Method for synthesizing apolipoprotein-containing hybrid nanoparticles

Abstract

The present invention relates to a method for synthesizing hybrid nanoparticles containing apolipoproteins. Specifically, the present invention relates to a method for synthesizing hybrid nanoparticles containing various functional proteins including apolipoproteins using a spiral microvortex device. By the production method of the present invention, nanoparticles having various biological activities, being small, uniform and stable can be produced.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing hybrid nanoparticles containing apolipoproteins. Specifically, the present invention relates to a method for synthesizing hybrid nanoparticles containing various functional proteins including apolipoproteins using a spiral micro-vortex device. By the production method of the present invention, nanoparticles having various bioactivities, being small, uniform, and stable can be produced.

Background Art

[0002] Nanoparticles mean particles having a particle size of less than micrometers. Because of their small size, they can move freely in the body and can be given various physical properties depending on the constituent materials, and thus are attracting attention as next-generation drug carriers.

[0003] Drug delivery nanoparticles are composed of various components such as phospholipids and polymers as needed, and have different properties depending on the constituent components. Nanoparticles made of inorganic substances are mainly used for diagnostic purposes, and polymer nanoparticles are mainly used as drug carriers for the purpose of adjusting the drug release rate and the in-vivo circulation time. Phospholipid nanoparticles have amphiphilicity, are easy to self-assemble, and have various molecular sieves, so various types of phospholipid nanoparticles are being studied as drug carriers.

[0004] In recent years, hybrid nanoparticles combining the advantages of such nanoparticles have been studied. However, since conventional nanoparticle production methods are mainly composed of non-standardized multi-step processes such as nanoprecipitation and emulsification-based solvent evaporation, there are many difficulties in mass-producing hybrid nanoparticles with a uniform particle size by conventional nanoparticle production methods. In particular, when incorporating polymers such as functional proteins into nanoparticles, it is difficult to synthesize them at once due to the complexity of the synthesis, and since they are produced through many processes, their uniformity and stability are lost, and there are many difficulties in mass production.

[0005] The present inventors have researched a synthesis method that allows functional proteins to be incorporated into polymer-lipid hybrid nanoparticles (PHNPs) using PLGA (poly(lactic-co-glycolic acid)), a polymer that is biodegradable and biocompatible and can protect drugs from degradation, and have completed the present invention. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Toth et al.,Robust manufacturing of lipid-polymer nanoparticles through feedback control of parallelized swirling microvortices.2017,Lab.Chip.,17.16: 2805-2813. [Non-Patent Document 2] Manon, R., et al.Optimal self-assembly of lipid nanoparticles(LNP) in a ring micromixer,Scientific reports,12:9483(2022) [Non-Patent Document 3] 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]

[0007] The present invention aims to provide a method for producing hybrid nanoparticles containing a mixture of hydrophilic and hydrophobic substances by mixing phospholipids and polymers under a swirling microvortex.

[0008] The present invention aims to provide a method for producing protein-containing hybrid nanoparticles, comprising the step of adding protein to the hybrid nanoparticles by causing the protein to collide with the hybrid nanoparticles under a swirling microvortex.

[0009] Furthermore, the present invention aims to provide protein-containing hybrid nanoparticles produced by the method described above. [Means for solving the problem]

[0010] The present invention provides a method for producing protein-containing hybrid nanoparticles, comprising the steps of: injecting hybrid nanoparticles into the first inlet and injecting a protein into the second inlet, and adding the protein to the hybrid nanoparticles by the swirling microvortex, in a vortex microvortex device having a first inlet, a second inlet and an outlet.

[0011] In one embodiment, the Reynolds number in the vortex micro-vortex device may be 50 to 300.

[0012] In one embodiment, the protein may be an apolipoprotein or a polymer having amphoteric properties.

[0013] In one embodiment, the apolipoprotein may be at least one selected from the group consisting of apolipoproteins A1, A2, E2, E3, J, and M.

[0014] In one embodiment, the synthetic weight ratio of the hybrid nanoparticles to the apolipoprotein may be 20:1 to 0.5:1, and is preferably 20:1 to 2:1.

[0015] In one embodiment, the manufacturing method may further include the step of recovering protein-containing nanoparticles from the outlet.

[0016] In one embodiment, the hybrid nanoparticles may be produced by a production method that includes the steps of injecting phospholipids into the first inlet and polymers into the second inlet of the vortex micro-vortex device, and mixing the phospholipids and polymers by the vortex micro-vortex.

[0017] The aforementioned phospholipids are 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), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), and 1-P Lumitoyl-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-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosph Phosphatidylethanolamine (DSPE), Distearoylphosphatidylethanolamine-Polyethylene Glycol (DSPE-PEG), 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 At least one 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, preferably DPPC and DSPE-PEG, and more preferably DPPC and DSPE-PEG 2000-5000.

[0018] In one embodiment, the ratio of the DPPC to the DSPE-PEG2000 may be 2.3:1 to 1:1.

[0019] In one embodiment, the polymer may be PLGA (poly(lactic-co-glycolic acid) copolymer).

[0020] In one embodiment, the hybrid nanoparticles may be polymer-lipid hybrid nanoparticles (PHNPs).

[0021] The present invention provides protein-containing hybrid nanoparticles produced by the above-described manufacturing method.

[0022] The present invention provides a method for producing protein-containing hybrid nanoparticles, which comprises, in a scroll micro-vortex device having a first injection port, a second injection port, and an outlet, a first step of producing hybrid nanoparticles, which includes injecting phospholipids into the first injection port and injecting a polymer into the second injection port, and mixing the phospholipids and the polymer by a scroll micro-vortex, and a second step of producing protein-containing hybrid nanoparticles, which includes injecting the hybrid nanoparticles obtained at the first injection port and injecting a protein into the second injection port.

[0023] In one embodiment, the protein may be apolipoprotein or a polymer having amphoteric properties.

[0024] In one embodiment, the synthetic compound weight ratio of the hybrid nanoparticles and the apolipoprotein may be 20:1 to 2:1.

[0025] In one embodiment, the polymer may be PLGA (poly(lactic-co-glycolic acid)).

[0026] In one embodiment, the hybrid nanoparticles may be polymer-lipid hybrid nanoparticles (PHNP).

Advantages of the Invention

[0027] The polymer-lipid hybrid nanoparticles (PHNP) produced by the production method of the present invention contain both hydrophilic substances and hydrophobic substances, and have a form in which the hydrophobic substances cover the center of the nanoparticles made of the polymer. Therefore, a high concentration of hydrophobic drugs can be effectively encapsulated and delivered inside.

[0028] Furthermore, the hybrid nanoparticles of the present invention can be used as a substance for delivering multiple drugs by utilizing the surface, intermediate shell, and core portions, respectively. For example, the hybrid nanoparticles of the present invention can be used as an effective carrier for anionic drugs such as genes.

[0029] Furthermore, polymers such as PEG located on the surface of hybrid nanoparticles prevent external substances from adsorbing to the particle surface or being removed by immune cells within the human body, thus enabling safer drug delivery. Therefore, when used for intracellular drug delivery, it can efficiently support treatment. [Brief explanation of the drawing]

[0030] [Figure 1] This is a schematic diagram showing the synthesis process of hybrid nanoparticles (PHNP-E3 / A1) containing both ApoE3 and ApoA1, and the morphology of PHNP-E3 / A1. [Figure 2] This graph compares the particle size of hybrid nanoparticles (PHNP-2000) at various synthetic weight ratios of DSPE-PEG2000 to DPPC. [Figure 3] Figure 2 shows TEM images of hybrid nanoparticles (PHNP-2000) at various synthetic weight ratios of DSPE-PEG2000 to DPPC. [Figure 4] This graph compares the particle size of hybrid nanoparticles (PHNPs) in DSPE-PEG at various PEG molecular weights. [Figure 5] Figure 4 shows TEM images of hybrid nanoparticles (PHNPs) of DSPE-PEG at various PEG molecular weights. [Figure 6] Figure 4 shows a graph illustrating the stability of each nanoparticle, obtained by measuring the particle size of the synthesized hybrid nanoparticles (PHNPs) on the day of synthesis and 3 days after synthesis. [Figure 7] This is a schematic diagram showing the synthesis process of ApoE3-containing hybrid nanoparticles (PHNP-E3) using a vortex micro-vortex flow apparatus, and the morphology of PHNP-E3. [Figure 8] This graph compares the particle sizes of PHNP-PEG2000 and PHNP-E3. [Figure 9] This is a TEM image of PHNP-E3. [Figure 10] This graph compares the particle sizes of PHNP-E3 synthesized under various Reynolds number conditions. [Figure 11] This graph shows the yield of ApoE3 contained in PHNP-E3. [Figure 12] This is a schematic diagram showing the synthesis process of ApoA1-containing hybrid nanoparticles (PHNP-A1) and the morphology of the hybrid nanoparticles (PHNP-A1). [Figure 13] This graph compares the particle sizes of PHNP-PEG2000 and PHNP-A1. [Figure 14] This is a TEM image of PHNP-A1. [Figure 15] This is a fluorescence image of rhodamine-labeled hybrid nanoparticles (PHNP-PEG2000) delivered into HAEC cells. [Figure 16] This is a fluorescence image of rhodamine-labeled hybrid nanoparticles (PHNP-A1) delivered into HAEC cells. [Figure 17] Figures 15 and 16 show graphs comparing the quantitative fluorescence intensity of rhodamine. [Figure 18] This graph compares the particle size and polydispersity index (PDI) of synthesized PHNP-A1(SMR) and PHNP-A1(BT) with and without a vortex micro-vortex device. [Figure 19] This graph compares the yields of ApoA1 produced by synthesizing PHNP-A1(SMR) and PHNP-A1(BT) at various synthesis weight ratios of PHNP relative to ApoA1, and then using ELISA. [Figure 20] This graph shows the yields of PHNP-A1(SMR) and PHNP-A1(BT) synthesized at different synthesis weight ratios of PHNP to ApoA1, followed by a comparison of the yields of ApoA1 using ELISA. [Figure 21]This image shows fluorescence images of PHNP-A1(SMR) and PHNP-A1(BT) delivered into iHBECs, along with a graph comparing the quantified fluorescence. [Figure 22] This graph shows the particle size distribution of synthesized PHNP-E3 / A1 at different blending weights of ApoE3 and ApoA1. [Figure 23] This figure shows the particle size and PDI results for PHNP-E3 / A1 on the day of synthesis, and 1, 3, 7, and 14 days later, indicating its stability. [Figure 24] This graph shows the fluorescence wavelengths of PHNP-PEG2000, PHNP-E3, PHNP-A1, and PHNP-E3 / A1. [Modes for carrying out the invention]

[0031] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the attached drawings so that those with ordinary skill in the art to which the present invention pertains can easily implement it. However, the present invention can be implemented in various forms and is not limited to the embodiments and examples described below.

[0032] When a specification states that a part of it "includes" a certain component, unless otherwise specified, this does not mean that other components are excluded, but rather that other components may be included.

[0033] In this invention, "hybrid nanoparticles" refers to nanomaterials formed by the coexistence of hydrophilic and hydrophobic materials, and are preferably polymer-lipid hybrid nanoparticles (PHNPs) in which polymers and lipids are mixed, and are also referred to as "PHNPs".

[0034] In this invention, "PLGA" refers to a polylactic acid-glycolic acid copolymer, which is a polymer that is biodegradable and biocompatible.

[0035] In this invention, "DPPC" refers to dipalmitoylphosphatidylcholine, which is a type of lipid.

[0036] In this invention, "DSPE-PEG" refers to a lipid in which distearoylphosphoethanolamine is bonded to polyethylene glycol (PEG), and the molecular weight of polyethylene glycol is preferably 2000 to 5000.

[0037] In this invention, "amphoteric polymers" refer to proteins that simultaneously possess both hydrophobic and hydrophilic properties. Examples include glycophorin, rhodopsin, CD36 (cluster of differentiation 36), seipin, glucose permease, cytochrome c, cupredoxins, high potential iron protein, adrenodoxin reductase, and flavoprotein.

[0038] In this invention, "phospholipids" specifically refer to 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-myristoyl phosphatidylcholine. (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-Diecocenoyl-sn-Glycero-3-Phosphocholine (DEPC), Palmitoyloleoylphosphatidylcholine (POPC), Lysophosphatidylcholine, Dilinoleoylphosphatidylcholine, Distea Roylphosphatidylethanolamine (DSPE), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)eth [L]-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 At least one 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, but not limited to these.

[0039] "Apolipoprotein E" means a mammalian protein encoded by the APOE gene or a functional variant thereof. In a preferred embodiment, apolipoprotein E is a human protein encoded by the human APOE gene on chromosome 19. Apolipoprotein E may be any isoform of the APOE gene product, e.g., apolipoprotein E2 (APOE2), apolipoprotein E3 (APOE3), or apolipoprotein E4 (APOE4). APOE is a polymorphism having three main alleles (epsilon 2, epsilon 3, and epsilon 4). Any allele may be used in various embodiments of the present invention. "Functional variant" means a variant of the mammalian protein encoded by the APOE gene that maintains the same or similar biological function as the APOE gene product. In some cases, the functional variant involves amino acid insertions, deletions, and / or substitutions compared to the protein encoded by the human APOE gene. In some cases, the functional variant is a fragment of the protein encoded by the human APOE gene.

[0040] In one embodiment, apolipoprotein E3 is a protein registered in GenBank under accession number ARQ79461.1, or has at least 95% sequence identity with it, preferably at least 98% or 99% sequence identity.

[0041] In one embodiment, the apolipoprotein E within the hybrid nanoparticles is a recombinant protein produced by genetic engineering or a synthetic protein produced by chemical synthesis.

[0042] "Apolipoprotein A1" means a mammalian protein encoded by the APOA1 gene or a functional variant thereof. In a preferred embodiment, apolipoprotein A1 is a human protein encoded by the human APOA1 gene located on chromosome 11. This "functional variant" means a variant of the mammalian protein encoded by the APOA1 gene that maintains the same or similar biological function as the APOA1 gene product. In some cases, the functional variant involves amino acid insertions, deletions, and / or substitutions compared to the protein encoded by the human APOA1 gene. In some cases, the functional variant is a fragment of the protein encoded by the human APOA1 gene.

[0043] In one embodiment, apolipoprotein A1 is a protein registered in GenBank under accession number AAS68227.1, or a protein having at least 95% sequence identity, preferably at least 98% or 99% sequence identity.

[0044] In one embodiment, the apolipoprotein A1 within the hybrid nanoparticles is a recombinant protein produced by genetic engineering or a synthetic protein produced by chemical synthesis.

[0045] In this invention, "apolipoprotein E" means that it includes fragments and functional variants thereof.

[0046] In this invention, "vortex microvortex flow" refers to a fluid flow in which small fluid particles rotate while flowing.

[0047] In this invention, "micro-vortex device" refers to a device that includes micro-channels and the like, which are arranged on a substrate made of various materials such as organic polymers, plastics, glass, metals, or silicon, so that a fluid can flow through it.

[0048] The present invention will be described in more detail below with reference to examples. These examples are for illustrative purposes only, and the present invention is not limited to these examples. [Examples]

[0049] Method for synthesizing apolipoprotein-containing hybrid nanoparticles using a vortex microvortex apparatus. Apolipoprotein-containing hybrid nanoparticles were synthesized. Specifically, the first step was to synthesize polymer-lipid hybrid nanoparticles (PHNPs), and the second step was to incorporate apolipoproteins into the hybrid nanoparticles. The same swirling microvortex device was used for all steps. A schematic diagram of the swirling microvortex device and synthesis method used for the synthesis of hybrid nanoparticles (PHNPs) in the present invention is shown in Figure 1.

[0050] The aforementioned vortex microvortex device includes two inlets and one outlet. The diameter and height of the device were optimized to effectively mix lipids and PLGA polymers by forming vortex microvortices (see Non-Patent Literature 1).

[0051] In the polymer-lipid hybrid nanoparticle (PHNP) synthesis step, a phospholipid mixture, DPPC (dipalmitoylphosphatidylcholine) and DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol), was injected into one of the two inlets, and a polymer, PLGA (poly(lactic-co-glycolic acid)), was injected into the other inlet on the opposite side, thereby synthesizing polymer-lipid hybrid nanoparticles (PHNP).

[0052] Subsequently, the polymer-lipid hybrid nanoparticles (PHNPs) synthesized as described above were injected into one inlet, and apolipoproteins were injected into the opposite inlet. The mixture was then mixed to ultimately synthesize apolipoprotein-containing hybrid nanoparticles. [Examples]

[0053] Synthesis of polymer-lipid hybrid nanoparticles at various weight ratios of DPPC:DSPE-PEG2000 Using the aforementioned vortex micro-vortex apparatus, polymer-lipid hybrid nanoparticles containing lipids (DPPC and DSPE-PEG2000) and polymers (PLGA) were synthesized by the following method. DSPE-PEG2000 contains PEG, which can prevent plasma proteins and other substances in the human body from adsorbing onto the nanoparticle surface and from being removed by immune cells; therefore, it was used to ensure the stability of the nanoparticles in the body.

[0054] A solution of PLGA was prepared by dissolving it in anhydrous acetonitrile (ACN), and DPPC and DSPE-PEG2000 were prepared as solutions of anhydrous ethanol. Synthesis was performed using a vortex microcurrent apparatus, and the final solution was immediately purified with deionized water (DW).

[0055] To produce stable polymer-lipid hybrid nanoparticles (PHNPs) according to the present invention, particle optimization was performed according to the lipid composition ratio in an anhydrous ethanol solution.

[0056] The particle sizes of nanoparticles synthesized under synthetic weight ratios of DPPC and DSPE-PEG2000 of 10:0, 9:1, 2.3:1, and 1:1 were measured. The particle sizes of the generated particles are shown in Figure 2. As shown in Figure 2, the particle sizes of the generated particles under synthetic weight ratios of 10:0, 9:1, 2.3:1, and 1:1 were confirmed to be 130 nm and 90 nm, respectively.

[0057] Furthermore, the particle size of the particles produced under synthetic blending weight ratios of 2.3:1 and 1:1 was 30 nm, and it was confirmed that the particle size was particularly homogeneous under the 1:1 synthetic blending weight ratio condition. Therefore, it can be seen that small, uniform nanoparticles are produced under synthetic blending weight ratios of 2.3:1 to 1:1.

[0058] Furthermore, Figure 3 shows the results of observing the morphology of the nanoparticles using TEM. As shown in Figure 3, it was confirmed that when the combined weight ratio of DPPC and DSPE-PEG2000 was 10:0 and 9:1, the particle size was not uniform, while when it was 7:3 and 5:5, the particle size was small and uniform. [Examples]

[0059] Synthesis of hybrid nanoparticles at various molecular weights of DSPE-PEG Using the vortex micro-vortex apparatus of Example 1, DSPE-PEG-containing hybrid nanoparticles (PHNPs) with various PEG molecular weights were fabricated.

[0060] PLGA was prepared as a solution of anhydrous acetonitrile (ACN), and DPPC and DSPE-PEG (550, 2000, and 5000) were prepared as solutions of anhydrous ethanol. The synthesis was carried out using a vortex micro-vortex apparatus with a combined weight ratio of DPPC to DSPE-PEG of 2.3:1. The final synthesized product was purified by deadwater filtration (DW).

[0061] Subsequently, the particle sizes of DSPE-PEGs with various PEG molecular weights were compared and are shown in Figure 4. As shown in Figure 4, it was confirmed that DSPE-PEG550-containing hybrid nanoparticles (PHNP-PEG550) produced non-uniform particles of 50 nm, while DSPE-PEG5000-containing hybrid nanoparticles (PHNP-PEG5000) produced particles of 27 nm. Furthermore, it was confirmed that DSPE-PEG2000-containing hybrid nanoparticles (PHNP-PEG2000) produced small, uniform particles of 18 nm.

[0062] Furthermore, as mentioned above, Figure 5 shows the results of observing the morphology of the generated hybrid nanoparticles using TEM. As shown in Figure 5, it can be seen that the particle size and morphology of the generated nanoparticles change depending on the molecular weight of PEG. In the case of PHNP-PEG550, the particle size was non-uniform, and measurements by DLS showed that the particle size ranged from 30 to 80 nm, while in the case of PHNP-PEG2000, it was confirmed that the particle size was uniform at 20 nm.

[0063] Furthermore, as mentioned above, to confirm the stability of the generated hybrid nanoparticles, the particle size was checked by DLS after 3 days. The results are shown in Figure 6. As shown in Figure 6, after 3 days, there was no change in the particle size of PHNP-PEG2000 and PHNP-PEG5000, confirming their stability.

[0064] Therefore, it can be seen that small, uniform, and stable particles can be obtained with PHNP-PEG2000 and PHNP-PEG5000. [Examples]

[0065] Synthesis of apolipoprotein-containing hybrid nanoparticles Using a vortex micro-vortex apparatus, polymer-lipid hybrid nanoparticles (PHNP-PEG2000) containing DPPC, DSPE-PEG2000, and PLGA were fabricated, and subsequently, apolipoprotein E3 (ApoE3)-containing hybrid nanoparticles (PHNP-E3) were fabricated. A schematic diagram of this process is shown in Figure 7.

[0066] Specifically, PLGA was prepared as an anhydrous acetonitrile (ACN) solution, and DPPC and DSPE-PEG2000 were prepared as anhydrous ethanol solutions. The synthesis ratio of DPPC:DSPE-PEG2000 was set to 2.3:1, and the synthesis was carried out using a vortex microcurrent apparatus. The final solution was then immediately purified by DW. Subsequently, the synthesized PHNP-PEG2000 and ApoE3 were injected into each inlet of the vortex microcurrent apparatus. Here, the weight ratio of PHNP-PEG2000 to ApoE3 was set to 4:1, and the Reynolds number was set to 250. The finally synthesized ApoE3-containing hybrid nanoparticles (PHNP-E3) were purified using an Amicon filter (MWCO 50kDa).

[0067] Figure 8 shows the results of DLS analysis of the particle sizes of PHNP-PEG2000 without ApoE3 and PHNP-E3 containing ApoE3. As shown in Figure 8, PHNP-PEG2000 was found to have a particle size of 21 nm, and PHNP-E3 was found to have a particle size of 24 nm. Furthermore, BCA assay analysis of the amount of ApoE3 during the purification process confirmed that 70% of the ApoE3 was contained in the final formed nanoparticles.

[0068] Furthermore, as shown in Figure 9, when the PHNP-E3 synthesized as described above was observed by TEM, a core-shell structure with a core inside the nanoparticle was observed.

[0069] Therefore, it can be seen that a small, uniform PHNP-E3 having a core-shell can be obtained by the above method. [Examples]

[0070] Synthesis of apolipoprotein-containing hybrid nanoparticles at each Reynolds number Using a vortex micro-vortex apparatus, DPPC, DSPE-PEG2000, and PLGA-containing hybrid nanoparticles (PHNP-PEG2000) were synthesized in the same manner as in Figure 7, and then ApoE3 was added to finally synthesize hybrid nanoparticles (PHNP-E3).

[0071] Similar to Example 4, PLGA was prepared as an anhydrous acetonitrile (ACN) solution, and DPPC and DSPE-PEG2000 were prepared as anhydrous ethanol solutions. The compounds were synthesized using a vortex microcurrent apparatus, and the final solution was purified by DW. The synthesized hybrid nanoparticles (PHNP-PEG2000) and ApoE3 protein were injected into each inlet according to the synthesis ratio. Here, the ratio of PHNP-PEG2000 to ApoE3 was 10:1.

[0072] PHNP-E3 particles were synthesized at various Reynolds numbers, and the particle size distribution was confirmed by DLS. The results are shown in Figure 10. As shown in Figure 10, under conditions where the Reynolds number was less than 50 (Re 10), the average particle size was 158 nm, and the measured particles were relatively larger compared to those under other conditions. This is because, under excessively slow flow velocity conditions, normal PHNP-E3 was not synthesized, and E3 was adsorbed onto the surface of PHNP-PEG2000, forming aggregates.

[0073] Furthermore, it was confirmed that PHNP-E3 exhibiting a normal particle size distribution could be synthesized under conditions of a Reynolds number of 300.

[0074] The mixing efficiency in a vortex micro-vortex device increases proportionally to the Reynolds number, and then, upon reaching the highly mixed regime, the mixing efficiency ceases to increase further and converges to its maximum value (Non-Patent Literature 2). Therefore, there is no need to increase the Reynolds number after reaching the highly mixed regime. Furthermore, an excessive Reynolds number negatively affects the three-dimensional structure of proteins. For example, in insulin, a shear rate of 200 s² -1 Or shear stress of 1000 dyne / cm 2 It is known that structural changes and aggregate formation occur based on this (Non-Patent Document 3).

[0075] In other words, under conditions where the Reynolds number is 300, the actual flow rate is 14.14 mL / min, and flow rate conditions higher than this place excessive pressure on the microvortex device and the protein. Therefore, it is preferable to use a Reynolds number of 50 to 300 as the condition for the present invention. [Examples]

[0076] Optimization of the synthesis and formulation ratio of apolipoprotein-containing hybrid nanoparticles PHNP-PEG2000 and ApoE3 were synthesized by changing the synthesis ratio (20:1, 10:1, 5:1, 2:1, 1:1, 0.5:1). In this study, 1000 μg of PHNP-PEG2000 was used, and the amount of ApoE3 is shown in Table 1.

[0077] [Table 1]

[0078] Similar to Example 4, PLGA was prepared as an anhydrous acetonitrile (ACN) solution, and DPPC and each DSPE-PEG2000 were prepared as anhydrous ethanol solutions. The mixtures were synthesized using a vortex microcurrent apparatus, and the final solution was then purified by DW. PHNP-PEG2000 and ApoE3 were injected into each inlet at the synthesis ratios shown in Table 1. The particle sizes of the resulting particles at each synthesis ratio of PHNP-PEG2000 and ApoE3 (20:1, 10:1, 5:1, 2:1, 1:1, 0.5:1) were confirmed by DLS. As shown in Table 1, PHNP-E3(20:1) has a particle size of 60.3 nm, PHNP-E3(10:1) has 64.2 nm, PHNP-E3(5:1) has 58.5 nm, and PHNP-E3(2:1) has 52.9 nm, and it was confirmed that all of them are stable with a polydispersity index (PDI) of 0.18 or less. A PDI value closer to 0 indicates that the nanoparticles exist with uniform particle size, and generally, nanoparticles have a PDI value of 0.3 to 0.4.

[0079] In contrast, nanoparticles (PHNP-E3(1:1)) had a particle size of 47.6 nm, and PHNP-E3(0.5:1) had a particle size of 27.7 nm. The PDI was found to be greater than 0.18, indicating an increase compared to the aforementioned particles.

[0080] Furthermore, the amount of ApoE3 contained in PHNP-E3 was confirmed using the BCA assay, a protein quantification method, for each synthetic blend weight ratio. As shown in Table 1 and Figure 11, it was confirmed that PHNP-E3(20:1) contained 31.4 μg of ApoE3, PHNP-E3(10:1) contained 55.6 μg of ApoE3, PHNP-E3(5:1) contained 112 μg of ApoE3, PHNP-E3(2:1) contained 231 μg of ApoE3, PHNP-E3(1:1) contained 545 μg of ApoE3, and PHNP-E3(0.5:1) contained 1100 μg of ApoE3. Therefore, it was confirmed that the amount of ApoE3 contained in PHNP-E3 is concentration-dependent according to the synthetic blend weight ratio.

[0081] Furthermore, while PHNP-PEG-2000 contains PEG-2000 in its nanoparticle shell, it was confirmed that when apolipoproteins are added during synthesis, the amount of apolipoproteins added stably in PHNP-PEG-2000 increases proportionally to the amount of apolipoproteins present. This is something that cannot be achieved with conventional synthesis methods and is obtained by adjusting the Reynolds number using a vortex micro-vortex device, which physically pushes the apolipoproteins into PHNP-PEG-2000 in an inelastic collision-like manner. The resulting PHNP-E3 is small, uniform, and stable. [Examples]

[0082] Method for synthesizing apolipoprotein (ApoA1)-containing hybrid nanoparticles Using a vortex micro-vortex apparatus, ApoA1-containing hybrid nanoparticles (PHNP-A1) were synthesized in the same manner as shown in Figure 12.

[0083] Specifically, PLGA was prepared as a solution in anhydrous acetonitrile (ACN), and DPPC and each DSPE-PEG2000 were prepared as a solution in anhydrous ethanol. The DPPC:DSPE-PEG2000 mixture was set to 2.3:1, and the synthesis was carried out using a vortex micro-vortex apparatus. Subsequently, the final solution was purified by DW to produce PHNP-PEG2000.

[0084] As described above, the synthesized PHNP-PEG2000 and apolipoprotein A1 (ApoA1) were injected into each inlet of the vortex microcurrent apparatus. Here, the Reynolds number was set to 250, and the finally synthesized ApoA1-containing hybrid nanoparticles (PHNP-A1) were purified using an Amicon filter (MWCO 50kDa).

[0085] Figure 13 shows the results of DLS analysis of the particle sizes of hybrid nanoparticles without ApoA1 (PHNP-PEG2000) and hybrid nanoparticles containing ApoA1 (PHNP-A1). As shown in Figure 13, PHNP-PEG2000 was found to have a particle size of 21 nm, and PHNP-A1 was found to have a particle size of 25 nm.

[0086] Furthermore, a BCA assay was performed to quantify ApoA1 during the purification process, and it was confirmed that 26% of ApoA1 was present in PHNP-PEG2000.

[0087] Furthermore, TEM observation of the morphology of PHNP-A1 revealed that, as shown in Figure 14, PHNP-A1 has a core-shell structure with a core inside. [Examples]

[0088] Confirmation of intracellular delivery of apolipoprotein (ApoA1)-containing hybrid nanoparticles. The intracellular delivery of PHNP-PEG2000 and PHNP-A1, labeled with rhodamine, to HAECs (P4) was observed using a confocal laser scanning microscope (CLSM).

[0089] Rhodamine equivalent to 30% of the mass of PLGA was added to the PLGA solution. HAEC cells were treated with PHNP-PEG2000 and PHNP-A1, respectively, and then cultured for 24 hours. The cells were then fixed with a 4% paraformaldehyde solution. Subsequently, the cells were stained with DAPI and observed using a confocal laser scanning microscope.

[0090] Images of intracellular delivery of PHNP-PEG2000 and PHNP-A1 are shown in Figures 15 and 16. As shown in Figures 15 and 16, it was confirmed that PHNP-A1 was delivered to HAEC cells in greater quantities than PHNP-PEG2000 due to its intracellular entry effect via the ApoA1 receptor.

[0091] Furthermore, Figure 17 shows the results of quantifying the fluorescence intensity of the images in Figures 15 and 16. Comparing the fluorescence intensities, it can be seen that PHNP-A1 is delivered to HAEC cells more than six times more efficiently than PHNP. In other words, PHNP-A1 has a superior effect on intracellular delivery. [Examples]

[0092] Comparative evaluation of hybrid synthesis methods containing apolipoprotein (ApoA1). Similar to Example 7, ApoA1-containing PHNP-A1 (SMR) was synthesized using a spiral microvortex flow (SMR) apparatus.

[0093] Furthermore, ApoA1-containing PHNP-A1(BT) was synthesized using a general bench-top (BT) method without employing the microvortex device of the present invention. A general magnetic stirring method was used for the bench-top method.

[0094] In the process of synthesizing PHNP-A1(SMR) and PHNP-A1(BT), the combined weight ratio of PHNP-PEG2000 to ApoA1 was set to 2:1. The results are shown in Figure 18.

[0095] As shown in Figure 18, PHNP-A1(SMR) showed an increased particle size compared to PHNP-PEG2000, while PHNP-A1(BT) was found to have a particle size similar to that of PHNP-PEG2000.

[0096] Furthermore, the polydispersity index (PDI) results also confirmed that PHNP-A1(SMR) had a lower PDI value than PHNP-A1(BT), indicating that PHNP-A1(SMR) has a more uniform particle size.

[0097] PHNP-2000 and ApoA1 were synthesized using weight ratios of 50:1, 10:1, and 2:1 to produce PHNP-A1(SMR) and PHNP-A1(BT). The results of ApoA1 quantification using ELISA are shown in Figure 19 as yield and in Figure 20 as yield.

[0098] As shown in Figures 19 and 20, when PHNP-A1(SMR) and PHNP-A1(BT) were synthesized in the same weight ratio of the synthetic formulation, PHNP-A1(SMR) was found to have higher values ​​in terms of ApoA1 yield and yield. Therefore, it can be seen that the synthesis method using a microvortex apparatus when incorporating apolipoproteins into nanoparticles is exceptionally superior.

[0099] Furthermore, human intrahepatic biliary epithelial cells (iHBECs) were treated with PHNP-2000, PHNP-A1(BT), and PHNP-A1(SMR), respectively. Confocal microscope images were taken 24 hours later, and the fluorescence intensity was quantified. As shown in Figure 21, fluorescence imaging confirmed that PHNP-A1(SMR) was delivered to the cells in the most abundant amounts, more than twice as much as PHNP-A1(BT).

[0100] This is because, as confirmed in Figures 19 and 20, PHNP-A1(SMR) contains a larger amount of apolipoprotein than PHNP-A1(BT).

[0101] In other words, the method using a microvortex apparatus for adding proteins to nanoparticles is significantly superior to conventional synthesis methods. [Examples]

[0102] Method for synthesizing apolipoprotein (ApoE3 and ApoA1)-containing hybrid nanoparticles Similar to Example 1, PHNP-PEG2000 containing DPPC, DSPE-PEG2000, and PLGA was prepared using a vortex micro-vortex apparatus, and then PHNP-E3 / A1 was synthesized by including all of ApoA1 and ApoE3.

[0103] To prepare PHNP-E3 / A1 containing both ApoE3 and ApoA1, ApoE3 and ApoA1 were dissolved in PBS (phosphate-buffered saline) in the weight ratios shown in Table 2 (ApoE3 (mg) / ApoA1 (mg)) and prepared as a mixed aqueous solution.

[0104] [Table 2]

[0105] A mixed aqueous solution of PHNP-PEG2000 and ApoE3 / ApoA1 was injected into each inlet of a microvortex apparatus to synthesize PHNP-E3 / A1. The Reynolds number was set to 250. The final synthesized PHNP-E3 / A1 was purified by centrifugation using an Amicon filter (MWCO 50kDa).

[0106] The particle size and polydispersity index (PDI) of PHNP-E3 / A1 were confirmed by DLS in a PBS (1% trehalose) buffer environment. As shown in Figure 22, PHNP-E3(500 / 0) had a particle size of 85.55±1.77 nm and a PDI of 0.23±0.03, PHNP-E3 / A1(250 / 25) had a particle size of 70.38±5.31 nm and a PDI of 0.08±0.03, PHNP-E3 / A1(250 / 50) had a particle size of 68.74±3.20 nm and a PDI of 0.09±0.02, and PHNP-E3 / A1(250 / 100) had a particle size of 96.58±12.16 nm and a PDI of 0.09±0.02. The PDI values ​​were 0.26±0.01, and it was confirmed that PHNP-E3 / A1(250 / 250) had a particle size of 73.03±5.45 nm and a PDI of 0.23±0.01, while PHNP-A1(0 / 500) had a particle size of 75.55±2.88 nm and a PDI of 0.26±0.03. Therefore, the particle size of less than 100 nm and the PDI results of less than 0.3 indicate that the generated PHNP-E3 / A1 contains a homogeneous and highly dispersible ApoE3 and ApoA1.

[0107] Figure 23 shows the results of DLS testing the dispersion stability of PHNP-E3 / A1 synthesized from ApoE3 and ApoA1 in their respective weight ratios under 4°C and PBS (1% trehalose) solution conditions. As shown in Figure 23, it was confirmed that by 14 days, all PHNP-E3 / A1 particles had a particle size of less than 80 nm and a PDI of less than 0.3. This means that PHNP-E3 / A1 maintains high dispersion stability for 14 days without any change in particle size.

[0108] Furthermore, fluorescence resonance energy transfer (FRET) confirmed that ApoE3 and ApoA1 are simultaneously introduced into PHNP-PEG2000 nanoparticles. Specifically, to confirm the fluorescence intensity, an Alexa Fluor 488 fluorescent molecule was bound to ApoE3 and an Alexa Fluor 568 fluorescent molecule was bound to ApoA1. When Alexa Fluor 488 bound to ApoE3 is given light energy at 470 nm, which is the wavelength at which it emits fluorescence, the Alexa Fluor 568 fluorescent molecule bound to ApoA1 emits light energy at 600 nm, which is its fluorescence wavelength. This indicates that ApoE3 and ApoA1 are simultaneously present within the nanoparticle.

[0109] To confirm this, Figure 24 shows the fluorescence wavelengths emitted when PHNP-PEG2000, PHNP-E3, PHNP-A1, and PHNP-E3 / A1 were given an energy of 470 nm. As shown in Figure 24, the control group PHNP-PEG2000 showed almost no fluorescence intensity, while PHNP-E3 showed the highest intensity at 520 nm because Alexa Fluor 488 emitted fluorescence. Furthermore, PHNP-A1 emitted fluorescence at 600 nm, but it was confirmed that the intensity was weaker than that of PHNP-E3. In PHNP-E3 / A1, it was confirmed that the fluorescence intensity of Alexa Fluor 488 bound to ApoE3 decreased compared to PHNP-E3 (520 nm, dark gray arrow), and the fluorescence intensity of Alexa Fluor 568 bound to ApoA1 increased compared to PHNP-A1 (600 nm, light gray arrow). By comparing the fluorescence spectra of PHNP-E3 / A1 with those of PHNP-E3 and PHNP-A1, respectively, the FRET effect was demonstrated by a decrease in the fluorescence intensity of Alexa Fluor 488 and an increase in the fluorescence intensity of Alexa Fluor 568, thus confirming the simultaneous presence of two types of proteins within PHNP-E3 / A1.

Claims

1. In a vortex micro-vortex device having a first inlet, a second inlet, and an outlet, the steps include injecting phospholipids into the first inlet and injecting polymers into the second inlet, The steps include mixing phospholipids and polymers using swirling microvortices, In a vortex microvortex device, the steps include injecting hybrid nanoparticles into the first injection port and injecting protein into the second injection port, The process includes a step in which proteins are added to hybrid nanoparticles by a swirling microvortex, The phospholipids are DPPC and DSPE-PEG, and the polymer is PLGA (poly(lactic acid / glycolic acid copolymer)), characterized in that A method for producing protein-containing hybrid nanoparticles.

2. A method for producing protein-containing hybrid nanoparticles according to claim 1, characterized in that the Reynolds number in the vortex microvortex device is 50 to 300.

3. A method for producing protein-containing hybrid nanoparticles according to claim 1, characterized in that the protein is an apolipoprotein or a polymer having amphoteric properties.

4. The method for producing protein-containing hybrid nanoparticles according to claim 1, characterized in that the apolipoprotein is at least one selected from the group consisting of apolipoproteins A1, A2, E2, E3, J, and M.

5. A method for producing protein-containing hybrid nanoparticles according to claim 3, characterized in that the synthetic blending weight ratio of the hybrid nanoparticles to the apolipoprotein is 20:1 to 0.5:

1.

6. A method for producing protein-containing hybrid nanoparticles according to claim 3, characterized in that the synthetic blending weight ratio of the hybrid nanoparticles to the apolipoprotein is 20:1 to 2:

1.

7. A method for producing protein-containing hybrid nanoparticles according to claim 1, further comprising the step of recovering protein-containing nanoparticles from the outlet.

8. A method for producing protein-containing hybrid nanoparticles according to claim 1, characterized in that the molecular weight of PEG in the DSPE-PEG is 2000 to 5000.

9. A method for producing protein-containing hybrid nanoparticles according to claim 1, characterized in that the synthetic blending weight ratio of DPPC and DSPE-PEG is 2.3:1 to 1:

1.

10. A method for producing protein-containing hybrid nanoparticles according to claim 1, characterized in that the hybrid nanoparticles are polymer-lipid hybrid nanoparticles (PHNPs).

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