Patent Application
20230398075
The present disclosure relates generally to particle synthesis, and more particularly, to nanoparticle synthesis via vortex flow focusing.
The use of liposomes and other lipid-based nanoparticles as nanoscale drug carriers for the controlled delivery of therapeutic agents has been harnessed for applications in cancer, infectious diseases, immune modulation, vaccine delivery, and beyond. Liposomal nanoparticles support the encapsulation of the full range of hydrophilic, amphipathic, and lipophilic drug compounds within unilamellar lipid vesicles, which protect the loaded drugs from degradation by the mononuclear phagocytic system (MPS) or endogenous enzymes. Liposome properties may also be modified during or after vesicle formation to engineer desirable biodistribution profiles via targeted cell delivery, thereby addressing issues of poor bioavailability, low plasma solubility, non-specific targeting, and high clearance rate often associated with free drug agents.
The efficacy and toxicity of liposome-based drug delivery systems can be influenced by liposome size. Smaller vesicles can exhibit more uniform pharmacodynamic characteristics and offer improved bioavailability through the enhanced permeability and retention (EPR) effect, which can allow smaller liposomal nanoparticles to exhibit increased accumulation within tumors due to higher vascular permeability within these tissues. In addition, liposomes below approximately 100 nm can pass the blood-brain barrier, while vesicles in the 30-40 nm range can enhance transdermal transport. Nanoparticle size can also affect blood circulation time, biodistribution, cell uptake, subcellular localization, and targeting efficiency. Size-dependent liposomal drug toxicity has also been reported, with higher toxicity resulting from larger liposomes due to their increased retention in healthy tissues. Thus, tuning nanoparticle size to a desired range while maintaining low polydispersity can help optimize, or at least improve, nanomedicine performance.
Continuous-flow microfluidic techniques have been explored to provide control over the microenvironment during lipid self-assembly. For example, in microfluidic flow focusing, lipids dissolved in a water-miscible polar solvent are injected into a microfluidic junction with aqueous buffer sheathing the lipid solution and hydrodynamically focusing the lipid solution into a narrow sheet. Diffusive transport of solvent and water in the laminar flow environment rapidly reduce lipid solubility during focusing to promote vesicle self-assembly. Due to the small lateral length scales of the focused lipid stream, smaller liposomes with decreased polydispersity can be achieved using this technique. Another microfluidic liposome synthesis method employs rapid mixing to achieve a rapid change in solubility and small diffusive length scales through increased interfacial area in a binary fluid system. Rapid mixing is achieved using periodic microstructures, such as a herringbone pattern, baffles, or toroidal or twisted microfluidic channels to generate localized chaotic advection at high flow velocity. Although microfluidic mixers can be simpler to operate than flow focusing devices, the resulting liposome populations tend to exhibit higher size variance and a more limited size range.
While the continuous-flow nature of these microfluidic techniques eliminates the need for multiple handling steps associated with batch methods, the small microchannel dimensions and laminar flow requirements constrain the throughput of the technology. Various modified flow focusing and micromixer designs have been developed to address this limitation, but with reduced size controllability and higher observed polydispersity due to the larger geometries required to support the increased buffer and lipid flow rates. Scaling these microfluidic platforms for high production throughput without sacrificing size control has thus proven to be challenging. Embodiments of the disclosed subject matter may address one or more of the above-noted problems and disadvantages, among other things.
Embodiments of the disclosed subject matter provide systems and methods for forming nanoparticles using vortex flow focusing. In some embodiments, a tangentially-directed flow in a microfluidic flow cell generates a primary vortex surrounding an axially-directed flow with constituent molecules for forming desired nanoparticles. The primary vortex subjects the axially-directed flow to simultaneous flow focusing and mixing, thereby allowing the formation of nanoparticles. In some embodiments, the constituent molecules can comprise inorganic molecules or organic molecules. In some embodiments, the sizes of the formed nanoparticles in the flow cell can be controlled by adjusting the respective flow rates of the tangentially-directed and axially-directed flows.
In one or more embodiments, a method for forming a plurality of nanoparticles can comprise (a) directing a first inlet flow along an axial direction in a hydrocyclonic flow cell. The first inlet flow can comprise first constituent molecules. The method can further comprise (b) at a same time as (a), directing one or more second inlet flows along a circumferential direction of the hydrocyclonic flow cell. Each second inlet flow can comprise a buffer solution. The directing of (a) and (b) can be such that the first inlet flow is subjected to flow focusing by a surrounding primary vortex formed by the one or more second inlet flows, so as to generate a flow comprising the plurality of nanoparticles at an outlet of the hydrocyclonic flow cell. Each nanoparticle can be formed by a respective plurality of the first constituent molecules.
In one or more embodiments, a nanoparticle synthesis system can comprise a hydrocyclonic flow cell, which can comprise a cylindrical section, an inlet nozzle, and a conical section. The cylindrical section can comprise a circumferentially-extending wall with one or more tangentially-oriented inlet ports. The inlet nozzle can be surrounded by the circumferentially-extending wall of the cylindrical section and can provide an axially-oriented inlet port. The conical section can extend from an axial end of the circumferentially-extending wall of the cylindrical section toward an axially-oriented outlet port. The conical section can be tapered along at least a portion of its length along an axial direction. The axially-oriented inlet port and the axially-oriented outlet port can be substantially co-axial. A radially-outer surface of the inlet nozzle can be tapered along at least a portion of its length along the axial direction.
Any of the various innovations of this disclosure can be used in combination or separately. This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. The foregoing and other objects, features, and advantages of the disclosed technology will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
Embodiments will hereinafter be described with reference to the accompanying drawings, which have not necessarily been drawn to scale. Where applicable, some elements may be simplified or otherwise not illustrated in order to assist in the illustration and description of underlying features. Throughout the figures, like reference numerals denote like elements.
For purposes of this description, certain aspects, advantages, and novel features of the embodiments of this disclosure are described herein. The disclosed methods and systems should not be construed as being limiting in any way. Instead, the present disclosure is directed toward all novel and nonobvious features and aspects of the various disclosed embodiments, alone and in various combinations and sub-combinations with one another. The methods and systems are not limited to any specific aspect or feature or combination thereof, nor do the disclosed embodiments require that any one or more specific advantages be present, or problems be solved. The technologies from any embodiment or example can be combined with the technologies described in any one or more of the other embodiments or examples. In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are exemplary only and should not be taken as limiting the scope of the disclosed technology.
Although the operations of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed methods can be used in conjunction with other methods. Additionally, the description sometimes uses terms like “provide” or “achieve” to describe the disclosed methods. These terms are high-level abstractions of the actual operations that are performed. The actual operations that correspond to these terms may vary depending on the particular implementation and are readily discernible by one skilled in the art.
The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person skilled in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods, as known to those skilled in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited. Whenever “substantially,” “approximately,” “about,” or similar language is explicitly used in combination with a specific value, variations up to and including 10% of that value are intended, unless explicitly stated otherwise.
Directions and other relative references may be used to facilitate discussion of the drawings and principles herein but are not intended to be limiting. For example, certain terms may be used such as “inner,” “outer,” “upper,” “lower,” “top,” “bottom,” “interior,” “exterior,” “left,” right,” “front,” “back,” “rear,” and the like. Such terms are used, where applicable, to provide some clarity of description when dealing with relative relationships, particularly with respect to the illustrated embodiments. Such terms are not, however, intended to imply absolute relationships, positions, and/or orientations. For example, with respect to an object, an “upper” part can become a “lower” part simply by turning the object over. Nevertheless, it is still the same part, and the object remains the same.
As used herein, “comprising” means “including,” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements unless the context clearly indicates otherwise.
Although there are alternatives for various components, parameters, operating conditions, etc. set forth herein, that does not mean that those alternatives are necessarily equivalent and/or perform equally well. Nor does it mean that the alternatives are listed in a preferred order, unless stated otherwise. Unless stated otherwise, any of the groups defined below can be substituted or unsubstituted.
Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one skilled in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Features of the presently disclosed subject matter will be apparent from the following detailed description and the appended claims.
The following are provided to facilitate the description of various aspects of the disclosed subject matter and to guide those skilled in the art in the practice of the disclosed subject matter.
Nanoparticle: A particle formed by one or more first constituent molecules and having a maximum cross-sectional diameter (e.g., diameter) less than about 1 μm, for example, in a range of 1-200 nm, inclusive. The particle can be formed of organic or inorganic molecules and can be a vesicle or solid particle. For example, in some embodiments, the nanoparticle comprises a shell formed of lipids (i.e., a liposome). Alternatively, in some embodiments, the nanoparticle is a solid lipid particle. In some embodiments, the nanoparticle can include and/or encapsulate one or more second constituent molecules (e.g., a drug).
Drug: Any substance designed for altering one or more bodily functions of a subject when ingested, inhaled, infused, or otherwise introduced into a subject (e.g., human or animal), for example, to treat a medical condition. In some embodiments, the drug can be an aqueous-soluble substance (e.g., molecule). Alternatively or additionally, in some embodiments, the drug can be a lipid-soluble substance (e.g. molecule).
Microfluidic: Refers to flow path features in a device (e.g., inlet port, outlet port, and/or flow channel in a flow cell) and/or fluid flows within a device that have a cross-sectional dimension along a direction substantially perpendicular to or crossing a direction of the respective fluid flow less than 1 mm, for example, less than or equal to 500 μm.
Hydrocyclonic flow cell: A microfluidic flow cell having one or more axially-oriented inlet ports for providing an axial flow(s), one or more tangentially-oriented inlet ports for forming a primary vortex surrounding the axial flow(s), and one or more axially-oriented outlet ports. In some embodiments, the hydrocyclonic flow cell has a cylindrical portion or section at an inlet end, and a conical portion or section that extends from the cylindrical portion to the outlet port(s). In some embodiments, the hydrocyclonic flow cell has a tapered nozzle in the cylindrical portion that defines at least one of the axially-oriented inlet ports.
Disclosed herein are systems and methods for forming nanoparticles using microfluidic vortex focusing (MVF). In some embodiments, one or more inlet flows 110 can be directed along a respective tangential direction 116 (e.g., tangent to a circumferential direction of the mixing volume) in an internal mixing volume 102 of a hydrocyclonic flow cell 100, as shown in
The primary vortex 112 can subject the one or more axially-directed flows 108 to simultaneous flow focusing (e.g., in both directions in a plane perpendicular to the axial direction) and mixing within the mixing volume 102, thereby facilitating formation of nanoparticles (e.g., via self-assembly) with precise size control as the inlet flows 108, 110 proceed along the axial direction toward axially-oriented outlet port 118. In some embodiments, the sizes of the formed nanoparticles can be tuned by adjusting the respective flow rates of the tangentially-directed and axially-directed flows. At the outlet port 118, the solution flows containing the formed nanoparticles can be collected for further processing and/or use. In some embodiments, the outlet port 118 and at least one of the one or more inlet ports 106 can be aligned, for example, substantially co-axial and/or centered on longitudinal axis 114. Alternatively or additionally, the outlet port 118 can be offset from at least one of the one or more inlet ports 106, for example, along a radial direction of the mixing volume 102.
The one or more axially-directed flows 108 can have constituent molecules for forming desired nanoparticles. In some embodiments, the constituent molecules can comprise inorganic molecules or organic molecules. For example, in some embodiments, lipids can be provided in the axially-directed flow 108 (e.g., solvated lipids) to form lipid nanoparticles and/or liposomes. In some embodiments, a tangential flow 110 of buffer solution (e.g., aqueous buffer) can create the primary vortex 112 around the axial stream 108 of solvated lipids in an axisymmetric mixing volume 102. Liposome self-assembly can occur within the mixing volume 102 through the simultaneous hydrodynamic focusing and rapid convective and diffusive mixing provided by the primary vortex 112.
Vesicle formation within the mixing volume 102 is a kinetic process controlled by the local solvent polarity. As lipid solubility decreases due to a combination of solvent convection, lipid advection, and solvent/lipid co-diffusion, the amphiphilic lipid molecules can spontaneously form planar disc-like micelles. These intermediate structures can grow in a reaction rate limited process until the line energy associated with the exposed hydrophobic lipid tails overcomes the elastic energy required to form spherical vesicles, at which point membrane closure becomes energetically favorable. Because the micelle growth rate and the elastic energy at closure can depend on lipid solubility, a sharp temporal solubility gradient can enable the formation of smaller liposomes by limiting the intermediate lipid structure growth time. In the hydrocyclonic flow cell 100, stretching and folding of the fluid interface under the influence of the primary vortex 112 contributes to rapid mixing of the miscible aqueous 110 and lipid 108 streams. Meanwhile, the sheathing by the primary vortex 112 helps to spatially constrain the mixing zone, thereby significantly reducing the diffusive length scale between the aqueous 110 and lipid 108 streams.
In some embodiments, the hydrocyclonic flow cell 100 can achieve reliable liposome synthesis and narrow size distributions, for example, with vesicle diameters ranging from 61 nm to 127 nm for a lipid composition without polyethylene glycol (PEG) or as small as 27 nm when introducing a PEG-conjugated lipid in the mixture. In some embodiments, the hydrocyclonic flow cell 100 can be operated at Reynolds numbers approaching the laminar limit, for example, to yield high throughput vesicle production. For example, in some embodiments, highly monodisperse populations (e.g., having a polydispersity index (PDI)≤0.2) of liposomes with sizes less than 100 nm (e.g., as small as 27 nm in diameter) can be produced at rates of over 20 g/h. Such precise control over vesicle size during nanoscale liposome synthesis can be helpful in defining the pharmaceutical properties of liposomal nanomedicines.
Although the description above and elsewhere herein explicitly addresses synthesis of liposomes, embodiments of the disclosed subject matter are not limited thereto. Rather, the systems and methods disclosed herein can be readily adapted to synthesis of other nanoparticles (e.g., solid lipid nanoparticles, inorganic nanoparticles, etc.), according to one or more contemplated embodiments. Indeed, the techniques disclosed herein can be employed in any particle synthesis system where rapid diffusive and convective transport within a continuous-flow setup may be advantageous.
In some embodiments, a nanoparticle synthesis system can include at least a hydrocyclonic flow cell 100 configured for MVF operation. In addition, the system can include additional modules or components to support and/or control continuous flow operation of the hydrocyclonic flow cell 100, such as, but not limited to, fluid reservoirs (e.g., supply volumes, collection volumes), fluid control components (e.g., pumps, valves, switches, conduits, etc.), detectors (e.g., particle size detectors, flow rate detectors, etc.), controllers, and/or processing systems (e.g., particle concentration systems).
For example,
In the illustrated example of
In the illustrated example of
In the discussion of
Alternatively, in some embodiments, the second constituent molecules can be provided with a separate reservoir and/or inlet flow volume into the flow cell 100. For example,
In the example of
In the discussion of
Alternatively, in some embodiments, the second constituent molecules can be provided with a separate reservoir and/or inlet flow volume into the flow cell 100. For example,
In some embodiments, for example, in any of the illustrated examples of
With reference to
A computing system may have additional features. For example, the computing environment 230 includes storage 260, one or more input devices 270, one or more output devices 280, and one or more communication connections 290. An interconnection mechanism (not shown) such as a bus, controller, or network interconnects the components of the computing environment 230. Typically, operating system software (not shown) provides an operating environment for other software executing in the computing environment 230, and coordinates activities of the components of the computing environment 230.
The tangible storage 260 may be removable or non-removable, and includes magnetic disks, magnetic tapes or cassettes, CD-ROMs, DVDs, or any other medium which can be used to store information in a non-transitory way, and which can be accessed within the computing environment 230. The storage 260 can store instructions for the software 232 implementing one or more innovations described herein.
The input device(s) 270 may be a touch input device such as a keyboard, mouse, pen, or trackball, a voice input device, a scanning device, or another device that provides input to the computing environment 230. The output device(s) 280 may be a display, printer, speaker, CD-writer, or another device that provides output from computing environment 230.
The communication connection(s) 290 enable communication over a communication medium to another computing entity. The communication medium conveys information such as computer-executable instructions, audio or video input or output, or other data in a modulated data signal. A modulated data signal is a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media can use an electrical, optical, radio-frequency (RF), or another carrier.
Any of the disclosed methods can be implemented as computer-executable instructions stored on one or more computer-readable storage media (e.g., one or more optical media discs, volatile memory components (such as DRAM or SRAM), or non-volatile memory components (such as flash memory or hard drives)) and executed on a computer (e.g., any commercially available computer, including smart phones or other mobile devices that include computing hardware). The term computer-readable storage media does not include communication connections, such as signals and carrier waves. Any of the computer-executable instructions for implementing the disclosed techniques as well as any data created and used during implementation of the disclosed embodiments can be stored on one or more computer-readable storage media. The computer-executable instructions can be part of, for example, a dedicated software application or a software application that is accessed or downloaded via a web browser or other software application (such as a remote computing application). Such software can be executed, for example, on a single local computer (e.g., any suitable commercially available computer) or in a network environment (e.g., via the Internet, a wide-area network, a local-area network, a client-server network (such as a cloud computing network), or any other such network) using one or more network computers.
For clarity, only certain selected aspects of the software-based implementations are described. Other details that are well known in the art are omitted. For example, it should be understood that the disclosed technology is not limited to any specific computer language or program. For instance, aspects of the disclosed technology can be implemented by software written in C++, Java™, Python®, and/or any other suitable computer language. Likewise, the disclosed technology is not limited to any particular computer or type of hardware. Certain details of suitable computers and hardware are well known and need not be set forth in detail in this disclosure.
It should also be well understood that any functionality described herein can be performed, at least in part, by one or more hardware logic components, instead of software. For example, and without limitation, illustrative types of hardware logic components that can be used include Field-programmable Gate Arrays (FPGAs), Program-specific Integrated Circuits (ASICs), Program-specific Standard Products (ASSPs), System-on-a-chip systems (SOCs), Complex Programmable Logic Devices (CPLDs), etc.
Furthermore, any of the software-based embodiments (comprising, for example, computer-executable instructions for causing a computer to perform any of the disclosed methods) can be uploaded, downloaded, or remotely accessed through a suitable communication means. Such suitable communication means include, for example, the Internet, the World Wide Web, an intranet, software applications, cable (including fiber optic cable), magnetic communications, electromagnetic communications (including RF, microwave, and infrared communications), electronic communications, or other such communication means. In any of the above-described examples and embodiments, provision of a request (e.g., data request), indication (e.g., data signal), instruction (e.g., control signal), or any other communication between systems, components, devices, etc. can be by generation and transmission of an appropriate electrical signal by wired or wireless connections.
Referring to
The hydrocyclonic flow cell 300 can further include an inlet nozzle 308 that defines an axially-oriented inlet port 310, for example, for introducing a flow of first constituent molecules (e.g., lipids in solution) along the longitudinal axis 330 of the flow cell 300 toward axially-oriented outlet port 318. In the illustrated example, the inlet port 310 and the outlet port 318 are coaxial and aligned with the longitudinal axis 330 of the flow cell 300. In some embodiments, the cylindrical portion 302 can have a circumferential wall 306 extending substantially parallel to the longitudinal axis 330 from a base wall 322 and surrounding the inlet nozzle 308 in plan view, thereby defining an annular inlet region to which a tangentially-oriented inlet port 316 opens. Fluid (e.g., provided via conduit 324) can exit inlet port 316 along a tangent 336 and flow through the annular inlet region of the cylindrical portion 302 along circumferential direction 334, thereby providing a primary vortex surrounding a flow (e.g., provided via conduit 326) exiting from inlet port 310.
In some embodiments, the nozzle 308 can have a tapered radially-outer surface 312 (e.g., along the axial direction), for example, to reduce a distance between the annular vortex generation zone and the axial flow from inlet port 310 for enhanced mixing. In the illustrated example of
In some embodiments, the cylindrical portion 302 can have a length, L1, along the axial direction (e.g., parallel to longitudinal axis 330), and the nozzle 308 can extend across the entire length, L1, or terminate before the conical portion 304. In some embodiments, L1 of the cylindrical portion 302 can be less than a length, L2, of the conical portion 304 along the axial direction. For example, L1 (e.g., about 1 mm) may be less than or equal to 20% of L2 (e.g., about 7.4 mm). In some embodiments, the annular inlet region formed between the wall 306 and the junction between the nozzle 308 and base wall 322 can have a width, W2, along the radial direction 332. In some embodiments, the width, W2, can be the same or different than d3, for example, less than or equal to about 0.5 mm (e.g., about 300 μm or less).
In some embodiments, the hydrocyclonic flow cell 300 can be integrated in a nanoparticle synthesis system 350, such as illustrated in
In some embodiments, system 350 with hydrocyclonic flow cell 300 can be used to synthesize liposomes. As shown in
The fluid mixing can occur when multiple fluid elements are brought into contact, allowing scalar concentrations within each volume to diffuse across the interface. In the case of a 2D steady vortex flow, local shear results in fluid elements located on different streamlines to separate, thereby stretching the fluid interface and folding adjacent elements around one another. For 3D vortex focusing generated by the hydrocyclonic flow cell 300 of
In the illustrated examples of
In the illustrated examples of
Each secondary inlet port 416a, 416b can be formed via respective internal axially-extending walls 414a, 414b, for example, extending substantially parallel to the longitudinal axis 330. Alternatively, in some embodiments, walls 414a, 414b can be tapered, for example in a manner similar to that of nozzle 408. In the illustrated example of
Alternatively, in some embodiments, the provision of secondary inlet ports can be extended to cover an entire circumference around the inlet nozzle, for example, by providing an annular port. For example,
The secondary inlet port 514 can be formed via internal axially-extending wall 504, for example, extending substantially parallel to the longitudinal axis 330. Alternatively, in some embodiments, wall 504 can be tapered, for example, in a manner similar to that of nozzle 508. In the illustrated example of
To study the combined mixing and focusing offered by the disclosed MVF technique, a numerical model was developed to investigate the distribution of solvent during both hydrodynamic flow focusing (HFF) and MVF, with results summarized in
Light scattering was employed to evaluate particle anisotropy as the solvent concentration was reduced. A sharp decrease in dissymmetry and increase in depolarization was observed at a solvent mole fraction of 0.5, consistent with the formation of disk-like structures around this value of ethanol concentration. Based on this observation, a mole fraction of 0.5 was adopted as an appropriate threshold below which vesicle formation begins to occur in the experiments. As shown in
Nanoparticle synthesis systems with miniature hydrocyclonic flow cell devices were fabricated and used for nanoscale liposome synthesis via the disclosed MVF technique. Devices were fabricated using a high-resolution stereolithography digital light processing (SLA-DLP) process. Each device had a total chamber diameter (e.g., d4 in
The resulting stereolithography (STL) file was converted into a mask layer stack for SLA-DLP printing with 25 μm z-step on a Perfactory 4 DLP-SLA instrument (sold by EnvisionTEC Inc., Dearborn, MI) using the EnvisionTEC Magics software. The stereolithography tool was equipped with a 75 mm objective lens for high-resolution printing, corresponding to a 74 mm×46 mm printable area at 1920×1200 pixel resolution. The STL file was oriented to align the axial center of the hydrocyclonic flow cell perpendicular to the print stage. After printing, the system was developed, processed (e.g., cleaned to remove residue) and cured (e.g., using an Otoflash UV curing unit sold by EnvisionTEC Inc., Dearborn, MI).
Print orientation was found to play a role in realizing the tapered annular structure that serves as a nozzle for the lipid inflow. The axial direction of the hydrocyclonic flow cell was thus aligned perpendicular to the print stage, with the upper ceiling of flow cell facing the print stage, allowing the thicker base of the tapered annular structure to be formed before patterning the tapering geometry. In addition, since mixing occurs at the junction of the annular vortex generation zone and the annular lipid inlet, nozzle asymmetry may affect the flow and mixing profiles during focusing. While orienting the devices perpendicular to the stage during printing helped to reduce asymmetry, nozzle deformation was further minimized by defining a fixed thickness for the nozzle tip, rather than allowing the geometry to taper to a point. A tip thickness (e.g., W1 in
During process optimization, the lipid injection channel diameter and wall thickness were minimized with the goal of reducing the radial mixing length scale during liposome formation. While channel dimensions as small as 150 μm were investigated, a diameter of 300 μm was selected for the final flow cells, since smaller ports were routinely found to be closed prior to the final development step. Designing the nozzle to have a length less than 1 mm also helped to avoid warping or clogging. However, smaller channels and/or longer nozzles should be possible via different fabrication systems and/or techniques. Finally, the light intensity during stereolithography was carefully controlled to improve device geometry and performance, in particular, by limiting exposure intensity to 97% of the nominal instrument level. Under the optimized processing conditions, device yield was approximately 50%, with full or partial clogging of the lipid channel being the primary failure mode. Surface roughness within the hydrocyclonic flow cell was measured by optical profilometry after cutting open the chamber using a low speed saw, with average roughness values of Ra=1.32 μm and Ra=0.45 μm observed in the axial and radial directions, respectively.
Characteristics of the resin used for device fabrication can also impact device performance. To support liposome synthesis, the material should be compatible with the solvent used as a lipid carrier and offer sufficient rigidity and mechanical strength to avoid deforming or breaking during high pressure operation. To ensure compatibility with ethanol as the lipid solvent, an acrylic-based photosensitive resin (HTM140, sold by EnvisionTEC of Dearborn, Michigan)) was selected. Secondary UV exposure to fully polymerize the resin after development can be used to prevent solvent-induced mechanical failure, such as cracking due to the presence of unreacted monomer, oligomers, or low molecular weight polymers within the solidified resin.
Particle diameter and size distribution can be important for controlling therapeutic effect and safety for nanocarrier systems. To emphasize the importance of controlling vesicle size and polydispersity for liposomal nanomedicines, consider a liposome population with known mean diameter and polydispersity index (PDI), defined as the particle size variance normalized by the square of the mean diameter. The volume of hydrophilic drug encapsulated within the liposome core for a given vesicle size range can be determined by integrating the product of the distribution probability density function and size-dependent particle volume. Given a log-normal particle size distribution with location and scale parameters derived from mean diameter and PDI values, a population with a mean diameter of 100 nm and PDI of 0.2 was found to have approximately 80% of the total drug encapsulated by the liposomes retained within particles larger than 100 nm, i.e., within a size range where delivery to the targeted tissues may not be optimal and accumulation in healthy organs can occur. In contrast, reducing the size to 80 nm and PDI to 0.05 significantly reduces the drug associated with these larger particles to below 28%. A similar analysis applies for the case of hydrophobic drug intercalated within the liposome membrane, where drug amount scales approximately with membrane area. In this case, 67% of the drug is found to be retained in vesicles above 100 nm for the larger and more polydisperse vesicle population, compared with only 21% for the smaller and more uniform particles.
The size of liposomes generated by MVF can be directly controlled by adjusting the relative flowrates of solvated lipid and aqueous buffer injected into the system. The impact of the buffer:lipid flow rate ratio (FRR) on liposome size and size distribution is shown in
The inverse relationship between FRR and vesicle size was similar to liposome formation using HFF or rapid micromixing; however, the MVF technique was found to yield lower polydispersity, with an average PDI value of 0.04 and nearly constant size variance over the full range of flow rate ratios (
Q=d−1PDI−1.
For the MVF platform, the Q values were generally in the range of 0.1-0.4. In contrast, conventional techniques such as ethanol injection or chaotic advection micromixing yielded Q values below 0.15.
A potential limitation of the MVF technique is the use of high FRR values to reduce vesicle size. High FRR may necessitate subsequent concentration of the liposome solutions for clinical use and/or may impact drug encapsulation efficiency. In practice, liposome synthesis techniques may employ downstream processing for buffer exchange, filtration, and concentration adjustment. To concentrate dilute suspensions of larger liposomes, ultracentrifugation can be employed. Smaller nanoliposomes may be concentrated by centrifugal filtration using a filter element with an appropriate cutoff size, which may also serve to remove solvent and free drug from the final liposome suspension.
When operating the MVF system at constant FRR, the overall liposome production throughput may be enhanced by increasing the total flow rate (TFR) of the combined lipid and buffer flows through the system. The impact of TFR on liposome size distribution is shown in
The impact of lipid concentration on liposome formation is also shown in
Long-term storage of liposomes is also a consideration for nanomedicine applications. Structural instability driven by thermodynamic perturbations can lead to degradation and structural reorganization of the vesicles. To evaluate the colloidal stability of vesicles generated by the MVF technique, three selected liposome populations formed under different total flow rates and flow rate ratios were stored at 4° C. for 99 days, with size distributions measured before and after storage. During this time period no detectable change in mean particle size or size variance was observed for any of the samples
Liposome surface modifications can also impact pharmacokinetic properties. For example, the attachment of polyethylene glycol (PEG) to the outer liposome surface allows the nanoparticles to avoid recognition by the MPS, thereby enabling longer blood circulation times, improved bioavailability, and higher levels of accumulation in tumor tissues. Because the presence of large PEG molecules imposes steric effects during liposome formation, it was desirable to understand the relationship between PEG content and vesicle size. The inclusion of PEG-conjugated lipids during nanoparticle formation can stabilize the resulting particles. While an inverse relationship between particle size and both PEG concentration and PEG chain length has been reported for the case of solid lipid nanoparticle nucleation, no significant change in liposome size was observed when adding increasing concentrations of PEG-lipids during liposome synthesis by HFF.
To investigate this issue for the MVF technique, a high concentration (10 mol %) of PE conjugated to PEG-2000 was introduced to the lipid feed solution before operating the hydrocyclonic flow cell at FRR=50 and 10 mM lipid concentration while varying the total flow rate. The resulting measurements of vesicle size are shown in
One of the advantages of the disclosed MVF technique is the ability to generate size-controlled liposomes by taking advantage of simultaneous flow focusing and vortical mixing, while operating at bulk flow rates that can be significantly higher than conventional microfluidic techniques, such as HFF or chaotic advection micromixer platforms. Maximum flow rates can be dictated by the need for laminar flow conditions within the flow cell to maintain efficient focusing. Based on the main chamber radius of the hydrocyclonic flow cell, the Reynolds number was found to approach the laminar limit when operating at a total flow rate of 60 mL/min, and begins to enter the transitional regime at the highest tested flow rate of 80 mL/min. Higher flow rates were thus avoided to improve throughput without inducing turbulent flow, which could otherwise destabilize the focusing zone and lead to higher polydispersity and an overall reduction in size control.
However, increasing the lipid feed concentration represents an alternative path to higher mass production rates independent of lipid and buffer flow conditions. To explore this alternative, liposomes were synthesized for each lipid mixture with concentrations approaching the lipid solubility limit while operating at the maximum flow rate of 80 mL/min. For the DCP-based lipid mixture, solubility in dehydrated ethanol was maintained for lipid concentrations up to 30 mM, while the PEGylated lipid mixture remained soluble up to 60 mM. The results of the these tests are shown in
The MVF technique thus combines the advantages of HFF and chaotic advection mixing to enable size-tunable liposome generation while operating at high levels of lipid flux. Moreover, unlike conventional microfluidic techniques, the MVF technique offers production rates compatible with manufacturing scale liposomal drug production lines, e.g., in a range of 1-60 g/h, while also providing greater control over both mean size and size variance for the resulting vesicles, particularly while operating at higher lipid mass transport rates. Nanoparticle systems employing the MVF technique can be operated as continuous-flow setup with minimal labor and infrastructure, while avoiding the sequential processing steps associated with conventional batch methods.
Any of the features illustrated or described herein, for example, with respect to
The present application claims the benefit of U.S. Provisional Application No. 63/366,240, filed Jun. 10, 2022, entitled “Microfluidic Vortex Focusing for High-Throughput Synthesis of Size-Tunable Liposomes,” and U.S. Provisional Application No. 63/377,178, filed Sep. 26, 2022, entitled “Microfluidic Vortex Focusing for High-Throughput Synthesis of Size-Tunable Liposomes,” each of which is hereby incorporated by reference herein in its entirety.
This invention was made with government support under R21HL159590 awarded by the National Institutes of Health (NIH), and under CMMI1562468 and CMMI1950234 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63377178 | Sep 2022 | US | |
| 63366240 | Jun 2022 | US |
