Microfluidic device for producing homogeneous nanoparticles

Abstract

The present invention relates to an apparatus for producing nanoparticles comprising a hydrophobic substance and a hydrophilic substance. Specifically, the apparatus according to the present invention is characterized in that it comprises: a plurality of inlet channels into which a hydrophobic substance and a hydrophilic substance are introduced, respectively; a mixing channel in which substances are mixed to produce nanoparticles; and an outlet channel through which the produced nanoparticles are discharged, in which the mixing channel includes a microcolumn capable of increasing the mixing efficiency of the substance. Accordingly, nanoparticles produced using the apparatus according to the present invention exhibit excellent particle uniformity and can be effectively used as a drug or a drug delivery carrier.

Description

Technical Field

[0001] This invention relates to a novel apparatus for producing nanoparticles composed solely of hydrophilic substances, as well as nanoparticles containing both hydrophobic and hydrophilic substances, and a method for producing uniform nanoparticles using the apparatus. The nanoparticles obtained according to this invention exhibit excellent particle uniformity and can be effectively used as drugs or drug delivery carriers. Background Technology

[0002] Numerous nanomedicines are being developed for targeted delivery of therapeutic and imaging agents, thereby treating and diagnosing major diseases including cancer, cardiovascular disease, diabetes, and Alzheimer's disease. Efficient drug delivery systems can improve the absorption of poorly soluble and unstable drugs, while increasing therapeutic efficacy and reducing toxicity. Furthermore, this has led to the discovery and development of more effective drugs to improve patient outcomes and quality of life.

[0003] With the development of nanomedicine technologies for drug delivery, numerous studies have been reported over the past decade, but only a very small number of therapeutic and diagnostic nanomedicines have received FDA approval. The low success rate of nanomedicines can be attributed to the low reproducibility of nanoparticles with the desired properties or efficacy, as well as batch-to-batch variations in physicochemical properties. The reproducibility and reliability of drug release profiles are crucial factors for successful drug delivery systems, and these largely depend on the size and uniformity of the nanoparticles. Therefore, there is a growing need to develop technologies that can stably and reproducibly ensure particle size and uniformity during the nanoparticle manufacturing process. In particular, since most drugs delivered in nanomedicines are hydrophobic, it is more difficult to uniformly synthesize hydrophilic substances (e.g., phospholipids) used as drug carriers, and thus their development has not yet been achieved. Accordingly, the inventors of this invention have developed a novel apparatus for producing nanoparticles composed solely of hydrophilic substances, as well as nanoparticles containing both hydrophobic and hydrophilic substances. Uniform nanoparticles have been successfully produced using this novel apparatus, thus completing this invention.

[0004] [Priority Document]

[0005] [Non-patent documents]

[0006] (Non-patent literature 1) Velencia, P. et al. Single-step assembly of homogeneous lipid-polymer and lipid-quantum dot nanoparticles enabled by microfluidic rapid mixing. ACS Nano, 4, 3, 1671-1679, (2010).

[0007] (Non-Patent Literature 2) Rhee, M. et al. Drop Mixing in a Microchannel for Lab-on-a-Chip Platforms. Langmuir, 24(2), 590-601, (2008).

[0008] (Non-patent literature 3) Rohit, K. et al. Microfluidic Platform for Controlled Synthesis of Polymeric Nanoparticles. Nano Letters, 8, 9, 2906-12, (2008).

[0009] (Non-Patent Literature 4) Johnson, BK et al. Mechanism for Rapid Self-Assembly of Block Copolymer Nanoparticles. Physical Review Letters, 91(11), 118302-1-4 (2003).

[0010] (Non-Patent Literature 5) Bekard, I. et al. The Effects of Shear Flow on Protein Structure and Function. Biopolymers, 95, 11, 733-745 (2011). Summary of the Invention

[0011] [The problem the invention aims to solve]

[0012] Traditional nanoparticle production methods primarily consist of non-standardized, multi-step processes (e.g., nanoprecipitation and emulsion-based solvent evaporation). Nanoprecipitation, accounting for over 50% of nanoparticle production methods, involves dropwise addition of a lipophilic or polymeric drug dissolved in another hydrophobic solvent to a hydrophilic solvent being stirred in the hydrophobic solvent, thereby forming nanoparticles from a colloidal suspension between the two solvent phases. The properties of the synthesized particles in this process can be controlled by the stirring rates of the two solvents, the degree of lipophilicity of the polymeric drug, and the polymeric drug drop rate. In this case, the particle size is determined by the non-linear and unpredictable flow at the stirring inlet, leading to polydispersity and significant batch-to-batch variations, making reproducible synthesis and production of nanoparticles difficult. Therefore, several additional steps are required to homogenize the synthesized nanoparticles.

[0013] Recently, microfluidics has been applied to the development of drug delivery systems to control intense microvortex flow, thereby effectively forming drug carriers. Microfluidics is widely used in chemical synthesis, chemical and biomolecular analysis, tissue engineering, and other applications because it allows for precise control of the microenvironment (e.g., the size, shape, and surface composition of nanoparticles) by manipulating small amounts of liquid. In particular, microfluidics can optimize nanomedicine delivery systems by reproducibly and continuously producing high-quality nanoparticles with various combinations of physicochemical properties, and can also facilitate clinical applications (e.g., improving or monitoring the effects of drug delivery, release, and elimination during patient treatment).

[0014] However, despite these advantages, most existing microfluidic nanomedicine synthesis methods still have limitations, such as low productivity due to relatively low flow rates during synthesis and difficulty in mixing different liquid substances. Furthermore, recent efforts have focused on overcoming these limitations by increasing flow rates in microfluidics, but these have been limited to forming uncontrolled microvortices through simple straight and curved structures. This limits the mixing of hydrophilic substances and makes it difficult to achieve efficient mixing of hydrophobic and hydrophilic substances. Therefore, the low productivity of synthesized nanoparticles, or the production of nanoparticles in the range of hundreds of nanometers to several micrometers, significantly limits their application as nanomedicines, as they must be produced uniformly to obtain sizes of 200 nanometers or smaller for effective drug delivery carriers.

[0015] [A device for solving a problem]

[0016] Therefore, the inventors of this invention have discovered a novel device that can control the size of the produced nanoparticles by adjusting the flow rate of the fluid, and can obtain high yields of nanoparticles of the same properties, thus completing this invention.

[0017] The present invention provides a novel apparatus for producing nanoparticles, comprising: a plurality of inlet channels through which hydrophobic and hydrophilic substances are respectively introduced; a mixing channel in which the substances are mixed to produce nanoparticles; and an outlet channel through which the produced nanoparticles are discharged, wherein the mixing channel includes microposts capable of enhancing the mixing efficiency of the introduced substances.

[0018] The term "mixing efficiency" used in this article eff ")" is a percentage value based on the fact that hydrophobic and hydrophilic substances, each with a mass fraction of 1, mix with each other in the channel and eventually converge to a mass fraction of 0.5, as calculated in the following formula.

[0019]

[0020] At this time, Mass fract This refers to the mass fraction. The closer the mass fraction is to 0.5, the higher the mixing efficiency proportionally.

[0021] As used herein, the term "device" or "microfluidic device" refers to a device that includes channels configured to allow fluid to flow over a substrate made of a variety of materials, including plastics, glass, metals, or silicon.

[0022] In the device of the present invention, hydrophobic and hydrophilic substances can be introduced separately through different inlet channels. Preferably, the hydrophobic substance can be introduced in the same direction as the flow direction of the fluid in the mixing channel, and the hydrophilic substance can be introduced in a direction different from the flow direction of the fluid in the mixing channel.

[0023] There may be one or more inlet channels for introducing hydrophobic substances, and there may be one or more inlet channels for introducing hydrophilic substances. Preferably, the device of the present invention may include one inlet channel for introducing hydrophobic substances and two inlet channels for introducing hydrophilic substances.

[0024] As used herein, the term "micro-column" refers to a structure that disrupts the straight flow of fluid within a mixing channel. The micro-column of this invention is a structure for efficiently mixing fluids introduced into a device by forming micro-vortices; it can be of any shape, preferably columnar. The micro-column can be designed to separate or bend fluid flows impacting it, or to merge them with different flows. The micro-column can be designed to maintain the main flow path and prevent stagnation, and can, for example, intersect the fluid at a right angle relative to the direction of the fluid flow.

[0025] The micropillars of the present invention have an edge shape capable of altering the fluid flow, and can be polyhedra, truncated polyhedra, polygonal pillars, or variations thereof, preferably square pillars. Furthermore, when viewed in a cross-section cut along the flow direction of the fluid within the mixing channel, it can be a polygon or a variation thereof, and preferably, it is rectangular, having one side perpendicular to the flow direction of the fluid (horizontal) and one side in the same direction as the flow direction of the fluid within the mixing channel (vertical).

[0026] In this invention, "the flow direction of the fluid in the mixing channel" or "the flow direction in the mixing channel" refers to the direction in which the fluid flows from the inlet channel to the outlet channel within the device. Preferably, a hydrophobic substance is introduced through an inlet channel arranged in the same direction as the flow direction in the mixing channel, and a hydrophilic substance is introduced through an inlet channel arranged in a direction perpendicular to the flow direction in the mixing channel.

[0027] The micropillars of the present invention can be arranged in one or more rows along the flow direction of the fluid in the mixing channel. Specifically, the micropillars can be arranged in one, two, three, four, five, or six or more rows along the direction of fluid flow, preferably in six rows.

[0028] The micropillars of the present invention can be arranged in one or more units, which are located in a single row in a direction different from (preferably orthogonal to) the flow direction of the fluid in the mixing channel. The number of micropillars present in each row can be the same, or can vary between rows.

[0029] In this invention, micropillars can be arranged in an alternating manner relative to micropillars in adjacent rows. As used herein, the term "arranged in an alternating manner" means that the micropillars in multiple rows are not aligned in a straight line in the direction of fluid flow, or are not arranged as a single parallel row in the direction of fluid flow. Instead, when viewed in the direction of fluid flow, micropillars arranged in a row can be positioned to partially or completely block the gaps between micropillars arranged in adjacent rows.

[0030] In this invention, the micropillars may have a height of 200 to 800 μm, and the micropillars may or may not be connected to the wall forming the mixing channel.

[0031] In this invention, "inlet volume velocity ratio" refers to the ratio of the inlet volume velocity of a fluid containing hydrophilic substances to the inlet volume velocity of a fluid containing hydrophobic substances.

[0032] In this invention, the term "flow blockage ratio" refers to the ratio of the width of the micropillars in the first row along the flow direction of the fluid in the mixing channel to the width of the entire channel.

[0033] Typically, the flow velocity within a device can be expressed using the Reynolds number (Re). The Reynolds number is a dimensionless number representing the ratio of inertial force to viscous force, and its calculation formula is as follows:

[0034]

[0035] Here, D h It is the hydraulic diameter within the microfluidic device, where ρ, μ, v, and Q represent the fluid density, dynamic viscosity, velocity, and flow rate, respectively. Therefore, the flow rate increases proportionally with the increase of the Reynolds number.

[0036] The Reynolds number in the apparatus of the present invention can be 500 or less, preferably 10 to 300, more preferably 12.5 to 200, and most preferably 50 to 100. When the Reynolds number exceeds 500, a turbulent-like flow is formed within the apparatus, making it impossible to generate controllable eddy current patterns, and the increased shear force between the solvent and the substance due to the high flow rate may lead to instability of the raw material drug substance and the generated nanoparticles. When the Reynolds number is 300 or more, uncontrollable chaotic flow begins to occur within the apparatus, making quality control for the production of uniform nanoparticles difficult. On the other hand, when the Reynolds number is as low as 10 or less, the process becomes diffusion-dependent, making it difficult to achieve effective mixing of hydrophobic and hydrophilic substances, and thus difficult to obtain a productivity superior to conventional nanoparticle synthesis methods.

[0037] The device of the present invention can adjust the particle size by changing the Reynolds number.

[0038] Furthermore, the present invention provides a method for producing nanoparticles comprising hydrophobic and hydrophilic substances using the device.

[0039] Specifically, the present invention provides a method for producing uniform nanoparticles comprising hydrophobic and hydrophilic substances using an apparatus comprising a plurality of inlet channels, a mixing channel and an outlet channel, the method comprising: (a) introducing the hydrophobic and hydrophilic substances into each of the inlet channels, (b) causing the substances to collide with micropillars in the mixing channel to form eddies, thereby generating nanoparticles, and (c) discharging the produced nanoparticles through the outlet channel.

[0040] The hydrophobic material of the present invention may be one or more selected from the group consisting of: polylactic acid-glycolic acid copolymer (PLGA), polyethylene glycol (PEG), distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauryl phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearate-sn-phosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPP) C) 1-Palmyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), 1,2-dicarachidoyl-sn-glycerol-3-phosphocholine (DBPC), 1,2-docosahexaenoyl-sn-glycerol-3-phosphocholine (DEPC), palmitoyloleylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleylphosphatidylcholine, distearylphosphatidylethanolamine (DSPE), dimyristoylphosphatidyl Ethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), Palmitoyloleoylphosphatidylethanolamine (POPE), Lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamoyl]ethyl]-3,4-di[oleoyloxy]-benzamide (VL-5), bis(octadecanoyl)glycylsper ... -3-Trimethylammonium propane (DOTAP), (1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dimyristoxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[2-(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanetrimonium bromide (GAP-DLRIE), N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropanediidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyl dioctadecylammonium bromide (DDAB), N4-cholesterolyl-spermine (GL67), 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA, and may include, but is not limited to, both high molecular weight hydrophobic substances and low molecular weight hydrophobic substances.

[0041] The hydrophilic material of the present invention can be a protein or a polymer, preferably an apolipoprotein, and more preferably an apolipoprotein A or E.

[0042] Nanoparticles comprising phospholipids, apolipoproteins, and polymers can be produced using the equipment of this invention and the production method according to this invention.

[0043] [Beneficial Effects]

[0044] The apparatus of the present invention and the method for producing nanoparticles using the apparatus have the following advantages: continuous synthesis of nanoparticles with uniform size in a single step, and large-scale production of nanoparticles with consistent properties due to low batch-to-batch variability. The resulting nanoparticles exhibit excellent particle uniformity and, because they do not contain additional components (such as surfactants), can be advantageously used as pharmaceuticals or pharmaceutical delivery carriers. Attached Figure Description

[0045] Figure 1 The fluid flow through the device of the present invention and the fluid flow around the micropillar are shown.

[0046] Figure 2 This is a schematic diagram of the device of the present invention.

[0047] Figure 3 The results of measuring mixing efficiency using the number of micropillar rows in the device according to the invention are shown.

[0048] Figure 4 The results of measuring mixing efficiency based on the number of micropillars present in a row in the device of the present invention are shown.

[0049] Figure 5 The results of measuring mixing efficiency by varying the Reynolds number in the device according to the invention are shown.

[0050] Figure 6 The results of measuring different mixing efficiencies by varying the channel height in the device according to the invention are shown.

[0051] Figure 7 The results of measuring mixing efficiency by the injection ratio of hydrophobic and hydrophilic substances in the device according to the present invention are shown.

[0052] Figure 8A method for calculating the residence time of a fluid is shown.

[0053] Figure 9 Fluid flow within the apparatus of the preparation examples 1 and 2 according to the present invention is shown.

[0054] Figure 10 The particle size distribution of rHDL nanoparticles is shown when rHDL nanoparticles are produced according to Preparation Examples 1 and 2 of the present invention.

[0055] Figure 11 The particle size distribution of lipid-polymer nanoparticles produced by the production method of the present invention is shown. Detailed Implementation

[0056] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings, so that those skilled in the art can readily implement the present invention. However, the present invention can be implemented in various forms and is not limited to the embodiments and examples described herein.

[0057] Throughout the specification, when a section is referred to as "including" a component, it does not mean that it excludes other components, but rather that it may include other components unless otherwise specifically stated.

[0058] The present invention relates to an apparatus for producing nanoparticles, characterized in that it includes an inlet channel, a mixing channel, and an outlet channel, wherein the mixing channel includes microcolumns.

[0059] Furthermore, the present invention relates to a method for producing nanoparticles using an apparatus for producing nanoparticles, characterized in that the apparatus includes an inlet channel, a mixing channel, and an outlet channel, wherein the mixing channel includes micropillars, and the method includes: introducing a hydrophobic substance and a hydrophilic substance into the inlet channel; forming a vortex between the micropillars and the substance in the mixing channel; and forming nanoparticles comprising the hydrophobic substance and the hydrophilic substance, and nanoparticles obtained by the method.

[0060] The inlet channel, mixing channel, and outlet channel can be arranged sequentially in the direction of fluid flow.

[0061] The inlet channel may include a channel for introducing hydrophilic substances and a channel for introducing hydrophobic substances, and the hydrophilic and hydrophobic substances may each be introduced into a separate inlet channel.

[0062] Hydrophobic substances can be introduced in the same direction as the fluid flow, while hydrophilic substances can be introduced in a direction different from the fluid flow. Preferably, hydrophobic substances can be introduced in the same direction as the fluid flow, while hydrophilic substances can be introduced in a direction perpendicular to the fluid flow.

[0063] The inlet volumetric flow rate ratio of hydrophobic to hydrophilic substances can be from 1:2.5 to 1:8.5 (v / v).

[0064] Hydrophobic substances may be one or more selected from the group consisting of: polylactic acid-glycolic acid copolymer (PLGA), polyethylene glycol (PEG), distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauryl phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearate-sn-phosphatidylcholine (DSPC), and 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC). 1-Palmyl-2-myristoylphosphatidylcholine (PMPC), 1-palmyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmylphosphatidylcholine (SPPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), 1,2-dicarachidoyl-sn-glycerol-3-phosphocholine (DBPC), 1,2-docosahexaenoyl-sn-glycerol-3-phosphocholine (DEPC), palmitoyloleylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleylphosphatidylcholine, distearylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine Amine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), Palmitoyloleoylphosphatidylethanolamine (POPE), Lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamoyl]ethyl]-3,4-di[oleoyloxy]-benzamide (VL-5), Dioctadecanoylglycylsper ... 3-Trimethylammonium propane (DOTAP), (1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dimyristoxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[2-(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanetrimonium bromide (GAP-DLRIE), N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropanediidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyl dioctadecylammonium bromide (DDAB), N4-cholesterolyl-spermine (GL67), 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA.

[0065] The hydrophilic substance can be a lipoprotein, transmembrane protein, peptide, or polymer, preferably apolipoprotein A or E. Furthermore, the polymer can be polylactic-co-glycolic acid copolymer (PLGA).

[0066] In the mixing channel, hydrophilic and hydrophobic substances can mix with each other.

[0067] The outlet channel can be a channel through which the produced nanoparticles are discharged.

[0068] The micropillars may or may not be connected to the walls forming the mixing channels, and may be at least one of the groups consisting of polyhedra, truncated polyhedra, polygonal pillars and their variants, preferably square pillars.

[0069] The micropillars can be arranged in one or more rows in the direction of fluid flow, and preferably in six or more rows in the direction of fluid flow. Alternatively, one or more micropillars can be arranged in a row in a direction opposite to the direction of fluid flow; preferably, one to six micropillars can be arranged in a row in a direction opposite to the direction of fluid flow; more preferably, one to six micropillars can be arranged in a row in a direction perpendicular to the direction of fluid flow; and most preferably, one to two micropillars can be arranged in a row in a direction perpendicular to the direction of fluid flow.

[0070] Multiple micropillars may not be arranged in a single row in the direction of fluid flow, and multiple micropillars may be arranged to partially or completely cover the gaps between micropillars arranged in adjacent rows when viewed in the direction of fluid flow.

[0071] The particle size can be adjusted by changing the Reynolds number of the fluid flow in the mixing channel. Furthermore, the Reynolds number of the fluid flow in the mixing channel can be from 12.5 to 200, and preferably from 50 to 100.

[0072] The channel can have a height of 200-800μm.

[0073] The flow blockage ratio in the device can be from 0.2 to 0.8, preferably from 0.35 to 0.65.

[0074] The polydispersity index of the produced nanoparticles can be 0.15 or less.

[0075] The invention will be described in more detail by way of the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0076] [Example 1]

[0077] Design of equipment for producing nanoparticles

[0078] Microfluidic devices are designed to produce reconstructed high-density nanoparticles by mixing hydrophobic and hydrophilic organic compounds. Figure 1 and Figure 2 The microfluidic device comprises three inlet channels and one outlet channel. A hydrophobic phospholipid or hydrophobic drug is introduced into the central channel, while hydrophilic proteins or hydrophilic drugs are introduced into the two side channels. Furthermore, micropillar structures are introduced within the device to efficiently mix lipids and proteins.

[0079] [Example 2]

[0080] The influence of a single micropillar structure

[0081] To determine the mixing efficiency of hydrophobic and hydrophilic substances based on the presence, number, and arrangement of micropillars placed inside the device, fluid dynamics techniques were used to analyze the distribution of hydrophobic substances inside the microfluidic device.

[0082] In this invention, the inlet volume flow rate ratio refers to the ratio of the inlet volume flow rate of the fluid containing hydrophilic substances to the inlet volume flow rate of the fluid containing hydrophobic substances, and the flow blockage ratio is defined as the ratio of the width of the micro-pillars in the first row along the flow direction of the fluid in the mixing channel to the width of the entire channel.

[0083] The results were validated under the following conditions: channel height of 200 μm, inlet volumetric flow rate ratio of 1:5.5 (hydrophobic:hydrophilic), Reynolds number (Re) of 50, flow blockage ratio of 0.5, and rectangular micropillar size of 1000 μm × 400 μm. Figure 2 , Figure 3 and Figure 4 As shown, the number of micropillars is designed to be evenly distributed across the channel width, while maintaining the size of the micropillars and keeping the width of the flow path between the micropillars constant.

[0084] like Figure 2 As shown, it was confirmed that hydrophobic and hydrophilic substances can be effectively mixed by micro-vortices generated when hydrophobic substances flow through the micropillar structure.

[0085] like Figure 3 and Figure 4 As shown, it was confirmed that the mixing efficiency was improved by approximately 1.5 to 4.0 times, depending on the number of micropillars distributed, compared to the case without micropillars.

[0086] [Table 1]

[0087] Mixing efficiency based on the number of microcolumns

[0088] Number of microcolumns Mixing efficiency 0 0.26 2 0.78 4 0.95 6 0.98

[0089] As mentioned above Figure 3 As shown in Table 1, the mixing efficiency increases with the number of rows, achieving an excellent mixing efficiency of 98% or higher when there are six or more rows.

[0090] [Table 2]

[0091] Mixing efficiency based on the number of micropillars in a row

[0092]

[0093]

[0094] As above Figure 4 As shown in Table 2, the mixing efficiency decreases with increasing number of micropillars within a single channel, with the optimal mixing efficiency achieved when the number is between 1 and 2. Therefore, it is confirmed that the mixing efficiency varies with the number and arrangement of micropillars within the device; in particular, the highest mixing efficiency is achieved when the individual micropillar structures are arranged in a staggered manner. The micropillars are arranged uniformly relative to the channel width while maintaining a constant width of the flow path between the micropillars.

[0095] [Example 3]

[0096] Optimize the available flow rate range and flow rate ratio range within the equipment.

[0097] To optimize the synthesis of nanoparticles based on the flow rate and injection ratio of hydrophobic and hydrophilic substances within the device, the mixing efficiency under different Reynolds numbers (Re) and flow rate ratios was analyzed.

[0098] To investigate the available flow rate range, the mixing efficiency was analyzed when the Reynolds number (Re) varied from 12.5 to 200, with a channel height of 200 μm and an injection ratio of 1:5.5 in the device. The results are as follows: Figure 5 As shown in Table 3.

[0099] [Table 3]

[0100] Reynolds number (Re) Mixing efficiency 12.5 0.92 2.5 0.97 50 0.98 100 0.98 200 0.95

[0101] As shown in Table 3 above, when the Reynolds number (Re) is in the range of 12.5 to 200, a mixing efficiency of 90% or higher is obtained, and when the Reynolds number (Re) is in the range of 50 to 100, a mixing efficiency of 98% is obtained.

[0102] Furthermore, the mixing efficiency was analyzed when the channel height varied from 200 μm to 800 μm, with an injection ratio of 1:5.5 and a Reynolds number (Re) of 50. The results are shown in [Figure / Table / Illustration]. Figure 6 And in Table 4.

[0103] [Table 4]

[0104] Channel height (μm) Mixing efficiency 200 0.98 400 0.98 600 0.97 800 0.95

[0105] As shown in Table 4 above, when the channel height is in the range of 200 to 800 μm, a mixing efficiency of 95% or higher is obtained.

[0106] Furthermore, the mixing efficiency was analyzed when the injection ratio was varied from 1:2.5 to 1:8.5, with a channel height of 200 μm and a Reynolds number (Re) of 50, and the results are shown in [Figure / Table / Illustration]. Figure 7 And in Table 5.

[0107] [Table 5]

[0108] Injection ratio Mixing efficiency 1:2.5 0.97 1:5.5 0.98 1:8.5 0.98

[0109] As shown in Table 5 above, a mixing efficiency of 97% or higher is achieved when the injection ratio is in the range of 1:2.5 to 1:8.5 μm.

[0110] [Example 4]

[0111] Research on how equipment height increases mixing efficiency

[0112] To maximize mixing efficiency based on the height of the mixing channel of the device, the mixing efficiency under different height conditions was analyzed.

[0113] During nanoparticle synthesis using this device, when the feedstock is injected at a relatively high flow rate (Re>50), strong shear stress is generated from the wall due to the narrow channel width.

[0114] In the case of proteins, some studies have reported that protein deformation (including aggregation, misfolding, and degradation) can be induced by shear stress from fluid flows generated in external devices. This deformation of precursor proteins can alter the structure, size, and even function of the produced nanoparticles. Typically, the range of induced deformation varies depending on the structure of the device generating the shear stress, as well as the type and size of the materials used. However, it has been reported that in the case of proteins (such as insulin, enzymes, and immunoglobulins) with sizes of 1–10 nm (mass range of 20–300 kDa), protein structures can reach approximately 1000 dyne / cm². 2Deformation under shear stress levels (Bekard, I. et al., The Effects of Shear Flow on Protein Structure and Function. Biopolymers, 95(11), 733-745, (2011)).

[0115] Therefore, in order to confirm whether the precursor material and nanoparticles are subject to shear stress (τ) generated by the fluid flow in the device of the present invention... w The variant, firstly, calculates the shear stress generated based on the height of each channel as follows.

[0116]

[0117] Here, Q is the flow rate, and μ, h, and w represent the fluid viscosity, the height of the channel cross-section, and the width of the channel cross-section, respectively.

[0118] [Table 6]

[0119]

[0120]

[0121] As shown in Table 6 above, it has been confirmed that as the channel height in the device of the present invention decreases, the shear stress increases exponentially, but remains below the protein structure deformation threshold of 1000 dyne / cm. 2 .

[0122] However, when the channel height is 100 μm, the shear stress reaches 1000 dyne / cm when Re increases to 300. 2 Furthermore, considering that the size of the nanoparticles to be produced is in the range of 10-100 nm, which is larger than the size of proteins, the following conclusion can be drawn: when the channel height is 200 μm or greater, the precursor material and nanoparticles will not deform due to shear stress.

[0123] Furthermore, as shown in Table 7 below, as the height of the mixing channel of the device increases, the volume increases, which in turn increases the residence time (τ) of the mixing precursor in the channel at the same flow rate. res This also increases the mixing time (τ) required to synthesize the precursor. mix The specific calculation method for the residence time of the mixture in the channel is as follows: Figure 8 As shown. The mixing time (τ) required for synthesizing the substance can be calculated based on the more dominant physical phenomena (convection or diffusion) in the device. mixAnd it can be obtained using the Pecklet number (Pe), which is a dimensionless number representing the ratio of convection time to diffusion time.

[0124]

[0125] In the device of this invention, the Peklay number (Pe) is in the range of 100-5000, based on which it is confirmed that convective mixing is dominant. Therefore, the convective mixing time (τ) within the channel is... mix,convection The calculations are as follows (Velencia, P. et al. Single-Step Assembly of Homogenous Lipid-Polymeric and Lipid-Quantum Dot Nanoparticles Enabled by Microfluidic Rapid Mixing. ACS Nano, 4, 3, 1671-1679 (2010), and Rhee, M. et al. Drop Mixing in a Microchannel for Lab-on-a-Chip Platforms. Langmuir, 24(2), 590-601 (2008)).

[0126]

[0127] Here, D represents the diffusion coefficient, and L and w represent the channel length and the width of the channel cross-section, respectively.

[0128] [Table 7]

[0129]

[0130]

[0131] At this point, the dwell time (τ) res It must be longer than the mixing time (τ) mix This allows the material to mix thoroughly within the channels. As shown in Table 7 above, because the residence time is longer than the mixing time at all channel heights, it can be confirmed that sufficient mixing time is ensured at a given channel height.

[0132] Furthermore, as shown in Table 7 above, the increase in mixing time at the maximum height of 800 μm, calculated based on the increase in channel height, is 53 ms, which falls within the aggregation time (τ). aggThe timeframe is in the range of 50 to 100 ms, during which the injected material begins to agglomerate and aggregate. Therefore, it can be considered the maximum channel height suitable for the device and production method of this invention (see Rohit, K. et al. Microfluidic Platform for Controlled Synthesis of Polymeric Nanoparticles. Nano Letters, 8, 9, 2906-12, (2008) and Johnson, BK et al. Mechanism for Rapid Self-Assembly of Block Copolymer Nanoparticles. Physical Review Letters, 91(11), 118302-1-4 pp. (2003)).

[0133] Therefore, as shown in Table 7 above, it can be seen that the optimal height of the mixing channel that can prevent aggregation and the generation of non-uniform nanoparticles when obtaining nanoparticles in the device is 200 to 800 μm.

[0134] [Example 5]

[0135] Fluid flow inside the equipment

[0136] Based on the channel height and microcolumn conditions, the device of the present invention was designed in two preparation examples, as shown in Table 8 below.

[0137] [Table 8]

[0138]

[0139] To generate preparation examples 1 and 2 and to confirm the mixed flow pattern of hydrophobic and hydrophilic substances, the distribution of the substances was visualized with ink and observed under a microscope.

[0140] Fluids containing hydrophobic substances were visualized using 6% ink in ethanol, and fluids containing hydrophilic substances were visualized using physiological saline. The results are shown in [the table / image / etc.]. Figure 9 middle.

[0141] like Figure 9 As shown, it can be seen that the hydrophobic and hydrophilic substances are effectively mixed by the micro-vortices generated when the hydrophobic substance flows through the micro-pillar structure.

[0142] [Example 6]

[0143] Preparation of recombinant high-density lipoprotein (rHDL) nanoparticles

[0144] Using Preparation Examples 1 and 2 of Example 5, rHDL nanoparticles containing phospholipids (DMPC) and apolipoproteins were prepared by the following method.

[0145] A solution of DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine) dissolved in anhydrous ethanol and a solution of apolipoprotein dissolved in PBS were prepared. Then, approximately 0.8 mL of the 0.83 mg / mL DMPC solution dissolved in anhydrous ethanol was placed into one syringe, and approximately 1.25 mL (total approximately 2.5 mL) of the 0.2 mg / mL apolipoprotein solution dissolved in PBS was placed into two other syringes in equal volumes. All air bubbles in the syringes were then removed.

[0146] Each syringe needle was connected to the inlet channel of the device of the present invention using tubing, and PBS was introduced at a rate of 1 mL / min using a syringe pump to clean the device. Subsequently, the injection flow rate of the DMPC solution was set to 0.8 mL / min, and the injection flow rate of the apolipoprotein solution was set to 2.2 mL / min using the syringe pump. The produced rHDL nanoparticles were obtained through the outlet of the device, mixed with PBS, centrifuged using a 10K filter, and purified three times at 4°C for 20 minutes each time.

[0147] After dissolving the obtained rHDL nanoparticles in PBS, the change in particle size distribution due to nanoparticle aggregation was measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS, and the results are shown in [Figure / Table / Insert Table ... Figure 10 middle.

[0148] like Figure 10 As shown, when the rHDL nanoparticles obtained using the apparatus of Preparation Examples 1 and 2 were dissolved in PBS and DLS data were measured, the sizes of the rHDL nanoparticles obtained in Preparation Examples 1 and 2 were measured to be 18.78 ± 4.58 nm and 13.06 ± 0.00 nm, respectively.

[0149] Therefore, it can be seen that the nanoparticles prepared using the equipment of the present invention in Example 2 have better uniformity compared with those prepared using conventional equipment in Example 1.

[0150] [Example 7]

[0151] Method for producing lipid-polymer nanoparticles using this equipment

[0152] Using Preparation Example 2, lipid-polymer nanoparticles comprising the polymer polylactic acid-glycolic acid copolymer (PLGA), phospholipid 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), and PEGylated phospholipid 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were prepared by the following method.

[0153] PLGA was prepared into a solution in anhydrous acetonitrile (ACN), and 5 mL of the solution was filled into a syringe. DPPC was dissolved in anhydrous ethanol, and DSPE-PEG2000 was prepared into an aqueous solution using deionized water (DW).

[0154] The two phospholipids were mixed at a ratio of DPPC:DSPE-PEG2000 = 1:2 (w / w), and the final solution was adjusted to contain 4% ethanol. 5 mL of this solution was then added to a syringe. Nanoparticles were obtained under the two synthesis conditions shown in Table 9 below. The obtained nanoparticles were purified using DW (deionized water; ultrapure water free of ions).

[0155] [Table 9]

[0156]

[0157] The particle size distribution of nanoparticles obtained under the two synthesis conditions is shown in the figure. Figure 11 The nanoparticles obtained under synthesis conditions 1 and 2 had particle sizes of 124 ± 5.3 nm and 56 ± 4.4 nm as determined by dynamic light scattering (DLS), respectively, and particle size uniformities measured by polydispersity index (PDI) were 0.05 and 0.13, respectively. Generally, a lower PDI indicates better uniformity, as uniformity is considered to be ensured when the PDI is 0.15 or less. Therefore, the nanoparticles produced under the above synthesis conditions exhibit uniformity.

[0158] Therefore, it can be seen that, using the equipment of the present invention, nanoparticles of different sizes can be produced according to the injection flow rate and the weight ratio of hydrophilic to lipophilic substances, and nanoparticles with uniform particle size composed of various phospholipids and proteins can be produced.

[0159] [Example 8]

[0160] Optimization of equipment structural design

[0161] To optimize mixing efficiency based on the dimensions of the channels and micropillars designed within the device, mixing efficiency was confirmed under various conditions, including: 1) the initial spacing of the micropillars; 2) the spacing between the micropillars; 3) the longitudinal length of the micropillars; and 4) the flow blockage ratio within the device structure.

[0162] The mixing efficiency was determined under the conditions of a fixed channel height of 0.2 mm, a flow rate ratio of 1:5.5, and a Reynolds number of 50. The results are as follows: Figure 12 and Figure 13 As shown.

[0163] [Table 10]

[0164]

[0165] As above Figure 12 As shown in Table 10, it has been confirmed that the flow pattern of the fluid through the micropillars designed in the channel does not change significantly regardless of the spacing and longitudinal length of the micropillars, and maintains a high mixing efficiency (>0.95) under all conditions.

[0166] [Table 11]

[0167] Flow blocking ratio Mixing efficiency 0.2 0.72 0.35 0.92 0.5 0.98 0.65 0.98 0.8 0.98

[0168] On the other hand, as mentioned above Figure 13 As shown in Table 11, mixing efficiency increases with increasing flow blockage ratio (the horizontal spacing ratio of micropillars within the channel). It has been confirmed that when the blockage ratio is 0.5, the mixing efficiency remains consistently at 0.98 or higher.

[0169] However, as the flow blockage ratio increases, the channel width also narrows, and correspondingly, the shear stress increases proportionally. As calculated above, when high shear stress exceeds a certain value, it may lead to the aggregation and deformation of organic matter present in the fluid (Bekard, I. et al. The Effects of Shear Flow on Protein Structure and Function. Biopolymers, 95(11), 733-745(2011)). Considering that when the flow rate increases to Re 300, the shear stress rises to 1000 dyne / cm2 or more when the blockage ratio exceeds 0.65, it can be seen that the flow blockage ratio of the channel can be 0.2 to 0.8, and preferably, when it is in the range of 0.35 to 0.65, high mixing efficiency can be maintained without being significantly affected by shear stress.

Claims

1. An apparatus for producing nanoparticles, comprising an inlet channel, a mixing channel, and an outlet channel. The mixing channel described therein comprises micropillars.

2. The device according to claim 1, wherein the inlet channel, the mixing channel, and the outlet channel are arranged sequentially in the direction of fluid flow.

3. The device according to claim 1, wherein the inlet channel includes a channel for introducing a hydrophilic substance and a channel for introducing a hydrophobic substance.

4. The device according to claim 3, wherein each of the hydrophilic substance and the hydrophobic substance is introduced into a different inlet channel.

5. The device according to claim 4, wherein the hydrophobic substance is introduced in the same direction as the flow direction of the fluid, and the hydrophilic substance is introduced in a direction different from the flow direction of the fluid.

6. The device according to claim 5, wherein the hydrophobic substance is introduced in the same direction as the flow direction of the fluid, and the hydrophilic substance is introduced in a direction perpendicular to the flow direction of the fluid.

7. The apparatus of claim 1, wherein the outlet channel is a channel for discharging the produced nanoparticles.

8. The device according to claim 3, wherein the hydrophilic substance and the hydrophobic substance are mixed with each other in the mixing channel.

9. The device of claim 1, wherein the micropillar is connected to or disconnected from the wall forming the mixing channel.

10. The device of claim 1, wherein the micropillar is selected from at least one of the group consisting of a polyhedron, a truncated polyhedron, a polygonal prism, and modified shapes thereof.

11. The device of claim 10, wherein the micropillar is a square pillar.

12. The device of claim 1, wherein the micropillars are arranged in one or more rows in the direction of fluid flow.

13. The device of claim 12, wherein the micropillars are arranged in six or more rows in the direction of fluid flow.

14. The device according to claim 12, wherein, One or more micropillars in a row are arranged in a direction different from the flow direction of the fluid.

15. The device according to claim 14, wherein, One to six micropillars in a row are arranged in a direction different from the flow direction of the fluid.

16. The device according to claim 15, wherein, One to six micropillars in a row are arranged in a direction perpendicular to the flow direction of the fluid.

17. The device according to claim 16, wherein, One or two micropillars in a row are arranged in a direction perpendicular to the flow direction of the fluid.

18. The device according to claim 12, wherein, Multiple micropillars in a row are not arranged in a straight line in the direction of fluid flow.

19. The device of claim 12, wherein the plurality of micropillars are arranged such that, when viewed along the flow direction of the fluid, they partially or completely cover the gaps between the micropillars arranged in adjacent rows.

20. The apparatus of claim 1, wherein the Reynolds number for the fluid flow in the mixing channel is from 12.5 to 200.

21. The apparatus of claim 20, wherein the Reynolds number for the fluid flow in the mixing channel is 50 to 100.

22. The apparatus of claim 1, wherein the particle size is controlled by changing the Reynolds number of the fluid flow in the mixing channel.

23. The device of claim 1, wherein the channel has a height of 200 to 800 μm.

24. The device according to claim 3, wherein the inlet volume flow rate ratio between the hydrophobic substance and the hydrophilic substance is 1:2.5 to 1:8.5 (v / v).

25. The device of claim 1, wherein the flow blockage ratio is 0.2 to 0.

8.

26. The device of claim 25, wherein the flow blockage ratio is from 0.35 to 0.

65.

27. The apparatus of claim 1, wherein the polydispersity index (PDI) of the produced nanoparticles is 0.15 or less.

28. The apparatus of claim 3, wherein the hydrophobic substance may be one or more selected from the group consisting of: polylactic acid-glycolic acid copolymer (PLGA), polyethylene glycol (PEG), distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauryl phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearate-sn-phosphatidylcholine (DSPC), 1-myristoyl-2-brown Palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), 1,2-dicarachidoyl-sn-glycerol-3-phosphocholine (DBPC), 1,2-docosahexaenoyl-sn-glycerol-3-phosphocholine (DEPC), palmitoyloleylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleylphosphatidylcholine, distearylphosphatidylethanolamine (DSPE), and di-... Myristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamoyl]ethyl]-3,4-di[oleoyloxy]-benzamide (VL-5), bis(octadecanoyl)glycyl spermine tetrafluoroacetate (DOGS), 3β-[N-(N',N'-dimethylaminoethyl)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2- Dioleoyl-3-trimethylammonium propane (DOTAP), (1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dimyristoxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[2-(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propanediamine trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanediamine bromide (GAP-DLRIE), N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropanediidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyl dioctadecylammonium bromide (DDAB), N4-cholesterolyl-spermine (GL67), 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA.

29. The device of claim 3, wherein the hydrophilic substance is a lipoprotein, transmembrane protein, peptide, or polymer.

30. The device of claim 29, wherein the hydrophilic substance is apolipoprotein A or apolipoprotein E.

31. The device according to claim 29, wherein the polymer is polylactic acid-glycolic acid copolymer (PLGA).

32. A method for producing nanoparticles, the method using an apparatus for producing nanoparticles, the apparatus including an inlet channel, a mixing channel, and an outlet channel, wherein the mixing channel includes micropillars, the method comprising: Hydrophobic and hydrophilic substances are introduced into the inlet channel; The micropillars induce eddies in the material within the mixing channel; as well as Nanoparticles comprising the hydrophobic and hydrophilic substances are formed.

33. The method of claim 32, wherein the inlet channel comprises a channel for introducing the hydrophilic substance and a channel for introducing the hydrophobic substance.

34. The method of claim 33, wherein the hydrophobic substance is introduced in the same direction as the flow direction of the fluid, and the hydrophilic substance is introduced in a direction different from the flow direction of the fluid.

35. The method of claim 32, wherein the Reynolds number for the fluid flow in the mixing channel is from 12.5 to 200.

36. The method of claim 35, wherein the Reynolds number for the fluid flow in the mixing channel is 50 to 100.

37. The method of claim 34, wherein the inlet volume flow rate ratio between the hydrophobic substance and the hydrophilic substance is 1:2.5 to 1:8.5 (v / v).

38. The method of claim 32, wherein the flow blockage ratio is 0.2 to 0.

8.

39. The method of claim 38, wherein the flow blockage ratio is from 0.35 to 0.

65.

40. Nanoparticles obtained by the method for producing nanoparticles according to any one of claims 32 to 39.

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