Method for synthesizing mixed nanoparticles containing apolipoprotein

Abstract

The invention relates to a synthetic method of mixed nanoparticles containing apolipoprotein. Specifically, the present invention relates to a method for synthesizing mixed nanoparticles, which may contain proteins having various functions, including apolipoprotein, by using a vortex micro-vortex device, and fine, uniform and stable nanoparticles having various physiological activities can be prepared by the preparation method of the present invention.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing hybrid nanoparticles containing apolipoprotein. Specifically, the present invention relates to a method for synthesizing hybrid nanoparticles using a vortex micro-vortex device, wherein the hybrid nanoparticles may contain proteins with various functions including apolipoprotein, and the preparation method of the present invention may be used to prepare small, uniform, and stable nanoparticles with various physiological activities. Background Art

[0002] Nanoparticles are particles with a size of less than a micron. They can move freely in the body due to their size and can be given various physical properties depending on the composition of the material, so they are attracting attention as the next generation of drug delivery carriers.

[0003] Nanoparticles used for drug delivery are composed of various components such as phospholipids and polymers according to the needs, and have different characteristics according to the components. Nanoparticles made of inorganic substances are mainly used for diagnostic purposes, and polymer nanoparticles are used as drug delivery carriers to mainly regulate the release rate of drugs and the circulation time in the body. Phospholipid nanoparticles are amphiphilic, easy to self-assemble, and have various molecular sieves, so various types of phospholipid nanoparticles are being studied as drug delivery carriers.

[0004] In recent years, a kind of hybrid nanoparticles combining the advantages of these nanoparticles is being studied. However, the existing method for preparing nanoparticles is mainly composed of non-standardized multi-step processes, such as nanoprecipitation and emulsification-based solvent evaporation, so there are many difficulties in using the existing method for preparing nanoparticles to mass produce the hybrid nanoparticles of uniform size. Particularly, in nanoparticles, polymers such as functional proteins are included, and due to the complexity of synthesis, it is difficult to synthesize once, and when prepared through many processes, it loses its uniformity and stability, bringing many difficulties to mass production.

[0005] The present inventors have completed the present invention by studying a method for synthesizing functional proteins in polymer-lipid hybrid nanoparticles (PHNPs) using poly(lactic-co-glycolic acid) (PLGA), which is a polymer that has biodegradability and biocompatibility and can protect drugs from degradation.

[0006] Prior art literature

[0007] Non-patent literature

[0008] 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. Summary of the invention

[0009] Technical issues

[0010] The present invention aims to provide a method for preparing mixed nanoparticles containing a mixture of a hydrophilic substance and a hydrophobic substance by mixing a phospholipid and a polymer in a vortex-type micro-vortex.

[0011] An object of the present invention is to provide a method for preparing hybrid nanoparticles containing a protein, which comprises the step of adding the protein to the hybrid nanoparticles by causing the protein to collide with the hybrid nanoparticles in a vortex-type micro-vortex.

[0012] Furthermore, an object of the present invention is to provide hybrid nanoparticles containing protein prepared by the method.

[0013] Technical Solution

[0014] The present invention provides a method for preparing mixed nanoparticles containing protein, which comprises: in a vortex-type micro-vortex device including a first injection port, a second injection port and an outlet, injecting mixed nanoparticles into the first injection port and injecting proteins into the second injection port; and adding proteins into the mixed nanoparticles by means of the vortex-type micro-vortex.

[0015] In one embodiment, the Reynolds number in the vortex micro-vortex device may be 50 to 300.

[0016] In one embodiment, the protein may be an apolipoprotein or a polymer having amphiphilic properties.

[0017] In one embodiment, the apolipoprotein may be one or more selected from the group consisting of apolipoprotein A1, A2, E2, E3, J and M.

[0018] In one embodiment, the synthetic compounding weight ratio of the hybrid nanoparticles to the apolipoprotein may be 20:1 to 0.5:1, preferably 20:1 to 2:1.

[0019] In one embodiment, the preparation method may further include a step of recovering the protein-containing nanoparticles from the outlet.

[0020] In one embodiment, the hybrid nanoparticles can be prepared by a preparation method comprising the following steps: in the vortex micro-vortex device, injecting phospholipids into the first injection port and injecting polymers into the second injection port; and mixing the phospholipids and polymers by vortex micro-vortex.

[0021] The phospholipids may be selected from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-distear ... 1,2-diazaoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diazaoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanol amine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylformamido)ethyl]-3,4-di[oleyl]-benzamide) (VL-5), dioctadecylamide glycol spermine 4 trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE) 、1,2-dimyristyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanium bromide (GAP-DLRIE), N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (EthylPC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesterol-spermine (GL67), 1,One or more of the group consisting of 2-dioleoyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA, preferably DPPC and DSPE-PEG, more preferably DPPC and DSPE-PEG 2000 to 5000.

[0022] In one embodiment, the ratio of dipalmitoylphosphatidylcholine to DSPE-PEG2000 may be 2.3:1 to 1:1.

[0023] In one embodiment, the polymer may be poly(lactic-co-glycolic acid) (PLGA).

[0024] In one embodiment, the hybrid nanoparticles may be polymer-lipid hybrid nanoparticles (PHNPs).

[0025] The present invention provides hybrid nanoparticles containing proteins prepared by the preparation method.

[0026] The present invention provides a method for preparing mixed nanoparticles containing proteins, which comprises: a first step of preparing mixed nanoparticles, comprising: in a vortex micro-vortex device comprising a first injection port, a second injection port and an outlet, injecting phospholipids into the first injection port and injecting polymers into the second injection port; and a step of mixing phospholipids and polymers by vortex micro-vortex; and a second step of preparing mixed nanoparticles containing proteins, comprising: in a vortex micro-vortex device comprising a first injection port, a second injection port and an outlet, injecting the obtained mixed nanoparticles into the first injection port and injecting proteins into the second injection port.

[0027] In one embodiment, the protein may be an apolipoprotein or a polymer having amphiphilic properties.

[0028] In one embodiment, the synthetic compounding weight ratio of the hybrid nanoparticles to the apolipoprotein may be 20:1 to 2:1.

[0029] In one embodiment, the polymer may be poly(lactic-co-glycolic acid) (PLGA).

[0030] In one embodiment, the hybrid nanoparticles may be polymer-lipid hybrid nanoparticles (PHNPs).

[0031] Effects of the Invention

[0032] The polymer-lipid hybrid nanoparticle (PHNP) prepared by the preparation method of the present invention contains both hydrophilic and hydrophobic substances, and thus has a morphology in which the hydrophobic substance surrounds the center of the nanoparticle made of a polymer, and can effectively encapsulate and deliver a high concentration of hydrophobic drugs inside.

[0033] Furthermore, the surface, the intermediate shell and the core of the hybrid nanoparticles of the present invention can be used as materials for delivering various drugs. For example, the hybrid nanoparticles of the present invention can be used as effective delivery carriers for anionic drugs such as genes.

[0034] Furthermore, polymers such as polyethylene glycol (PEG) on the surface of hybrid nanoparticles can prevent foreign substances in the human body from being adsorbed onto the particle surface or removed by immune cells, thereby achieving safer drug delivery. Therefore, it can be used for intracellular drug delivery and other purposes to help effective treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the synthesis process of hybrid nanoparticles containing both ApoE3 and ApoA1 (PHNP-E3 / A1) and the morphology of PHNP-E3 / A1.

[0036] Figure 2 This is a graph comparing the sizes of hybrid nanoparticles (PHNP-2000) according to the synthetic blending weight ratio of DPPC and DSPE-PEG2000.

[0037] Figure 3 Based on Figure 2 TEM (transmission electron microscope) photograph of hybrid nanoparticles (PHNP-2000) obtained by synthesizing DPPC and DSPE-PEG2000 with a weight ratio of 1.547 W / m2.

[0038] Figure 4 is a graph comparing the sizes of hybrid nanoparticles (PHNPs) according to the PEG molecular weight of DSPE-PEG.

[0039] Figure 5 Based on Figure 4 TEM images of hybrid nanoparticles (PHNPs) obtained with DSPE-PEG and PEG molecular weight.

[0040] Figure 6 To measure Figure 4 Figure 3 is a graph showing the stability of each nanoparticle by comparing the sizes of the hybrid nanoparticles (PHNPs) synthesized on the day of synthesis and 3 days after synthesis.

[0041] Figure 7 Schematic diagram of the process of synthesizing hybrid nanoparticles (PHNP-E3) containing ApoE3 using a vortex micro-vortex device and the morphology of PHNP-E3.

[0042] Figure 8 A chart comparing the sizes of PHNP-PEG2000 and PHNP-E3.

[0043] Fig. 9 TEM image of PHNP-E3.

[0044] Fig.10 This is a graph comparing the sizes of PHNP-E3 synthesized under various Reynolds number conditions.

[0045] Fig.11 It is a graph showing the production amount of ApoE3 contained in PHNP-E3.

[0046] Fig.12 Schematic diagram of the synthesis process of hybrid nanoparticles (PHNP-A1) containing ApoA1 and the morphology of the hybrid nanoparticles (PHNP-A1).

[0047] Fig.13 A chart comparing the sizes of PHNP-PEG2000 and PHNP-A1.

[0048] Fig.14 TEM image of PHNP-A1.

[0049] Fig.15 The fluorescence photographs show that hybrid nanoparticles (PHNP-PEG2000) labeled with rhodamine were delivered into HAECs cells.

[0050] Fig.16 Fluorescence photograph showing the delivery of hybrid nanoparticles (PHNP-A1) labeled with rhodamine into HAECs cells.

[0051] Fig.17 For quantitative comparison Fig.15 and Fig.16 Graph of rhodamine fluorescence intensity.

[0052] Fig.18Figure 3 is a graph comparing the size and polydispersity index (PDI) of PHNP-A1(SMR), PHNP-A1(BT) synthesized by using or not using a vortex microfluidization device.

[0053] Fig.19 This is a graph comparing the production amounts of ApoA1 by ELISA (enzyme-linked immunosorbent assay) after synthesizing PHNP-A1(SMR) and PHNP-A1(BT) according to the synthesis weight ratio of ApoA1 to PHNP.

[0054] Fig. 20 This is a graph comparing the yields of ApoA1 using ELISA after synthesizing PHNP-A1(SMR) and PHNP-A1(BT) according to the synthesis weight ratio of ApoA1 to PHNP.

[0055] Fig.21 Fluorescence photographs and graphs of fluorescence were provided for quantitative comparison of PHNP-A1(SMR) and PHNP-A1(BT) delivered into iHBECs.

[0056] Fig. 22 It is a size distribution chart of PHNP-E3 / A1 synthesized according to each blending weight of ApoE3 and ApoA1.

[0057] Fig.23 The size and PDI results on the day of synthesis, 1 day, 3 days, 7 days, and 14 days after synthesis show the stability of PHNP-E3 / A1.

[0058] Fig.24 It is a graph showing the fluorescence wavelengths of PHNP-PEG2000, PHNP-E3, PHNP-A1, and PHNP-E3 / A1. DETAILED DESCRIPTION

[0059] Hereinafter, the embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily implement the present invention. However, the present invention can be implemented in various forms and is not limited to the embodiments and examples described herein.

[0060] Throughout the specification of the present invention, when a part is described as “comprising” a certain structural element, it does not mean that other structural elements are excluded but means that other structural elements may be further included unless there is a particular description to the contrary.

[0061] The term "hybrid nanoparticles" used in the present invention refers to nanoparticles formed by the coexistence of hydrophilic substances and hydrophobic substances, preferably, polymer-lipid hybrid nanoparticles (PHNPs), also called "PHNPs".

[0062] The term "PLGA" used in the present invention refers to poly(lactic-co-glycolic acid), which is a biodegradable and biocompatible polymer.

[0063] The term "DPPC" used in the present invention refers to dipalmitoylphosphatidylcholine, which is a type of lipid.

[0064] The term "DSPE-PEG" used in the present invention refers to a lipid in which polyethylene glycol (PEG) is bonded to distearoylphosphoethanolamine. Preferably, the molecular weight of the polyethylene glycol may be 2,000 to 5,000.

[0065] The term "polymer with amphiphilic properties" used in the present invention refers to proteins that are both hydrophobic and hydrophilic, and examples include glycophorin, rhodopsin, CD36 (cluster of differentiation 36), seipin, glucose permease, cytochrome c, cupredoxins, high potential iron protein, adrenoxin reductase and flavoprotein, etc.

[0066] The term "phospholipid" used in the present invention can be specifically selected from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (DPPC), Palmitoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diazaoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphospholipids choline, distearoylphosphatidylethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylcarboxamido)ethyl 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleyl-3-trimethylammonium propane (DOTAP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORI E), 1,2-dimyristyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanium bromide (GAP-DLRIE), N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesterol-spermine (GL67), 1,One or more of the group consisting of 2-dioleoyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA, but not limited thereto.

[0067] The term "apolipoprotein E" refers to a mammalian protein encoded by the APOE gene or its functional variant. In a preferred embodiment, apolipoprotein E is a human protein encoded by the human APOE gene on chromosome 19. Apolipoprotein E can be one of the isoforms of the APOE gene product, such as apolipoprotein E2 ("APOE2"), apolipoprotein E3 ("APOE3"), and apolipoprotein E4 (APOE4). APOE is a polymorph with three major alleles (ε2, ε3, and ε4). Any allele can be used in various embodiments of the present invention. Its "functional variant" refers to a variant of a mammalian protein encoded by the APOE gene that retains the same or similar biological function as the APOE gene product. In some cases, compared with the protein encoded by the human APOE gene, the functional variant includes amino acid insertions, deletions, and / or substitutions. In some cases, the functional variant is a protein fragment encoded by the human APOE gene.

[0068] In some embodiments, apolipoprotein E3 is the protein disclosed in GenBank under accession number ARQ79461.1 or a protein having at least 95% sequence identity thereto, preferably at least 98% or 99% sequence identity thereto.

[0069] In some embodiments, the apolipoprotein E in the hybrid nanoparticles is a recombinant protein produced by genetic engineering, or a synthetic protein produced by chemical synthesis.

[0070] The term "apolipoprotein A1" refers to 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. Its "functional variant" refers to a variant of the mammalian protein encoded by the APOA1 gene, which retains the same or similar biological function as the APOA1 gene product. In some cases, the functional variant includes 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 protein fragment encoded by the human APOA1 gene.

[0071] In some embodiments, apolipoprotein A1 is the protein disclosed in GenBank under accession number AAS 68227.1 or a protein having at least 95% sequence identity thereto, preferably at least 98% or 99% sequence identity thereto.

[0072] In some embodiments, the apolipoprotein A1 in the hybrid nanoparticles is a recombinant protein produced by genetic engineering, or a synthetic protein produced by chemical synthesis.

[0073] The term "apolipoprotein E" used in the present invention is intended to include fragments and functional variants thereof.

[0074] The term "vortex-type micro-vortex" used in the present invention refers to a situation in which a small fluid flows while performing a rotational motion in a fluid flow.

[0075] The term "micro-vortex device" used in the present invention refers to a device including a microchannel, etc., wherein the microchannel is configured to enable a fluid to flow on a substrate made of plastic including organic polymer substances, glass, metal or various materials including silicon.

[0076] Hereinafter, the present invention will be described in more detail by way of examples, but the following examples are only for illustrative purposes and are not intended to limit the scope of the present invention.

[0077] Example 1

[0078] Method for synthesizing hybrid nanoparticles containing apolipoprotein using a vortex micro-vortex device

[0079] The hybrid nanoparticles containing apolipoprotein are synthesized. Specifically, the first step is the step of synthesizing polymer-lipid hybrid nanoparticles (PHNP), and the second step is the step of including apolipoprotein in the hybrid nanoparticles, and each step uses the same swirling microvortex device. The schematic diagram of the swirling microvortex device and the synthesis method for synthesizing the hybrid nanoparticles (PHNP) of the present invention is shown in FIG. Figure 1 shown.

[0080] The vortex micro-vortex device includes two inlets and one outlet. In order to effectively mix lipids and PLGA polymers by forming a vortex micro-vortex, the device is designed by optimizing the diameter and height (see Toth et al., Robust manufacturing of lipid-polymer nanoparticles through feedback control of parallelized swirling microvortices. 2017, Lab. Chip., 17.16: 2805-2813).

[0081] In the polymer-lipid hybrid nanoparticle (PHNP) synthesis step, a phospholipid mixture DPPC (dipalmitoyl phosphatidylcholine) and DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol) is injected into one of the two inlets, and a poly(lactic-co-glycolic acid) (PLGA) polymer is injected into an inlet located on the opposite side to synthesize polymer-lipid hybrid nanoparticles (PHNP).

[0082] Then, the synthesized polymer-lipid hybrid nanoparticles (PH NP) are injected into one inlet, and apolipoprotein is injected into the opposite inlet, and then mixed, and finally hybrid nanoparticles containing apolipoprotein are synthesized.

[0083] Example 2

[0084] Synthesis of polymer-lipid hybrid nanoparticles according to the weight ratio of DPPC: DSPE-PEG2000

[0085] The vortex micro-vortex device was used to synthesize polymer-lipid hybrid nanoparticles containing lipids (DPPC and DSPE-PEG2000) and polymers (PLGA) as follows. DS PE-PEG2000 contains PEG, which can prevent plasma proteins in the human body from being adsorbed to the surface of nanoparticles or being removed by immune cells, and is therefore used to ensure the stability of nanoparticles in vivo.

[0086] A solution of PLGA dissolved in anhydrous acetonitrile (ACN) was prepared, and DPPC and DSPE-PEG2000 were prepared as anhydrous ethanol solutions. After synthesis using a vortex microfluidizer, the final solution was immediately purified with DW (deionized water).

[0087] In order to prepare the stable polymer-lipid hybrid nanoparticles (PHNPs) of the present invention, particle optimization was performed according to the lipid formulation ratio in anhydrous ethanol solution.

[0088] The size measurement results of the nanoparticles synthesized under the conditions of the synthetic compounding weight ratio of DPPC to DSPE-PEG2000 being 10:0, 9:1, 2.3:1, and 1:1 are shown in the table below. Figure 2 In. Figure 2 As shown, it was confirmed that the sizes of a plurality of particles produced under the conditions of the synthetic compounding weight ratios of 10:0, 9:1, 2.3:1, and 1:1 were 130 nm and 90 nm, respectively.

[0089] Furthermore, it was confirmed that the size of the plurality of particles produced under the conditions of the synthetic compounding weight ratio of 2.3:1 and 1:1 was 30 nm, and the size of the plurality of particles was particularly uniform under the conditions of the synthetic compounding weight ratio of 1:1. Therefore, it can be seen that small and uniform nanoparticles are produced under the conditions of the synthetic compounding weight ratio of 2.3:1 to 1:1.

[0090] Furthermore, the morphology of the nanoparticles was observed using TEM, and the results are shown in Figure 3 As described Figure 3 As shown, it was confirmed that when the weight ratio of DPPC to DSPE-PEG2000 was 10:0 and 9:1, the particle size was non-uniform, and when the weight ratio was 7:3 and 5:5, the particle size was small and uniform.

[0091] Example 3

[0092] Synthesis of hybrid nanoparticles according to the molecular weight of DSPE-PEG

[0093] The vortex microfluidic device of Example 1 was used to prepare hybrid nanoparticles (PHNPs) containing DSPE-PEG with different PEG molecular weights.

[0094] PLGA was prepared as an acetonitrile (ACN) solution, and DPPC and DSPE-PEG (550, 2000, and 5000) were prepared as an ethanol solution, and the synthesis was performed using a vortex micro-vortex device with a synthesis weight ratio of DPPC to DSPE-PEG of 2.3:1. The final synthesis product was purified by DW.

[0095] Then, the sizes of particles containing DSPE-PEG with different PEG molecular weights were compared and the results are shown in Figure 4 As described Figure 4 As shown, in the case of the mixed nanoparticles (PHNP-PEG550) containing DSPE-PEG550, it was confirmed that a plurality of non-uniform particles of 50 nm were generated, and in the case of the mixed nanoparticles (PHNP-PEG5000) containing DSPE-PEG5000, it was confirmed that a plurality of particles of 27 nm were generated. In addition, in the case of the mixed nanoparticles (PHNP-PEG2000) containing DSPE-PEG2000, it was confirmed that a plurality of small and uniform particles of 18 nm were generated.

[0096] Furthermore, the morphology of the generated hybrid nanoparticles was observed using TEM, and the results are shown in Figure 5 As described Figure 5 As shown, it can be seen that the size and morphology of the generated nanoparticles vary depending on the molecular weight of PEG. In the case of PHNP-PEG550, the size of the particles is not uniform, and the results of DLS (dynamic light scattering) measurement confirm that the size is 30 to 80 nm, and in the case of PHNP-PEG2000, a uniform particle size of 20 nm is confirmed.

[0097] In order to confirm the stability of the generated hybrid nanoparticles, the particle size was confirmed by DLS after 3 days. The results are shown in Figure 6 As described Figure 6 As shown, after 3 days, in the case of PHNP-PEG2000 and PHNP-PEG5000, the particle size was confirmed to be stable without changing.

[0098] Therefore, it can be seen that in the case of PHNP-PEG2000 and PHNP-PEG5000, small, uniform and stable particles can be obtained.

[0099] Example 4

[0100] Synthesis of hybrid nanoparticles containing apolipoprotein

[0101] A vortex micro-vortex device was used to prepare polymer-lipid hybrid nanoparticles (PHNP-PEG2000) containing DPPC, DSPE-PEG2000 and PLGA, and then hybrid nanoparticles (PHNP-E3) containing apolipoprotein E3 (ApoE3) were prepared, as shown in the schematic diagram. Figure 7 middle.

[0102] Specifically, PLGA was prepared as an anhydrous acetonitrile (ACN) solution, and DPPC and DSPE-PEG2000 were prepared as an anhydrous ethanol solution. After synthesis using a vortex micro-vortex device with a synthesis ratio of DPPC: DSPE-PEG2000 of 2.3:1, the final solution was immediately purified with DW. Then, the synthesized PHNP-PEG2000 and ApoE3 were injected into each inlet of the vortex micro-vortex device. In this case, the weight ratio of PHNP-PEG2000 and ApoE3 was set to 4:1, and the Reynolds number was set to 250. The final synthesized mixed nanoparticles (PHNP-E3) containing ApoE3 were purified using an Amicon filter (MWCO 50kDa).

[0103] The sizes of PHNP-PEG2000 without ApoE3 and PHNP-E3 containing ApoE3 were confirmed by DLS. The results are shown in Figure 8 As described Figure 8 As shown, the particle size of PHNP-PEG2000 was confirmed to be 21 nm, and the particle size of PHNP-E3 was confirmed to be 24 nm. In addition, during the purification process, the amount of ApoE3 was confirmed by BCA assay (protein quantification kit), and the result confirmed that 70% of ApoE3 was contained in the finally formed nanoparticles.

[0104] And, if Fig. 9 As shown, the result of observing the synthesized PHNP-E3 using TEM showed that the nanoparticles had a core-shell structure with a core in the middle.

[0105] Therefore, it can be seen that small and uniform PHN P-E3 with core-shell was obtained by the method.

[0106] Example 5

[0107] Synthesis of hybrid nanoparticles containing apolipoprotein according to Reynolds number

[0108] Using vortex micro-vortex device to Figure 7 The same method was used to synthesize hybrid nanoparticles (PHNP-PEG2000) containing DPPC, DS PE-PEG 2000 and PLGA, and then ApoE3 was added to finally synthesize hybrid nanoparticles (PHNP-E3).

[0109] PLGA was prepared as an acetonitrile (ACN) solution in the same manner as in Example 4, and DPPC and DSPE-PEG2000 were prepared as an ethanol solution, and the final solution was purified by DW after synthesis by a vortex micro-vortex device. The synthesized hybrid nanoparticles (PHNP-PEG2000) and ApoE3 protein were injected into each inlet according to the synthesis ratio. In this case, the ratio of PHNP-PEG2000 to ApoE3 was set to 10:1.

[0110] Particles of PHNP-E3 were synthesized at different Reynolds numbers, and the resulting particle size distribution was confirmed by DLS. The results are shown in Fig.10 As described Fig.10 As shown in the figure, under the condition of Reynolds number less than 50 (Re 10), the average particle size was 158 nm, and a relatively large number of particles were measured compared with other conditions. This may be because under the condition of too slow flow rate, normal PHNP-E3 was not synthesized, and E3 was adsorbed to the surface of PHNP-PEG2000 and formed aggregates.

[0111] Furthermore, it was confirmed that PHNP-E3 showing a normal particle size distribution was synthesized under the condition of a Reynolds number of 300.

[0112] In a vortex microfluidic device, the mixing efficiency increases proportionally with the Reynolds number, but when the highly mixed regime is reached, the mixing efficiency no longer increases and converges to a maximum value (Manon, R., et al. Optimal self-assembly of lipid nanoparticles (LNP) in a ring micromixer, Scientific reports, 12: 9483 (2022)). Therefore, after reaching the highly mixed regime, there is no need to increase the Reynolds number. Moreover, too high a Reynolds number may have a negative impact on the three-dimensional structure of the protein. For example, in the case of insulin, it is well known that structural changes and aggregates are based on 200s -1 Shear rate or 1000 dyne / cm 2of shear stress (Bekard, I., et al. The Effects of Shear Fl ow on Protein Structure and Function. Biopolymers, 95, 11, 733-745 (2011)).

[0113] Finally, under the condition of a Reynolds number of 300, the actual flow rate is 14.14 mL / min. Since flow rates above this flow rate may exert excessive pressure on the microvortex device and the protein, it can be seen that the Reynolds number of the present invention preferably has a condition of 50 to 300.

[0114] Example 6

[0115] Optimizing the synthesis ratio of hybrid nanoparticles containing apolipoprotein

[0116] PHNP-E3 was prepared by changing the synthetic compounding ratio of PHNP-PEG2000 to ApoE3 (20:1, 10:1, 5:1, 2:1, 1:1, 0.5:1). In this case, 1000 μg of PHNP-PEG2000 was used, and the amount of ApoE3 was as shown in Table 1 below.

[0117] Table 1

[0118]

[0119]

[0120] Using the same method as in Example 4, PLGA was prepared as an acetonitrile (ACN) solution, and DPPC and each DSPE-PEG2000 were prepared as an ethanol solution, and after synthesis by a vortex micro-vortex device, the final solution was purified by DW. PH NP-PEG2000 and ApoE3 were injected into each inlet at the synthesis ratio of Table 1. The particle size generated according to the synthesis ratio of PHNP-PEG2000 and ApoE3 (20:1, 10:1, 5:1, 2:1, 1:1, 0.5:1) was confirmed by DLS. As shown in Table 1, it was confirmed that the particle size of PHNP-E3 (20: 1) was 60.3 nm, the particle size of PHNP-E3 (10: 1) was 64.2 nm, the particle size of PHNP-E3 (5: 1) was 58.5 nm, and the particle size of PHNP-E3 (2: 1) was 52.9 nm, and the polydispersity index (PDI) was stable below 0.18. The closer the PDI is to 0, the more uniform the size of the nanoparticles, and the value of the nanoparticles is usually 0.3 to 0.4. The closer the PDI is to 0, the more uniform the size of the nanoparticles, and the value of the nanoparticles is usually 0.3 to 0.4.

[0121] In contrast, it was confirmed that the particle size of the nanoparticles (PHNP-E3 (1:1)) was 47.6 nm, the particle size of PHNP-E3 (0.5:1) was 27.7 nm, and the PDI exceeded 0.18, which was increased compared with the plurality of particles.

[0122] In addition, the amount of ApoE3 contained in PHNP-E3 according to the synthetic blending weight ratio was confirmed by BCA assay, which is a protein quantification method. Fig.11 As shown, 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 was contained in a concentration-dependent manner according to the synthetic compounding weight ratio.

[0123] Furthermore, PHNP-PEG-2000 contains PEG-2000 in the shell of the nanoparticle, but when synthesized by adding apolipoprotein, it is confirmed that it is stably contained in PHNP-PEG-2000 in proportion to the amount of apolipoprotein added. This cannot be obtained by existing synthesis methods, but can be obtained by adjusting the Reynolds number using a vortex micro-eddy flow device, and physically pushing the apolipoprotein into PHNP-PEG-2000, for example, by inelastic collision, and it can be seen that the obtained PHNP-E3 has small, uniform and stable particles.

[0124] Example 7

[0125] Method for synthesizing hybrid nanoparticles containing apolipoprotein (ApoA1)

[0126] Using vortex micro-vortex device to Fig.12 The same method was used to synthesize hybrid nanoparticles containing ApoA1 (PHNP-A1).

[0127] Specifically, PLGA was prepared as an acetonitrile (ACN) solution, and DPPC and each DSPE-PEG2000 were prepared as an ethanol solution. After synthesis using a vortex micro-vortex device with a mixing ratio of DPPC: DSPE-PEG2000 of 2.3:1, the final solution was purified using DW to prepare PHNP-PEG2000.

[0128] The synthesized PHNP-PEG2000 and apolipoprotein A1 (ApoA1) were injected into each inlet of a vortex microfluidizer. In this case, the Reynolds number was set to 250, and the final synthesized hybrid nanoparticles (PHNP-A1) containing ApoA1 were purified using an Amicon filter (MWCO 50 kDa).

[0129] The sizes of the hybrid nanoparticles not containing ApoA1 (PHNP-PEG2000) and the hybrid nanoparticles containing ApoA1 (PHNP-A1) were confirmed by DLS. The results are shown in Fig.13 As described Fig.13 As shown, it was confirmed that the particle size of PHNP-PEG2000 was 21 nm and the particle size of PHNP-A1 was 25 nm.

[0130] Furthermore, during the purification process, ApoA1 was quantified by BCA assay (protein quantification kit), and it was confirmed that 26% of ApoA1 was contained in PHNP-PEG2000.

[0131] Furthermore, the morphology of PHNP-A1 was observed using TEM. Fig.14 As shown, it was confirmed that PHNP-A1 has a core-shell structure having a core in the middle.

[0132] Example 8

[0133] Confirmation of intracellular delivery of hybrid nanoparticles containing apolipoprotein (ApoA1)

[0134] Confocal Laser Scanning Microscope (CLSM) was used to observe the intracellular delivery of PHNP-PEG2000 and PHNP-A1 labeled with rhodamine into HAECs (P4).

[0135] 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 cultured for 24 hours, and then fixed with 4% paraformaldehyde solution. The cells were then stained with DAPI (4',6-diamidino-2-phenylindole) and observed using a confocal laser scanning microscope.

[0136] The intracellular delivery images of PHNP-PEG2000 and PHNP-A1 are shown in Fig.15 and Fig.16 As described Fig.15 and Fig.16 As shown, it was confirmed that more PHNP-A1 was delivered into HAECs cells compared with PHNP-PEG2000 due to the effect of ApoA1 receptor entering the cells.

[0137] and, Fig.15 and Fig.16 The quantitative results of the fluorescence intensity of the images are shown in Fig.17 By comparing the fluorescence intensity results, it can be seen that the intracellular delivery of PHNP-A1 to HAEC is more than 6 times higher than that of PHNP. Finally, it can be seen that PHNP-A1 has an excellent intracellular delivery effect.

[0138] Example 9

[0139] Comparative evaluation of hybrid synthesis methods involving apolipoprotein (ApoA1)

[0140] The same method as in Example 7 was used to synthesize PHNP-A1 (SMR) containing ApoA1 using a vortex microfluidizer (SMR).

[0141] Also, PHNP-A1 (BT) containing ApoA1 was synthesized using a common bench-top (BT) method without using the microvortex device of the present invention. The bench-top method uses common magnetic stirring.

[0142] In the process of synthesizing PHNP-A1(SMR) and PHNP-A1(BT), the synthetic weight ratio of PHNP-PEG2000 to ApoA1 was set to 2:1. The results are shown in Fig.18 middle.

[0143] As stated Fig.18 As shown, it was confirmed that the particle size of PHNP-A1(SMR) was larger than that of PHNP-PEG2000, while PHNP-A1(BT) had a particle size similar to that of PHNP-PEG2000.

[0144] Furthermore, the polydispersity index (PDI) results also showed that PHNP-A1(SMR) had a lower PDI value than PHNP-A1(BT), indicating that PHNP-A1(SMR) had a uniform particle size.

[0145] PHNP-A1(SMR) and PHNP-A1(BT) were synthesized with a weight ratio of PHNP-2000 to ApoA1 of 50:1, 10:1, and 2:1. The results of quantification of ApoA1 by enzyme-linked immunosorbent assay (ELISA) are shown in Figure 2. Fig.19 The production and Fig. 20 The yield is shown.

[0146] As stated Fig.19 and Fig. 20 As shown in the figure, when PHNP-A1(SMR) and PHNP-A1(BT) were synthesized at the same synthesis weight ratio, it was confirmed that PHNP-A1(SMR) had higher values ​​in terms of the yield and productivity of ApoA1. Therefore, it can be seen that the synthesis method using the microvortex device is significantly superior in terms of including apolipoprotein in nanoparticles.

[0147] In addition, human intrahepatic biliary epithelial cells (iHBEC) were treated with PHNP-2000, PHNP-A1(BT) and PHNP-A1(SMR), respectively, and confocal microscopy images were taken 24 hours later and their fluorescence intensity was quantified. Fig.21 As shown in the results of fluorescence imaging, it was confirmed that PHNP-A1(SMR) had the highest intracellular delivery amount, and its delivery amount was more than twice that of PHNP-A1(BT).

[0148] like Fig.19 and Fig. 20 It can be seen that this is because PHNP-A1(SMR) contains a larger amount of apolipoprotein than PHNP-A1(BT).

[0149] Ultimately, it can be seen that the method using the microvortex device as a synthetic method for adding proteins to nanoparticles is significantly superior to existing synthetic methods.

[0150] Example 10

[0151] Method for synthesizing hybrid nanoparticles containing apolipoproteins (ApoE3 and ApoA1)

[0152] In the same manner as in Example 1, PHNP-PEG2000 containing DPP C, DSPE-PEG2000 and PLGA was prepared using a vortex micro-vortex device, and then ApoA1 and ApoE3 were added simultaneously to synthesize PHNP-E3 / A1.

[0153] In order to prepare PHNP-E3 / A1 containing both ApoE3 and ApoA1, a mixed aqueous solution was prepared by dissolving ApoE3 and ApoA1 in PBS (phosphate buffered saline) at a weight ratio (ApoE3 (mg) / ApoA1 (mg)) shown in Table 2 below.

[0154] Table 2

[0155]

[0156]

[0157] PHNP-E3 / A1 was synthesized by injecting a mixed aqueous solution of PHNP-PEG2000 and ApoE3 / Apo A1 into each inlet of the microvortex device. In this case, the Reynolds number was set to 250. The synthesized final PHNP-E3 / A1 was centrifuged and purified through an Amicon filter (MWCO 50 kDa).

[0158] The particle size and polydispersity index (PDI) of PHNP-E3 / A1 were confirmed by DLS in a PBS (1% trehalose) buffer solution environment. Fig. 22As shown, the size of PHNP-E3 (500 / 0) was confirmed to be 85.55±1.77nm, the PDI was 0.23±0.03, and the PHNP The size of P-E3 / A1 (250 / 25) was 70.38±5.31nm and PDI was 0.08±0.03, the size of PHNP-E3 / A1 (250 / 50) was 68.74±3.20nm and PDI was 0.09±0.02, the size of PHNP-E3 / A1 (250 / 100) was 96.58±12.16nm and PDI was 0.26±0.01, the size of PHNP-E3 / A1 (250 / 250) was 73.03±5.45nm and PDI was 0.23±0.01, and the size of PHNP-A1 (0 / 500) was 75.55±2.88nm and PDI was 0.26±0.03. Therefore, through the size of less than 100 nm and the PDI result of less than 0.3, it can be seen that in the produced PHNP-E3 / A1, ApoE3 and ApoA1 are uniform and highly dispersed.

[0159] The results of confirming the dispersion stability of PHNP-E3 / A1 synthesized with ApoE3 and ApoA1 according to the synthetic blending weight ratio under the conditions of PBS (1% trehalose) solution at 4°C by DLS are shown in FIG. Fig.23 As described Fig.23 As shown, it was confirmed that all PHNP-E3 / A1 had a size of less than 80 nm and a PDI of less than 0.3 until day 14. This means that PHNP-E3 / A1 maintained high dispersion stability without size change within 14 days.

[0160] Furthermore, fluorescence resonance energy transfer (FRET) was used to confirm that ApoE3 and ApoA1 were simultaneously introduced into the nanoparticles of PHNP-PEG2000. That is, in order to confirm the fluorescence intensity, the Alexa Fluor 488 fluorescent molecule was attached to ApoE3, and the Alexa Fluor 568 fluorescent molecule was attached to ApoA1 for confirmation. When the Alexa Fluor 488 attached to ApoE3 was applied with 470nm light energy that can release fluorescence, if the Alexa Fluor 568 fluorescent molecule attached to ApoA1 released light energy with a fluorescence wavelength of 600nm, it can be considered that ApoE3 and ApoA1 are simultaneously present in the nanoparticles.

[0161] To confirm this, the fluorescence wavelengths released when 470 nm light energy was applied to PHNP-PEG2000, PHNP-E3, PHNP-A1, and PHNP-E3 / A1 are shown in Fig.24 As described Fig.24 As shown, the control group PHNP-PEG2000 has almost no fluorescence intensity, and PHNP-E3 shows the highest intensity at 520nm because Alexa Fluor 488 emits fluorescence. In addition, it is confirmed that PHNP-A1 emits fluorescence at 600nm, but the intensity is weaker than the fluorescence of PHNP-E3. It is confirmed that in PHNP-E3 / A1, the fluorescence intensity of Alexa Fluor 488 attached to ApoE3 is reduced than PHNP-E3 (520nm, dark gray arrow), and the fluorescence intensity of Alexa Fluor 568 attached to ApoA1 is increased than PHNP-A1 (600nm, light gray arrow). The result of comparing the fluorescence spectrum of PHNP-E3 / A1 with that of PHNP-E3 and PHNP-A1, the fluorescence resonance energy transfer (FRET) effect is proved by the reduction of the fluorescence intensity of Alexa Fluor 488 and the increase of the fluorescence intensity of Alexa Fluor 568, thus confirming that two proteins are present in PHNP-E3 / A1 at the same time.

Claims

1. A method for preparing hybrid nanoparticles containing protein, characterized in that: include: In a vortex-type micro-vortex device including a first injection port, a second injection port and an outlet, the step of injecting mixed nanoparticles into the first injection port and injecting protein into the second injection port; as well as Steps for adding proteins to hybrid nanoparticles by vortexing microfluidization.

2. The method for preparing hybrid nanoparticles containing protein according to claim 1, characterized in that: The Reynolds number in the vortex-type micro-vortex device is 50 to 300.

3. The method for preparing hybrid nanoparticles containing protein according to claim 1, characterized in that: The protein is an apolipoprotein or a polymer having amphiphilic properties.

4. The method for preparing hybrid nanoparticles containing protein according to claim 1, characterized in that: The apolipoprotein is one or more selected from the group consisting of apolipoprotein A1, A2, E2, E3, J and M.

5. The method for preparing hybrid nanoparticles containing protein according to claim 3, characterized in that: The synthetic compounding weight ratio of the mixed nanoparticles to the apolipoprotein is 20:1 to 0.5:

1.

6. The method for preparing hybrid nanoparticles containing protein according to claim 3, characterized in that: The synthetic compounding weight ratio of the mixed nanoparticles to the apolipoprotein is 20:1 to 2:

1.

7. The method for preparing hybrid nanoparticles containing protein according to claim 1, characterized in that: Also included is the step of recovering the protein-containing nanoparticles from the outlet.

8. The method for preparing hybrid nanoparticles containing protein according to claim 1, characterized in that: The hybrid nanoparticles are prepared by a preparation method comprising the following steps: In the vortex-type micro-vortex device, injecting phospholipid into the first injection port and injecting polymer into the second injection port; and The phospholipids and polymer were mixed by vortexing.

9. The method for preparing hybrid nanoparticles containing protein according to claim 8, characterized in that: The phospholipids are selected from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, egg phosphatidylcholine, dilauroyl phosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1,2-Di-erucyl-sn-glycero-3-phosphocholine, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, palmitoyloleoylphosphatidylethanolamine, lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino] 1,2-dioleyl-3-trimethylammonium propane, (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide, 1,2-dimyristyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide, 2,3-dioleyloxy-N-[2 One or more of the group consisting of [(spermidine formylamino)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate, N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanium bromide, N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine, ethylphosphocholine, dimethyldioctadecylammonium bromide, N4-cholesterol-spermamine, 1,2-dioleoyloxy-3-dimethylaminopropane, D-Lin-MC3-DMA, DLin-KC2-DMA and DLin-DMA.

10. The method for preparing hybrid nanoparticles containing protein according to claim 9, characterized in that: The phospholipids are dipalmitoylphosphatidylcholine and 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-polyethylene glycol.

11. The method for preparing hybrid nanoparticles containing protein according to claim 10, characterized in that: The molecular weight of the polyethylene glycol in the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol is 2,000 to 5,000.

12. The method for preparing hybrid nanoparticles containing protein according to claim 10, characterized in that: The synthetic compounding weight ratio of the dipalmitoylphosphatidylcholine to 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-polyethylene glycol is 2.3:1 to 1:

1.

13. The method for preparing hybrid nanoparticles containing protein according to claim 8, characterized in that: The polymer is poly(lactic-co-glycolic acid).

14. The method for preparing hybrid nanoparticles containing protein according to claim 8, characterized in that: The hybrid nanoparticles are polymer-lipid hybrid nanoparticles. 15 . Hybrid nanoparticles containing a protein produced by the method for producing hybrid nanoparticles containing a protein according to any one of claims 1 to 14 .

16. A method for preparing hybrid nanoparticles containing protein, characterized in that: include: The first step of preparing the hybrid nanoparticles comprises: in a vortex-type micro-vortex device comprising a first injection port, a second injection port and an outlet, injecting phospholipid into the first injection port and injecting polymer into the second injection port; and the step of mixing the phospholipid and the polymer by vortexing; and The second step of preparing mixed nanoparticles containing protein includes the steps of injecting the obtained mixed nanoparticles into the first injection port and injecting protein into the second injection port in a vortex micro-vortex device including a first injection port, a second injection port and an outlet.

17. The method for preparing hybrid nanoparticles containing protein according to claim 16, characterized in that: The protein is an apolipoprotein or a polymer having amphiphilic properties.

18. The method for preparing hybrid nanoparticles containing protein according to claim 16, characterized in that: The synthetic compounding weight ratio of the mixed nanoparticles to the apolipoprotein is 20:1 to 2:

1.

19. The method for preparing hybrid nanoparticles containing protein according to claim 16, characterized in that: The phospholipids are selected from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, egg phosphatidylcholine, dilauroyl phosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1,2-Di-erucyl-sn-glycero-3-phosphocholine, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, palmitoyloleoylphosphatidylethanolamine, lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino] 1,2-dioleyl-3-trimethylammonium propane, (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide, 1,2-dimyristyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide, 2,3-dioleyloxy-N-[2 One or more of the group consisting of [(spermidine formylamino)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate, N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanium bromide, N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine, ethylphosphocholine, dimethyldioctadecylammonium bromide, N4-cholesterol-spermamine, 1,2-dioleoyloxy-3-dimethylaminopropane, D-Lin-MC3-DMA, DLin-KC2-DMA and DLin-DMA.

20. The method for preparing hybrid nanoparticles containing protein according to claim 16, characterized in that: The polymer is poly(lactic-co-glycolic acid).

21. The method for preparing hybrid nanoparticles containing protein according to claim 16, characterized in that: The hybrid nanoparticles are polymer-lipid hybrid nanoparticles.

Images