Branching mixer and its use and manufacturing method

Abstract

To provide new microfluidic mixers that enable inexpensive manufacture.SOLUTION: Disclosed herein are fluidic mixers having a bifurcated fluidic flow through toroidal mixing elements. The mixers operate, at least partially, by Dean vortexing. Accordingly, the mixers are referred to as Dean Vortex Bifurcating Mixers ("DVBMs"). The DVBMs utilize Dean vortexing and asymmetric bifurcation of the fluidic channels that form the mixers in order to achieve the goal of optimized microfluidic mixing. The disclosed DVBM mixers can be incorporated into any fluidic (e.g., microfluidic) device known to those of skill in the art where mixing two or more fluids is desired. The disclosed mixers can be combined with any fluidic elements known to those of skill, including syringes, pumps, inlets, outlets, non-DVBM mixers, heaters, assays, detectors, and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 275,630, filed January 6, 2016, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] In recent years, high-performance microfluidic mixers have been developed for use in nanoparticle production at industrially relevant flow rates (e.g., 10–12 mL / min). While these mixers have been widely adopted in the drug development market, currently used mixers are difficult to manufacture and have certain performance limitations. At the same time, there is a market for mixers capable of operating at much smaller volumes (on the order of 100 μl). In addition to volume loss, the high flow rates required to operate existing mixers make them unsuitable for such applications. One solution would be to miniaturize existing technologies, such as the Staggered Herringbone Mixer (SHM), to achieve smaller dimensions. However, such devices require features smaller than 50 μm, which can be difficult to fabricate using tooling traditionally used for machining injection molding tools (the preferred mass production method for plastic microfluidic devices).

[0003] Given the difficulties inherent in miniaturizing conventional microfluidic mixers, new mixer designs that allow for inexpensive manufacturing are needed to continue the commercial expansion of their use. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2009 / 096558 [Patent Document 2] WO98 / 22489 [Patent Document 3] WO98 / 39352 [Patent Document 4] WO99 / 14226 [Patent Document 5] U.S. Patent No. 6,251,666 [Patent Document 6] U.S. Patent No. 5,753,789 [Patent Document 7] EP0540742 [Patent Document 8] U.S. Patent No. 5,432,272 [Patent Document 9] U.S. Patent No. 5,644,048 [Patent Document 10] U.S. Patent No. 5,386,023 [Patent Document 11] U.S. Patent No. 5,637,684 [Patent Document 12] U.S. Patent No. 5,602,240 [Patent Document 13] U.S. Patent No. 5,216,141 [Patent Document 14] U.S. Patent No. 4,469,863 [Patent Document 15] U.S. Patent No. 5,235,033 [Patent Document 16] U.S. Patent No. 5,034,506 [Patent Document 17] U.S. Patent Application No. 10 / 290672 [Patent Document 18] U.S. Patent No. 6,433,134 [Patent Document 19] WO92 / 20702 [Patent Document 20] WO92 / 20703 [Patent Document 21] U.S. Patent No. 5,539,082 [Patent Document 22] U.S. Patent No. 5,527,675 [Patent Document 23] U.S. Patent No. 5,623,049 [Patent Document 24] U.S. Patent No. 5,714,331 [Patent Document 25] U.S. Patent No. 5,718,262 [Patent Document 26] U.S. Patent No. 5,736,336 [Patent Document 27] U.S. Patent No. 5,773,571 [Patent Document 28] U.S. Patent No. 5,766,855 [Patent Document 29] U.S. Patent No. 5,786,461 [Patent Document 30] U.S. Patent No. 5,837,459 [Patent Document 31] U.S. Patent No. 5,891,625 [Patent Document 32] U.S. Patent No. 5,972,610 [Patent Document 33] U.S. Patent No. 5,986,053 [Patent Document 34] U.S. Patent No. 6,107,470 [Patent Document 35] WO96 / 04000 [Patent Document 36] U.S. Patent Application No. 13 / 464690 [Patent Document 37] U.S. Patent Application Serial No. 14 / 353,460 [Patent Document 38] PCT / US2014 / 029116 [Patent Document 39] PCT / US2014 / 041865 [Patent Document 40] PCT / US2014 / 060961 [Patent Document 41] U.S. Provisional Patent Application No. 62 / 120,179 [Patent Document 42] U.S. Provisional Patent Application No. 62 / 154,043 [Non-patent literature]

[0005] [Non-licensed Document 1] Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology, pages 385~394, CRC Press, Boca Raton, Fla. [Non-licensed Document 2] Loakes, NAR 2001, Volume 29: Pages 2437~2447 [Non-licensed Document 3] Seela, NAR 2000, Volume 28: Pages 3224~3232 [Non-licensed Document 4] Asseline, (1991), Nucl. Acids Res. 19:4067~74 pages [Non-licensed Document 5] Garbesi, (1993), Nucl. Acids Res. 21:4159-65. [Non-licensed Document 6] Fujimori, (1990), J. Amer. Chem. Soc. 112:7435 pages [Non-licensed Document 7] Urata, (1993), Nucleic Acids Symposium Ser. 29: 69-70 pages [Non-licensed Document 8] Kornberg and Baker, (1992), DNA Replication, 2nd edition, Freeman, San Francisco, Calif. [Non-licensed Document 9] Beaucage, Tetrahedron 49(10):1925 (1993) [Non-licensed Document 10] Letsinger, J. Org. Chem. 35:3800 (1970) [Non-licensed Document 11] Sprinzlä, Eur. J. Biochem. 81:579 (1977) [Non-licensed Document 12] Letsinger, Nucl. Acids Res. 14:3487 (1986) [Non-Patent Document 13] Sawai et al., Chem. Lett. 805 (1984) [Non-Patent Document 14] Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988) [Non-Patent Document 15] Pauwels et al., Chemica Scripta 26:141 91986 [Non-Patent Document 16] Mag et al., Nucleic Acids Res. 19:1437 (1991) [Non-Patent Document 17] Briu et al., J. Am. Chem. Soc. 111:2321 (1989) [Non-Patent Document 18] Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press. [Non-Patent Document 19] Dempcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995) [Non-Patent Document 20] Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991) [Non-Patent Document 21] Letsinger et al., Nucleoside & Nucleotide 13:1597 (194): Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. YS Sanghui and P. Dan Cook. [Non-Patent Document 22] Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994) [Non-Patent Document 23] Jeffs, J. Biomolecular NMR 34:17 (1994) [Non-licensed Document 24] Tetrahedron Lett. 37:743 (1996) [Non-licensed Document 25] Jenkins, Chem. Soc. Rev. (1995) pages 169~176 [Non-licensed Document 26] Rawls, C & E News, June 2, 1997, page 35 [Non-licensed Document 27] Lagriffoulら, Bioorganic & Medicinal Chemistry Letters, 4: 1081-1082 (1994) [Non-licensed Document 28] Petersen, Bioorganic & Medicinal Chemistry Letters, 6: 793-796 (1996) [Non-licensed Document 29] Diderichsen, Tett. Lett. 37: 475-478 (1996) [Non-licensed Document 30] Fujii, Bioorg. Med. Chem. Lett. 7: 637-627 (1997) [Non-licensed Document 31] Jordan, Bioorg. Med. Chem. Lett. 7: 687-690 (1997) [Non-licensed Document 32] Krotz, Tett. Lett. 36: 6941-6944 (1995) [Non-licensed Document 33] Diederichsen, U., Bioorganic & Medicinal Chemistry Letters, 7: 1743-1746 (1997) [Non-licensed Document 34] Lowe, J. Chem. Soc. Perkin Trans. 1, (1997)1: 539-546 [Non-licensed Document 35] Lowe et al., J. Chem. Soc. Perkin Trans. 11: 547-554 (1997) [Non-Patent Document 36] Howarth et al., J. Org. Chem. 62: 5441-5450 (1997) [Non-Patent Document 37] Altmann, KH et al., Bioorganic & Medicinal Chemistry Letters, 7: 1119-1122 (1997) [Non-Patent Document 38] Diederichsen et al., Angew. Chem. Int. Ed., 37: 302-305 (1998) [Non-Patent Document 39] Cantin et al., Tett. Lett., 38: 4211-4214 (1997) [Non-Patent Document 40] Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997) [Non-Patent Document 41] Lagriffoule et al., Chem. Eur. J., 3: 912-919 (1997) [Non-Patent Document 42] Kumar et al., Organic Letters 3(9): 1269-1272 (2001) Summary of the Invention [Problem to be solved by the invention]

[0006] This Summary is provided to introduce selected concepts that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0007] In certain embodiments, disclosed herein are new configurations of microfluidic devices that act as efficient mixers. In certain embodiments, these new mixers can be fabricated using injection molding tools, allowing for inexpensive and efficient manufacturing of the devices. [Means for solving the problem]

[0008] In one aspect, a mixer is provided that operates by forming a Dean vortex to mix at least a first liquid and a second liquid, the mixer including an inlet channel leading to a plurality of annular mixing elements arranged in series, the plurality of annular mixing elements including a first annular mixing element downstream of the inlet channel and a second annular mixing element in fluid communication with the first annular mixing element through a first neck region, the first annular mixing element defining a first neck angle between the inlet channel and the first neck region.

[0009] In another aspect, a method of using a mixer disclosed herein is provided. In one embodiment, the method includes mixing a first liquid with a second liquid by flowing (e.g., pushing or drawing) the first liquid and the second liquid through a mixer disclosed herein to produce a mixed solution.

[0010] In another aspect, a method of manufacturing a mixer is provided, hi one embodiment, the method includes forming a master mold using an end mill, the master mold configured to form a DVBM mixer according to an embodiment disclosed herein.

[0011] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0012] [Figure 1]1 is a photomicrograph of an exemplary Dean vortex diverging mixer ("DVBM") mixing two liquids according to embodiments disclosed herein. [Figure 2] 1 is an illustration of a portion of a DVBM mixer according to an embodiment disclosed herein. [Figure 3] 1 is an illustration of a portion of a DVBM mixer according to an embodiment disclosed herein. [Figure 4] 1 is an illustration of a portion of a DVBM mixer according to an embodiment disclosed herein. [Figure 5] FIG. 2 is a diagram of an exemplary DVBM mixer according to an embodiment disclosed herein. [Figure 6] 1 is an illustration of a portion of a DVBM mixer according to an embodiment disclosed herein. [Figure 7] 1 shows a graph of measured mixing times for an exemplary DVBM with various neck angles. [Figure 8] 1 shows a graph of measured mixing times for an exemplary DVBM mixer and a comparative DVBM mixer. [Figure 9] 1 shows a graph comparing particle size and polydispersity index ("PDI") for a staggered herringbone mixer and two exemplary DVBM mixers. [Figure 10] A micrograph of the DVBM mixer before mixing. Images like this serve as "templates" for image analysis. [Figure 11] 1 is a micrograph of a DVBM mixer in operation, where a clear liquid and a blue liquid are mixing to form a yellow liquid at the far right of the image (i.e., mixing is complete). [Figure 12] 1 is a micrograph showing circles detected using the Hough Circle Transform. [Figure 13A] 1 is a processing template and data image of a mixer. [Figure 13B] 1 is a processing template and data image of a mixer. [Figure 13C] 1 is a processing template and data image of a mixer. [Figure 14] This is a template image with a mask applied. [Figure 15] This is the data (mixed) image with the mask applied. [Figure 16] This is a data (mixed) image in which white pixels are counted. [Figure 17] 1 shows a graph of the size and PDI characteristics of liposomes produced by an exemplary DVBM according to embodiments disclosed herein. [Figure 18] 1 shows a graph of the size and PDI characteristics of emulsion-encapsulated therapeutic agent particles produced by a representative DVBM according to embodiments disclosed herein, as well as a comparison with non-therapeutic-containing emulsion particles having a similar composition except that they do not contain therapeutic agent particles. [Figure 19] 1 shows a graph of the size and PDI properties of exemplary DVBM-produced polymer nanoparticles according to embodiments disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0013] When a fluid flows through a curved channel, the fluid toward the center of the channel is pushed outward by the centripetal force and the higher velocity of the fluid at the bend (caused by the no-slip boundary condition). The action of these forces causes the fluid to rotate perpendicular to the channel in a manner known as Dean vortex formation.

[0014] Disclosed herein is a fluid mixer having bifurcated fluid flow through an annular mixing element. The mixer operates, at least in part, by Dean vortex formation. Therefore, the mixer is referred to as a Dean vortex bifurcating mixer ("DVBM"). The DVBM utilizes Dean vortex formation and asymmetric bifurcating of fluid channels to form a mixer to achieve the goal of optimized microfluidic mixing. The disclosed DVBM mixer can be incorporated into any fluidic (e.g., microfluidic) device known to those skilled in the art where mixing of two or more fluids is desired. The disclosed mixer can be combined with any fluidic element known to those skilled in the art, including syringes, pumps, inlets, outlets, non-DVBM mixers, heaters, assays, detectors, etc.

[0015] The provided DVBM mixers include a plurality of annular mixing elements (also referred to herein as "annular mixers"). As used herein, "annulus" refers to a generally circular structure having two "leg" channels that define the periphery of the annulus between the inlet and outlet of the annular mixer. In certain embodiments, the annular mixer is circular. In other embodiments, the annular mixer may not be perfectly circular, but instead have an elliptical or irregular shape.

[0016] In one aspect, a mixer is provided that operates by forming a Dean vortex to mix at least a first liquid and a second liquid, the mixer including an inlet channel leading to a plurality of annular mixing elements arranged in series, the plurality of annular mixing elements including a first annular mixing element downstream of the inlet channel and a second annular mixing element in fluid communication with the first annular mixing element through a first neck region, the first annular mixing element defining a first neck angle between the inlet channel and the first neck region.

[0017] In DVBM, two (or more) fluids enter the mixer through two (or more) separate inlets, e.g., via inlet channels, each flowing into one of the two (or more) fluids to be mixed. The two fluids initially combine in one region, but then reach a branch in the flow path that splits into two meandering channels of different lengths. These two meandering channels are referred to herein as the "legs" of the annular mixer. The different lengths have different impedances (impedance is defined herein as pressure / flow rate (e.g., (PSI*min) / mL)). In one embodiment, the impedance ratio of the first leg compared to the second leg is about 1:1 to about 10:1. This imbalance results in more fluid entering one leg than the other. The impedance imbalance results in a volume ratio between the two legs, which is very close to the impedance ratio. Thus, in one embodiment, the ratio of volumetric flow in the first leg compared to the second leg is about 1:1 to about 10:1. Impedance (or impedance per length * viscosity) is clearly independent of the operation of the device.

[0018] If the legs have the same cross-sectional area, the different lengths result in different impedances and mixing occurs. If there is a true 1:1 impedance, the volume is divided equally between the legs, but mixing still occurs due to Dean vortex formation. However, in such a situation, the benefits of bifurcation are not fully utilized.

[0019] An exemplary DVBM with a series of four annular mixers is shown in FIG.

[0020] In one embodiment, the channels (e.g., legs) of the mixer have a substantially uniform latitudinal cross-sectional area (e.g., height and width). Channels can be defined using standard width and height measurements. In one embodiment, the channels have a width of about 100 to about 500 μm and a height of about 50 to about 200 μm. In one embodiment, the channels have a width of about 200 to about 400 μm and a height of about 100 to about 150 μm. In one embodiment, the channels have a width of about 100 μm to about 1 mm and a height of about 100 μm to about 1 mm. In one embodiment, the channels have a width of about 100 μm to about 2 mm and a height of about 100 μm to about 2 mm.

[0021] In other embodiments, the channel area varies within individual rings or ring pairs. Hydrodynamic diameter is often used to characterize microfluidic channel dimensions. As used herein, hydrodynamic diameter is defined as (2*width*height) / (width+height), using channel width and height dimensions. In one embodiment, the mixer channels have a hydrodynamic diameter of about 20 μm to about 2 mm. In one embodiment, the mixer channels have a hydrodynamic diameter of about 20 μm to about 1 mm. In one embodiment, the mixer channels have a hydrodynamic diameter of about 20 μm to about 300 μm. In one embodiment, the mixer channels have a hydrodynamic diameter of about 113 μm to about 181 μm. In one embodiment, the mixer channels have a hydrodynamic diameter of about 150 μm to about 300 μm. In one embodiment, the mixer channels have a hydrodynamic diameter of about 1 mm to about 2 mm. In one embodiment, the mixer channels have a hydrodynamic diameter of about 500 μm to about 2 mm.

[0022] In one embodiment, the mixer is a microfluidic mixer and the legs of the annular mixing element have microfluidic dimensions.

[0023] To maintain laminar flow, keep the behavior of the solutions within the microfluidic device predictable, and keep the method repeatable, the system is designed to accommodate flow at low Reynolds numbers. In one embodiment, the first mixer is sized and configured to mix the first and second solutions at a Reynolds number of less than 2000. In one embodiment, the first mixer is sized and configured to mix the first and second solutions at a Reynolds number of less than 1000. In one embodiment, the first mixer is sized and configured to mix the first and second solutions at a Reynolds number of less than 900. In one embodiment, the first mixer is sized and configured to mix the first and second solutions at a Reynolds number of less than 500.

[0024] 2 and 3, an exemplary device is shown to better illustrate the embodiments disclosed herein. FIG. 2 graphically illustrates the impedance differences obtained by varying the channel length of the DVBM. In this case, there are four different path lengths: the path length L of path A; a , the path length of path B, L b , the path length L of path C c and the path length L of path D d Therefore, the impedance ratio of the first annular body is L b :L a and L c :L d Figure 3 shows the impedance difference obtained by varying the channel width of the DVBM. In this case, there are four different channel widths: the channel width w a , the channel width of path B, w b , the channel width of path C, w c and the channel width w of route D d Therefore, the impedance ratio of the first pair of annular bodies is (approximately) w a :w b and w c :w d This becomes:

[0025] The illustrated mixer contains two annular mixing elements, each defined by four "legs" (A-D). Fluid flows through these legs along four "paths" (A-D) of fluid. Due to impedance imbalances resulting from the paths formed within the device, more fluid passes through path A (in leg A) than through path B (in leg B). These meandering channels are designed to induce Dean vortex formation. Upon exiting these meandering channels, the fluids again combine and split by a second branch. As before, this split results in two channels with different impedances, but this time the impedance ratio is reversed. In Figure 2, path C (through leg C) has a lower impedance than path D (through leg D) and is equal to the impedance of path A. Similarly, path D and path B are matched. As a result, path C contains fluid from both paths A and B. When this alternating impedance branching pattern is repeated multiple times, the two fluids are "kneaded" together (as visually indicated by the color changes in Figure 1, for example), resulting in an increased contact area between the two fluids and therefore a shorter mixing time. This kneading is the same mechanism used in a staggered herringbone mixer (SHM), but with a simpler, planar structure.

[0026] As shown in Figure 2, the combined lengths of the two legs of an annular mixing element sum to the circumference of an annulus defined through the centerlines of the channel widths of the two legs. The two points where the legs meet (e.g., the beginning and end of the flow path of the annular mixing element) are defined by where the centerline through the inlet, outlet, or neck intersects with the annulus. Referring to Figure 2, the "combined flow" line intersects with the "path."

[0027] The pressure drop across a given length of the channel is given by:

[0028]

number

[0029] is given by During the ceremony,

[0030]

number

[0031] and

[0032]

number

[0033] and Width w and height h(h <w)のチャネルについては、

[0034]

number

[0035] and where μ is the fluid viscosity and L is the channel length. From this equation, it is clear that if h is held constant, the impedance ratio can be obtained by varying either L (FIG. 2) or w (FIG. 3).

[0036] The term "inner radius" (R) is defined as the inner radius of an annular feature. Figure 4 shows a schematic of the inner radius (R) of an annular mixing element.

[0037] The outer radius of a toroid is defined as the inner radius plus the width of the leg channel from which the radius is measured. As described elsewhere herein, in certain embodiments, the two legs of the toroid have the same width. In other embodiments, the two legs have different widths. Thus, a single toroid may have different radii depending on the measurement location. In such embodiments, the outer radius may be defined by the average of the outer radii around the toroid. The maximum radius of a variable radius toroid is defined as half the length of a line connecting the farthest points opposite the center of the toroid.

[0038] In one embodiment, the mixer includes a plurality of annular mixing elements ("rings"). In one embodiment, all of the plurality of rings have approximately the same radius. In one embodiment, not all of the rings have approximately the same radius. In one embodiment, the mixer includes one or more pairs of rings. In one embodiment, two rings of a ring pair have approximately the same radius. In another embodiment, the two rings have different radii. In one embodiment, the mixer includes a first pair and a second pair. In one embodiment, the radius of the rings in the first pair is approximately the same as the radius of the rings in the second pair. In another embodiment, the radius of the rings in the first pair is not approximately the same as the radius of the rings in the second pair.

[0039] The mixers disclosed herein contain two or more rings to adequately mix two or more liquids moving through the mixer. In certain embodiments, the mixer includes a basic structure of two rings connected together as a pair (e.g., as shown in FIG. 5). The two rings are connected by a neck at a specific neck angle. In one embodiment, the mixer includes 1 to 10 pairs of rings (i.e., 2 to 20 rings), where a pair is defined as having approximately the same characteristics in terms of impedance, structure, and mixing capacity (although the two rings in each pair may have different characteristics). In one embodiment, the mixer includes 2 to 8 pairs of rings. In one embodiment, the mixer includes 2 to 6 pairs of rings.

[0040] In another embodiment, the mixer contains 2 to 20 rings, whether or not the rings are arranged in pairs.

[0041] Figure 5 shows a representative mixer that includes a repeating series of annulus pairs, i.e., eight annulus total in four pairs. In each pair, the first annulus has "legs" of lengths a and b, and the legs of the second annulus have lengths c and d. In one embodiment, lengths a and c are equal, and b and d are equal. In another embodiment, the ratio a:b is equal to c:d. The mixer of Figure 5 is an example of a mixer with uniform channel width, annulus radius, neck angle (120 degrees), and neck length.

[0042] The length of the legs of the rings can be the same or different between pairs of rings. With reference to Figures 2 and 6, the two legs of at least one ring are different, resulting in a particular neck angle. In one embodiment, the leg of the first ring in the mixer is between 0.1 mm and 2 mm. In another embodiment, all of the legs of the rings in the mixer are within this range.

[0043] In its simplest form, a mixer utilizing Dean vortex formation includes a series of annuli with no "necks" between them. However, this overly simplistic concept results in sharp "knife-edge" features where two annuli meet. It would be impossible to machine a mold for such a feature using standard machining techniques. The two simplest means to overcome this would be to introduce a specific radius into the feature (where the radius is the same as the radius of the end mill used) or to create a channel region, or "neck," between the annuli. Both of these modifications result in a decrease in mixing performance, as shown by the mixing rate measurements (see the Exemplary Device Testing and Results section below). This performance loss is believed to be due to the losses during the sharp change in direction the fluid is forced to enter the next annulus. To overcome this performance loss, the DVBM uses angled "necks" between the annuli.

[0044] The neck angle is defined as the smallest angle formed with respect to the center of each annulus, defined by a line passing through the centers of the inlet and outlet channels of each annulus. Figure 6 shows a schematic of how the neck angle is measured in the disclosed embodiments.

[0045] Each pair of annulus is configured according to a neck angle between the pair: for an annulus adjacent to an inlet or outlet channel (i.e., an annulus at the beginning or end of a plurality of annulus), the neck angle is the angle defined by considering the inlet or outlet channel to be the neck of that annulus.

[0046] In one embodiment, the neck angle is approximately the same for each annulus of the device, hi another embodiment, there are multiple neck angles and not each annulus has the same neck angle.

[0047] In one embodiment, the neck angle is 0 to 180 degrees. In another embodiment, the neck angle is 90 to 180 degrees. In another embodiment, the neck angle is 90 to 150 degrees. In another embodiment, the neck angle is 100 to 140 degrees. In another embodiment, the neck angle is 110 to 130 degrees. In another embodiment, the neck angle is 120 degrees.

[0048] Referring to Figure 6, neck length is defined as the distance between points on adjacent annuli where the direction of the curve changes.

[0049] In one embodiment, the neck length is at least twice the radius of curvature of the end mill used to make the mixer. In one embodiment, the neck has a length of at least 0.05 mm. In one embodiment, the neck has a length of at least 1 mm. In one embodiment, the neck has a length of at least 0.2 mm. In one embodiment, the neck has a length of at least 0.25 mm. In one embodiment, the neck has a length of at least 0.3 mm. In one embodiment, the neck has a length of 0.05 to 2 mm. In one embodiment, the neck has a length of 0.2 to 2 mm.

[0050] Regarding the material used to form the mixer, any known or future developed material that can be used to form fluidic devices may be used. In one embodiment, the mixer comprises a polymer selected from the group consisting of polypropylene, polycarbonate, COC, COP, PDMS, polystyrene, nylon, acrylic, HDPE, LDPE, other polyolefins, and combinations thereof. Non-polymeric materials can also be used to fabricate the mixer, including inorganic glasses such as conventional silica-based glasses, metals, and ceramics.

[0051] In certain embodiments, multiple mixers are included on the same "chip" (i.e., a single substrate containing a compound mixer). In such embodiments, a DVBM mixer can be considered to be multiple annular mixing elements arranged in series, beginning and ending with inlet and outlet channels, respectively. Thus, a chip with a compound mixer includes embodiments with compound DVBM mixers (each mixer containing multiple annular mixing elements) arranged in a parallel or serial configuration. In another embodiment, multiple mixers include one or more DVBM mixers and one non-DVBM mixer (e.g., SHM). By combining mixer types, the advantages of each type of mixer can be utilized in a single device.

[0052] How to use In another aspect, methods of using the mixers disclosed herein are provided. In one embodiment, the method includes mixing a first liquid with a second liquid by flowing (e.g., pushing or drawing) the first and second liquids through a mixer (i.e., a DVBM) disclosed herein to produce a mixed solution. Such methods are described in detail elsewhere herein in the context of defining DVBM devices and their performance. The disclosed mixers can be used in any mixing application known to those skilled in the art in which vapors of two or more liquids are mixed in relatively small amounts (e.g., at the microfluidic level).

[0053] In one embodiment, the mixer is incorporated into a larger device containing multiple mixers (including DVBM), and the method further includes flowing the first liquid and the second liquid through the multiple mixers to form the mixed solution. This embodiment relates to parallelization of mixers to generate higher mixing volumes on a single device. Such parallelization is discussed in the patent documents incorporated by reference.

[0054] In one embodiment, the first liquid comprises a first solvent. In one embodiment, the first solvent is an aqueous solution. In one embodiment, the aqueous solution is a buffer solution at a predetermined pH.

[0055] In one embodiment, the first liquid comprises one or more polymers in a first solvent.

[0056] In one embodiment, the polymer is a nucleic acid. In another embodiment, the polymer is a protein. In a further embodiment, the polymer is a polypeptide.

[0057] In one embodiment, the first liquid comprises one or more low molecular weight compounds in a first solvent.

[0058] In one embodiment, the second liquid comprises a lipid particle-forming material in a second solvent.

[0059] In one embodiment, the second liquid comprises a polymer particle-forming material in a second solvent.

[0060] In one embodiment, the second liquid comprises a lipid particle-forming material and one or more polymers in a second solvent.

[0061] In one embodiment, the second liquid comprises a lipid particle-forming material and one or more low molecular weight compounds in a second solvent.

[0062] In one embodiment, the second liquid comprises a polymer particle-forming material and one or more polymers in a second solvent.

[0063] In one embodiment, the second liquid comprises a polymer particle-forming material and one or more low molecular weight compounds in a second solvent.

[0064] In one embodiment, the mixed solution comprises particles generated by mixing the first liquid and the second liquid, hi one embodiment, the particles are selected from the group consisting of lipid nanoparticles and polymer nanoparticles.

[0065] Manufacturing method In another aspect, a method of manufacturing a mixer is provided. In one embodiment, the method includes forming a master mold using an end mill, the master mold configured to form a DVBM mixer according to an embodiment disclosed herein. In certain embodiments, an end mill is used to create the master, while in other embodiments, the master is formed using techniques including lithography or electroforming. In such embodiments, R is the smallest feature size permitted by the particular technique.

[0066] If the device is fabricated using injection molding and the injection-molded insert is fabricated by milling, the inner radius (R) of the annular mixing element will be equal to or greater than the radius of the end mill used to fabricate the mold for forming the mixer. For mass production, whether by embossing, casting, molding, or any other replication technique, a master (e.g., a mold) must be created. Such a master is most easily fabricated using a precision mill. During milling, a high-speed rotating cutting tool known as an end mill is driven through a solid piece of material (such as a steel plate) to remove specific sections and form the desired features. The radius of the end mill therefore defines the minimum radius of any features that will be formed. Masters may also be fabricated by other techniques, such as lithography or electroforming, in which case the resolution of the selected technique will define the minimum inner radius of the annular body. In one embodiment, the inner radius of the mixer is between 0.1 mm and 2 mm. In one embodiment, the inner radius of the mixer is between 0.1 mm and 1 mm.

[0067] definition Microfluidics As used herein, the term "microfluidic" refers to a system or device for manipulating (e.g., flowing or mixing) fluid samples that includes at least one channel having microscale dimensions (i.e., dimensions less than 1 mm).

[0068] therapeutic substance As used herein, the term "therapeutic agent" is defined as a substance intended to provide pharmacological activity or to have a direct effect in the diagnosis, cure, mitigation, understanding, treatment, or prevention of disease, or to have a direct effect in the restoration, correction, or improvement of physiological function. Therapeutic agents include, but are not limited to, small molecule drugs, nucleic acids, proteins, peptides, polysaccharides, inorganic ions, and radionuclides.

[0069] nanoparticles As used herein, the term "nanoparticle" is defined as a homogeneous particle comprising two or more component materials (e.g., lipids, polymers, etc.) used to encapsulate a therapeutic agent and having a minimum dimension of less than 250 nm. Nanoparticles include, but are not limited to, lipid nanoparticles and polymer nanoparticles. In one embodiment, a device is configured to form lipid nanoparticles. In one embodiment, a device is configured to form polymer nanoparticles. In one embodiment, a method for forming lipid nanoparticles is provided. In one embodiment, a method for forming polymer nanoparticles is provided.

[0070] lipid nanoparticles In one embodiment, the lipid nanoparticles are (a) a core; and (b) a shell surrounding the core, the shell comprising phospholipids Includes:

[0071] In one embodiment, the core comprises a lipid (e.g., a fatty acid triglyceride) and is solid. In another embodiment, the core is liquid (e.g., aqueous) and the particle is a vesicle such as a liposome. In one embodiment, the shell surrounding the core is a single layer.

[0072] As mentioned above, in one embodiment, the lipid core comprises a fatty acid triglyceride. Suitable fatty acid triglycerides include C8 to C20 fatty acid triglycerides. In one embodiment, the fatty acid triglyceride is oleic acid triglyceride.

[0073] Lipid nanoparticles have a core surrounded by a shell comprising phospholipids. Suitable phospholipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside. In one embodiment, the phospholipid is a C8-C20 fatty acid diacylphosphatidylcholine. An exemplary phospholipid is 1-palmitoyl-2-oleoylphosphatidylcholine (POPC).

[0074] In certain embodiments, the ratio of phospholipids to fatty acid triglycerides is from 20:80 (mol:mol) to 60:40 (mol:mol). Preferably, triglycerides are present in a proportion of more than 40% and less than 80%.

[0075] In certain embodiments, the nanoparticles further comprise a sterol. Exemplary sterols include cholesterol. In one embodiment, the ratio of phospholipid to cholesterol is 55:45 (mol:mol). In exemplary embodiments, the nanoparticles comprise 55-100% POPC and up to 10 mol% PEG-lipid.

[0076] In other embodiments, the lipid nanoparticles of the present disclosure may contain one or more other lipids, including phosphoglycerides, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.Other compounds that lack phosphorus, such as sphingolipids and glycosphingolipid families, are also useful.Triacylglycerols are also useful.

[0077] Representative nanoparticles of the present disclosure have a diameter of about 10 to about 100 nm, with the lower diameter limit being about 10 to about 15 nm.

[0078] The lipid nanoparticles of the present disclosure can contain one or more low molecular weight compounds used as therapeutic and / or diagnostic agents. These agents are typically contained within the particle core. The nanoparticles of the present disclosure can contain a wide variety of therapeutic and / or diagnostic agents.

[0079] Suitable low molecular weight compound drugs include chemotherapeutic agents (i.e., antitumor agents), anesthetics, beta-adrenergic blocking agents, antihypertensive agents, antidepressants, anticonvulsants, antiemetics, antihistamines, antiarrhythmic agents, and antimalarials.

[0080] Representative antitumor drugs include doxorubicin, daunorubicin, mitomycin, bleomycin, streptozocin, vinblastine, vincristine, mechlorethamine hydrochloride, melphalan, cyclophosphamide, triethylenethiophosphoramide, carmustine, lomustine, semustine, fluorouracil, hydroxyurea, thioguanine, cytarabine, floxuridine, dacarbazine, cisplatin, procarbazine, vinorelbine, rubin, ciprofloxacin, norfloxacin, paclitaxel, docetaxel, etoposide, bexarotene, teniposide, tretinoin, isotretinoin, sirolimus, fulvestrant, valrubicin, vindesine, leucovorin, irinotecan, capecitabine, gemcitabine, mitoxantrone hydrochloride, oxaliplatin, adriamycin, methotrexate, carboplatin, estramustine, and pharmaceutically acceptable salts thereof.

[0081] In another embodiment, the lipid nanoparticle is a nucleic acid-lipid nanoparticle.

[0082] The term "nucleic acid-lipid nanoparticle" refers to a lipid nanoparticle containing a nucleic acid. The lipid nanoparticle comprises one or more cationic lipids, one or more second lipids, and one or more nucleic acids.

[0083] Cationic lipid. Lipid nanoparticles comprise cationic lipid. As used herein, the term "cationic lipid" refers to lipid that is cationic or becomes cationic (protonated) when pH is lower than the pK of the ionizable group of lipid, but gradually approaches neutral at higher pH values.At pH values ​​lower than pK, lipid can associate with negatively charged nucleic acid (for example, oligonucleotide).As used herein, the term "cationic lipid" includes zwitterionic lipid that becomes positively charged when pH is decreased.

[0084] The term "cationic lipid" refers to any of several lipid species that have a net positive charge at a selected pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). In addition, several commercially available formulations of cationic lipids are available that can be used in the present disclosure. These commercially available formulations include, for example, LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) from GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol from Promega, Madison, WI). The following lipids are cationic and have a positive charge at sub-physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0085] In one embodiment, the cationic lipid is an amino lipid.Suitable amino lipids useful in the present disclosure include the amino lipids described in WO2009 / 096558, the entire contents of which are incorporated herein by reference.Representative amino lipids include 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA·Cl), 1,2-di These include linoleoyl-3-trimethylaminopropane chloride (DLin-TAP·Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0086] Suitable amino lipids include those having the formula:

[0087] [ka]

[0088] and wherein R and R are the same or different and independently represent optionally substituted C 10 ~C 24 Alkyl, optionally substituted C 10 ~C 24Alkenyl, optionally substituted C 10 ~C 24 Alkynyl, or optionally substituted C 10 ~C 24 It is acyl; R3 and R4 are the same or different and independently represent an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl, or R3 and R4 may be linked to form an optionally substituted heterocycle of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R5 is absent or present, and if present, is hydrogen or C1-C6 alkyl; m, n, and p are the same or different and independently 0 or 1, with the proviso that m, n, and p cannot simultaneously be 0; q is 0, 1, 2, 3, or 4; Y and Z are the same or different and independently O, S, or NH.

[0089] In one embodiment, R1 and R2 are each linoleyl and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[0090] Representative useful dilinoleyl amino lipids have the formula:

[0091] [ka]

[0092] and wherein n is 0, 1, 2, 3, or 4.

[0093] In one embodiment, the cationic lipid is DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA as described above, where n is 2).

[0094] Other suitable cationic lipids, in addition to those specifically mentioned above, include cationic lipids having a net positive charge at near physiological pH, such as N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl-N,NN-triethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt (DOTAP·Cl); 3β-(N—(N′,N′-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP·Cl); Cationic lipids include (methylaminoethane) carbamoyl) cholesterol (DC-Chol); N-(1-(2,3-dioleoyloxy) propyl)-N-2-(sperminecarboxamido) ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA); dioctadecylamidoglycylcarboxyspermine (DOGS); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); N,N-dimethyl-2,3-dioleoyloxy) propylamine (DODMA); and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).In addition, some commercially available preparations of cationic lipids can be used, for example, LIPOFECTIN (including DOTMA and DOPE available from GIBCO / BRL) and LIPOFECTAMINE (including DOSPA and DOPE available from GIBCO / BRL).

[0095] The cationic lipid is present in the lipid particle in an amount of about 30 to about 95 mol %. In one embodiment, the cationic lipid is present in the lipid particle in an amount of about 30 to about 70 mol %. In one embodiment, the cationic lipid is present in the lipid particle in an amount of about 40 to about 60 mol %.

[0096] In one embodiment, the lipid particle comprises (“consists of”) only one or more cationic lipids and one or more nucleic acids.

[0097] Second lipids. In certain embodiments, the lipid nanoparticles comprise one or more second lipids. Suitable second lipids stabilize the formation of nanoparticles during nanoparticle formation.

[0098] The term "lipid" refers to a group of organic compounds that are esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three types: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0099] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0100] Neutral lipid. The term "neutral lipid" refers to any one of several lipid species that exist in uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0101] Exemplary lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4- These include (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (trans DOPE).

[0102] In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0103] Anionic lipid. The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups linked to neutral lipids.

[0104] Other suitable lipids include glycolipids (e.g., monosialoganglioside GM1). Other suitable second lipids include sterols, such as cholesterol.

[0105] Polyethylene glycol-lipid. In certain embodiments, the second lipid is a polyethylene glycol-lipid. Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol) 2000 )carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG.

[0106] In certain embodiments, the second lipid is present in the lipid particle in an amount of about 0.5 to about 10 mol %. In one embodiment, the second lipid is present in the lipid particle in an amount of about 1 to about 5 mol %. In one embodiment, the second lipid is present in the lipid particle in an amount of about 1 mol %.

[0107] Nucleic Acids. The lipid nanoparticles of the present disclosure are useful for the systemic or local delivery of nucleic acids. As described herein, nucleic acids are incorporated into the lipid particles during formation.

[0108] As used herein, the term "nucleic acid" is meant to include any oligonucleotide or polynucleotide. Fragments containing up to 50 nucleotides are generally referred to as oligonucleotides, while longer fragments are referred to as polynucleotides. In specific embodiments, oligonucleotides of the present disclosure are 20 to 50 nucleotides in length. In the context of this disclosure, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers containing non-naturally occurring monomers or portions thereof that function similarly. Such modified or substituted oligonucleotides are often preferred over natural forms due to properties such as enhanced cellular uptake and increased stability in the presence of nucleases. Oligonucleotides are classified as deoxyribooligonucleotides or ribooligonucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose, covalently linked to phosphate at the 5' and 3' carbons of the five-carbon sugar, forming an alternating, unbranched polymer. Ribooligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose. The nucleic acids present in the lipid particles of the present disclosure include any known form of nucleic acid. The nucleic acids used herein may be single-stranded DNA or RNA, double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viruses or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNA, mRNA, and triplex-forming oligonucleotides.

[0109] In one embodiment, the polynucleic acid is an antisense oligonucleotide. In certain embodiments, the nucleic acid is an antisense nucleic acid, a ribozyme, a tRNA, a snRNA, a snoRNA, a siRNA, a shRNA, a saRNA, a tRNA, a rRNA, a piRNA, an ncRNA, a miRNA, an mRNA, an lncRNA, a sgRNA, a tracrRNA, a pre-enriched DNA, an ASO, or an aptamer.

[0110] The term "nucleic acid" also refers to ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, which may be single-stranded or double-stranded and, where appropriate, may contain portions of both double-stranded and single-stranded sequence.

[0111] As used herein, the term "nucleotide" generally encompasses the following terms: nucleotide base, nucleoside, nucleotide analog, and universal nucleotide, as defined below.

[0112] As used herein, the term "nucleotide base" refers to a substituted or unsubstituted parent aromatic ring(s). In some embodiments, the aromatic ring(s) contain at least one nitrogen atom. In some embodiments, the nucleotide base is capable of forming Watson-Crick and / or Hoogsteen hydrogen bonds with an appropriately complementary nucleotide base. Exemplary nucleotide bases and their analogs include, but are not limited to, purines, such as 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-2-isopentenyladenine (6iA), N6-2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidines, 6-thioguanine (6sG), hypoxanthine, and O6-methylguanine; 7-deaza-purine derivatives. nucleotides such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidine derivatives such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indole derivatives such as nitroindole and 4-methylindole; pyrrole derivatives such as nitropyrrole; nebularine; and base (Y). In some embodiments, the nucleotide base is a universal nucleotide base. Additional exemplary nucleotide bases can be found in Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla., and references cited therein. Further examples of universal bases can be found, for example, in Loakes, NAR 2001, 29:2437-2447, and Seela, NAR 2000, 28:3224-3232.

[0113] As used herein, the term "nucleoside" refers to a compound having a nucleotide base covalently linked to the C-1' carbon of a pentose sugar. In some embodiments, this linkage is through a heteroaromatic ring nitrogen. Exemplary pentose sugars include, but are not limited to, pentoses in which one or more of the carbon atoms are each independently substituted with one or more of the same or different -R, -OR, -NRR, or halogen groups, where each R is independently hydrogen, (C1-C6) alkyl, or (C5-C14) aryl. The pentose sugar can be saturated or unsaturated. Exemplary pentose sugars and their analogs include, but are not limited to, ribose, 2'-deoxyribose, 2'-(C1-C6)alkoxyribose, 2'-(C5-C14)aryloxyribose, 2',3'-dideoxyribose, 2',3'-didehydroribose, 2'-deoxy-3'-halolibose, 2'-deoxy-3'-fluororibose, 2'-deoxy-3'-chlororibose, 2'-deoxy-3'-aminoribose, 2'-deoxy-3'-(C1-C6)alkylribose, 2'-deoxy-3'-(C1-C6)alkoxyribose, and 2'-deoxy-3'-(C5-C14)aryloxyribose. See also, for example, 2'-O-methyl, 4'-α-anomeric nucleotides, 1'-α-anomeric nucleotides (Asseline, (1991), Nucl. Acids Res. 19:4067-74), 2'-4'- and 3'-4'-linkages, and other "locked" or "LNA" bicyclic sugar modifications (WO 98 / 22489; WO 98 / 39352; WO 99 / 14226). LNA, or "locked nucleic acid," is a DNA analogue that is conformationally locked, with the ribose ring constrained by a methylene linkage between the 2'-oxygen and the 3'- or 4'-carbon. The conformational restriction imparted by the linkage increases the binding affinity for complementary sequences and often enhances the thermal stability of such duplexes.

[0114] Sugars include modifications at the 2' or 3' position, such as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro, chloro, and bromo. Nucleosides and nucleotides include natural D-configuration isomers (D-forms) and L-configuration isomers (L-forms) (Beigelman, U.S. Pat. No. 6,251,666; Chu, U.S. Pat. No. 5,753,789; Shudo, EP 0540742; Garbesi, (1993), Nucl. Acids Res. 21:4159-65; Fujimori, (1990), J. Amer. Chem. Soc. 112:7435; Urata, (1993), Nucleic Acids Symposium Ser. 29:69-70). When the nucleobase is a purine, e.g., A or G, a ribose sugar is added to the N9 position of the nucleobase. If the nucleobase is a pyrimidine, e.g., C, T, or U, a pentose sugar is added to the N1 position of the nucleobase (Kornberg and Baker, (1992), DNA Replication, 2nd ed., Freeman, San Francisco, Calif.).

[0115] One or more of the pentose carbons of the nucleoside may be substituted with a phosphate ester. In some embodiments, the phosphate ester is attached to the 3'- or 5'-carbon of the pentose. In some embodiments, the nucleoside is a nucleoside in which the nucleotide base is a purine, a 7-deazapurine, a pyrimidine, a universal nucleotide base, a specific nucleotide base, or an analog thereof.

[0116] As used herein, the term "nucleotide analog" refers to embodiments in which one or more of the pentose sugar and / or nucleotide base, and / or phosphate ester of a nucleoside can be replaced with their respective analogs. In some embodiments, exemplary pentose sugar analogs are those described above. In some embodiments, a nucleotide analog has a nucleotide base analog described above. In some embodiments, exemplary phosphate ester analogs include, but are not limited to, alkyl phosphonates, methyl phosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates, phosphoroamidates, and boronophosphates, and exemplary phosphate ester analogs may include associated counterions. Other nucleic acid analogs and bases include, for example, intercalating nucleic acids (INA, as described in Christensen and Pedersen, 2002) and AEGIS bases (Eragen, US Pat. No. 5,432,272). Additional descriptions of various nucleic acid analogs can be found in, for example, (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 (1986)), phosphorothioates (Mag et al., Nucleic Acids Res. 19:1437 (1986)), and thiol- and thiol-esters (Mag et al., Nucleic Acids Res. 19:1437 (1986)). (1991); as well as U.S. Pat. No. 5,644,048.Other nucleic acid analogs include phosphorodithioates (Briu et al., J. Am. Chem. Soc. 111:2321 (1989)), O-methyl phosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), analogs with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995)); analogs with non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1996)). (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (194): Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research," Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and analogs with non-ribose backbones (U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research" Ed. Y.S. Sanghui and P. Dan Cook). Modifications in Antisense Research, Ed. Y.S. Sanghui and P. Dan Cook). Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp. 169-176).Some nucleic acid analogs are also described in Rawls, C & E News, June 2, 1997, p. 35.

[0117] As used herein, the term "universal nucleotide base" or "universal base" refers to an aromatic ring moiety, which may or may not contain a nitrogen atom. In some embodiments, a universal base may be covalently attached to the C-1' carbon of a pentose sugar to form a universal nucleotide. In some embodiments, a universal nucleotide base does not specifically hydrogen bond with another nucleotide base. In some embodiments, a universal nucleotide base hydrogen bonds with nucleotide bases, including up to all nucleotide bases in a particular target polynucleotide. In some embodiments, a nucleotide base may interact with adjacent nucleotide bases on the same nucleic acid strand by hydrophobic stacking. Universal nucleotides include, but are not limited to, deoxy-7-azaindole triphosphate (d7AITP), deoxyisocarbostyril triphosphate (dICSTP), deoxypropynylisocarbostyril triphosphate (dPICSTP), deoxymethyl-7-azaindole triphosphate (dM7AITP), deoxyImPy triphosphate (dImPyTP), deoxyPP triphosphate (dPPTP), or deoxypropynyl-7-azaindole triphosphate (dP7AITP). Further examples of such universal bases can be found, inter alia, in published U.S. patent application Ser. No. 10 / 290,672 and U.S. Patent No. 6,433,134.

[0118] As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single- and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA), linked by internucleotide phosphodiester bonds, e.g., 3'-5' and 2'-5', inverted linkages, e.g., 3'-3' and 5'-5', branched structures, or internucleotide analogs. Polynucleotides have associated counterions, e.g., H, NH, trialkylammonium, Mg, Na. Polynucleotides may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Polynucleotides may be composed of internucleotide analogs, nucleobase analogs, and / or sugar analogs. Polynucleotides typically range in size from a few monomeric units, e.g., 3-40 (in which case the polynucleotide is often referred to in the art as an oligonucleotide) to thousands of monomeric nucleotide units. Unless otherwise specified, whenever a polynucleotide sequence is depicted, the nucleotides will be understood to be in 5' to 3' order from left to right, and unless otherwise noted, "A" will be understood to represent deoxyadenosine, "C" will represent deoxycytosine, "G" will represent deoxyguanosine, and "T" will represent thymidine.

[0119] As used herein, "nucleobase" refers to naturally occurring and non-naturally occurring heterocyclic moieties that are well known to those who utilize nucleic acid technology or peptide nucleic acid technology to create polymers of sequences capable of specifically binding to nucleic acids. Non-limiting examples of suitable nucleobases include adenine, cytosine, guanine, thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine), and N8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitable nucleobases include those shown in Figures 2(A) and 2(B) of Buchardt et al. (WO92 / 20702 or WO92 / 20703).

[0120] As used herein, "nucleobase sequence" refers to any segment of a polymer comprising nucleobase-containing subunits, or an assembly of two or more segments (e.g., an assembly of two or more oligomer blocks, a nucleobase sequence). Non-limiting examples of suitable polymers or polymer segments include oligodeoxynucleotides (e.g., DNA), oligoribonucleotides (e.g., RNA), peptide nucleic acids (PNAs), PNA chimeras, PNA combinatorial oligomers, nucleic acid analogs, and / or nucleic acid mimics.

[0121] As used herein, "polynucleobase strand" means an entire single polymeric strand comprising nucleobase subunits. For example, a single nucleic acid strand of a double-stranded nucleic acid is a polynucleobase strand.

[0122] As used herein, a "nucleic acid" is a nucleobase sequence-containing polymer, or polymer segment, having a backbone formed from nucleotides or their analogs.

[0123] Preferred nucleic acids are DNA and RNA.

[0124] As used herein, nucleic acid may also refer to "peptide nucleic acid" or "PNA," which refers to any oligomer or polymer segment (e.g., block oligomer) that contains two or more PNA subunits (residues) but does not contain any nucleic acid subunits (or analogs thereof). Examples of "peptide nucleic acids" or "PNAs" include, but are not limited to, those disclosed in U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,714,332, 5,714,333, 5,714,334, 5,714,335, 5,714,336, 5,714,337, 5,714,338, 5,714,339 ... This includes any of the oligomeric or polymeric segments referred to or claimed as peptide nucleic acids in US Pat. Nos. 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053, and 6,107,470, all of which are incorporated herein by reference. The term "peptide nucleic acid" or "PNA" is used in the following publications: Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters, 4: 1081-1082 (1994); Petersen et al., Bioorganic & Medicinal Chemistry Letters, 6: 793-796 (1996); Diderichsen et al., Tett. Lett. 37: 475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7: 687-690 (1997); Krotz et al., Tett. Lett. 36: 6941-6944 (1995); Lagriffoul et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994);Diederichsen, U., Bioorganic & Medicinal Chemistry Letters, 7: 1743-1746 (1997);Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997)1: 539-546;Lowe et al., J. Chem. Soc. Perkin Trans.11: 547-554 (1997);Lowe et al., J. Chem. Soc. Perkin Trans. 11:555-560 (1997);Howarth et al., J. Org. Chem. 62: 5441-5450 (1997);Altmann, KH et al., Bioorganic & Medicinal Chemistry Letters, 7: 1119-1122 (1997);Diederichsen, U., Bioorganic & Med. Chem. Lett., 8: 165-168 (1998);Diederichsen et al., Angew. Chem. Int. Ed., 37: 302-305 (1998);Cantin et al., Tett. Lett., 38: 4211-4214 (1997); Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997); Lagriffoule et al., Chem. Eur. J., 3: 912-919 (1997); Kumar et al., Organic Letters 3(9): 1269-1272 (2001); and any oligomeric or polymeric segment containing two or more subunits of a nucleic acid mimic, as described in Peptide-Based Nucleic Acid Mimetics (PENAMS) by Shah et al., as disclosed in WO 96 / 04000.

[0125] polymer nanoparticles The term "polymeric nanoparticles" refers to polymeric nanoparticles containing a therapeutic agent. Polymeric nanoparticles have been developed using a wide range of materials, including, but not limited to, synthetic homopolymers such as polyethylene glycol, polylactide, polyglycolide, poly(lactide-co-glycolide), polyacrylate, polymethacrylate, polycaprolactone, polyorthoester, polyanhydride, polylysine, polyethyleneimine, etc.; synthetic copolymers such as poly(lactide-co-glycolide), poly(lactide)-poly(ethylene glycol), poly(lactide-co-glycolide)-poly(ethylene glycol), poly(caprolactone)-poly(ethylene glycol); natural polymers such as cellulose, chitin, and alginate; and polymer-therapeutic agent conjugates.

[0126] As used herein, the term "polymer" refers to a compound, usually having a high molecular weight, constructed primarily or entirely from many similar units linked together. Such polymers include any of a number of natural, synthetic, and semi-synthetic polymers.

[0127] The term "natural polymer" refers to any of a number of polymer species found in nature, including, but not limited to, polysaccharides, cellulose, chitin, and alginates.

[0128] The term "synthetic polymer" refers to any of a number of synthetic polymer species not found in nature, including, but not limited to, synthetic homopolymers and synthetic copolymers.

[0129] Synthetic homopolymers include, but are not limited to, polyethylene glycol, polylactide, polyglycolide, polyacrylate, polymethacrylate, polycaprolactone, polyorthoester, polyanhydride, polylysine, and polyethyleneimine.

[0130] "Synthetic copolymer" refers to any of a number of synthetic polymer species composed of two or more synthetic homopolymer subunits, including, but not limited to, poly(lactide-co-glycolide), poly(lactide)-poly(ethylene glycol), poly(lactide-co-glycolide)-poly(ethylene glycol), and poly(caprolactone)-poly(ethylene glycol).

[0131] The term "semi-synthetic polymer" refers to any number of polymers obtained by chemical or enzymatic treatment of natural polymers, including, but not limited to, carboxymethylcellulose, acetylated carboxymethylcellulose, cyclodextrin, chitosan, and gelatin.

[0132] As used herein, the term "polymer conjugate" refers to a compound prepared by covalently or non-covalently conjugating one or more molecular species to a polymer. Such polymer conjugates include, but are not limited to, polymer-therapeutic agent conjugates.

[0133] A polymer-therapeutic agent conjugate refers to a polymer conjugate in which one or more of the conjugated molecular species is a therapeutic agent. Such polymer-therapeutic agent conjugates include, but are not limited to, polymer-drug conjugates.

[0134] "Polymer-drug conjugate" refers to any number of polymer species conjugated to any number of drug species. Such polymer-drug conjugates include, but are not limited to, acetylmethylcellulose-polyethylene glycol-docetaxol.

[0135] As used herein, the term "about," unless otherwise indicated, indicates that the associated value may be modified by plus or minus five percent (+ / -5%) to remain within the scope of the disclosed embodiments.

[0136] Incorporation by Reference The following references disclose compatible microfluidic mixing methods and devices: The mixers disclosed herein can be incorporated into any of the mixing devices disclosed in the following references, or can be used to mix any of the compositions disclosed in the following references: (1) U.S. Patent Application No. 13 / 464690, a continuation of PCT / CA2010 / 001766, filed November 4, 2010, which claims the benefit of U.S. Patent Application No. 61 / 280510, filed November 4, 2009; (2) U.S. Patent Application No. 14 / 353,460, a continuation of PCT / CA2012 / 000991, filed October 25, 2012, which claims the benefit of U.S. Patent Application No. 61 / 551,366, filed October 25, 2011; (3) PCT / US2014 / 029116, filed March 14, 2014 (published October 23, 2014 as WO2014 / 172045), which claims the benefit of U.S. Patent Application No. 61 / 798,495, filed March 15, 2013; (4) PCT / US2014 / 041865, filed July 25, 2014 (published January 29, 2015 as WO2015 / 013596), which claims the benefit of U.S. Patent Application No. 61 / 858,973, filed July 26, 2013; (5) PCT / US2014 / 060961, which claims the benefit of U.S. Patent Application No. 61 / 891,758, filed October 16, 2013; (6) U.S. Provisional Patent Application No. 62 / 120,179, filed February 24, 2015; and (7) U.S. Provisional Patent Application No. 62 / 154,043, filed April 28, 2015, the entire disclosure of which is incorporated herein by reference.

[0137] The following examples are intended to illustrate, but not limit, the described embodiments. [Example]

[0138] Example 1 DVBM Device Testing and Results For testing, devices with two fluid inlets and one outlet were fabricated. Four different concepts were tested. The four designs are summarized in Table 1 below. In the case of mixer types 1 to 3, the impedance imbalance is created by varying the width of the two sides of the annulus (Figure 3). DVBM achieves the impedance imbalance by varying the path length through the annulus. All test devices had an inlet channel width of 140 μm and a height of 105 μm (hydrodynamic diameter of 120 μm; approximate impedance / length*viscosity: 6.9*10^-5 / um^4).

[0139] [Table 1A]

[0140] [Table 1B]

[0141] To optimize performance, a set of four exemplary DVBM mixers was fabricated with offset angles of 120°, 140°, 160°, and 180°. Mixing rates were measured optically for a range of flow rates (Figure 7). This testing confirmed that offset angle is a parameter for improving mixing rates, with 120° being the optimal angle. Therefore, the 120° DVBM was used for comparison with Mixer Types 1-3.

[0142] Samples were imaged using a bright field stereoscope. 125 mM NaAc and 1 M NaOH containing bromothymol blue ("BTB") were used as reagents to visualize mixing. Mixing time was calculated by imaging the mixer with a color CCD and identifying the point at which a uniform yellow color distribution existed throughout the channel. The mixing time of the device was then measured as the time required for the fluid to be introduced and reach this point of complete mixing. See the appendix for further details regarding the experimental techniques used to measure mixing time.

[0143] Figure 8 shows that the performance of Types 1-3 and the exemplary DVBM differs across a range of input flow rates (measured by mixing time). Below 10 ml / min, both Mixer Types 1 and 3 mix slower than Type 2 or the exemplary DVBM (as expected). Interestingly, the exemplary DVBM with a 120° offset not only approaches the performance of the Type 2 mixer, but actually exceeds it at low flow rates. This is unexpected and non-trivial.

[0144] Both the 120° and 180° exemplary DVBM mixers were used to compound lipid nanoparticles (of the type formed in the references incorporated in the following section). Briefly, a lipid composition of POPC and cholesterol was dissolved in ethanol at a molar ratio of 55:45. The final lipid concentration was 16.9 mM. Flow rates of 2 to 10 ml / min were tested for the commercially available NanoAssemblr Benchtop microfluidic cartridge (using SHM), the 120° exemplary DVBM, and the 180° exemplary DVBM. The results are shown in Figure 9 below. Both exemplary DVBM devices exhibited the same size-to-flow ratio as the cartridge. However, at lower flow rates, the exemplary DVBM mixer produced smaller, less polydisperse particles than the cartridge.

[0145] Figure 9 compares particle size and PDI for the Staggered Herringbone mixer and two DVBM designs. It can be seen that the exemplary DVBM mixer performs similarly to the SHM mixer, especially at higher flow rates.

[0146] Mixing Time Calculation The following equipment was used: AmScope camera AmScope microscope White / black backplate PTFE piping 1 / 32'' Dean vortex mixing device (PDMS on a glass slide) Petri dish Stainless steel weights

[0147] Data were collected using an AmScope microscope equipped with a 56-LED illuminator and a white baseplate. A Petri dish with weights was also placed in the recording area to facilitate device positioning. 1 M NaOH with 125 mM NaAc and BTB was mixed in a 3:1 ratio. Complete mixing was determined when the solution turned yellow with a uniform intensity distribution. All images were acquired at the same flow rate without moving the Dean vortex mixer (see "Processing Methods"). To better detect color changes, the image processing software was manually adjusted to maximize saturation. Figure 10 shows a micrograph of the DVBM mixer before mixing.

[0148] Figure 11 is a micrograph of the DVBM mixer in operation, where the clear and blue liquids are mixing to form a yellow liquid at the far right of the image (i.e., mixing is complete).

[0149] Processing method The original images were placed in a folder, and a program using Python and OpenCV 3.0 was used to rotate, center, and stitch the original images. The template image was first processed (using the Hough circle transform (see Figure 12) to detect circles in the image and use them as a reference for the transformation calculation), and then the same transformation as the template was performed on subsequent images. During this process, the radius was also calculated and used to determine the pixel area of ​​the image in micrometers. 13A-13C are processing templates and data images of a mixer. Fig. 13A is a DVBM template image. Fig. 13B is a DVBM image during mixing. Fig. 13C is a template image of a non-DVBM mixer.

[0150] Calculation methods and algorithms The template image channels were detected by checking each pixel's value against a specific color threshold (in this case, blue intensity) and then changing the pixel color to black if the values ​​were not within the threshold range. This method was applied to a mask containing only the mixer's channels. The mixed image was then uploaded and the same mask was applied to it. The mixing point was visually confirmed, and then a calculation range was entered. The number of pixels in the channel up to this range were counted and colored white. The volume was calculated from the previously determined pixel area and the channel height within the device. After calculating the total mixing volume, it was divided by the flow rate of the device during mixing to determine the mixing time.

[0151] Figure 14 is a template image with a mask applied, Figure 15 is a data (mixed) image with a mask applied, and Figure 16 is a data (mixed) image with white pixels counted.

[0152] Liposome generation using DVBM We have produced liposomal vesicles with a size of less than 100 nm and a narrow PDI, as summarized in Figure 17. Figure 17 shows a graph of the size and PDI characteristics of liposomes produced by a representative DVBM according to embodiments disclosed herein. The data was generated in a DVBM device with a neck length of 0.25 mm, a neck angle of 120 degrees, an inner radius of 0.16, a channel width and height of 80 μm, and a flow ratio of approximately 2:1 (aqueous:lipid). The lipid composition was either pure POPC liposomes or liposomes containing POPC:cholesterol (55:45). The initial lipid mixture concentration was 50 mM. The aqueous phase contained PBS buffer.

[0153] Materials and Methods: POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was obtained from Avanti Polar Lipids, Inc., USA. Cholesterol, triolein, C-6 (coumarin-C6), DMF (dimethylformamide), PVA, [poly(vinyl alcohol)], Mowiol® 4-88], and PBS (Dulbecco's phosphate buffered saline) were obtained from Sigma-Aldrich, USA. Ethanol was obtained from Greenfield Specialty Alcohols, Inc., Canada. PLGA, polylactic-co-glycolic acid, was obtained from PolyciTech, USA.

[0154] The following solutions were dispensed into each well in the cartridge: 36 μL of PBS was placed in the aqueous reagent well, 48 μL of PBS was placed in the collection well, and finally, 12 μL of a 50 mM lipid mixture in ethanol was placed in the organic reagent well before mixing across the chip. The reagent solutions were micromixed. The generated particles were diluted 1:1 with PBS.

[0155] Emulsion generation using DVBM We have produced emulsions with a narrow PDI, sub-100 nm in size, as summarized in Figure 18. Figure 18 ("POPC:Triolein (60:40)") shows a graph of the size and PDI characteristics of liposomes produced by a representative DVBM according to embodiments disclosed herein. The data was generated in a DVBM device with a neck length of 0.25 mm, a neck angle of 120 degrees, an inner radius of 0.16, a channel width and height of 80 μm, and a flow ratio of approximately 2:1 (aqueous:lipid mixture). The lipid composition was POPC:triolein (60:40). The initial lipid mixture concentration was 50 mM. The aqueous phase contained PBS buffer.

[0156] Materials and Methods: Same as described above for liposomes.

[0157] Therapeutic Encapsulation in Emulsions Using DVBM We generated a model hydrophobic drug, coumarin-6, encapsulated during emulsion formation with particle size less than 100 nm and a narrow PDI, as shown in Figure 18. Figure 18 ("POPC-triolein (60:40):C6") graphically illustrates the size and PDI characteristics of an encapsulated therapeutic agent, coumarin-6, generated by a representative DVBM according to embodiments disclosed herein, as well as a comparison with another similarly composed emulsion particle containing non-therapeutic drug particles. This data was generated in a DVBM device with a neck length of 0.25 mm, a neck angle of 120°, an inner radius of 0.16, a channel width and height of 80 μm, and a flow ratio of approximately 2:1 (aqueous:lipid mixture). The lipid mixture composition was 50 mM POPC:triolein (60:40) and coumarin-6 in DMF with a D / L (drug / lipid) ratio of 0.024 wt / wt. The aqueous phase contained PBS buffer. "Emulsion only" nanoparticles formed without coumarin-6 have essentially the same size and PDI.

[0158] Materials and Methods: Same as described above for liposomes.

[0159] Polymer nanoparticles formed using DVBM We have produced emulsions with sizes below 200 nm and narrow PDIs, as summarized in Figure 19. Figure 19 shows a graph of the size and PDI characteristics of polymer nanoparticles produced by a representative DVBM according to embodiments disclosed herein. The data was generated in a DVBM device with a neck length of 0.25 mm, a neck angle of 120 degrees, an inner radius of 0.16, a channel width and height of 80 μm, and a flow ratio of approximately 2:1 (aqueous:polymer mixture). The polymer mixture included 20 mg / mL polylactic-co-glycolic acid ("PLGA") in acetonitrile. The aqueous phase included PBS buffer.

[0160] Materials and Methods: Same materials as described above for liposomes. The following solutions were dispensed into each well in the cartridge: 36 μL of 2% PVA w / v in MilliQ water was placed in the aqueous reagent well, 48 μL of MilliQ water was placed in the collection well, and finally, 12 μL of 20 mg / mL PLGA in acetonitrile was placed in the organic reagent well just before mixing across the chip. The reagent solutions were micromixed. The generated particles were diluted 1:1 with MilliQ water.

[0161] Although exemplary embodiments have been particularly illustrated and described, it will be understood that various changes can be made within the scope of the invention without departing from the spirit and scope of the invention.

Claims

1. 1. A method of mixing a first liquid with a second liquid, comprising: flowing the first liquid and the second liquid into an inlet channel of a mixer to form a combined stream; bifurcating the combined stream into a first curved stream and a second curved stream around a first annular mixer of the mixer; recombining the first curved stream and the second curved stream into a combined stream at a neck region downstream of the first annular mixer; bifurcating the combined stream into a third curved stream and a fourth curved stream around a second annular mixer of the mixer; and recombining the third meandering stream and the fourth meandering stream into a combined stream downstream of the second annular mixer to form a mixed solution; Including, a first ratio of the volume of the first curved stream to the volume of the second curved stream is different from a second ratio of the volume of the third curved stream to the volume of the fourth curved stream; The method wherein the first annular mixer defines a first neck angle between a centerline of the inlet channel and a centerline of the neck region of between 100 and 150 degrees.

2. 10. The method of claim 1, wherein the mixer is incorporated into a microfluidic device comprising a plurality of mixers, and the method further comprises flowing the first liquid and the second liquid through the plurality of mixers to form the mixed solution.

3. The method of claim 1 , wherein the first liquid comprises nucleic acids in a first solvent.

4. 10. The method of claim 1, wherein the second liquid comprises a substance in a second solvent configured to form lipid particles in the mixed solution.

5. The method of claim 1 , wherein the mixed solution comprises particles generated by mixing a first liquid and a second liquid.

6. 6. The method of claim 5, wherein the particles are selected from the group consisting of lipid nanoparticles and polymer nanoparticles.

7. 2. The method of claim 1, wherein the first ratio is from 1:1 to 10:

1.

8. The method of claim 1 , wherein the first curved flow and the second curved flow have different lengths.

9. 10. The method of claim 1, wherein the first and third curved streams have different lengths and are located on different sides of the mixer.

10. 10. The method of claim 1, wherein the first curved stream passes through a first channel of the first annular mixer and the second curved stream passes through a second channel of the first annular mixer, each of the first and second channels having a longitudinal cross-sectional area along each of the first and second channels defining a channel height and a channel width, and wherein the first and second channels have different channel widths.

11. the first curved stream passes through a first channel of the first annular mixer, the second curved stream passes through a second channel of the first annular mixer, the third curved stream passes through a third channel of the second annular mixer, and the fourth curved stream passes through a fourth channel of the second annular mixer; each of the first, second, third, and fourth channels having a longitudinal cross-sectional area along each of the channels that defines a channel height and a channel width; The method of claim 1 , wherein a channel width of the first channel is different from at least one of a channel width of the second channel or a channel width of the third channel.

12. The method of claim 1 , wherein the first curved stream and the third curved stream are located on different sides of a mixer.

13. The method of claim 1 , wherein the first curved stream and the third curved stream have the same volume.

14. 10. The method of claim 1, wherein the first annular mixer and the second annular mixer are generally circular or elliptical in configuration, the first curved stream and the second curved stream have a first combined length equal to the circumference of the first annular mixer, and the third curved stream and the fourth curved stream have a second combined length equal to the circumference of the second annular mixer.

15. 1. A method of mixing a first liquid with a second liquid, comprising: flowing the first liquid and the second liquid into an inlet channel of a mixer to form a combined stream; bifurcating the combined stream into a first curved stream and a second curved stream around a first annular mixer of the mixer; and recombining the first curved stream and the second curved stream into a combined stream at a neck region downstream of the first annular mixer; wherein the first annular mixer defines a neck angle between a centerline of the inlet channel and a centerline of the neck region of 100 to 150 degrees.

16. branching the combined stream into a third curved stream and a fourth curved stream around a second annular mixer of the mixers downstream of the first annular mixer; and recombining the third curved stream and the fourth curved stream into a combined stream downstream of the second annular mixer.

16. The method of claim 15, further comprising:

Images