Microfluidic apparatus for the production of uniform nanoparticles

Abstract

The present invention relates to an apparatus useful for producing nanoparticles containing hydrophobic and hydrophilic substances. Specifically, the apparatus of the present invention includes a plurality of inflow channels into which hydrophobic and hydrophilic substances flow, a mixing channel into which the substances are mixed to produce nanoparticles, and an outflow channel through which the produced nanoparticles flow out, wherein the mixing channel includes microposts that can enhance the mixing efficiency of the substances. Therefore, nanoparticles produced using the apparatus of the present invention have excellent particle uniformity and can be usefully used as drugs or drug delivery bodies.

Description

[Technical Field]

[0001] This invention relates to a novel apparatus useful for producing nanoparticles containing both hydrophobic and hydrophilic substances, as well as nanoparticles consisting solely of hydrophilic substances, and to a method for producing uniform nanoparticles using the same. The nanoparticles obtained by this invention have excellent particle uniformity and can therefore be usefully used as drugs or drug delivery bodies. [Background technology]

[0002] Many nanopharmaceuticals are being developed for targeted delivery of therapeutic and contrast agents for the treatment and diagnosis of major diseases, including cancer, cardiovascular disease, diabetes, and Alzheimer's disease. Effective drug delivery systems can improve the absorption of poorly soluble and unstable drugs while enhancing their therapeutic effects and reducing their toxic effects, ultimately leading to the discovery and development of more effective drugs for improving patient prognosis and quality of life.

[0003] With the development of such nanopharmaceutical technologies for drug delivery, many studies have been reported over the past decade, but only a very small number of therapeutic and diagnostic nanopharmaceuticals have received FDA approval. The low success rate of nanopharmaceuticals is attributed to the low reproducibility of nanoparticles with the desired properties or efficacy, and the differences in physicochemical properties between batches. The reproducibility and reliability of the drug release profile are crucial elements of a successful drug delivery system, and this depends primarily on the size and uniformity of the nanoparticles. Therefore, there is an increasing demand for the development of technologies that can stably and reproducibly ensure particle size and uniformity during the nanoparticle manufacturing process. In particular, since the drugs delivered in nanopharmaceuticals are often hydrophobic, uniform synthesis with hydrophilic substances (such as phospholipids) used as drug delivery bodies is even more difficult, and development in this area has not been pursued. Therefore, the inventors have developed a novel apparatus useful for manufacturing nanoparticles containing both hydrophobic and hydrophilic substances, as well as nanoparticles consisting solely of hydrophilic substances, and have succeeded in manufacturing uniform nanoparticles using this novel apparatus, thus completing the present invention. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] 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) [Non-Patent Document 2] Rhee, M. et al. Drop Mixing in a Microchannel for Lab-on-a-Chip Platforms. Langmuir, 24 (2), 590-601 (2008) [Non-Patent Document 3] Rohit, K et al. Microfluidic Platform for Controlled Synthesis of Polymeric Nanoparticles. Nano Letters, 8, 9, 2906-12, (2008) [Non-Patent Document 4] Johnson, BK et al. Mechanism for Rapid Self-Assembly of Block Copolymer Nanoparticles. Physical Review Letters, 91, 11,118302-1 -4 (2003) [Non-Patent Document 5] Bekard, I., et al. The Effects of Shear Flow on Protein Structure and Function. Biopolymers, 95, 11, 733-745 (2011) [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Existing nanoparticle manufacturing methods mainly consist of unstandardized multi-step processes such as nanoprecipitation and emulsification-based solvent evaporation. Nanoprecipitation accounts for more than 50% of nanoparticle manufacturing methods. n) is a method for forming nanoparticles from a colloidal suspension between two solvent phases by dropping a lipophilic or polymeric drug dissolved in another hydrophobic solvent onto a stirring body of a hydrophilic solvent. The properties of the particles synthesized in this process can be controlled by the agitation speed of the two solvents, the degree of lipophilicity of the polymeric drug, and the polymer drip rate. However, the particle size is determined by the nonlinear and unpredictable flow of the stirring body, resulting in polydispersity and large batch-to-batch differences. This makes reproducible synthesis and production of nanoparticles difficult and requires various additional steps to homogenize the synthesized nanoparticles.

[0006] Recently, microfluidic technology has been applied to the development of drug delivery systems to control strong microvortex flow and effectively form drug carriers. Microfluidic technology allows for precise control of the microenvironment, such as the size, pattern, and surface composition of nanoparticles, by manipulating small amounts of liquid, and is therefore widely used in chemical synthesis, chemical and biomolecular analysis, tissue engineering, and other application fields. In particular, by using microfluidic technology to reproducibly and continuously produce high-quality nanoparticles with diverse combinations of physicochemical properties, nano-drug delivery systems can be optimized, and clinical applications can be facilitated, such as promoting and monitoring the effects of drug delivery, release, and removal during the patient's treatment process.

[0007] However, despite these advantages, most existing microfluidic methods for synthesizing nanopharmaceuticals still suffer from limitations such as low productivity due to relatively low flow rates during synthesis and difficulty in mixing different liquid substances. Recently, there have been efforts to overcome these limitations by increasing the flow rate within the microfluidic, but these have been limited to the formation of uncontrolled microvortices using simple linear and curved structures, and are restricted to the mixing of hydrophilic substances, making it difficult to achieve effective mixing of hydrophobic and hydrophilic substances. As a result, the production yield of synthesized nanoparticles is low, or the size of the produced nanoparticles is several hundred nanometers to several micrometers, which is a relatively large limitation for application as nanopharmaceuticals, where particle size must be uniformly produced to be 200 nanometers or less in order to be an effective drug delivery system.

[0008] Therefore, the inventors have discovered a novel apparatus that can adjust the size of the generated nanoparticles by adjusting the fluid flow rate, thereby enabling the production of homogeneous nanoparticles with high yield, and have completed the present invention. [Means for solving the problem]

[0009] The present invention provides a novel apparatus for producing nanoparticles, comprising a plurality of inflow channels through which hydrophobic substances and hydrophilic substances flow in, a mixing channel through which these substances are mixed to produce nanoparticles, and an outflow channel through which the produced nanoparticles flow out, wherein the mixing channel includes microposts capable of increasing the mixing efficiency of the incoming substances.

[0010] The term "mixing efficiency" used in this invention eff ) is a value that expresses the ratio as a percentage, taking advantage of the fact that hydrophobic and hydrophilic substances, each having a mass fraction of 1, are mixed with each other in a channel and eventually converge to 0.5. The calculation formula is as follows:

[0011]

number

[0012] At this time, Mass fract is the mass fraction, and the closer the mass fraction is to 0.5, the higher the synthesis efficiency is proportionally.

[0013] The term "device" or "microfluidic device" used in the present invention means a device including channels provided on a substrate made of various materials including plastic, glass, metal or silicone so that fluid can flow.

[0014] In the device of the present invention, the hydrophobic substance and the hydrophilic substance can each flow in through different inflow channels. Here, preferably, the hydrophobic substance can flow in from the mixing channel in the same direction as the fluid flow direction, and the hydrophilic substance can flow in from the mixing channel in a direction different from the fluid flow direction.

[0015] The number of inflow channels through which the hydrophobic substance flows in is one or more, and the number of inflow channels through which the hydrophilic substance flows in can be one or more. Preferably, the device of the present invention can include one inflow channel through which the hydrophobic substance flows in and two inflow channels through which the hydrophilic substance flows in.

[0016] The term "micropost" used in this specification means a structure that obstructs the straight flow of fluid in the mixing channel. The micropost of the present invention is a structure for forming a microvortex in the fluid flowing into the device to achieve efficient mixing, and can include all forms, and preferably can be columnar. The micropost can be designed to separate, meander, or merge different flows of the fluid colliding with the post. The micropost can be designed to maintain the main flow of the fluid so that the flow does not stagnate. For example, it can be made to collide with the fluid at a right angle with respect to the fluid flow direction.

[0017] The microposts of the present invention have a shape with corners capable of changing the fluid flow, and may be a polyhedron, a truncated polyhedron, a prism, or a modified shape thereof, and preferably may be a quadrangular prism. Further, in a cross-section cut along the fluid flow direction in the mixing channel, it may be a polygon or a modified shape thereof, and preferably it may be a rectangle having one side in a direction orthogonal (horizontal) to the fluid flow direction and one side in the same direction (vertical) as the fluid flow direction in the mixing channel.

[0018] In the present invention, the "fluid flow direction in the mixing channel" or the "flow direction in the mixing channel" means the direction in which the fluid flows from the inflow channel to the outflow channel in the device. Preferably, the hydrophobic substance flows in through an inflow channel arranged in the same direction as the flow direction in the mixing channel, and the hydrophilic substance flows in through an inflow channel arranged in a direction orthogonal to the flow direction in the mixing channel.

[0019] The microposts of the present invention can be arranged in one or more rows along the fluid flow direction in the mixing channel. Specifically, the microposts can be arranged in 1, 2, 3, 4, 5, or 6 or more rows along the fluid flow direction, and preferably can be arranged in 6 rows.

[0020] The microposts of the present invention can be arranged in one or more in a direction different (preferably orthogonal) to the fluid flow direction in the mixing channel in one row. The number of microposts present in each row may be constant or different from each other.

[0021] In the present invention, the microposts can be arranged alternately with the microposts in adjacent rows. In the present invention, "arranged alternately" means that the microposts in the plurality of rows are not arranged in a straight line or parallel to each other in the fluid flow direction, and the microposts arranged in one row can be arranged so as to partially or completely cover the gaps between the microposts arranged in the adjacent rows when viewed from the fluid flow direction.

[0022] In the present invention, the microposts have a height of 200 to 800 μm, and the microposts may or may not be connected to the walls forming the mixing channel.

[0023] In this invention, "inlet volume flow rate ratio" means the ratio of the inlet volume flow rate of a fluid containing a hydrophilic substance to the inlet volume flow rate of a fluid containing a hydrophobic substance.

[0024] In this invention, the "flow blockage ratio" is the ratio of the width of the microposts to the total channel width in the first row along the fluid flow direction in the mixing channel.

[0025] Generally, the flow rate within a device can be expressed by the Reynolds number Re, a dimensionless number representing the ratio of inertial force to viscous force, and its calculation formula is as follows:

[0026]

number

[0027] At this time, D h ρ represents the hydraulic diameter within the microfluidic device, while ρ, μ, v, and Q represent the fluid density, kinematic viscosity, velocity, and flow velocity, respectively. Therefore, the flow velocity increases proportionally as the Reynolds number increases.

[0028] In the apparatus of the present invention, the Reynolds number may be 500 or less, preferably 10 to 300, more preferably 12.5 to 200, and most preferably 50 to 100. If the Reynolds number exceeds 500, turbulence-like flow is formed in the apparatus, preventing the generation of a controllable vortex pattern. This can lead to instability of the raw material drug substance and the generated nanoparticles due to increased shear force between the solvent and the substance at high flow velocities. If the Reynolds number is 300 or higher, uncontrollable chaotic flow begins to occur in the apparatus, making quality control for the production of uniform nanoparticles difficult. Conversely, if the Reynolds number is low, such as 10 or less, it becomes difficult to effectively mix hydrophobic and hydrophilic substances due to diffusion dependence, making it difficult to achieve superior productivity compared to existing nanoparticle synthesis methods.

[0029] The apparatus of the present invention can adjust the particle size by changing the Reynolds number.

[0030] Furthermore, the present invention provides a method for producing nanoparticles containing hydrophobic and hydrophilic substances using the apparatus of the present invention.

[0031] Specifically, the present invention relates to a device including a plurality of inflow channels, mixing channels and outflow channels, wherein (a) a hydrophobic substance and a hydrophilic substance are introduced through the inflow channels, respectively. The present invention provides a method for producing uniform nanoparticles containing hydrophobic and hydrophilic substances, comprising the steps of (b) introducing a substance, (c) the substance colliding with microposts in a mixing channel to form vortices and produce nanoparticles, and (d) the produced nanoparticles flowing out through an outflow channel.

[0032] The hydrophobic substances of the present invention include poly-co-glycolic acid (PLGA), polyethylene glycol (PEG), DSPE-PEG, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimryristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), and 1-myristoyl-2-palmitoyl phosphatidyl Choline (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-diicosanoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (PO PC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3 ,4-di[oleyloxy]-benzamide (VL-5), dioctadecylamideglycylspermine 4-trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-Dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneaminium bromide (GAP-DLRIE), Nt-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), Ethyl phosphocholine (Ethyl It may be one or more selected from the group consisting of PC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl-spermine (GL67), 1,2-dioleyloxy-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.

[0033] The hydrophilic substance of the present invention may be a protein or a polymer, preferably an apolipoprotein, and more preferably apolipoprotein A or E.

[0034] The apparatus and manufacturing method of the present invention can be used to produce nanoparticles containing phospholipids, apolipoproteins, and polymers. [Effects of the Invention]

[0035] The apparatus of the present invention and the method for producing nanoparticles using the same have the advantage of being able to continuously synthesize nanoparticles of uniform size in a single step, and because of the low variability between batches, it is possible to produce large quantities of nanoparticles with consistent properties. The nanoparticles obtained in this way have excellent particle uniformity and do not contain additional components such as surfactants, so they can be usefully used as drugs and drug delivery bodies. [Brief explanation of the drawing]

[0036] [Figure 1] This shows the fluid flow using the apparatus of the present invention and the fluid flow around the micropost. [Figure 2] This is a schematic diagram of the apparatus of the present invention. [Figure 3] The results of measuring the mixing efficiency based on the number of rows of microposts in the apparatus of the present invention are shown. [Figure 4] The results of measuring the mixing efficiency based on the number of microposts present in a single row using the apparatus of the present invention are shown. [Figure 5] The results of measuring the mixing efficiency with respect to the change in Reynolds number in the apparatus of the present invention are shown. [Figure 6] The results of measuring the mixing efficiency due to changes in channel height in the apparatus of the present invention are shown. [Figure 7] The results of measuring the mixing efficiency based on the injection ratio of hydrophobic and hydrophilic substances in the apparatus of the present invention are shown. [Figure 8] This section describes a method for calculating the residence time of a fluid. [Figure 9] The fluid flow within the apparatus according to manufacturing examples 1 and 2 of the present invention is shown. [Figure 10] The particle size distribution of nanoparticles during the production of rHDL nanoparticles according to Production Examples 1 and 2 of the present invention is shown. [Figure 11] This shows the particle size distribution of lipid-polymer nanoparticles produced by the manufacturing method of the present invention. [Modes for carrying out the invention]

[0037] Hereinafter, embodiments and examples of the present application will be described in detail with reference to the attached drawings so that they can be easily implemented by a person with ordinary skill in the art to which the present invention pertains. However, the present invention can be realized in various forms and is not limited to the embodiments and examples described herein.

[0038] Throughout the specification of the present invention, when a part of the specification "includes" a certain component, this means that, unless otherwise stated, it may include other components rather than excluding them.

[0039] The present invention relates to an apparatus for producing nanoparticles, comprising an inflow channel, a mixing channel, and an outflow channel, wherein the mixing channel includes microposts.

[0040] The present invention also relates to a nanoparticle manufacturing apparatus comprising an inflow channel, a mixing channel, and an outflow channel, wherein the mixing channel includes microposts, and includes a method for manufacturing nanoparticles comprising the steps of: introducing a hydrophobic substance and a hydrophilic substance into the inflow channel; the substances forming a vortex due to the microposts in the mixing channel; and forming nanoparticles containing the hydrophobic substance and the hydrophilic substance, and nanoparticles obtained by the manufacturing method.

[0041] The inflow channel, mixing channel, and outflow channel may be arranged sequentially in the direction of fluid flow.

[0042] The inflow channel may include a channel through which a hydrophilic substance flows and a channel through which a hydrophobic substance flows, and the hydrophilic substance and the hydrophobic substance can each flow into different inflow channels.

[0043] The hydrophobic material can flow in the same direction as the fluid flow direction, while the hydrophilic material can flow in a direction different from the fluid flow direction. Preferably, the hydrophobic material can flow in the same direction as the fluid flow direction, and the hydrophilic material can flow in a direction perpendicular to the fluid flow direction.

[0044] The inlet volume flow rate ratio of the hydrophobic substance to the hydrophilic substance can be 1:2.5 to 1:8.5 (v / v).

[0045] The hydrophobic substances include poly-co-glycolic acid (PLGA), polyethylene glycol (PEG), DSPE-PEG, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimryristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), and 1-myristoyl-2-palmitoyl phosphatidylcholine. Phosphorus (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-diicosanoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POP C) Lysophosphatidylcholine, Dilinoleoylphosphatidylcholine, Distearoylphosphatidylethanolamine (DSPE), Dimyristoylphosphatidylethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), Palmitoyloleoylphosphatidylethanolamine (POPE), Lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3, 4-di[oleyloxy]-benzamide (VL-5), dioctadecylamideglycylspermine 4-trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-Dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneaminium bromide (GAP-DLRIE), Nt-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), Ethyl phosphocholine (Ethyl It may be one or more selected from the group consisting of PC, dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl-spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA.

[0046] The hydrophilic substance may be a lipoprotein, a transmembrane protein, a peptide, or a polymer, and is preferably an apolipoprotein A or E. The polymer may also be PLGA (poly-co-glycolic acid).

[0047] In the aforementioned mixing channel, hydrophilic and hydrophobic substances can be mixed with each other.

[0048] The aforementioned outflow channel may be a channel through which the generated nanoparticles are released.

[0049] The micropost may or may not be connected to the wall forming the mixed channel, and may be one or more selected from the group consisting of a polygonal pyramid, a frustum of a polygon, a polygonal prism, and variations thereof, and is preferably a quadrangular prism.

[0050] The microposts can be arranged in one or more columns in the direction of fluid flow, preferably in six or more columns in the direction of fluid flow. In addition, one or more microposts can be arranged in a direction different from the direction of fluid flow within a single column, preferably 1 to 6 microposts can be arranged in a direction different from the direction of fluid flow within a single column, more preferably 1 to 6 microposts can be arranged in a direction perpendicular to the direction of fluid flow within a single column, and most preferably 1 to 2 microposts can be arranged in a direction perpendicular to the direction of fluid flow within a single column.

[0051] The plurality of microposts do not necessarily have to be arranged in a line in the direction of fluid flow; the plurality of microposts can be arranged so as to partially or completely cover the gaps between microposts arranged in adjacent rows when viewed from the direction of fluid flow.

[0052] The particle size can be adjusted by changing the Reynolds number with respect to the fluid flow in the mixing channel. The Reynolds number with respect to the fluid flow in the mixing channel may be 12.5 to 200, preferably 50 to 100.

[0053] The height of the channel may be 200 to 800 μm.

[0054] In the above-mentioned apparatus, the flow blockage ratio may be 0.2 to 0.8, preferably 0.35 to 0.65.

[0055] The polydispersity index of the manufactured nanoparticles may be 0.15 or less. [Examples]

[0056] The present invention will be described in more detail below with reference to examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0057] [Example 1] Design of equipment for nanoparticle manufacturing A microfluidic device was designed for producing reconstructed high-density nanoparticles by mixing hydrophobic and hydrophilic organic materials (Figures 1 and 2). The microfluidic device includes three inlet channels and one outlet channel. Of the three inlet channels, the central channel is used to supply hydrophobic phospholipids or hydrophobic drugs, while the two channels on either side are used. A hydrophilic protein or hydrophilic drug was added to the channel. Furthermore, a micropost structure was introduced inside the apparatus to effectively mix the lipids and proteins.

[0058] [Example 2] The effect of a single micropost structure To determine the mixing efficiency of hydrophobic and hydrophilic materials based on the presence, number, and arrangement of microposts placed inside the device, the distribution of hydrophobic materials within the microfluidic device was analyzed using fluid dynamics techniques.

[0059] In this invention, the inlet volume flow rate ratio refers to the ratio of the inlet volume flow rate of a fluid containing a hydrophilic substance to the inlet volume flow rate of a fluid containing a hydrophobic substance, and the flow blockage ratio is the ratio of the width of the microposts to the width of the entire channel in the first column along the fluid flow direction in the mixing channel.

[0060] The results were confirmed under the following conditions: channel height of 200 μm, inlet volume flow rate ratio of 1:5.5 (hydrophobic material:hydrophilic material), Re of 50, flow blockage ratio of 0.5, and rectangular micropost size of 1000 μm × 400 μm. The results are shown in Figures 2, 3, and 4. The number of microposts was designed to be evenly distributed across the channel width while maintaining the micropost size and keeping the flow width between microposts constant.

[0061] As shown in Figure 2, we confirmed that hydrophobic and hydrophilic substances are efficiently mixed by microvortexes generated when hydrophobic substances flow through the micropost structure.

[0062] As shown in Figures 3 and 4, we confirmed that the mixing efficiency improved by approximately 1.5 to 4.0 times when microposts were present compared to when they were absent, depending on the distribution of microposts.

[0063] [Table 1]

[0064] As shown in Figure 3 and Table 1, the mixing efficiency increases as the number of columns increases, and it can be seen that when there are 6 or more columns, an excellent mixing efficiency of 98% or more is observed.

[0065] [Table 2]

[0066] As shown in Figure 4 and Table 2, the mixing efficiency actually decreases as the number of microposts located within the channel in a single row increases, and the best mixing efficiency is observed when the number is 1 to 2. Therefore, the mixing efficiency changes depending on the number and arrangement of microposts placed inside the device, and the highest mixing efficiency is observed when single micropost structures are arranged alternately. The microposts were evenly distributed across the channel width, and the width through which the flow passes between the placed microposts was kept constant.

[0067] [Example 3] Optimization of the usable flow velocity range and flow velocity ratio range within the device. To optimize nanoparticle synthesis by controlling the flow rates of hydrophobic and hydrophilic materials within the apparatus and their injection ratios, mixing efficiency was analyzed under various Reynolds number (Re) and flow rate ratio conditions.

[0068] To investigate the usable flow velocity range, the mixing efficiency was analyzed in the apparatus when the Reynolds number (Re) was varied from 12.5 to 200 with a channel height of 200 μm and an injection ratio of 1:5.5. The results are shown in Figure 5 and Table 3.

[0069] [Table 3]

[0070] As shown in Table 3 above, it was confirmed that a mixing efficiency of 90% or higher was obtained in all cases when the Reynolds number (Re) was between 12.5 and 200, and a mixing efficiency of 98% was obtained when the Reynolds number (Re) was 50 and 100.

[0071] Furthermore, the mixing efficiency was analyzed when the channel height was varied from 200 to 800 μm at an injection ratio of 1:5.5 and a Reynolds number (Re) of 50, and the results are shown in Figure 6 and Table 4.

[0072] [Table 4]

[0073] As shown in Table 4 above, it was confirmed that the mixing efficiency was 95% or higher when the channel height was 200 to 800 μm.

[0074] Furthermore, the mixing efficiency was analyzed in the apparatus when the injection ratio was changed from 1:2.5 to 1:8.5 at a channel height of 200 μm and a Reynolds number (Re) of 50, and the results are shown in Figure 7 and Table 5.

[0075] [Table 5]

[0076] As shown in Table 5 above, it was confirmed that the mixing efficiency was 97% or higher when the injection ratio was 1:2.5 to 1:8.5.

[0077] [Example 4] Research on apparatus height for increasing mixing efficiency To maximize the mixing efficiency by adjusting the height of the mixing channel in the device, the mixing efficiency was analyzed under conditions of different heights.

[0078] When raw materials are injected at a relatively high flow rate (Re>50) during the nanoparticle synthesis process using the apparatus, strong shear stress is generated from the walls due to the narrow width of the channel.

[0079] In the case of proteins, various studies have reported that shear stress from fluid flow generated from external equipment can induce protein deformation, including aggregation, folding, and degradation. Such deformation of precursor proteins alters the structure, size, and function of the manufactured nanoparticles. Generally, the range of deformation induced varies depending on the structure of the equipment generating the shear stress and the shape and size of the materials used. However, for proteins with a size of 1-10 nm (mass range of 20 kDa-300 kDa), such as insulin, enzymes, and immunoglobulins, a shear stress of 1000 dyne / cm² is sufficient. 2It has been reported that the structure of proteins can be deformed within this range (Bekard, I., et al. The Effects of Shear Flow on Protein Structure and Function. Biopolymers, 95, 11, 733-745(2011))。

[0080] Therefore, in the apparatus of the present invention, the shear stress (τ) generated by the fluid flow w To confirm whether or not the precursor material and nanoparticles are deformed by ) first, according to the height of each channel, The shear stress was calculated as follows.

[0081]

number

[0082] In this case, Q is the flow rate, and μ, h, and w represent the kinematic viscosity of the fluid, the height of the channel cross-section, and the horizontal length of the channel cross-section, respectively.

[0083] [Table 6]

[0084] As shown in Table 6 above, in the apparatus of the present invention, the shear force increases exponentially as the channel height decreases, but the deformation of the protein structure is 1000 dyne / cm. 2 We confirmed that it did not exceed the specified range.

[0085] However, with a channel height of 100 μm, increasing the flow velocity to Re300 results in 1000 dyne / cm². 2 This range is reached. Furthermore, considering that the size of the nanoparticles to be manufactured is in the range of 10-100 nm, which is larger than the size of the protein, it can be seen that when the channel height is 200 μm or more, the precursor material and nanoparticles do not deform due to shear stress.

[0086] Also, as shown in Table 7 below, when the height of the mixing channel of the device increases, the volume increases, and for the same flow rate, the residence time (τ res ) during which the precursor to be mixed stays in the channel and the mixing time (τ mix ) required for the synthesis of the precursor increase. The specific calculation method for the residence time during which the substance to be mixed stays in the channel is shown in Figure 8. The mixing time (τ mix ) required for the synthesis of the substance can be calculated by the more dominant physical phenomenon between convection and diffusion in the device, and can be calculated as follows using the Peclet number (Pe), which is a dimensionless number indicating the ratio of the convection time to the diffusion time.

[0087]

Equation

[0088] In the device of the present invention, the Peclet number (Pe) has a value of 100 to 5000. Based on this, it can be seen that mixing by convection is dominant. Therefore, the mixing time (τ mix、convection ) by convection in the channel was calculated as follows (Valencia, 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)).

[0089]

Equation

[0090] In this case, D represents the diffusion coefficient, and L and w represent the channel length and the horizontal length of the channel cross-section, respectively.

[0091] [Table 7]

[0092] In this case, basically, for the substance to be sufficiently mixed within the channel, the residence time (τ) is necessary. res ) is the mixing time (τ mix ) must be longer than the mixing time, but as shown in Table 7 above, since the residence time is longer than the mixing time at all channel heights, it can be confirmed that sufficient mixing time has been secured at the given channel height.

[0093] Furthermore, the increase in mixing time due to the increase in channel height is calculated to be 53 ms at the largest height of 800 μm, as shown in Table 7 above. This is because the aggregation time (τ) is the time it takes for the injected substances to gather and aggregate with each other. agg This is the time when the 50-100 ms range begins to be entered, which can be considered the maximum height for applying the apparatus and manufacturing method of the present 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 (2003)).

[0094] Therefore, as shown in Table 7 above, the optimal height of the mixing channel that can prevent aggregation of nanoparticles obtained in the apparatus and the generation of non-uniform nanoparticles to the greatest extent possible is 200 to 800 μm.

[0095] [Example 5] Fluid flow within the device Depending on the channel height and micropost conditions, the apparatus of the present invention was designed in two manufacturing examples as shown in Table 8 below.

[0096] [Table 8]

[0097] The materials from Production Example 1 and Production Example 2 were manufactured, and the distribution of the materials was visualized using ink and observed under a microscope to confirm the mixed flow pattern of the hydrophobic and hydrophilic materials.

[0098] Fluids containing hydrophobic substances were visualized using 6% ethanol ink, and fluids containing hydrophilic substances were visualized using physiological saline solution. The results are shown in Figure 9.

[0099] As shown in Figure 9, it can be seen that the hydrophobic material is efficiently converted into a hydrophilic material by the microvortex generated when the hydrophobic material flows through the micropost structure.

[0100] [Example 6] Manufacturing of reconstituted high-density lipoprotein (rHDL) nanoparticles Using Production Examples 1 and 2 of Example 5, rHDL nanoparticles containing phospholipids (DMPC) and apolipoproteins were produced by the following method.

[0101] A solution of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) in anhydrous ethanol and an apolipoprotein solution in PBS were prepared. Then, approximately 0.8 mL of the DMPC solution at a concentration of 0.83 mg / ml in anhydrous ethanol was filled into one syringe, and approximately 1.25 mL (total of approximately 2.5 mL) of the same volume of the apolipoprotein solution at a concentration of 0.2 mg / ml in PBS was filled into two different syringes, and then the bubbles were removed from all syringes.

[0102] Using tubing, the needles of each syringe were connected to the inflow channels of the apparatus of the present invention, and PBS was flowed through the apparatus at an outflow rate of 1 mL / min using a syringe pump to clean it. 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 apparatus, and after mixing the obtained nanoparticles with PBS, they were centrifuged using a 10K filter and purified three times at 4°C for 20 minutes each time.

[0103] After dissolving the obtained rHDL nanoparticles in PBS, the change in particle size distribution due to nanoparticle aggregation was measured using dynamic light scattering (DLS) with a Zetasizer Nano ZS, and the results are shown in Figure 10.

[0104] As shown in Figure 10, after dissolving the rHDL nanoparticles obtained using the apparatus in Production Example 1 and 2 in PBS, the DLS data was measured, and the sizes of the rHDL nanoparticles obtained in Production Example 1 and 2 were measured to be 18.78 ± 4.58 nm and 13.06 ± 0.00 nm, respectively.

[0105] Therefore, it can be seen that the nanoparticles produced in Production Example 2 using the apparatus of the present invention have better uniformity than the nanoparticles produced in Production Example 1 using an existing apparatus.

[0106] [Example 7] Method for producing lipid-polymer nanoparticles using the apparatus Lipid-polymer nanoparticles containing polymer PLGA (poly-co-glycolic acid), phospholipid (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), and PEGylated phospholipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], DSPE-PEG2000) were produced using the following method with Production Example 2.

[0107] PLGA was prepared in anhydrous acetonitrile (ACN) solution, and 5 mL was injected into a syringe. DPPC was an anhydrous ethanol solution, and DSPE-PEG2000 was prepared as an aqueous solution using ultrapure distilled water (DW).

[0108] The mixing ratio of the two phospholipids was DPPC:DSPE-PEG2000 = 1:2 (by weight), and 5 mL of the final solution was injected into a syringe to obtain a 4% ethanol solution. Nanoparticles were obtained under the two synthesis conditions shown in Table 9 below. The obtained nanoparticles were purified with DW (deionized water, pure water completely free of ions).

[0109] [Table 9]

[0110] Figure 11 shows the particle size distribution of the nanoparticles obtained under the two synthesis conditions described above. The particle sizes of the nanoparticles obtained under synthesis conditions 1 and 2 were measured using dynamic light scattering and were found to be 124±5.3 nm and 56±4.4 nm, respectively. The polydispersity index (PSI) was measured to determine the uniformity of the particle size and was found to be 0.05 and 0.13, respectively. Generally, a lower PSI indicates better uniformity, and a PSI of 0.15 or less indicates that uniformity is ensured. Therefore, the nanoparticles produced under the above synthesis conditions exhibit uniformity.

[0111] Therefore, it can be seen that by using the apparatus of the present invention, nanoparticles of different sizes can be produced depending on the injection flow rate and the weight ratio of hydrophilic to lipophilic substances, and nanoparticles with a uniform particle size composed of various phospholipids and proteins can be produced.

[0112] [Example 8] Optimization of the structural design within the device To optimize the channel size and micropost mixing efficiency within the device, the mixing efficiency was verified under various conditions within the device structure, including 1) the starting interval of microposts, 2) the spacing between microposts, 3) the vertical length of the microposts, and 4) the flow blockage ratio.

[0113] The mixing efficiency for each variable is calculated with a fixed channel height of 0.2 mm and a flow rate ratio of 1:5.5. This was confirmed under the condition of a Reynolds number of 50. The results are shown in Figures 12 and 13.

[0114] [Table 10]

[0115] As shown in Figure 12 and Table 10, we confirmed that the fluid flow morphology through the microposts designed within the channel remained largely unchanged regardless of the post spacing and longitudinal orientation, maintaining high mixing efficiency (>0.95) under all conditions.

[0116] [Table 11]

[0117] On the other hand, as shown in Figure 13 and Table 11, in the case of the occlusion ratio, which is the ratio of the lateral spacing of microposts in the channel, it was confirmed that the mixing efficiency increases as the value increases, and when the occlusion ratio is 0.5, the mixing efficiency is maintained at a constant level of 0.98 or higher.

[0118] However, as the flow occlusion rate increases, the channel width also narrows, and this proportionally increases the shear stress. High shear stress, when it exceeds a certain value as previously calculated, can cause 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)). When the flow rate is increased to Re300 under conditions where the occlusion rate exceeds 0.65, the shear stress becomes 1000 dyne / cm². 2 When considering the above increase, the channel flow occlusion rate may be 0.2 to 0.8, and preferably in the range of 0.35 to 0.65, it can be seen that high mixing efficiency is not greatly affected by shear stress.

Claims

1. Including inflow channels, mixing channels and outflow channels, The apparatus for producing nanoparticles is characterized in that the mixing channel includes microposts.

2. The apparatus for producing nanoparticles according to claim 1, characterized in that the inlet channel, the mixing channel, and the outlet channel are sequentially arranged in the direction of fluid flow.

3. The apparatus for producing nanoparticles according to claim 1, characterized in that the inflow channel includes a channel through which a hydrophilic substance flows and a channel through which a hydrophobic substance flows.

4. The apparatus for producing nanoparticles according to claim 3, characterized in that the hydrophilic substance and the hydrophobic substance each flow into different inflow channels.

5. The nanoparticle manufacturing apparatus according to claim 4, characterized in that the hydrophobic substance flows in in the same direction as the fluid flow direction, and the hydrophilic substance flows in in a direction different from the fluid flow direction.

6. The nanoparticle manufacturing apparatus according to claim 5, characterized in that the hydrophobic substance flows in in the same direction as the fluid flow direction, and the hydrophilic substance flows in in a direction perpendicular to the fluid flow direction.

7. The nanoparticle manufacturing apparatus according to claim 1, characterized in that the outflow channel is a channel through which the generated nanoparticles flow out.

8. The apparatus for producing nanoparticles according to claim 3, characterized in that a hydrophilic substance and a hydrophobic substance are mixed with each other in the mixing channel.

9. The apparatus for producing nanoparticles according to claim 1, characterized in that the microposts are connected to or not connected to a wall forming a mixing channel.

10. The nanoparticle manufacturing apparatus according to claim 1, characterized in that the micropost is one or more selected from the group consisting of a polygonal pyramid, a frustum of a polygon, a polygonal prism, and a modified shape thereof.

11. The apparatus for producing nanoparticles according to claim 10, characterized in that the micropost is a rectangular prism.

12. The nanoparticle manufacturing apparatus according to claim 1, characterized in that the microposts are arranged in one or more columns in the direction of fluid flow.

13. The nanoparticle manufacturing apparatus according to claim 12, characterized in that the microposts are arranged in six or more columns in the direction of fluid flow.

14. The nanoparticle manufacturing apparatus according to claim 12, characterized in that one or more microposts are arranged in a row in a direction different from the fluid flow direction.

15. The nanoparticle manufacturing apparatus according to claim 14, characterized in that one to six microposts are arranged in a row in a direction different from the fluid flow direction.

16. In a single row, 1 to 6 microposts are arranged perpendicular to the direction of fluid flow. The apparatus for producing nanoparticles according to claim 15, characterized in that it is able to do so.

17. The nanoparticle manufacturing apparatus according to claim 16, characterized in that one or two microposts are arranged in a row perpendicular to the direction of fluid flow.

18. The nanoparticle manufacturing apparatus according to claim 12, characterized in that the plurality of microposts are not arranged in a line in the direction of fluid flow.

19. The nanoparticle manufacturing apparatus according to claim 12, characterized in that the plurality of microposts are arranged to partially or completely cover the gaps between microposts arranged in adjacent rows when viewed from the direction of fluid flow.

20. The apparatus for producing nanoparticles according to claim 1, characterized in that the Reynolds number for the fluid flow in the mixing channel is 12.5 to 200.

21. The nanoparticle manufacturing apparatus according to claim 20, characterized in that the Reynolds number for the fluid flow in the mixing channel is 50 to 100.

22. The nanoparticle manufacturing apparatus according to claim 1, characterized in that the particle size can be adjusted by changing the Reynolds number with respect to the fluid flow in the mixing channel.

23. The nanoparticle manufacturing apparatus according to claim 1, characterized in that the height of the channel is 200 to 800 μm.

24. The apparatus for producing nanoparticles according to claim 3, characterized in that the injection volume flow rate ratio of the hydrophobic substance to the hydrophilic substance is 1:2.5 to 1:8.5 (v / v).

25. The apparatus for producing nanoparticles according to claim 1, characterized in that the flow blockage ratio is 0.2 to 0.

8.

26. The apparatus for producing nanoparticles according to claim 25, characterized in that the flow blockage ratio is 0.35 to 0.

65.

27. The nanoparticle manufacturing apparatus according to claim 1, characterized in that the polydispersity index of the manufactured nanoparticles is 0.15 or less.

28. The hydrophobic substances include poly-co-glycolic acid (PLGA), polyethylene glycol (PEG), DSPE-PEG, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimryristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), and 1-palmitoyl-2 -Myristoylphosphatidylcholine (PMPC), 1-Palmitoyl-2-Stearoylphosphatidylcholine (PSPC), 1-Stearoyl-2-Palmitoylphosphatidylcholine (SPPC), 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DAPC), 1,2-Diarachidoyl-sn-Glycero-3-Phosphocholine (DBPC), 1,2-Diicosanoyl-sn-Glycero-3-Phosphocholine (DEPC), Palmitoyloleoylphosphatidylcholine (POPC), Lysophosphatidylcholine, Dilinoleoylphosphatidylcholine, Distea Roylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (VL-5), dioctadecylamideglycylspermine 4-trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTM) A) 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneammonium bromide (GAP-DLRIE), N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl The apparatus for producing nanoparticles according to claim 3, characterized in that it is one or more selected from the group consisting of PC, dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl-spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3,DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA.

29. The apparatus for producing nanoparticles according to claim 3, characterized in that the hydrophilic substance is a lipoprotein, a transmembrane protein, a peptide, or a polymer.

30. The apparatus for producing nanoparticles according to claim 29, characterized in that the hydrophilic substance is apolipoprotein A or apolipoprotein E.

31. The apparatus for producing nanoparticles according to claim 29, characterized in that the polymer is PLGA (poly-co-glycolic acid).

32. An apparatus for producing nanoparticles, comprising an inflow channel, a mixing channel, and an outflow channel, wherein the mixing channel includes microposts, The steps include introducing a hydrophobic substance and a hydrophilic substance into the inflow channel, The step of the substance forming a vortex by the microposts in the mixing channel, A method for producing nanoparticles, comprising the step of forming nanoparticles containing the hydrophobic substance and the hydrophilic substance.

33. The method for producing nanoparticles according to claim 32, characterized in that the inflow channel includes a channel through which the hydrophilic substance flows and a channel through which the hydrophobic substance flows.

34. The method for producing nanoparticles according to claim 33, characterized in that the hydrophobic substance flows in in the same direction as the fluid flow direction, and the hydrophilic substance flows in in a direction different from the fluid flow direction.

35. The method for producing nanoparticles according to claim 32, characterized in that the Reynolds number for the fluid flow in the mixing channel is 12.5 to 200.

36. The method for producing nanoparticles according to claim 35, characterized in that the Reynolds number for the fluid flow in the mixing channel is 50 to 100.

37. A method for producing nanoparticles according to claim 34, characterized in that the injection volume flow rate ratio of the hydrophobic substance to the hydrophilic substance is 1:2.5 to 1:8.5 (v / v).

38. A method for producing nanoparticles according to claim 32, characterized in that the flow blockage ratio is 0.2 to 0.

8.

39. The apparatus for producing nanoparticles according to claim 38, characterized in that the flow blockage ratio is 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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