Fluidic modules comprising membranes and associated methods

Abstract

Fluidic modules (100; 200; 300; 600) and methods employing fluidic modules to contact liquids are generally described. In some embodiments, a fluidic module described herein is particularly suitable for synthesizing particles (e.g., microparticles and / or nanoparticles) and / or a method described herein comprises synthesizing particles.

Description

[0001] FLUIDIC MODULES COMPRISING MEMBRANES AND ASSOCIATED METHODS

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 652,835, filed May 29, 2024, and entitled “Fluidic Modules Comprising Membranes and Associated Methods,” which is incorporated herein by reference in its entirety for all purposes.

[0004] FIELD

[0005] Fluidic modules comprising channels separated by a membrane are generally described. Some fluidic modules described herein may be suitable for synthesizing nanoparticles, such as lipid nanoparticles, and associated methods for such synthesis are also described.

[0006] BACKGROUND

[0007] Some fluidic devices may be employed to mix liquids. However, some such fluidic devices may mix liquids in a manner that is unsuitable for applications in which improved results are observed when one or more aspects of the mixing is maintained within certain parameters.

[0008] Accordingly, new fluidic modules and methods for flowing samples through membranes would be advantageous.

[0009] SUMMARY

[0010] The present disclosure generally describes fluidic modules and associated methods. The subject matter described herein involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0011] In some embodiments, a fluidic module is provided. The fluidic module comprises a first channel comprising a first-channel inlet, a second channel comprising a second-channel inlet and a second-channel outlet, and a membrane positioned between the first and second channels. The first and second channels are in fluidic communication with each other through the membrane. The fluidic module is configured such that liquid flowing in the second channel directly adjacent to the membrane experiences a substantially constant shear rate over an area of the membrane.

[0012] A dimension of the first channel perpendicular to the membrane decreases from a first portion of the channel proximal to the first-channel inlet to a second portion of the channel distal from the first-channel inlet. A dimension of the second channel perpendicular to the membrane increases from a first portion of the channel distal to the second-channel outlet to a second portion of the channel proximal to the second-channel outlet.

[0013] In some embodiments, a fluidic module comprises a first channel comprising a first- channel inlet, a second channel comprising a second-channel inlet and a second-channel outlet, and a membrane positioned between the first and second channels. The first and second channels are in fluidic communication with each other through the membrane. The membrane comprises laterally isolated pores. The membrane has a pore size of greater than or equal to 0.01 micron and less than or equal to 30 microns.

[0014] In some embodiments, a method is provided. The method comprises flowing a first liquid through a first channel, through a membrane, and into a second liquid flowing in a second channel. This method may be performed in a fluidic module having the characteristics described in either or both of the preceding two paragraphs.

[0015] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures: FIG. 1 shows one non-limiting example of a fluidic module comprising a first channel, a second channel, and a membrane positioned between the first and second channels, in accordance with some embodiments;

[0018] FIG. 2 shows one non-limiting example of a fluidic module in which a first channel is disposed in a first layer and a second channel is disposed in a second layer, in accordance with some embodiments;

[0019] FIG. 3 shows one non-limiting example of a fluidic module in which a first adhesive is positioned between a first layer and a membrane and a second adhesive is positioned between a second layer and the membrane, in accordance with some embodiments;

[0020] FIG. 4 shows one non-limiting example of a fluidic module comprising a plurality of pairs of first and second channels, in accordance with some embodiments;

[0021] FIGs. 5 and 6 depict further non-limiting examples of fluidic modules, in accordance with some embodiments;

[0022] FIGs. 7 and 8 depict exemplary designs for first channels, in accordance with some embodiments;

[0023] FIG. 9 depicts and exemplary design for a second channel, in accordance with some embodiments;

[0024] FIGs. 10 and 11 are further examples of exemplary first and second channel designs, in accordance with some embodiments;

[0025] FIG. 12 shows profiles of the dimensions perpendicular to a membrane therebetween of exemplary first and second channels, in accordance with some embodiments;

[0026] FIG. 13 shows one non-limiting example of a method, in accordance with some embodiments;

[0027] FIG. 14 shows a photograph of an experimental setup, in accordance with some embodiments;

[0028] FIGs. 15-22 show experimental data, in accordance with some embodiments;

[0029] FIG. 23 shows photographs of an experimental setup, in accordance with some embodiments;

[0030] FIGs. 24-27 show experimental data, in accordance with some embodiments;

[0031] FIG. 28 shows a photograph of an exemplary fluidic module, in accordance with some embodiments; FIGs. 29-30 show experimental data, in accordance with some embodiments;

[0032] FIG. 31 shows various calculated profiles for the dimension of a first channel perpendicular to a membrane in a fluidic module having a Hydrodynamic Design that further comprises a second channel having the profile also shown therein, in accordance with some embodiments;

[0033] FIG. 32 shows experimental data, in accordance with some embodiments;

[0034] FIGs. 33 and 34 show mechanical drawings of an exemplary fluidic module, in accordance with some embodiments;

[0035] FIG. 35 shows a photograph of an exemplary fluidic module, in accordance with some embodiments; and

[0036] FIGs. 36-38 show mechanical drawings of exemplary fluidic modules, in accordance with some embodiments.

[0037] DETAILED DESCRIPTION

[0038] Fluidic modules and methods employing fluidic modules to contact liquids are generally described. In some embodiments, a fluidic module described herein is particularly suitable for synthesizing particles (e.g., microparticles and / or nanoparticles) and / or a method described herein comprises synthesizing particles.

[0039] In some embodiments, a fluidic module described herein has one or more properties that promote contact between two liquids in a manner that yields particles (e.g., microparticles and / or nanoparticles) that are relatively small, have a relatively low polydispersity index, and / or are produced at a relatively high rate. In some embodiments, a fluidic module described herein has one or more properties that allow for the production of particles to be readily scaled. For instance, the fluidic module may have one or more properties that allow for facilely increasing the rate of production of particles without substantially affecting some or all of the particle properties.

[0040] As one example, a fluidic module may have a structure such that two liquids are contacted by flowing a first liquid in a first channel through a membrane and into a second liquid flowing in a second channel, and the first channel and / or the second channel may have one or more properties that promote contact between the first and second liquid in a manner that is advantageous for synthesizing particles (e.g., microparticles and / or nanoparticles). For instance, the first channel and / or the second channel may have a design that results in the flow of liquid through the second channel in a manner such that it exhibits a substantially constant shear rate across one or more surfaces. As two examples, liquid may flow through the second channel such that liquid flowing directly adjacent to the membrane experiences a substantially constant shear rate over the area of the membrane (and / or one or more portions thereof) and / or liquid flowing directly adjacent to the surface of the second channel opposite the membrane experiences a substantially constant shear rate over this surface (and / or one or more portions thereof). It is also possible for all (and / or substantially all) of the liquid flowing in the second channel at any given distance from the membrane to experience a substantially constant shear rate.

[0041] Advantageously, fluidic modules having such designs may exhibit flow of liquid from the first channel, through the membrane, and into the second channel that is relatively uniform over the area of the membrane, which may advantageously enhance the stability of particles (e.g., microparticles and / or nanoparticles) formed during such flow. Additionally, fluidic modules having such designs may be capable of being facilely scaled up from the laboratory scale to industrial scales without appreciably affecting fluidic module operation.

[0042] In some embodiments, a fluidic module has a structure such that liquid flowing from the first-channel inlet, through the membrane, and to the second-channel outlet experiences a substantially constant pressure drop over the area of the membrane. A substantially constant pressure drop over the membrane area may be advantageous because it may promote flow through the membrane that is substantially constant over the membrane area. This may cause each portion of the membrane to be used equally, which, when a fluidic module is employed for particle (e.g., microparticle and / or nanoparticle) synthesis, may cause the particles to be generated at a relatively uniform rate across the portion of the second channel contacting the membrane. It may also cause the membrane to foul and / or become clogged in a relatively homogenous manner. By contrast, a pressure drop that varies over the membrane area may result in uneven flow through the membrane, resulting in enhanced generation of particles in some areas of the channel and reduced generation of particles in other areas of the channel. Additionally, such a pressure drop may result in locations where the pressure drop is lower fouling first and clogging, which may cause premature membrane failure, reduced yield of particles, and / or reduced performance. In some embodiments, a fluidic module comprises a membrane that has one or more desirable properties. For instance, a fluidic module may comprise a membrane that has a pore structure that is advantageous for forming particles (e.g., microparticles and / or nanoparticles). As three examples, the membrane may have a pore size in a range that is particularly suited to this purpose, may comprise pores that are relatively uniform in size, and / or may comprise laterally isolated pores. Pore sizes within a particular range (e.g., particularly small pores), pores that are relatively uniform in size, and laterally isolated pores may also promote flow through the membrane that is substantially constant over the membrane area. Thus, it may be beneficial for the same reasons provided in the preceding paragraph.

[0043] As noted above, certain embodiments described herein relate to forming particles (e.g., microparticles and / or nanoparticles), such as lipid nanoparticles. Such methods may comprise flowing one or more liquids through a fluidic module described herein, such as flowing a first liquid through a first channel, through a membrane, and into a second channel in which a second liquid is flowing. In some embodiments, if such a method is performed, particles may be generated upon contact and / or mixing between the first and second liquids. For instance, the first liquid may comprise one or more species that precipitates to form particles upon contact and / or mixing with the second liquid (and / or vice versa). In some embodiments, such precipitation may also comprise precipitation of one or more species from the second liquid. In such methods, particles may be formed that comprise at least one species from the first liquid and at least one species from the second liquid.

[0044] FIG. 1 shows one non-limiting example of a fluidic module 100 comprising a first channel 102, a second channel 104, and a membrane 106 positioned between the first and second channels. As can be seen in FIG. 1, the first channel also comprises a first-channel inlet 108 and the second channel comprises a second-channel inlet 110 and a second-channel outlet 112. As shown in FIG. 1, the membrane may be directly adjacent to the first channel and / or the second channel (e.g., it may be positioned directly between the first channel and the second channel), form a wall of the first channel, and / or form a wall of the second channel. In some embodiments, the first channel and the second channel are in fluidic communication (e.g., directly) through the membrane.

[0045] As used herein, where two species are “directly adjacent,” there is no intervening species positioned therebetween. It should be understood that FIG. 1 is a schematic depiction of a fluidic module and that the fluidic modules described herein may have some differences with FIG. 1 and / or may have some similarities thereto.

[0046] As an example, a fluidic module may comprise channels that have different shapes than those shown in FIG. 1. It is also possible for channels present in a fluidic module to have different relative dimensions with respect to each other, a membrane positioned therebetween, the inlets, and / or the outlet.

[0047] As another example, a fluidic module may comprise channels that are arranged with respect to each other in a manner like that shown in FIG. 1 or other than that shown in FIG. 1. For instance, a fluidic module may comprise channels that are arranged such that the first channel is disposed above the second channel (e.g., as shown in FIG. 1), such that the first channel is disposed below the second channel, such that the first and second channels are side- by-side, such that the first channel is disposed (partially or fully) concentrically around the second channel, such that the second channel is disposed (partially or fully) concentrically around the second channel, etc.

[0048] As a third example, a fluidic module may comprise inlets and / or outlets having designs that are similar to those shown in FIG. 1 and / or differ from those shown in FIG. 1 in one or more ways. For instance, channel inlets may feed a channel horizontally (e.g., as shown in FIG. 1) or vertically (e.g., from above the channel from below the channel) and channel outlets may receive liquid from a channel horizontally (e.g., as shown in FIG. 1) or vertically. In some embodiments, a channel inlet comprises a channel (or other component) that is in fluidic communication with a source of a liquid to be introduced into the channel via the channel inlet. It is also possible for a channel inlet to take the form of a port (or other opening) in a channel. Similarly, a channel outlet may comprise a channel (or other component) that is in fluidic communication with a location to which liquid flowing out of the fluidic module may be directed or to take the form of a port (or other opening) in the channel.

[0049] In some embodiments, a module comprises one or more layers in which channels are disposed. For instance, a fluidic module may comprise a first layer in which a first channel is disposed and a second layer in which a second channel is disposed. In such embodiments, the layer may form one or more walls of the channel and / or may, together with a membrane, fully enclose one or more portions (or all) of the channel. FIG. 2 shows one non-limiting example of a fluidic module 200 in which the first channel is disposed in a first layer 214 and the second channel 206 is disposed in a second layer 216. As shown in FIG. 2, in such embodiments, a membrane (e.g., the membrane 206) may be positioned between the first and the second layers. In some embodiments, like the embodiment shown in FIG. 2, the membrane may extend laterally beyond the channels (e.g., it may be between portions of the first and second layer into which the first channel does not extend and / or the second channel does not extend).

[0050] As shown in FIG. 2, a membrane positioned between two layers may be positioned directly between such layers. It is also possible for one or more components (e.g., one or more intervening layers) to be positioned therebetween. In some embodiments, an adhesive is positioned between a first layer and a membrane and / or an adhesive is positioned between (e.g., directly between) a second layer and a membrane. In such embodiments, the adhesive may adhere together the membrane and the layer between which it is positioned.

[0051] FIG. 3 shows one non-limiting example of such an embodiment. In FIG. 3, in the fluidic module 300, a first adhesive 318 is positioned between the first layer 314 and the membrane 306 and a second adhesive 320 is positioned between the second layer 316 and the membrane 306. As shown in FIG. 3, adhesives positioned between layers (and / or membranes) may take the form of adhesive layers. Such layers may, in some embodiments, comprise one or more openings, holes, and / or discontinuities. For instance, as shown in FIG. 3, some adhesive layers do not extend into channels (e.g., they are disposed only on portions of the layer and membrane where the layer contacts the membrane). With respect to FIG. 3, the adhesive layer 318 does not extend into the channel 302 (i.e., it is not positioned between the channel 302 and the membrane 306) and the adhesive layer 320 does not extend into the channel 304 (i.e., it is not positioned between the channel 304 and the membrane 306). It is also possible for an adhesive to be positioned between a layer and a membrane in a manner such that it does not form a layer.

[0052] In some embodiments in which a first channel is disposed in a first layer and a second channel is disposed in a second layer positioned therebeneath, the fluidic module may have a design that allows for liquid to be introduced into both the first and second channels through a common surface. For instance, in some embodiments, a fluidic module may have a design that allows for liquid to be introduced into both the first and second channels through an upper surface of the first layer. In some embodiments, a fluidic module may have a design such that liquid can be removed from a second channel through this same surface. One example of a fluidic module having this design is shown schematically in FIG. 4. In FIG. 4, it can be seen that the upper surface of the first layer includes first-channel inlets taking the form of ports through which liquids can be delivered to channels in a first layer (i.e., the ports through which liquids comprising lipids can be delivered thereto, labeled as “LIPID IN”), second-channel inlets taking the form of ports through which liquids can be delivered to channels in a second layer (i.e., the ports through which liquids comprising a buffer can be delivered thereto, labeled as “BUFFER IN”), and second-channel outlets ports through which liquids can be recovered from channels in the second layer (i.e., the ports labeled “LNP OUT”). In FIG. 4, the “Top channel” corresponds to the first channel described herein and the “Bottom channel” corresponds to the second channel described herein. As can also be seen in FIG. 4, in some embodiments, a second-channel inlet and / or a second-channel outlet passes through the first layer and any other components positioned between the second layer and the upper surface of the first layer (e.g., a membrane, one or more adhesives).

[0053] FIG. 4 further shows other exemplary fluidic module components, including adhesives (labeled as “Top PSA” and “Bottom PSA”), a membrane, and dowel pins that hold together these components.

[0054] In some embodiments, like the embodiment shown in FIG. 4, first and second channels in a fluidic module may have different lengths. This may be particularly beneficial when a second- channel inlet and / or second-channel outlet passes through which a layer in which the first channel is disposed. In such embodiments, if the second channel extends beyond the first- channel inlet in an upstream direction therefrom, the second-channel inlet may pass through a portion of the first layer that is not in fluidic communication with the first channel. Similarly, if the second channel extends beyond the first channel terminus in a downstream direction therefrom, the second-channel outlet may pass through a portion of the first layer that is not in fluidic communication with the first channel.

[0055] A further example of a fluidic module having this design is shown schematically in FIG. 5. As can be seen in FIG. 5, the second channel extends beyond both the first-channel inlet and the terminus of the first channel. It extends beyond the first channel in both the upstream and downstream directions. In FIG. 5, the port through which “Buffer / mRNA” can flow is the second-channel inlet, the port through which “Lipid / Ethanol” can flow is the first-channel inlet, and the port through which “LNP” can flow is the second-channel outlet. Additionally, in FIG. 5, the “Mixing region” is located in the second channel proximal to the membrane.

[0056] In some embodiments, like the embodiment shown in FIG. 4, a single fluidic module may comprise more than one pair of first and second channels. For instance, with reference to FIG. 4, it can be seen that the fluidic module comprises four pairs of first and second channels. Such channels may differ from each other in one or more ways, which may facilitate the testing of different conditions and / or assist with employing the fluidic module to generate particles (e.g., microparticles and / or nanoparticles) at different rates. With reference to FIG. 4, it can be seen that the pairs of first and second channels exhibit decreasing widths from left to right. It is also possible for a fluidic module to comprise two or more pairs of first and second channels that are identical to each other or only differ in ways that do not affect particle production, which may facilitate the use of the fluidic module to generate particles of a single type at higher rates (e.g., by employing identical conditions in each such pair, thereby multiplying the rate of particle production by the number of such pairs).

[0057] When a fluidic module comprises more than one pair of first and second channels, the pairs of first and second channels, and their components, may have a variety of suitable arrangements with respect to each other. For instance, different first channels in a common fluidic module may comprise first-channel inlets that are in fluidic communication with each other through the fluidic module (e.g., that are fed by a common port and / or a common channel positioned within the fluidic module) or that are fluidically isolated from each other within the fluidic module (i.e., that lack a pathway through which they are in fluidic communication with each other that passes through the fluidic module). Similarly, different second channels in a common fluidic module may comprise second-channel inlets that are in fluidic communication with each other through the fluidic module (e.g., that are fed by a common port and / or a common channel positioned within the fluidic module) or that are fluidically isolated from each other within the fluidic module. As a third example, different second channels in a common fluidic module may comprise second-channel outlets that are in fluidic communication with each other through the fluidic module (e.g., that feed a common port and / or a common outlet) or that are fluidically isolated from each other within the fluidic module. FIG. 4 shows one non-limiting example of a fluidic module comprising a plurality of pairs of first and second channels for which the first-channel inlets, second-channel inlets, and second-channel outlets are fluidically isolated from each other within the fluidic module.

[0058] Without wishing to be bound by any particular theory, it is believed that a plurality of pairs of first and second channels for which the first-channel inlets, second-channel inlets, and second-channel outlets that are fluidically isolated from each other within the fluidic module (e.g., as shown in FIG. 4) may be desirable for fluidic modules that are employed to generate particles (e.g., microparticles and / or nanoparticles) under different conditions, having different morphologies, and / or having different compositions in the different pairs. It is also believed that a plurality of pairs of first and second channels for which the first-channel inlets, second-channel inlets, and / or second-channel outlets are in fluidic communication with each other through the fluidic module (e.g., inlets that are fed by a common port and / or channel, outlets that feed a common port and / or channel) may be desirable for fluidic modules employed to make use of the pairs of first and second channels to generate particles (e.g., microparticles and / or nanoparticles) under the same conditions, having the same morphologies, and / or having the same compositions in the different pairs. For instance, such fluidic modules may be desirable when the pairs of first and second channels are employed to generate particles that are substantially identical to each other at relatively high rates.

[0059] As described above, in some embodiments, a fluidic module has one or more features that promote the formation of particles (e.g., microparticles and / or nanoparticles) having one or more desirable properties. As one example, in some embodiments, a fluidic module is configured such that liquid flowing in the second channel directly adjacent to the membrane experiences a substantially constant shear rate over an area of the membrane. This area of the membrane may be an entirety of the area of the membrane directly adjacent to the second channel, an entirety of an area of the membrane positioned directly between the first channel and the second channel, and / or a subset of either of these two areas. As another example, in some embodiments, a fluidic module is configured such that liquid flowing in the second channel directly adjacent to a surface of the second channel opposite the membrane experiences a substantially constant shear rate over an area of the surface of the second channel opposite the membrane. This area of the surface of the second channel opposite the membrane may be an entirety of the area of surface of the second channel opposite the membrane, an entirety of an area of the surface of the second channel opposite the membrane and positioned beneath the first channel, and / or a subset of either of these two areas.

[0060] FIG. 6 illustrates some of the relevant areas identified in the preceding paragraph with enhanced clarity. In FIG. 6, the module 600 comprises a first channel 602, a second channel 604, and a membrane 606. The area of the membrane directly adjacent to the second channel is the portion of the membrane enclosed by the box 622, the area of the membrane positioned directly between the first channel and the second channel is the portion of the membrane enclosed by the box 624, the surface of the second channel opposite the membrane is the surface of the second channel enclosed by the box 626, and the surface of the second channel opposite the membrane and positioned beneath the first channel is the surface of the second channel enclosed by the box 628.

[0061] In some embodiments, a shear rate that is substantially constant in a location varies by a relatively small amount over that location. For instance, a shear rate that is substantially constant may vary by less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10%, less than or equal to 7.5%, less than or equal to 5%, less than or equal to 2%, or less than or equal to 1%. A shear rate that is substantially constant may vary by greater than or equal to 0%, greater than or equal to 1%, greater than or equal to 2%, greater than or equal to 5%, greater than or equal to 7.5%, greater than or equal to 10%, greater than or equal to 20%, or greater than or equal to 30%. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0% and less than or equal to 40%, greater than or equal to 0% and less than or equal to 10%, or greater than or equal to 30% and less than or equal to 40%). Other ranges are also possible. In some embodiments, a shear rate that is substantially constant over a location varies by 0% over that location.

[0062] When there are two or more locations in a fluidic module in which the shear rate is substantially constant, the shear rate in each such location may independently vary by an amount in one or more of the above-described ranges over each such location.

[0063] A channel shape for a second channel suitable for obtaining a particular shear rate and / or a particular rate of shear rate variation for a liquid flowing in one or more portions of a fluidic module described herein may be determined by performing the following steps: (1) Determine flow rates for liquid flowing through the first channel and the second channel; (2) Determine liquids to be flowed in the first and second channel; (3) Based on the preceding information, calculate the anticipated flow rate of liquid in the first and second channels; and (4) Use the shear-rate equation for a channel with a rectangular cross-section to determine a profile for the dimension of the second channel perpendicular to the membrane that will result in the desired shear rate and / or shear rate variation.

[0064] In some embodiments, a second channel has a channel shape suitable for obtaining a shear rate that is substantially constant for liquid flowing in one or more of the locations described elsewhere herein (e.g., liquid flowing in the second channel directly adjacent to the membrane, liquid flowing in the second channel directly adjacent to a surface of the second channel opposite the membrane) when liquid is flowing through the first and second channels at particular flow rates. For instance, a second channel may have such a shape when the ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel is greater than or equal to 1, greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, or greater than or equal to 9. The second channel may have such a shape when the ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel is less than or equal to 10, less than or equal to 9, less than or equal to 8, less than or equal to 7, less than or equal to 6, less than or equal to 5, less than or equal to 4, less than or equal to 3, or less than or equal to 2. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 1 and less than or equal to 10, greater than or equal to 1 and less than or equal to 5, or greater than or equal to 1 and less than or equal to 3). Other ranges are also possible. In some embodiments, the ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel is identically one of the endpoints of the ranges provided below (e.g., identically 1, identically 3, or identically 10).

[0065] In some embodiments, a fluidic module is configured such that liquid flowing from the first-channel inlet to the second-channel outlet experiences a substantially constant pressure drop over an area of the membrane. This may be an entirety of the area of the membrane directly adjacent to the second channel, an entirety of an area of the membrane positioned directly between the first channel and the second channel, and / or a subset of either of these two areas.

[0066] In some embodiments, a pressure drop that is substantially constant in a location varies by a relatively small amount over that location. For instance, a pressure drop that is substantially constant may vary by less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10%, less than or equal to 7.5%, less than or equal to 5%, less than or equal to 2%, or less than or equal to 1%. A pressure drop that is substantially constant may vary by greater than or equal to 0%, greater than or equal to 1%, greater than or equal to 2%, greater than or equal to 5%, greater than or equal to 7.5%, greater than or equal to 10%, greater than or equal to 20%, or greater than or equal to 30%. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0% and less than or equal to 40%, greater than or equal to 0% and less than or equal to 10%, or greater than or equal to 30% and less than or equal to 40%). Other ranges are also possible. In some embodiments, a pressure drop that is substantially constant over a location varies by 0% over that location.

[0067] When there are two or more locations in a fluidic module in which the pressure drop is substantially constant, the pressure drop in each such location may independently vary by an amount in one or more of the above-described ranges over each such location.

[0068] When a fluidic module is configured both so that liquid flowing therethrough exhibits a particular shear rate in one or more locations and exhibits a particular pressure drop over an area of a membrane, channel shapes for first and second channels suitable for this purpose may be determined by performing the following steps: (1) Perform steps ( 1 )-(4) for determining a channel shape for a second channel suitable for obtaining a particular shear rate for a liquid flowing in one or more portions of a fluidic module described herein; (2) Use the pressure-flow rate equation for a channel with a rectangular cross-section to determine a pressure profile in the second channel assuming a uniform flux of liquid across the membrane area from the first channel to the second channel through the membrane; (3) Determine a pressure profile in the first channel assuming a known transmembrane pressure and assuming that the pressure drop is constant over the membrane area; and (4) Based on the preceding information, use the pressure- flow-rate equation to determine a profile for the dimension of the first channel perpendicular to the membrane that will result in the desired pressure drop.

[0069] A fluidic module comprising first and second channels having channel shapes calculated as described in the preceding paragraph may be referred to elsewhere herein as a fluidic module having a “hydrodynamic design.”

[0070] In some embodiments, a first channel and / or a second channel has one or more geometric features that facilitate the above-described substantially constant shear rate and / or substantially constant pressure drop. As one example, in some embodiments, the dimension of a first channel perpendicular to the membrane exhibits a profile that is particularly advantageous. For clarity, with respect to FIG. 6, the dimension 630 of the first channel 602 is perpendicular to the membrane 606.

[0071] In some embodiments, a dimension of a first channel that is perpendicular to a membrane decreases from a first portion of the first channel proximal to a first-channel inlet to a second portion of the first channel distal from the first-channel inlet. One example of a first channel having such a design is shown in FIG. 7. In FIG. 7, the dimension 730 of the first channel 702 decreases from a first portion 732 proximal to the first-channel inlet 708 to a second portion 734 distal to the first-channel inlet 708. As can be seen in FIG. 7, the dimension 730 is perpendicular to the membrane 706. As can also be seen in FIG. 7, in some embodiments, the first channel is a dead-end channel. In other words, the channel may not include any outlet. In such embodiments, all liquid exiting the first channel does so through the membrane (e.g., and into the second channel). In some embodiments, the first portion 732 is directly adjacent to the first- channel inlet and the first channel is a dead-end channel. In such embodiments, the dimension of the first channel that is perpendicular to the membrane decreases over the entirety of the first channel excepting the first-channel inlet.

[0072] In some embodiments, a first channel comprises two portions, each having a different rate of decrease of a dimension perpendicular to a membrane. One example of a first channel having such a geometry is shown schematically in FIG. 8. In FIG. 8, the first channel 802 comprises a first portion 832 proximal to the first-channel inlet 808, a second portion 834 distal to the first-channel inlet 808, and a third portion 836 positioned between the first and second portions 832 and 834. As can be seen in FIG. 8, the dimension 830 is perpendicular to the membrane 806. As can also be seen in FIG. 8, the rate of decrease of the dimension 830 from the first portion 832 to the third portion 836 is different from the rate of decrease of the dimension 830 from the third portion 836 to the second portion 834. In some embodiments, like the embodiment shown in FIG. 8, the rate of decrease of the dimension perpendicular to the membrane between the third portion and the second portion is greater than the rate of decrease of this dimension between the first portion and the third portion. Additionally, in some embodiments, like the embodiment shown in FIG. 8, these different rates of decrease are relatively constant between the first portion and the third portion and between the third portion and the second portion (e.g., they may vary by less than or equal to 6%, less than or equal to 5%, less than or equal to 4%, less than or equal to 2%, or less than or equal to 1% and / or greater than or equal to 0%).

[0073] When a first channel has a geometry similar to that shown in FIG. 7 or FIG. 8, the locations of the first and second (and, optionally, third) portions of the first channel may be selected as desired. In some embodiments, the first portion of the first channel is the portion of the first channel directly adjacent to the first-channel inlet. In some embodiments, the second portion of the first channel is the terminus of the first channel.

[0074] When a first channel has a geometry similar to that shown in FIG. 8, the ratio of the distance from the first portion of the first channel to the third portion of the first channel to the distance from the third portion of the first channel to the second portion of the first channel may be selected as desired. In some embodiments, the distance from this first portion to this third portion is greater than the distance from this third portion to this second portion. In some embodiments, this ratio may be approximately 3 (e.g., greater than or equal to 2.5 and less than or equal to 3.5, or greater than or equal to 2 and less than or equal to 4).

[0075] In some embodiments, a dimension of a second channel perpendicular to a membrane increases from a first portion of the channel distal to the second-channel outlet to a second portion of the channel proximal to the second-channel outlet. One example of a second channel having this geometry is shown schematically in FIG. 9. In FIG. 9, the dimension 938 increases from the first portion 940 distal to the second-channel outlet 912 to the second portion 942 proximal to the second-channel outlet 912. As can be seen in FIG. 9, the dimension 938 is perpendicular to the membrane 906. In some embodiments, like the embodiment shown in FIG. 9, the rate of increase of the dimension perpendicular to the membrane is relatively constant between the first portion and the second portion (e.g., it may vary by less than or equal to 1.2%, less than or equal to 1%, less than or equal to 0.75%, less than or equal to 0.5%, less than or equal to 0.2%, or less than or equal to 0.1% and / or greater than or equal to 0%).

[0076] When a first channel has a geometry similar to that shown in FIG. 9, the locations of the first and second portions of the second channel may be selected as desired. In some embodiments, the first portion of the second channel is the portion of the first channel directly adjacent to the second-channel inlet. In some embodiments, the first portion of the second channel is the portion of the second channel positioned beneath the portion of the first channel directly adjacent to the first-channel inlet. In some embodiments, the second portion of the second channel is the portion of the second channel directly adjacent to the second-channel outlet. In some embodiments, the second portion of the second channel is the portion of the second channel positioned beneath the terminus of the first channel. As one example, in some embodiments, the surface of the second channel opposite the membrane and positioned beneath the first channel extends from the first portion of the second channel to the second portion of the second channel.

[0077] FIGs. 10 and 11 are further examples of exemplary first and second channel designs. FIG. 12 shows profiles of the dimensions perpendicular to a membrane therebetween of exemplary first and second channels.

[0078] In some embodiments, a fluidic module comprises first and second channels that are vertically oriented and / or oriented such that liquid flows vertically therethrough (e.g., primarily vertically, exclusively vertically). In such embodiments, a membrane positioned between the first and second channels may be oriented vertically (e.g., it may be oriented such that the direction along which liquid flows therethrough and / or the direction along which its thickness is measured forms a 90° angle with respect to the vertical direction). It is also possible that a fluidic module comprises first and second channels that are horizontally oriented and / or oriented such that liquid flows horizontally therethrough (e.g., primarily horizontally, exclusively horizontally). In such embodiments, a membrane positioned between the first and second channels may be oriented horizontally (e.g., it may be oriented such that the direction along which liquid flows therethrough and / or the direction along which its thickness is measured forms a 0° angle with respect to the vertical direction). In some embodiments, a fluidic module comprises first and second channels that are oriented such that liquid flows both vertically and horizontally therethrough.

[0079] In some embodiments, a fluidic module has a design such that it is capable of being employed and / or is configured to be employed with a pipette (e.g., a manual pipette, a dualchannel pipette), a pipette tip (e.g., a dual-channel pipette tip), a syringe (e.g., a dual-channel syringe), and / or a liquid handler (e.g., an automated liquid handler, an automated pipette). This may be accomplished by, e.g., the presence of one or more components (e.g., a fitting therein, a compressible component therein) in the fluidic module that is sized and shaped to interact mechanically with one or more components of the pipette, pipette tip, syringe, and / or liquid handler (e.g., a fitting therein, a compressible component therein). In some embodiments, the fluidic module may comprise one or more components that mates and / or mechanically fixes the fluidic module to the pipette, pipette tip, syringe, and / or liquid handler.

[0080] As noted above, some embodiments relate to methods. In some embodiments, a method comprises flowing one or more liquids through a fluidic module described herein. FIG. 13 shows one non-limiting example of a method 1344. The method 1344 comprises the step 1346 of flowing a first liquid through a first channel, through a membrane, and into a second liquid flowing in a second channel. Such a method may be performed in a fluidic module described herein. The method 1344 may further comprise one or more additional, optional, steps. Nonlimiting examples of such steps are further shown in FIG. 13. The first such and second such optional steps are the optional steps 1348 and 1350, which comprise flowing the first liquid into the first channel and flowing the second liquid into the second channel, respectively. This may be performed by flowing the first liquid through the first-channel inlet and the second liquid through the second-channel inlet. The third such optional step is the optional step 1352, which comprises mixing the first and second liquids in the second channel. The fourth such optional step is the optional step 1354, which comprises forming nanoparticles in the second channel.

[0081] The fifth such optional step is the optional step 1356, which comprises recovering the nanoparticles. This may be accomplished via a second-channel outlet (e.g., by flowing a liquid in which the nanoparticles are suspended therethrough). In some embodiments, additionally or alternatively, steps like the optional steps 1354 and / or 1356 may be performed with respect to a type of particles other than nanoparticles (e.g., microparticles).

[0082] In some embodiments, a method may be performed during which liquid flowing in the second channel directly adjacent to the membrane experiences a substantially constant shear rate over an area of the membrane, liquid flowing in the second channel directly adjacent to surface of the second channel opposite the membrane experiences a substantially constant shear rate over the surface of the second channel opposite the membrane, liquid flowing in the second channel at any given distance from the membrane to experience a substantially constant shear rate, and / or liquid flowing from the first-channel inlet to the second-channel outlet experiences a substantially constant pressure drop over an area of the membrane.

[0083] Pressure that causes first and second liquids to flow through the first and second channels may be applied in a variety of suitable manners. As one example, gravity may cause first and second liquids to flow through the first and second channels if the first and second channels are oriented vertically or at an angle to the vertical such that gravity would cause such flow. As further examples, pumps (e.g., syringe pumps), plungers, and / or other sources of pressure may be employed to apply pressure to liquids in the first and second channels, or liquids upstream from the first and second channels, to cause first and second liquids to flow through the first and second channels. Such components may be operated manually and / or via automation.

[0084] Pressure from a single source may cause first and second liquids to flow through the first and second channels and / or pressure may be applied to one channel but not the other. As an example of the former, pressure may be applied to first and second liquids in the first and second channel by gravity, a single plunger, two plungers mechanically coupled to a single depressible component, and / or a pump in fluidic communication with both channels. As an example of the latter, in some embodiments, one channel is in fluidic communication with a source of pressure that the other is not (e.g., a plunger may be positioned in one channel but not the other, a pump may be in fluidic communication with one channel but not the other).

[0085] In some embodiments, first and second liquids flow through both of the first and second channels simultaneously and / or substantially simultaneously. For instance, first and second liquids may be introduced into the first and second channels simultaneously (and / or substantially simultaneously) and allowed to flow therethrough under the influence of pressure that is applied to the liquid in both channels simultaneously (and / or substantially simultaneously), such as pressure applied by a single source.

[0086] It is also possible for one liquid to be flowed intentionally through one channel without liquid flowing through the other (e.g., prior to simultaneous flow of first and second liquids through first and second channels). Such a process may advantageously result in the trapping of fewer bubbles in the channel through which the liquid is flowed. For instance, flowing a liquid through only one channel (e.g., a first channel, a channel disposed on another, a channel through which a liquid comprising a lipid flows) may push air bubbles that would otherwise be present in that channel through the membrane and into the other channel (e.g., a second channel, a channel disposed beneath the channel through which the liquid is flowed, a channel through which a liquid comprising mRNA flows). This air, along with the other air initially present in the other channel (e.g., the second channel, the channel beneath the channel through which the liquid is flowed, the channel through which a liquid comprising mRNA flows), may then be pushed out of the other channel when a liquid is subsequently flowed therethrough. As one example, in some embodiments, a source of pressure may be employed to cause a liquid to flow through one channel but not the other. This may occur, for instance, in a fluidic module comprising first and second channels that are horizontally oriented. As one non-limiting example, in some embodiments, a source of pressure may be employed to prime one of the channels (e.g., a first channel, such as a first channel that is disposed above a second channel) by filling it with a first liquid and / or flowing a first liquid therethrough. Then, after the channel is primed, a source of pressure (e.g., a different source of pressure) may be employed to also cause a liquid (e.g., a different liquid) to flow through the other channel.

[0087] In some embodiments, an inlet to a first channel and / or an inlet to a second channel has a design that causes liquid to flow into and / or through one channel before flowing into and / or through the other. For instance, in some embodiments, a fluidic module comprises one inlet that has a larger volume than the other. Liquid of comparable volumes and / or flowing at comparable flow rates into these inlets will take longer to flow through the inlet of larger volume. This can delay entry of this liquid into the associated channel, allowing liquid flowing through the inlet having smaller volume to enter its associated channel first.

[0088] For instance, in some embodiments, a fluidic module comprises a first-channel inlet and a second-channel inlet, and the first-channel inlet has a smaller volume than the second-channel inlet. In such embodiments, if a first liquid and a second liquid are introduced simultaneously into the first-channel inlet and the second-channel inlet, respectively, the first liquid will exit the first-channel inlet and enter the first channel before the second liquid exits the second-channel inlet and enters the second-channel. This may allow for first and second liquids to be simultaneously introduced into the first- and second-channel inlets, but for the first liquid to still flow through the first channel before the second liquid begins flowing through the second channel. This may be advantageous for gravity -driven flow, where it may be challenging to apply different pressures to first and second liquids flowing through the first and second channels.

[0089] In some embodiments, a fluidic module comprises a second-channel inlet that has a larger volume than the volume of the first channel (and / or the sum of the volumes of the first channel and the first-channel inlet). This may allow a first liquid and a second liquid to be introduced into both inlets simultaneously, but for the first liquid to flow fully through the first channel before the second liquid enters the second channel. Such flow may expel air from the first channel into the second channel before the second liquid enters the second channel.

[0090] In some embodiments, the ratio of the flow rates of liquid in the first and second channels can be selected to produce particles (e.g., microparticles and / or nanoparticles) having a particular size and / or poly dispersity index. For instance, the ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel may be in one or more of the ranges provided elsewhere herein. In some embodiments, the rate of particle (e.g., microparticle and / or nanoparticle) generation can be adjusted by adjusting this ratio and / or by adjusting the widths of the first and / or second channels. In some embodiments, the rate of particle generation can be selected without substantially varying the particle properties by employing a hydrodynamic design as described herein. Total channel volumes may be as low as 250 microliters.

[0091] The total flow rate of liquid in both the first and second channels combined during particle generation (e.g., the generation of microparticles and / or nanoparticles) may be selected as desired. In some embodiments, the total flow rate of liquid is greater than or equal to 0.2 mL / min, greater than or equal to 0.5 mL / min, greater than or equal to 0.75 mL / min, greater than or equal to 1 mL / min, greater than or equal to 2 mL / min, greater than or equal to 5 mL / min, greater than or equal to 7.5 mL / min, greater than or equal to 10 mL / min, greater than or equal to 20 mL / min, greater than or equal to 50 mL / min, greater than or equal to 75 mL / min, greater than or equal to 100 mL / min, greater than or equal to 125 mL / min, greater than or equal to 150 mL / min, or greater than or equal to 175 mL / min. In some embodiments, the total flow rate of liquid is less than or equal to 200 mL / min, less than or equal to 175 mL / min, less than or equal to 150 mL / min, less than or equal to 125 mL / min, less than or equal to 100 mL / min, less than or equal to 75 mL / min, less than or equal to 50 mL / min, less than or equal to 20 mL / min, less than or equal to 10 mL / min, less than or equal to 7.5 mL / min, less than or equal to 5 mL / min, less than or equal to 2 mL / min, less than or equal to 1 mL / min, less than or equal to 0.75 mL / min, or less than or equal to 0.5 mL / min. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.2 mL / min and less than or equal to 200 mL / min). Other ranges are also possible.

[0092] During performance of the method shown in FIG. 13, the first and second liquids can be selected as desired. In some embodiments, one of the liquids comprises a lipid and the other liquid comprises mRNA. Upon contact and / or mixing between such liquids, particles (e.g., microparticles and / or nanoparticles) comprising the lipid and the mRNA (e.g., lipid nanoparticles) may form. When such mixing and / or contact occurs in the second channel, the particles may form in the second channel (e.g., in one or more portions of the second channel proximal to the membrane). It is possible for the first liquid to comprise the lipid and the second liquid to comprise the mRNA, or for the first liquid to comprise the mRNA and for the second liquid to comprise the lipid. The liquid comprising the lipid may be an organic liquid. The liquid comprising the mRNA may be an aqueous liquid, such as an aqueous liquid comprising a buffer (e.g., a citrate buffer).

[0093] A variety of suitable lipid formulations may be employed, non-limiting examples of which include: (1) SM-102, DSPC, cholesterol, DMG-PEG2000; and (2) ALC-0315, DSPC, cholesterol, and ALC-0159.

[0094] The membranes described herein may have a variety of suitable designs. In some embodiments, a membrane described herein comprises laterally isolated pores. It is also possible for the pores in a membrane to consist of and / or to consist essentially of laterally isolated pores. In some embodiments, a membrane described herein comprises cylindrical pores (e.g., cylindrical, laterally isolated pores). It is also possible for the pores in a membrane to consist of and / or to consist essentially of cylindrical pores (e.g., cylindrical, laterally isolated pores). One non-limiting example of a suitable membrane is a track-etched membrane (e.g., a track-etched polycarbonate membrane).

[0095] The pores present in a membrane may have a variety of suitable sizes. In some embodiments, a membrane has a pore size of greater than or equal to 0.01 micron, greater than or equal to 0.02 microns, greater than or equal to 0.05 microns, greater than or equal to 0.075 microns, greater than or equal to 0.1 micron, greater than or equal to 0.2 microns, greater than or equal to 0.5 microns, greater than or equal to 0.75 microns, greater than or equal to 1 micron, greater than or equal to 2 microns, greater than or equal to 5 microns, greater than or equal to 7.5 microns, greater than or equal to 10 microns, greater than or equal to 15 microns, greater than or equal to 20 microns, or greater than or equal to 25 microns. In some embodiments, a membrane has a pore size of less than or equal to 30 microns, less than or equal to 25 microns, less than or equal to 20 microns, less than or equal to 15 microns, less than or equal to 10 microns, less than or equal to 7.5 microns, less than or equal to 5 microns, less than or equal to 2.5 microns, less than or equal to 1 micron, less than or equal to 0.75 microns, less than or equal to 0.5 microns, less than or equal to 0.2 microns, less than or equal to 0.1 micron, less than or equal to 0.075 microns, less than or equal to 0.05 microns, or less than or equal to 0.02 microns. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.01 micron and less than or equal to 30 microns). Other ranges are also possible.

[0096] In some embodiments, the size of a pore is equivalent to the diameter of the smallest circular particle that is unable to pass through the pore.

[0097] In some embodiments, a membrane has a number-average pore size in one or more of the above-referenced range (i.e., the “pore size” is a number-average pore size). In some embodiments, a membrane has a mode pore size in one or more of the above-referenced ranges (i.e., the “pore size” is the mode pore size). In some embodiments, the largest pore in a membrane has a size in one or more of the above-referenced ranges (i.e., the “pore size” is the maximum pore size). In some embodiments, the pores present in a membrane consist of and / or consist essentially of pores having sizes that fall in one or more of the above-referenced ranges.

[0098] As noted above, in some embodiments, a fluidic module described herein comprises an adhesive. A variety of suitable adhesive may be employed. In some embodiments, a fluidic module comprises a pressure-sensitive adhesive (e.g., positioned between a first layer and a membrane, positioned between a second layer and a membrane). One non-limiting example of a suitable pressure-sensitive adhesive is a double-sided silicone pressure-sensitive adhesive.

[0099] In some embodiments, an adhesive may not extend such that it is positioned between a channel and a membrane or such that a minimal amount of the adhesive is positioned between the channel and the membrane. Adhesives may be cut to form an appropriate shape via, e.g., die cutting, laser cutting, and / or use of a desktop cutting plotter.

[0100] The channels described herein may have a variety of suitable designs. In some embodiments, a channel has a rectangular cross-sectional profile (e.g., a rectangular profile perpendicular to liquid flow therethrough).

[0101] In some embodiments, a first channel has a width (e.g., a dimension perpendicular to the dimension perpendicular to the membrane and perpendicular to the direction of liquid flow through the channel) of greater than or equal to 0.5 mm, greater than or equal to 0.75 mm, greater than or equal to 1 mm, greater than or equal to 1.5 mm, greater than or equal to 2 mm, greater than or equal to 2.5 mm, greater than or equal to 3 mm, greater than or equal to 4 mm, greater than or equal to 4.5 mm, greater than or equal to 5 mm, greater than or equal to 6 mm, greater than or equal to 7 mm, greater than or equal to 8 mm, or greater than or equal to 9 mm. In some embodiments, a first channel has a width of less than or equal to 10 mm, less than or equal to 9 mm, less than or equal to 8 mm, less than or equal to 7 mm, less than or equal to 6 mm, less than or equal to 5 mm, less than or equal to 4.5 mm, less than or equal to 4 mm, less than or equal to 3.5 mm, less than or equal to 3 mm, less than or equal to 2.5 mm, less than or equal to 2 mm, less than or equal to 1.5 mm, less than or equal to 1 mm, or less than or equal to 0.75 mm. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.5 mm and less than or equal to 10 mm, or greater than or equal to 0.5 mm and less than or equal to 5 mm). Other ranges are also possible.

[0102] In some embodiments, a second channel has a width of greater than or equal to 0.5 mm, greater than or equal to 0.75 mm, greater than or equal to 1 mm, greater than or equal to 1.5 mm, greater than or equal to 2 mm, greater than or equal to 2.5 mm, greater than or equal to 3 mm, greater than or equal to 4 mm, greater than or equal to 4.5 mm, greater than or equal to 5 mm, greater than or equal to 5.5 mm, greater than or equal to 6 mm, greater than or equal to 7 mm, greater than or equal to 8 mm, greater than or equal to 9 mm, or greater than or equal to 10 mm. In some embodiments, a first channel has a width of less than or equal to 10.5 mm, less than or equal to 10 mm, less than or equal to 9 mm, less than or equal to 8 mm, less than or equal to 7 mm, less than or equal to 6 mm, less than or equal to 5.5 mm, less than or equal to 5 mm, less than or equal to 4.5 mm, less than or equal to 4 mm, less than or equal to 3.5 mm, less than or equal to 3 mm, less than or equal to 2.5 mm, less than or equal to 2 mm, less than or equal to 1.5 mm, less than or equal to 1 mm, or less than or equal to 0.75 mm. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 0.5 mm and less than or equal to 10.5 mm, or greater than or equal to 0.5 mm and less than or equal to 5.5 mm). Other ranges are also possible.

[0103] In some embodiments, a fluidic module comprises a second channel having a width that is greater than the width of the first channel also therein. Without wishing to be bound by any particular theory, it is believed that this may facilitate assembly of the fluidic module.

[0104] Channels may have a variety of suitable thicknesses (e.g., average thicknesses, maximum thicknesses, minimum thicknesses). The thickness may be equivalent to the dimension perpendicular to the membrane (e.g., the average thickness may be the average value of the dimension perpendicular to the membrane, etc.). In some embodiments, a first channel has a thickness of about 11.5 mm and / or a second channel has a thickness of about 2 mm.

[0105] Channels may be formed in a variety of suitable manners. In some embodiments, channels are formed by machining (e.g., via CNC).

[0106] The layers described herein may have a variety of suitable compositions. In some embodiments, a layer comprises a plastic, such as PMMA. Layers may be joined together in a variety of suitable manners, such as by threading (e.g., via 1 / 4-28 UNF thread interfacing).

[0107] Layers, and / or other fluidic module components, may be assembled in a variety of suitable manners, such as via pressure lamination.

[0108] As noted above, in some embodiments, a method may comprise employing a fluidic module described herein to synthesize particles, such as microparticles and / or nanoparticles (e.g., lipid nanoparticles). In some embodiments, the particles may have relatively small z-average diameters (e.g., less than 200 nm) and / or poly dispersity indices (e.g., less than 0.2, less than 0.1).

[0109] EXAMPLE 1

[0110] This Example describes the synthesis of lipid nanoparticles in a fluidic module described herein.

[0111] A first liquid comprising a lipid (an ALC lipid formulation) and a second liquid comprising mRNA were flowed through first and second channels, respectively, of fluidic modules having a design described herein. The first liquid flowed through the first channel, through a membrane positioned between the first and second channels, and into the second channel. A mixture of the first and second liquids flowed out of the second channel through a second-channel outlet.

[0112] The fluidic modules had the design shown in FIG. 4. One pair of first and second channels in each such fluidic module were employed. This pair included a first channel having a width of 500 microns and a depth of 500 microns, a second channel having a width of 1000 microns and a depth of 500 microns, and a track-etched membrane. Four different track-etched membranes were employed, each having a different pore size (of 0.2 microns, 1 micron, 5 microns, or 10 microns). The ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel was 3. FIG. 14 shows a photograph of the experimental setup. The liquid flowing out of the second channel of the fluidic module included lipid nanoparticles formed upon contact between the first and second liquids in the second channel. These lipid nanoparticles had desirably low z-average diameters (of less than 200 nm) and poly dispersity indices (of less than 0.2). The z-average diameters and poly dispersity indices of the nanoparticles generated under various total flow rates of liquid in the first and second channels and for various pore sizes of the membrane (identified by the different-colored bars) are shown in FIG. 15. As can be seen in FIG. 15, employing a total flow rate of liquid in the first and second channels of greater than or equal to 10 mL / min and less than or equal to 15 mL / min generated the nanoparticles having the smallest diameters and employing a total flow rate of liquid in the first and second channels of greater than or equal to 5 mL / min and less than or equal to 20 mL / min generated the nanoparticles having the lowest polydispersity indices.

[0113] FIGs. 16-19 show this same data, and shows further data comparing the nanoparticle z- average diameter and poly dispersity index before and after dialysis for these nanoparticles. The box showing a micron value in each such Figure indicates the pore size of the track-etched membrane in the fluidic module, and the z-average and poly dispersity index axes refer the z- average diameter and poly dispersity index, respectfully, of the nanoparticles formed therein. From these Figures, it can be seen that dialysis caused an increase in the z-average diameter of the nanoparticles but a decrease in the poly dispersity indices.

[0114] RiboGreen assays were conducted on these lipid nanoparticles. All lipid nanoparticles generated exhibited encapsulation efficiencies of 97% or higher. 17 / 20 experiments performed exhibited RNA recovery above 80%. The total liquid flow rates in the first and second channels, measured encapsulation efficiencies via RiboGreen assay, and concentration of RNA are shown below in Table 1.

[0115] Table 1.

[0116]

[0117] EXAMPLE 2

[0118] This Example describes the effect of the ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel on the properties of nanoparticles formed in a fluidic module.

[0119] A first liquid comprising a lipid (SM-102) and a second liquid an aqueous buffer were flowed through first and second channels, respectively, of a fluidic module having a design described herein. The first liquid flowed through the first channel, through a membrane positioned between the first and second channels, and into the second channel. A mixture of the first and second liquids flowed out of the second channel through a second-channel outlet.

[0120] The same fluidic module employed in Example 1 was also employed here. A track- etched membrane having a pore size of 1 micron was positioned between the first and second channels. The total flow rate of liquid in both the first and second channels combined was 1.2 mL / min.

[0121] FIG. 20 shows the z-average nanoparticle diameter and poly dispersity index for nanoparticles formed by this process. As can be seen from FIG. 20, improved performance was observed when the ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel was greater than or equal to 2 and less than or equal to 3.

[0122] EXAMPLE 3 This Example describes the effect of the total flow rate of liquid in both the first and second channels combined on the properties of nanoparticles formed in a fluidic module having a design described herein.

[0123] A first liquid comprising a lipid and a second liquid an aqueous buffer were flowed through first and second channels, respectively, of a fluidic module having a design described herein. The first liquid flowed through the first channel, through a membrane positioned between the first and second channels, and into the second channel. A mixture of the first and second liquids flowed out of the second channel through a second-channel outlet.

[0124] The same fluidic module employed in Example 1 was also employed here. A track- etched membrane having a pore size of 1 micron was positioned between the first and second channels. The ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel was 3.

[0125] In a first experiment, a liquid comprising a first type of lipid (SM-102) was flowed through the first channel of the fluidic module and an aqueous buffer was flowed through the second channel. FIG. 21 shows the z-av erage nanoparticle diameter and poly dispersity index for nanoparticles formed by this process. As can be seen from FIG. 21, improved performance was observed as the total flow rate of liquid in both the first and second channels increased.

[0126] In a second experiment, a liquid comprising a second type of lipid (an ALC lipid formulation) was flowed through the first channel of the module and an aqueous buffer was flowed through the second channel. FIG. 22 shows the z-av erage nanoparticle diameter and poly dispersity index for nanoparticles formed by this process. As can be seen from FIG. 22, small-diameter nanoparticles having low poly dispersity indices were formed across the various total flow rates of liquid in the first and second channels.

[0127] EXAMPLE 4

[0128] This Example describes the use of exemplary fluidic modules to generate nanoparticles.

[0129] A first liquid comprising a lipid and a second liquid an aqueous buffer were flowed through first and second channels, respectively, of fluidic modules having designs described herein. The first liquid flowed through the first channel, through a membrane positioned between the first and second channels, and into the second channel. A mixture of the first and second liquids flowed out of the second channel through a second-channel outlet. One set of fluidic modules had a design in which the dimension perpendicular to the membrane is constant (“a regular design”). The other set of fluidic modules had a hydrodynamic design. The widths of these channels are shown below in Table 2. A track-etched membrane was positioned therebetween. The ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel was 3. FIG. 23 shows two photographs of the experimental setup.

[0130] Table 2.

[0131] The first liquid included ALC-0315, DSPC, cholesterol, and ALC-0159 in the following molar ratio: 46.3:9.4:42.7:1.6. The second liquid was a 10 mM citrate buffer.

[0132] FIGs. 24 and 25 show the z-average diameters and poly dispersity indices for nanoparticles generated in the fluidic modules having the regular and hydrodynamic designs, respectively. FIG. 26 further shows the poly dispersity index after collecting the synthesized nanoparticles from each fluidic module. From these Figures, it can be seen that both types of fluidic modules are capable of generating nanoparticles with desirably low z-average diameters and poly dispersity indices. It can also be seen that the fluidic modules having the hydrodynamic design generated nanoparticles having comparable z-average diameters to and lower poly dispersity indices than the nanoparticles generated by the fluidic modules having the regular design.

[0133] EXAMPLE 5

[0134] This Example describes the use of exemplary fluidic modules to generate nanoparticles. A first liquid comprising a lipid and a second liquid an aqueous buffer were flowed through first and second channels, respectively, of fluidic modules having hydrodynamic designs described herein. The first liquid flowed through the first channel, through a membrane positioned between the first and second channels, and into the second channel. A mixture of the first and second liquids flowed out of the second channel through a second-channel outlet.

[0135] The channels in the fluidic modules had widths of 1.5 mm. A track-etched membrane was positioned therebetween. The ratio of the flow rate of liquid in the second channel to the flow rate of liquid in the first channel was 3. The total flow rate of liquid through both the first and the second channels varied between 5 and 120 mL / min. The experimental set-up is shown in the right-hand portion of FIG. 23.

[0136] The first liquid included ALC-0315, DSPC, cholesterol, and ALC-0159 in the following molar ratio: 46.3:9.4:42.7: 1.6. The second liquid was a 10 mM citrate buffer.

[0137] FIG. 27 shows the z-average diameter and poly dispersity indices of the nanoparticles generated at the various total flow rates of liquid through both the first and the second channels. As can be seen in FIG. 27, nanoparticles having low z-average diameters and poly dispersity indices were generated. Additionally, total flow rates of liquid through both the first and the second channels of greater than or equal 40 mL / min resulted in lower z-average diameters than lower such flow rates. The poly dispersity indices of the nanoparticles generated decreased with increasing total flow rate of liquid through both the first and the second channels when this total flow rate was less than or equal to 20 mL / min, remained relatively constant when this total flow rate was increased to 30 mL / min, and then remained below 0.2 at total flow rates of up to 100 mL / min for this fluidic module.

[0138] EXAMPLE 6

[0139] This Example describes an exemplary fluidic module.

[0140] The exemplary fluidic module is shown in FIG. 28. The volume of each channel is shown below in Table 3.

[0141] Table 3.

[0142] EXAMPLE 7

[0143] This Example describes the calculation of permeate velocity and shear rate in two locations for exemplary fluidic modules.

[0144] Permeate velocity, shear rate near the bottom of the second channel, and shear rate in the second channel near the membrane were calculated for two types of fluidic modules using COMSOL. One type of fluidic module had a “Regular Design,” in which both the first and second channels exhibited a constant cross-section along the length of the channel. The other type of fluidic module had a “Hydrodynamic Design,” and accordingly had a hydrodynamic design as described elsewhere herein.

[0145] Table 4 shows the dimensions perpendicular to the membrane and widths for first and second channels for fluidic modules having a Regular Design.

[0146] Table 4.

[0147] FIG. 29 shows the calculated permeate velocity and shear rates at various total flow rates of liquid in the first and second channels and in two locations for modules having Design No. 1 and including membranes having various pore sizes (identified in the boxes in the top row). As can be seen from FIG. 29, such designs exhibit shear rates that vary with location along the direction of flow. Additionally, the permeate velocities vary with location along the direction of flow for the modules including the membranes having larger pore sizes.

[0148] FIG. 30 shows the calculated permeate velocity and shear rates at various total flow rates of liquid in the first and second channels and in two locations for the designs shown in Table 4 and including membranes having a pore size of 5 microns. As can be seen in FIG. 30, such designs exhibit shear rates and permeate velocities that vary with location along the direction of flow.

[0149] FIG. 31 shows various calculated profiles for the dimension of a first channel perpendicular to a membrane in a fluidic module having a Hydrodynamic Design that further comprises a second channel having the profile also shown therein. These various profiles correspond to different values of the dimension of the first channel perpendicular to the membrane at a location directly adjacent to the first-channel inlet, dimension of the second channel perpendicular to the membrane at a location directly adjacent to the second-channel inlet, and the widths of the first and second channels. These parameters are shown below in Table 5. As can be seen in FIG. 31, as the dimension for the first channel perpendicular to the membrane at a location directly adjacent to the first-channel inlet increases, and as the width of the first channel increases, the profile for the dimension of the first channel perpendicular to the membrane converges.

[0150] Table 5.

[0151] Additionally, fluidic modules having a Hydrodynamic Design were compared to similar fluidic modules having a Regular Design. For both types of fluidic modules, the first channel had a width of 5 mm, the second channel had a width of 5.5 mm, and the membrane was a track- etched membrane having a pore size of 10 microns. FIG. 32 shows the calculated permeate velocity and shear rates in two locations for both designs at various flow rates of liquid in the first and second channels. As can be seen from FIG. 32, the fluidic module having a hydrodynamic design exhibited calculated permeate velocities and shear rates that were relatively constant along the flow while the fluidic module having the regular design exhibited calculated permeate velocities and shear rates that varied appreciably along the flow.

[0152] EXAMPLE 8

[0153] This Example describes an exemplary fluidic module that is configured for use with a pipette.

[0154] FIG. 33 shows a machine drawing of the fluidic module, FIG. 34 shows an exploded view of the fluidic module, and FIG. 35 is a photograph showing the fluidic module mechanically coupled to a dual -channel syringe.

[0155] EXAMPLE 9

[0156] This Example describes another exemplary fluidic module that is configured for use with a pipette.

[0157] FIGs. 36-38 show cross-sectional schematic drawings of fluidic modules including inlets having different volumes. The ratio of the volume of the inlet on the left to the inlet on the right increases from FIG. 36 to FIG. 37 to FIG. 38.

[0158] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0159] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0160] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0161] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0162] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0163] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0164] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0165] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi -closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMSWhat is claimed is:

1. A fluidic module, comprising: a first channel comprising a first-channel inlet; a second channel comprising a second-channel inlet and a second-channel outlet; and a membrane positioned between the first and second channels, wherein: the first and second channels are in fluidic communication with each other through the membrane, the fluidic module is configured such that liquid flowing in the second channel directly adjacent to the membrane experiences a substantially constant shear rate over an area of the membrane, a dimension of the first channel perpendicular to the membrane decreases from a first portion of the channel proximal to the first-channel inlet to a second portion of the channel distal from the first-channel inlet, and a dimension of the second channel perpendicular to the membrane increases from a first portion of the channel distal to the second-channel outlet to a second portion of the channel proximal to the second-channel outlet.

2. A fluidic module, comprising: a first channel comprising a first-channel inlet; a second channel comprising a second-channel inlet and a second-channel outlet; and a membrane positioned between the first and second channels, wherein: the first and second channels are in fluidic communication with each other through the membrane, the membrane comprises laterally isolated pores, the membrane has a pore size of greater than or equal to 0.01 micron and less than or equal to 30 microns.

3. A method, compri sing : in the fluidic module of claim 1 : flowing a first liquid through the first channel, through the membrane, and into a second liquid flowing in the second channel.

4. A method, comprising: in the fluidic module of claim 2: flowing a first liquid through the first channel, through the membrane, and into a second liquid flowing in the second channel.

5. A fluidic module or method as in any preceding claim, wherein the first liquid comprises a lipid.

6. A fluidic module or method as in any preceding claim, wherein the second liquid comprises mRNA.

7. A fluidic module or method as in any preceding claim, wherein the first liquid comprises mRNA.

8. A fluidic module or method as in any preceding claim, wherein the second liquid comprises a lipid.

9. A fluidic module or method as in any preceding claim, wherein the liquid comprising the lipid is an organic liquid.

10. A fluidic module or method as in any preceding claim, wherein the liquid comprising mRNA is an aqueous liquid.

11. A fluidic module or method as in any preceding claim, wherein the liquid comprising mRNA comprises a buffer.

12. A fluidic module or method as in any preceding claim, further comprising forming nanoparticles comprising the lipid and mRNA.

13. A fluidic module or method as in any preceding claim, further comprising mixing the first and second liquids in the second channel.

14. A fluidic module or method as in any preceding claim, further comprising forming lipid nanoparticles in the second channel.

15. A fluidic module or method as in any preceding claim, wherein the lipid nanoparticles comprise the mRNA.

16. A fluidic module or method as in any preceding claim, wherein the membrane has a pore size of greater than or equal to 0.01 micron and less than or equal to 30 microns.

17. A fluidic module or method as in any preceding claim, wherein the fluidic module is configured such that liquid flowing from the first-channel inlet to the second-channel outlet experiences a substantially constant pressure drop over an area of the membrane.

18. A fluidic module or method as in any preceding claim, wherein the membrane comprises laterally isolated pores.

19. A fluidic module or method as in any preceding claim, the pores in the membrane consist of laterally isolated pores and / or consist essentially of laterally isolated pores.

20. A fluidic module or method as in any preceding claim, wherein the membrane is a track- etched membrane.

21. A fluidic module or method as in any preceding claim, _wherein the first channel is disposed in a first layer, the second channel is disposed in a second layer, and the membrane is positioned between the first and second layers.

22. A fluidic module or method as in any preceding claim, wherein a first adhesive is positioned between the first layer and the membrane.

23. A fluidic module or method as in any preceding claim, wherein a second adhesive is positioned between the second layer and the membrane.

24. A fluidic module or method as in any preceding claim, wherein the first adhesive is a pressure-sensitive adhesive and / or the second adhesive is a pressure-sensitive adhesive.

25. A fluidic module or method as in any preceding claim, wherein the first channel is a dead-end channel.

26. A fluidic module or method as in any preceding claim, wherein the first channel comprises a third portion positioned between the first portion and the second portion.

27. A fluidic module or method as in any preceding claim, wherein the dimension of the first channel perpendicular to the membrane decreases at a first rate between the first portion thereof and the third portion thereof and decreases at a second, different, rate between the third portion thereof and the second portion thereof.

28. A fluidic module or method as in any preceding claim, wherein the second rate is greater than the first rate.

29. A fluidic module or method as in any preceding claim, wherein the first rate varies by less than or equal to 2%.

30. A fluidic module or method as in any preceding claim, wherein the second rate varies by less than or equal to 6%.

31. A fluidic module or method as in any preceding claim, wherein the first portion is directly adjacent to the first-channel inlet.

32. A fluidic module or method as in any preceding claim, wherein a distance from the second portion is a terminus of the first channel.

33. A fluidic module or method as in any preceding claim, wherein the dimension of the second channel perpendicular to the membrane increases at a rate that varies by less than or equal to 1.2%.

34. A fluidic module or method as in any preceding claim, wherein the first channel has a width of greater than or equal to 0.5 mm and less than or equal to 10 mm.

35. A fluidic module or method as in any preceding claim, wherein the second channel has a width of greater than or equal to 0.5 mm and less than or equal to 10.5 mm.

36. A fluidic module or method as in any preceding claim, wherein the fluidic module is configured such that liquid flowing in the second channel directly adjacent to a surface of the second channel opposite the membrane experiences a substantially constant shear rate over the surface of the second channel opposite the membrane.

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