. The microscale droplet can have a volume of from about 100 picoliters to about 100 microliters, from about 1 nanoliter to about 50 microliters, or from about 500 nanoliters to about 10 microliters. In one example, the microscale droplet can have a volume of from about 1 microliter to about 5 microliters. These ranges are provided as examples and are not intended to limit the scope of the invention. Other droplet volumes suitable for the system or application may also be used within the capabilities described herein.

[0062] The method 300 can further include introducing a light beam to the NTA sample location through an unobstructed opening 320. The light beam can be from a laser or other suitable light source. In some examples, the light beam can be a light sheet. In further examples, the light beam can be sufficient to illuminate particles within the droplet, producing particle light scattering. The light beam can be sequentially introduced at multiple different intensities to the NTA sample location to improve particle light scattering for different sized particles. As previously mentioned, in nanoparticle tracking analysis, particles are detected via light scattering. There are many factors that determine that amount of scattered light, with the most important factor being particle size. Scattering intensity scales approximately with the cross-sectional area of the particle, which means that small particles scatter dramatically less light while large particles can easily saturate an imaging device. Therefore, light beams with low intensity are best for large particles and highly scattering materials, such as metals and dense polymers, while light beams with high intensity are best for small particles and weak scatterers, such as extracellular vesicles (EVs), lipid nanoparticles (LNPs), lipoproteins, and protein aggregates. As a specific example, light intensities can range from 0.04 to 250 mW / mm2, with 10 to 25 mW / mm2tending to work well for relatively smaller polymer particles (50-200 nm) or weak scatterers, while 0.4 to 10 mW / mm2tending to work well for larger polymer particles (200-400 nm), but other rangesare contemplated. Thus, by sequentially introducing varying light intensities, the scattering of light can be enhanced for nanoparticles of different sizes, size ranges, and material composition. The method 300 can also include capturing particle light scattering from the illuminated particles within the droplet 330. This can be done by using an imaging device, such as a standard optical microscope, CCD camera, CMOS camera, or a combination thereof.

[0063] Additionally, the method 300 can include collecting data and analyzing nanoparticles in the droplet 340. Some specific, but not limiting, examples of analyzing nanoparticles include analysis of the sizing and quantification of the nanoparticles. In some examples, the NTA sample location can further include at least one cell culture site. Cell culture sites are where cells can be maintained, grown, or manipulated. In some examples, the cell culture site can be used to generate extracellular vesicles (EVs). EVs are small, membrane-bound particles released by cells into their surrounding environment. EVs act as a fundamental mechanism for cell-to-cell communication. For example, EVs can bind to receptors on target cells, fuse with the cell membrane, and can be internalized via endocytosis. EVs play an important role in immune regulation, tissue repair and regeneration, neural communication, as well as in development and homeostasis. In particular, EV secretion from immune cells, such as T cells, can change dramatically in response to cellular activation states, making EV secretion a potentially useful indicator of cell activation. For example, activated T cells may secrete greater numbers and distinct populations of EVs compared to their resting counterparts. Measuring these differences at microscale volumes can provide valuable insights into immune cell states and functions.

[0064] In some examples, the process of collecting data and analyzing nanoparticles can include using particle light scattering to analyze EVs or lipid nanoparticles (LNPs). LNPs are nanoscale synthetic particles predominantly composed of lipids, specifically engineered to encapsulate and deliver nucleic acids, such as messenger RNA (LNP-mRNA), into target cells. This technology can serve as a component in drug delivery applications. Compared to EVs, which are naturally secreted nanoparticles made by cells, LNPs are designed to mimic some functions of EVs. In some examples, the biogenesis and secretion of EVs from cells can be modulated by treatment of the cells with LNP-mRNA formulations. In some examples, cells treated with LNP-mRNAs can secrete EVs that appear capable of influencingthe efficiency with which cells subsequently internalize LNPs. Without being bound by theory, such EVs may interfere with or otherwise alter cellular uptake mechanisms, thus affecting the potency of LNP-mediated transfection. Notably, experimental observations indicate reduced LNP uptake by cells previously exposed to EVs derived from LNP-treated cells. Thus, the extent of EV secretion may serve as an indirect indicator for evaluating LNP potency, with formulations eliciting minimal EV secretion potentially being advantageous. In some examples, capturing particle light scattering from EVs secreted following treatment with different LNP candidates allows for comparative evaluation and potency assessment among those candidates. In addition, EV secretion data can be paired with complementary cell-based assay data, such as direct measurements of LNP uptake and protein expression, as well as physical characterization data of the LNPs collected prior to treatment, including particle size and concentration. Together, these combined data provide enhanced capability for screening and evaluating multiple LNP candidates comprising various lipid formulations and nucleic acid cargo compositions.

[0065] In some examples, the method 300 can further include manipulating the droplet using DMF. In one example, the NTA sample location can be oriented at a periphery of a DMF chip. The manipulation can be accomplished by applying electric fields, which accumulates charges, creating an electrostatic handle. This electrostatic handle can thus manipulate the droplets. The manipulating can include measuring precise volumes, mixing samples or reagents, and transferring droplets for analysis and retrieval. The DMF-NTA platform 220 can facilitate automated collection of multiple related datasets. These datasets include physical characterization data for LNPs used to treat cells and for EVs subsequently secreted by the treated cells, including particle size and concentration, as well as cell-based assay data to evaluate potency effects, such as uptake efficiency of the LNPs and the resulting protein expression arising from successful delivery of the nucleic acid cargo. Collectively, these combined data sets obtained using the DMF-NTA platform 220 provide an enhanced capability for screening and evaluating multiple LNP candidates comprising various lipid formulations and nucleic acid cargo compositions.

[0066] In still other optional examples, the method and system can incorporate fluorescence. For instance, DMF can be utilized to manipulate sample droplets containing nanoparticles (e.g., extracellular vesicles) and reagent droplets containing specific or non-specific fluorescent labeling agents such as lipid dyes and fluorescently conjugated antibodies. The DMF system can precisely mix these droplets to label the nanoparticles in the sample volume. This labeling process can prepare the sample for subsequent fluorescence-based optical analysis. Further, the light beam can be chosen to excite fluorescence in specific target entities within the sample volume, where the fluorescence light emission is at a different wavelength from the excitation light (e.g. the light beam or light sheet). An optical spectral filter can be placed in the path between the sample volume and the detector to enable the detection of primarily the fluorescence emission from the target entities.

[0067] Examples

[0068] Example 1

[0069] Referring back to FIG. 1A, an NTA system 100 was formed using a 430 nm 4000 mW multi-spatial mode laser 112 which was coupled to a 400 micron core fiber 113, terminating with a fiber collimator (f = 4.6 mm) 115. A spherical lens (118) was used to minimize residual beam divergence. The quasi-collimated beam next passed through a cylindrical lens (120), which was used to concentrate the beam along one axis onto a horizontal slit (117) with a 100 micron opening. A circular aperture (122) was used to clean up the beam from diffractive artifacts. The beam path was folded by mirror (124) before being directed to a second cylindrical lens (126) to create the light sheet under a 2f-2f imaging condition (unity conjugate). Following lens 126, the beam path was folded again by a second mirror (128) before reaching the sample region 113. Finally, the focused light sheet passed through a small droplet in the 360 pm gap between two glass slides, where the light sheet was measured to be 250 pm tall and 4.6 mm wide. An Evident DP23M camera 116 was positioned at a 90-degree detection angle to detect particle light scattering through a Newport 20X, 0.4 NA microscope objective coupled to the camera via an optical extension tube.

[0070] A sample was prepared by mixing an amount of NIST-traceable polystyrene size standards having particle sizes of 100 nm and 400 nm. FIG. 4 is a graph of relative frequency (density) as a function of diameter (nm) for a 10 microliter sample of the mixture analyzed by the NTA system in FIG. 1 A. FIG. 5 is a graph of relative frequency (density) as a function of diameter (nm) for a 1 mL sample of the mixture analyzed by a commercial NTA system.This shows that the NTA system in FIG. 1A can perform equivalent measurements of a commercial NTA system with 100-fold reduction in sample volume, including the capacity to differentiate sizes of nanoscale particles.

[0071] Example 2

[0072] FIGs. 6A, 6B, and 6C are examples of NTA analysis of various nanoparticles and dilutions. FIG. 6A shows analysis of polymeric particles of various sizes, specifically NIST-traceable polystyrene particles. The size distributions generally align with expected particle diameters of 50 nm, 100 nm, 200 nm, 300 nm, and 400 nm, respectively. FIG. 6B shows analysis of various dilutions of EVs secreted from a leukemia cell line, and FIG. 6C shows a similar analysis of LNP-mRNA. In each analysis, the dilutions were automatically generated using the DMF-NTA platform 220 of FIG. 2B with sample measurement volumes of approximately 3 microliters. Collectively, these results demonstrate that the DMF-NTA platform 220 can automatically generate appropriate sample dilutions from arbitrary initial particle concentrations, facilitating calibration with known particle standards and ensuring measurements fall within the linear quantification range. This capability enables robust and quantitative nanoparticle tracking analysis at microliter-scale volumes without labor-intensive manual dilution steps. This represents a significant improvement over conventional benchtop NTA systems, which typically require approximately 100- to 1000-fold greater sample volumes and manual sample preparation. Additionally, detection of EVs and LNP-mRNA further demonstrates that the platform is sufficiently sensitive for biological nanoparticles that exhibit weaker scattering. These capabilities highlight the suitability of the DMF-NTA platform for diverse nanoparticle characterization applications, including studies in EV biology and high-throughput screening of lipid nanoparticles.

[0073] Example 3

[0074] FIGs. 7A, 7B, 7C, 7D, and 7E demonstrate illustrative examples of cell-based assay data obtained by the DMF-NTA platform 220 to evaluate the potency of LNP-mRNA, such as uptake efficiency of the LNPs and the resulting protein expression arising from successful delivery of the nucleic acid cargo. Specifically, retinal epithelial cells cultured in the cell culture region 236 in microscale droplets of approximately 300 nanoliters are treated with DiD-labeled lipid nanoparticles encapsulating GFP mRNA for four hours, washed, and allowed to secrete EVs. FIG. 7A shows fluorescence-based analysis of GFP expression as ameasure of transfection efficiency and DiD labeling as an indicator of LNP uptake. FIG. 7B shows a comparative analysis of GFP expression resulting from transfection with three different formulations of LNP-mRNA, with formulation LNP1 demonstrating the highest potency. FIGs. 7C and 7D plot the single-cell fluorescence of GFP expression and DiD-labeled LNP uptake data from FIG. 7A. FIG. 7E quantitatively compares single-cell GFP protein expression levels measured as fluorescent intensity from FIG. 7B, clearly showing that cells treated with formulation LNP1 have significantly higher GFP expression compared to formulations LNP2, LNP3, and the negative control. The secreted EVs from the cultured cells, following LNP treatment, are automatically diluted in the dilution region 238 and subsequently analyzed in the particle analysis region 240 to determine the effects of LNP -induced EV secretion.

[0075] Example 4

[0076] FIGs. 8A, 8B, 8C, 8D, and 8E demonstrate that LNP treatment induces elevated EV secretion, and that those EVs appear to reduce the efficiency with which cells subsequently internalize LNPs. FIG. 8A plots EV secretion from retinal epithelial cells treated with LNPs for four hours, washed, and allowed to secrete EVs for 20 hours, showing significantly higher EV counts relative to baseline or untreated cells. FIG 8B shows a trend toward further increased EV secretion induced by LNP treatment when cells are pre-exposed to EVs secreted from LNP -treated cells. FIG. 8C compares cells pre-exposed to the EVs and cells without EV pre-exposure, analyzing GFP expression and LNP uptake resulting from LNP treatment. FIGs 8D and 8E plot the single-cell fluorescence of GFP expression and DiD-labeled LNP uptake data from FIG. 8C, demonstrating that EV -pretreatment reduces the potency of LNPs. Thus, the extent of EV secretion may serve as an indirect indicator for evaluating LNP potency, with formulations eliciting minimal EV secretion potentially being advantageous. Collectively, these analyses demonstrate that the integrated microscale workflow enables robust, quantitative, and high-throughput screening of multiple LNP formulations with significantly reduced volumes compared to conventional cell-based assays typically performed in multiwell plates.

[0077] Example 5

[0078] FIGs. 9A, 9B, and 9C demonstrate that the integrated DMF-NTA platform 220 can be used to quantitatively assess EV secretion as a proxy for immune cell activation atmicroscale volumes. Specifically, Jurkat T cells were cultured in droplets of approximately 300 nanoliters at the cell culture region 236 on the DMF-NTA platform 220, and activated using CD3 / CD28 T-cell activator reagents or left untreated as controls. EV secretion was quantified using NTA at various discrete time points from independent microscale droplet experiments, spanning from 2 hours up to 24 hours following activation. FIG. 9A shows the time course of EV release from activated and control Jurkat cells. Activated cells progressively secrete more EVs overtime, with clearly elevated secretion compared to nonactivated control cells becoming pronounced at the 24-hour time point. FIG. 9B specifically quantifies EV secretion at 24 hours, demonstrating a statistically significant increase in EV secretion from activated Jurkat cells relative to controls. FIG. 9C illustrates differences in EV size distributions from activated and control Jurkat cells at 24 hours. EVs derived from activated cells exhibit distinct and larger size populations, with clear peaks at approximately 146 nm, 311 nm, and 555 nm. In contrast, EVs secreted from control cells show smaller and distinctly different peaks at approximately 91 nm, 221 nm, and 458 nm, indicating an activation-dependent shift in EV subpopulation composition. Collectively, these data demonstrate that EV concentration and size distributions measured using the integrated DMF-NTA platform 220 provide quantitative, sensitive, and microscale indicators of Jurkat T-cell activation. The capability to detect activation-dependent changes in EV secretion profdes at low sample volumes highlights the utility of the DMF-NTA platform 220 for a broad range of immune-cell-related applications. Such applications include high-throughput screening of immunomodulatory agents, assessment of immune cell functionality, and realtime monitoring of immune responses, all within an integrated microscale workflow.

[0079] While the flowcharts presented for this technology may imply a specific order of execution, the order of execution may differ from what is illustrated. For example, the order of two more blocks may be rearranged relative to the order shown. Further, two or more blocks shown in succession may be executed in parallel or with partial parallelization. In some configurations, one or more blocks shown in the flow chart may be omitted or skipped. Any number of counters, state variables, warning semaphores, or messages might be added to the logical flow for purposes of enhanced utility, accounting, performance, measurement, troubleshooting or for similar reasons.Reference was made to the examples illustrated in the drawings and specific language was used herein to describe the same. It will nevertheless be understood that no limitation of the scope of the technology is thereby intended. Alterations and further modifications of the features illustrated herein and additional applications of the examples as illustrated herein are to be considered within the scope of the description.

[0080] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more examples. In the preceding description, numerous specific details were provided, such as examples of various configurations to provide a thorough understanding of examples of the described technology. It will be recognized, however, that the technology may be practiced without one or more of the specific details, or with other methods, components, devices, etc. In other instances, well-known structures or operations are not shown or described in detail to avoid obscuring aspects of the technology.

[0081] For example, various features and elements of the above-described invention can be used alone or in various combinations, where elements described in one example can be utilized in other described examples. More specifically, the invention can be generally described by the following clauses:

[0082] Clause 1. A microscale nanoparticle tracking analysis (NT A) system, comprising: a tracking volume, including an NTA sample location, configured to accommodate a microscale droplet, wherein at least one wall of the tracking volume includes an unobstructed opening; an excitation light source configured to introduce a light beam to the NTA sample location through the unobstructed opening of the tracking volume sufficient to illuminate particles within the microscale droplet to produce a particle light scattering; and an imaging device configured to capture the particle light scattering from the illuminated particles within the microscale droplet to be used for NTA data collection and analysis.

[0083] Clause 2. The system of any of clauses 1-13, wherein the tracking volume is formed between two plates.

[0084] Clause 3. The system of any of clause 2, wherein the two plates further form a microfluidic array.Clause 4. The system of any of clause 3, wherein the microfluidic array is a digital microfluidics (DMF) chip and the NTA sample location is oriented at a periphery of the DMF chip.

[0085] Clause 5. The system of any of clauses 1-13, wherein the NTA sample location has a gap size from about 100 to about 400 microns.

[0086] Clause 6. The system of any of clauses 1-13, wherein the at least one wall includes a single unobstructed opening.

[0087] Clause 7. The system of any of clauses 1-13, wherein the at least one wall of the tracking volume further includes a transparent window oriented between the illuminated particles and the imaging device so as to allow the imaging device to capture at least a portion of the particle light scattering.

[0088] Clause 8. The system of clause 7, wherein the at least one wall further includes a second transparent window oriented opposite the transparent window that reduces reflection of the particle light scattering.

[0089] Clause 9. The system of any of clauses 1-13, wherein the at least one wall has a hydrophobic coating.

[0090] Clause 10. The system of any of clauses 1-13, wherein the light beam is a light sheet. Clause 11. The system of clause 10, wherein the light beam is shaped to have a maximum dimension from about 50% to about 90% of a maximum dimension of the NTA sample location.

[0091] Clause 12. The system of clause 10, wherein the light beam is uniformly illuminated across the microscale droplet without hitting the at least one wall.

[0092] Clause 13. The system of any of clauses 1-12, further comprising a DMF chip having microscale channels formed by parallel plates, wherein the tracking volume and NTA sample location are oriented at a periphery of the parallel plates, and wherein the DMF chip enables droplet-based fluid manipulation, including measuring precise volumes, mixing samples or reagents, and transferring droplets for analysis or retrieval.

[0093] Clause 14. A method of tracking and analyzing nanoparticles, comprising: dispensing a microscale droplet into an NTA sample location; introducing a light beam to the NTA sample location through an unobstructed opening, wherein the light beam is sufficient to illuminate particles within the microscale droplet to produce a particle light scattering;capturing the particle light scattering from the illuminated particles within the microscale droplet using an imaging device; and collecting data and analyzing nanoparticles in the microscale droplet.

[0094] Clause 15. The method of any of clauses 14-27, wherein the microscale droplet is a volume of from about 1 microliter to about 10 microliters.

[0095] Clause 16. The method of any of clauses 14-27, wherein the analyzing includes sizing and quantification of the nanoparticles.

[0096] Clause 17. The method of any of clauses 14-27, further comprising manipulating the microscale droplet using digital microfluidics, wherein the NTA sample location is oriented at a periphery of a digital microfluidics chip.

[0097] Clause 18. The method of any of clauses 14-27, wherein the light beam is sequentially introduced at multiple different intensities to the NTA sample location to improve particle light scattering for nanoparticles of different sizes and compositions.

[0098] Clause 19. The method of any of clauses 14-27, wherein the NTA sample location further comprises at least one cell culture site where cells are maintained, grown, or manipulated.

[0099] Clause 20. The method of any of clauses 14-27, wherein the NTA sample location further comprises one or more dilution regions configured to automatically generate appropriate sample dilutions from arbitrary initial particle concentrations, facilitating calibration with known particle standards and ensuring measurements fall within the linear quantification range.

[0100] Clause 21. The method of any of clauses 19-27, wherein the collecting data and analyzing nanoparticles includes correlating the particle light scattering with extracellular vesicles (EVs).

[0101] Clause 22. The method of any of clauses 19-27, wherein the collecting data and analyzing nanoparticles includes correlating the particle light scattering with lipid nanoparticles (LNPs).

[0102] Clause 23. The method of clause 21, wherein the EVs are secreted by cells cultured at the cell culture site.Clause 24. The method of clause 22, wherein the LNPs are used to treat cells cultured at the cell culture site, and wherein EV secretion from the cells treated with LNPs is used to evaluate the potency of the LNPs.

[0103] Clause 25. The method of clause 24, wherein evaluating potency of LNPs further comprises analyzing particle size and quantification data of the LNPs prior to treating the cells and performing cell-based assays to evaluate protein expression and LNP uptake.

[0104] Clause 26. The method of clause 25, wherein LNP potency evaluation comprises screening multiple LNP candidates in an integrated, microscale platform, and wherein orthogonal datasets collected from particle analysis of the LNPs and EVs secreted by cells treated with the LNPs, and cell-based assays are combined to produce a comprehensive potency metric for comparative evaluation among the LNP candidates.

[0105] Clause 27. The method of clause 23, wherein EV secretion measured from immune cells cultured at the cell culture site is used as an indirect indicator of immune cell activation state, and wherein EV concentration and / or EV size distribution provides quantitative information relating to immune cell activation.

[0106] Although the subject matter has been described in language specific to structural features and / or operations, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features and operations described above. Rather, the specific features and acts described above are disclosed as example forms of implementing the claims. Numerous modifications and alternative arrangements may be devised without departing from the spirit and scope of the described technology.

Claims

CLAIMSWhat is claimed is:

1. A microscale nanoparticle tracking analysis (NTA) system, comprising:a tracking volume, including an NTA sample location, configured to accommodate a microscale droplet, wherein at least one wall of the tracking volume includes an unobstructed opening;an excitation light source configured to introduce a light beam to the NTA sample location through the unobstructed opening of the tracking volume sufficient to illuminate particles within the microscale droplet to produce a particle light scattering; andan imaging device configured to capture the particle light scattering from the illuminated particles within the microscale droplet to be used for NTA data collection and analysis.

2. The system of claim 1, wherein the tracking volume is formed between two plates.

3. The system of claim 2, wherein the two plates further form a microfluidic array.

4. The system of claim 3, wherein the microfluidic array is a digital microfluidics (DMF) chip and the NTA sample location is oriented at a periphery of the DMF chip.

5. The system of claim 1, wherein the NTA sample location has a gap size from about 100 to about 400 microns.

6. The system of claim 1, wherein the at least one wall includes a single unobstructed opening.

7. The system of claim 1, wherein the at least one wall of the tracking volume further includes a transparent window oriented between the illuminated particles and the imaging device so as to allow the imaging device to capture at least a portion of the particle light scattering.

8. The system of claim 7, wherein the at least one wall further includes a second transparent window oriented opposite the transparent window that reduces reflection of the particle light scattering.

9. The system of claim 1, wherein the at least one wall has a hydrophobic coating.

10. The system of claim 1, wherein the light beam is a light sheet.

11. The system of claim 10, wherein the light beam is shaped to have a maximum dimension from about 50% to about 90% of a maximum dimension of the NTA sample location.

12. The system of claim 10, wherein the light beam is uniformly illuminated across the microscale droplet without hitting the at least one wall.

13. The system of claim 1, further comprising a DMF chip having microscale channels formed by parallel plates, wherein the tracking volume and NTA sample location are oriented at a periphery of the parallel plates, and wherein the DMF chip enables droplet-based fluid manipulation, including measuring precise volumes, mixing samples or reagents, and transferring droplets for analysis or retrieval.

14. A method of tracking and analyzing nanoparticles, comprising:dispensing a microscale droplet into an NTA sample location; introducing a light beam to the NTA sample location through an unobstructed opening, wherein the light beam is sufficient to illuminate particles within the microscale droplet to produce a particle light scattering;capturing the particle light scattering from the illuminated particles within the microscale droplet using an imaging device; andcollecting data and analyzing nanoparticles in the microscale droplet using an integrated microscale platform.

15. The method of claim 14, wherein the microscale droplet is a volume of from about 1 microliter to about 10 microliters.

16. The method of claim 14, wherein the analyzing includes sizing and quantification of the nanoparticles.

17. The method of claim 14, further comprising manipulating the microscale droplet using digital microfluidics, wherein the NTA sample location is oriented at a periphery of a digital microfluidics chip.

18. The method of claim 14, wherein the light beam is sequentially introduced at multiple different intensities to the NTA sample location to improve particle light scattering for nanoparticles of different sizes and compositions.

19. The method of claim 14, wherein the NTA sample location further comprises at least one cell culture site where cells are maintained, grown, or manipulated in microscale volumes.

20. The method of claim 19, wherein the collecting data and analyzing nanoparticles includes correlating the particle light scattering with extracellular vesicles (EVs) secreted by the cells cultured at the cell culture site.

21. The method of claim 19, wherein the collecting data and analyzing nanoparticles includes correlating the particle light scattering with lipid nanoparticles (LNPs) used to treat the cells cultured at the cell culture site.

22. The method of claim 21, wherein EV secretion from the cells treated with the LNPs is used as an indicator for evaluating potency or suitability of the LNPs.

23. The method of claim 22, wherein evaluating potency of the LNPs further comprises analyzing particle size and quantification of the LNPs prior to treatment and performing cell-based assays evaluating protein expression and LNP uptake in the treated cells.

24. The method of claim 23, wherein evaluating potency comprises screening multiple LNP candidates using the integrated microscale platform.

25. The method of claim 24, wherein orthogonal datasets collected from particle analysis of the LNPs and EVs secreted by cells treated with the LNPs, and from the cell-based assays, are combined to produce a comprehensive potency metric for comparative evaluation among the LNP candidates.

26. The method of claim 20, wherein the cells cultured at the cell culture site are immune cells, and wherein the EV secretion measured from the immune cells is used as an indirect indicator of immune cell activation state, wherein EV concentration and / or EV size distribution provides quantitative information relating to immune cell activation.

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