Synthesis method of 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 vortex microvortex device. By the production method of the present invention, nanoparticles having various biological activities, 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 controlling 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 inventors of the present invention have studied a synthesis method capable of incorporating a functional protein into a polymer-lipid hybrid nanoparticle (PHNP) using PLGA (poly(lactic-co-glycolic acid)), a polymer having biodegradability and biocompatibility and capable of protecting a drug from degradation, and have completed the present invention.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0007] The object of the present invention is to provide a method for producing hybrid nanoparticles in which hydrophilic and hydrophobic substances are mixed by mixing phospholipids and polymers under a swirling micro vortex flow.

[0008] The object of the present invention is to provide a method for producing protein-containing hybrid nanoparticles, which includes a step of adding a protein to the hybrid nanoparticles by colliding the protein with the hybrid nanoparticles under a swirling micro vortex flow.

[0009] Furthermore, the object of the present invention is to provide protein-containing hybrid nanoparticles produced by the above method.

Means for Solving the Problems

[0010] The present invention provides a method for producing protein-containing hybrid nanoparticles, which includes a step of injecting hybrid nanoparticles into the first injection port and injecting a protein into the second injection port in a swirling micro vortex flow device having a first injection port, a second injection port, and an outlet, and a step of adding the protein to the hybrid nanoparticles by the swirling micro vortex flow.

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

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

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

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

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

[0016] In one embodiment, the hybrid nanoparticles may be produced by a production method including a step of injecting a phospholipid into the first injection port and injecting a polymer into the second injection port in the spiral micro-vortex device, and a step of mixing the phospholipid and the polymer by spiral micro-vortices.

[0017] The phospholipids are 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyl oleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine (DSPE), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleoyloxy]-benzamide (VL-5), dioctadecylamidoglycyl spermine 4 trifluoroacetate (DOGS), 3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleoyloxypropyl)-3 dimethylhydroxyethylammonium bromide (DORIE), 1,2-Dimyristyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(speminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanammonium bromide (GAP-DLRIE), N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl-spemine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA, and may be at least one selected from the group consisting of, and preferably DPPC and DSPE-PEG, and more preferably DPPC and DSPE-PEG2000 to 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)).

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

[0021] The present invention provides protein-containing hybrid nanoparticles produced by the above production 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 the 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 amphiphilic 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 simultaneously contain hydrophilic substances and hydrophobic substances, and have a form in which the hydrophobic substance covers the center of the nanoparticles made of the polymer. Therefore, a high concentration of hydrophobic drug can be effectively encapsulated and delivered inside.

[0028] In addition, in the hybrid nanoparticles of the present invention, the surface, the intermediate shell portion, and the core portion can be utilized respectively and used as a substance for delivering a plurality of drugs. 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 the hybrid nanoparticles prevent the adsorption of external substances to the particle surface and the removal by immune cells in the human body, thus enabling safer drug delivery. Therefore, when used for intracellular drug delivery and the like, treatment can be efficiently supported.

Brief Description of the Drawings

[0030]

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Mode for Carrying Out the Invention

[0031] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings so that those having ordinary knowledge in the technical field 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] Throughout the specification, when a certain part states that a certain component "includes", this means that, unless otherwise specified, it does not exclude other components, but may further include other components.

[0033] The "hybrid nanoparticle" in the present invention means a nanoparticle formed by the coexistence of a hydrophilic substance and a hydrophobic substance, and is preferably a nanoparticle in which a polymer and a lipid are mixed (polymer-lipid hybrid nanoparticle; PHNP), and is also referred to as "PHNP".

[0034] The "PLGA" in the present invention means a poly(lactic acid-glycolic acid) copolymer, which is a polymer having biodegradability and biocompatibility.

[0035] "DPPC" in the present invention means dipalmitoylphosphatidylcholine, which is a kind of lipid.

[0036] "DSPE-PEG" in the present invention means a lipid in which polyethylene glycol (PEG) is conjugated to distearoylphosphoethanolamine, and the molecular weight of the polyethylene glycol is preferably from 2000 to 5000.

[0037] "Polymer having amphoteric properties" in the present invention means a protein having both hydrophobicity and hydrophilicity, and examples thereof include glycophorin, rhodopsin, CD36 (cluster of differentiation 36), Seipin, glucose permease, cytochrome c, cupredoxins, high potential iron protein, adrenodoxin reductase, flavoprotein, and the like.

[0038] The "phospholipid" in the present invention specifically includes 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyl oleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine (DSPE), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleoyloxy]-benzamide (VL-5), dioctadecylamidoglycyl spermine 4 trifluoroacetate (DOGS), 3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3 dimethylhydroxyethylammonium bromide (DORIE), 1,At least one selected from the group consisting of 2-dimyristyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanammonium bromide (GAP-DLRIE), N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethyl phosphocholine (Ethyl 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 thereto.,

[0039] The term "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, such as apolipoprotein E2 (APOE2), apolipoprotein E3 (APOE3), apolipoprotein E4 (APOE4). APOE is a polymorphism having three main alleles (epsilon 2, epsilon 3 and epsilon 4). Any allele is used in various embodiments of the present invention. The term "functional variant" means a variant of a 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 includes insertions, deletions and / or substitutions of amino acids as 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 a protein having at least 95% sequence identity, preferably at least 98% or 99% sequence identity thereto.

[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 a 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 includes insertions, deletions and / or substitutions of amino acids 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 thereto.

[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] "Apolipoprotein E" in the present invention means those including fragments and functional variants thereof.

[0046] The "swirling microvortex" in the present invention means that in the flow of a fluid, small fluid flows while rotating.

[0047] The "microvortex device" in the present invention means a device including a microchannel and the like provided such that a fluid flows on a substrate made of various materials such as plastic, glass, metal or silicon including organic polymer substances.

[0048] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for explaining the present invention, and the present invention is not limited to these examples.

Example

[0049] Method for synthesizing apolipoprotein-containing hybrid nanoparticles using a swirling microvortex device Apolipoprotein-containing hybrid nanoparticles were synthesized. Specifically, the first step is to synthesize polymer-lipid hybrid nanoparticles (PHNP), and the second step is to include apolipoprotein in the hybrid nanoparticles. The same swirling microvortex device was used in each step. A schematic diagram of the swirling microvortex device and the synthesis method used for synthesizing the hybrid nanoparticles (PHNP) of the present invention is shown in Fig. 1.

[0050] The swirling microvortex device includes two inlets and one outlet. In order to form a swirling microvortex and effectively mix lipids and PLGA polymers, the diameter and height of the device were optimized and designed (see Non-Patent Document 1).

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

[0052] After that, the polymer-lipid hybrid nanoparticles (PHNP) synthesized as described above were injected into one inlet, apolipoprotein was injected into the inlet on the opposite side, and then they were mixed to finally synthesize apolipoprotein-containing hybrid nanoparticles.

Example

[0053] Synthesis of polymer-lipid hybrid nanoparticles at each compounding weight ratio of DPPC:DSPE-PEG2000 Using the spiral micro-vortex device, polymer-lipid hybrid nanoparticles containing lipids (DPPC and DSPE-PEG2000) and polymers (PLGA) were synthesized by the following method. DSPE-PEG2000 contains PEG and can prevent plasma proteins in the human body from adsorbing on the nanoparticle surface and being removed by immune cells, so it was used for the stability of in vivo nanoparticles.

[0054] A solution in which PLGA was dissolved in anhydrous acetonitrile (ACN) was prepared, and DPPC and DSPE-PEG2000 were prepared as an anhydrous ethanol solution. They were synthesized using the spiral micro-vortex device, and then the final solution was immediately purified with DW (deionized water).

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

[0056] The particle sizes of the nanoparticles synthesized under the conditions of the synthetic compound weight ratios of DPPC and DSPE-PEG2000 being 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, it was confirmed that the particle sizes of the particles generated under the conditions of the synthetic compound weight ratios of 10:0, 9:1, 2.3:1, and 1:1 were 130 nm and 90 nm, respectively.

[0057] Also, the particle sizes of the particles generated under the conditions of the synthetic compound weight ratios of 2.3:1 and 1:1 were 30 nm. In particular, it was confirmed that the particle sizes were very homogeneous under the condition of the synthetic compound weight ratio of 1:1. Therefore, it can be seen that nanoparticles with small and uniform particle sizes are generated under the conditions of the synthetic compound weight ratio of 2.3:1 to 1:1.

[0058] In addition, the results of observing the morphology of the nanoparticles by TEM are shown in Figure 3. As shown in Figure 3, when the synthetic compound weight ratios of DPPC and DSPE-PEG2000 were 10:0 and 9:1, the particle sizes were not uniform, and when they were 7:3 and 5:5, it was confirmed that the particle sizes were small and uniform.

Example

[0059] Synthesis of Hybrid Nanoparticles at Each Molecular Weight of DSPE-PEG Using the vortex microvortex device of Example 1, DSPE-PEG-containing hybrid nanoparticles (PHNPs) having various PEG molecular weights were prepared.

[0060] PLGA was prepared as an absolute acetonitrile (ACN) solution, DPPC and DSPE-PEG (550, 2000, and 5000) were prepared as absolute ethanol solutions, and synthesis was performed using a vortex microvortex device with the synthetic compound weight ratio of DPPC and DSPE-PEG being 2.3:1. The final synthetic product was purified with DW.

[0061] After that, the particle sizes of those containing DSPE-PEG with various PEG molecular weights were compared and are shown in Fig. 4. As shown in Fig. 4, in the case of DSPE-PEG550-containing hybrid nanoparticles (PHNP-PEG550), it was confirmed that 50-nm heterogeneous particles were generated, and in the case of DSPE-PEG5000-containing hybrid nanoparticles (PHNP-PEG5000), it was confirmed that 27-nm particles were generated. Also, in the case of DSPE-PEG2000-containing hybrid nanoparticles (PHNP-PEG2000), it was confirmed that small and uniform 18-nm particles were generated.

[0062] Furthermore, Fig. 5 shows the results of observing the morphology of the hybrid nanoparticles generated as described above by TEM. As shown in Fig. 5, it can be seen that the particle size and morphology of the generated nanoparticles change depending on the molecular weight of PEG. In PHNP-PEG550, the particle size was heterogeneous, and as a result of measurement by DLS, it had a particle size of 30 to 80 nm, and in PHNP-PEG2000, it was confirmed to have a uniform particle size of 20 nm.

[0063] Also, in order to confirm the stability of the hybrid nanoparticles generated as described above, after 3 days, the particle size was confirmed by DLS. The results are shown in Fig. 6. As shown in Fig. 6, after 3 days, in PHNP-PEG2000 and PHNP-PEG5000, it was confirmed that there was no change in the particle size and they were stable.

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

Example

[0065] Synthesis of Apolipoprotein-Containing Hybrid Nanoparticles Using a vortex micro-vortex device, polymer-lipid hybrid nanoparticles (PHNP-PEG2000) containing DPPC, DSPE-PEG2000, and PLGA were prepared, and then hybrid nanoparticles (PHNP-E3) containing apolipoprotein E3 (ApoE3) were prepared. A schematic diagram thereof is shown in Fig. 7.

[0066] Specifically, PLGA was prepared as an anhydrous acetonitrile (ACN) solution, and DPPC and DSPE-PEG2000 were prepared as anhydrous ethanol solutions. The synthetic mixing ratio of DPPC:DSPE-PEG2000 was 2.3:1, and it was synthesized using a vortex micro-vortex device, and then 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. Here, the compounding weight ratio of PHNP-PEG2000 and ApoE3 was 4:1, and the Reynolds number was 250. Finally, the synthesized ApoE3-containing hybrid nanoparticles (PHNP-E3) were purified by an Amicon filter (MWCO 50 kDa).

[0067] The results of confirming the particle sizes of PHNP-PEG2000 without ApoE3 and PHNP-E3 containing ApoE3 by DLS are shown in Fig. 8. As shown in Fig. 8, it was confirmed that PHNP-PEG2000 had a particle size of 21 nm and PHNP-E3 had a particle size of 24 nm. Also, as a result of confirming the amount of ApoE3 by BCA assay during the purification process, it was confirmed that 70% of ApoE3 was contained in the finally formed nanoparticles.

[0068] Furthermore, as shown in Fig. 9, as a result of observing the PHNP-E3 synthesized as described above by TEM, a core-shell structure with a core in the nanoparticles was observed.

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

Example

[0070] Synthesis of Apolipoprotein-Containing Hybrid Nanoparticles at Each Reynolds Number Using a scroll micro-vortex device, similar to Figure 7, hybrid nanoparticles containing DPPC, DSPE-PEG2000, and PLGA (PHNP-PEG2000) were synthesized, and then finally hybrid nanoparticles (PHNP-E3) were synthesized including ApoE3.

[0071] Similar to Example 4, PLGA was prepared as an acetonitrile (ACN) solution, DPPC and DSPE-PEG2000 were prepared as ethanol solutions, synthesized using a scroll micro-vortex device, and then the final solution was purified with 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] Particles of PHNP-E3 were synthesized at various Reynolds numbers, and the resulting particle size distribution was confirmed by DLS. The results are shown in Figure 10. As shown in Figure 10, under the condition where the Reynolds number is less than 50 (Re 10), the average particle size is 158 nm, and the measured particles were relatively large compared to those under other conditions. This is because under the condition of an overly slow flow rate, normal PHNP-E3 is not synthesized, and E3 adsorbs on the surface of PHNP-PEG2000 to form aggregates.

[0073] Also, it was confirmed that PHNP-E3 showing a normal particle size distribution was synthesized under the condition where the Reynolds number is 300.

[0074] The mixing efficiency within the scroll micro-vortex device increases proportionally to the Reynolds number. After reaching the highly mixed regime, the mixing efficiency no longer increases further and converges to a maximum value (Non-Patent Document 2). Therefore, after reaching the highly mixed regime, it is not necessary to increase the Reynolds number. Also, an excessive Reynolds number has a negative impact on the three-dimensional structure of proteins. For example, in the case of insulin, it is known that structural changes and aggregate formation occur based on a shear rate of 200 s -1 or a shear stress of 1000 dyne / cm 2 (Non-Patent Document 3).

[0075] That is, under the condition that the Reynolds number is 300, the actual flow rate is 14.14 mL / min. Flow rate conditions higher than this apply excessive pressure to the micro-vortex device and the protein. It can be seen that it is preferable to set the Reynolds number of the present invention to be in the range of 50 to 300.

Example

[0076] Optimization of the synthesis formulation ratio of apolipoprotein-containing hybrid nanoparticles PHNP-E3 was prepared by changing the synthesis formulation ratio (20:1, 10:1, 5:1, 2:1, 1:1, 0.5:1) of PHNP-PEG2000 and ApoE3. Here, the amount of PHNP-PEG2000 used was 1000 μg, and the amount of ApoE3 was as shown in Table 1.

[0077]

Table 1

[0078] Similar to Example 4, PLGA was prepared as an acetonitrile (ACN) solution, DPPC and each DSPE-PEG2000 were prepared as ethanol solutions, synthesized using a vortex micromixer, and then the final solution was purified with DW. PHNP-PEG2000 and ApoE3 were injected into each inlet at the synthetic mixing ratios shown in Table 1. The particle sizes of the particles generated at each synthetic mixing 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) had a particle size of 60.3 nm, PHNP-E3 (10:1) had a particle size of 64.2 nm, PHNP-E3 (5:1) had a particle size of 58.5 nm, and PHNP-E3 (2:1) had a particle size of 52.9 nm, and all were confirmed to be stable with a polydispersity index (PDI) of 0.18 or less. The closer the PDI is to 0, the more uniformly sized the nanoparticles are present. Generally, nanoparticles have a numerical value of 0.3 - 0.4. The closer the PDI is to 0, the more uniformly sized the nanoparticles are present. Generally, nanoparticles have a numerical value of 0.3 - 0.4.

[0079] In contrast, the 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 greater than 0.18 and was confirmed to increase compared to the particles described above.

[0080] Also, using the BCA assay, a protein quantification method, the amount of ApoE3 contained in PHNP-E3 at each synthetic mixing weight ratio was confirmed. As shown in Table 1 and Figure 11, 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) was confirmed to contain 1100 μg of ApoE3. Therefore, it was confirmed that the amount of ApoE3 contained in PHNP-E3 was concentration-dependent according to the synthetic mixing weight ratio.

[0081] In addition, PHNP-PEG-2000 contains PEG-2000 in the shell of the nanoparticles. However, when synthesized by adding apolipoprotein, it was confirmed that PHNP-PEG-2000 was stably contained in proportion to the amount of apolipoprotein added. This cannot be obtained by conventional synthesis methods. By using a spiral micro-vortex device and adjusting the Reynolds number, it is obtained in the process of physically pushing apolipoprotein into PHNP-PEG-2000 like an inelastic collision. It can be seen that the obtained PHNP-E3 has small, uniform and stable particles.

Example

[0082] Synthesis method of apolipoprotein (ApoA1)-containing hybrid nanoparticles Using a spiral micro-vortex device, similar to Figure 12, apolipoprotein A1 (ApoA1)-containing hybrid nanoparticles (PHNP-A1) were synthesized.

[0083] Specifically, PLGA was prepared as an anhydrous acetonitrile (ACN) solution, and DPPC and each DSPE-PEG2000 were prepared as anhydrous ethanol solutions. The mixing ratio of DPPC:DSPE-PEG2000 was 2.3:1, and it was synthesized using a spiral micro-vortex device. Then, the final solution was purified with DW to prepare PHNP-PEG2000.

[0084] The PHNP-PEG2000 and apolipoprotein A1 (ApoA1) synthesized as described above were injected into each inlet of the spiral micro-vortex device. Here, the Reynolds number was 250, and the finally synthesized apolipoprotein A1 (ApoA1)-containing hybrid nanoparticles (PHNP-A1) were purified by an Amicon filter (MWCO 50 kDa).

[0085] The results of confirming the particle sizes of apolipoprotein A1 (ApoA1)-free hybrid nanoparticles (PHNP-PEG2000) and ApoA1-containing hybrid nanoparticles (PHNP-A1) by DLS are shown in Fig. 13. As shown in Fig. 13, it was confirmed that PHNP-PEG2000 had a particle size of 21 nm and PHNP-A1 had a particle size of 25 nm.

[0086] In addition, as a result of confirmation by BCA assay to quantify ApoA1 during the purification process, it was confirmed that 26% of ApoA1 was contained in PHNP-PEG2000.

[0087] Furthermore, as a result of observing the morphology of PHNP-A1 by TEM, as shown in Fig. 14, it was confirmed that PHNP-A1 had a core-shell structure with a core inside.

Example

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

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

[0090] The images of intracellular delivery of PHNP-PEG2000 and PHNP-A1 are shown in Figs. 15 and 16. As shown in Figs. 15 and 16, it was confirmed that compared with PHNP-PEG2000, PHNP-A1 was delivered more into HAECs cells due to the intracellular entry effect via the receptor of ApoA1.

[0091] In addition, the results of quantifying the fluorescence intensities of the images in FIGS. 15 and 16 are shown in FIG. 17. As a result of comparing the fluorescence intensities, it can be seen that the intracellular delivery of PHNP-A1 into HAEC cells is more than six times that of PHNP. That is, it can be seen that PHNP-A1 has an excellent effect on intracellular delivery.

Example

[0092] Comparative evaluation of the method for synthesizing hybrid containing apolipoprotein (ApoA1) Similar to Example 7, ApoA1-containing PHNP-A1 (SMR) was synthesized using a spiral micro vortex device (SMR).

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

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

[0095] As shown in FIG. 18, it was confirmed that the particle size of PHNP-A1 (SMR) increased compared to PHNP-PEG2000, while PHNP-A1 (BT) had a particle size comparable to that of PHNP-PEG2000.

[0096] In addition, in the results of the polydispersity index (PDI), it was also confirmed that PHNP-A1 (SMR) had a lower PDI value than PHNP-A1 (BT), so it can be seen that PHNP-A1 (SMR) has a uniform particle size.

[0097] The synthetic compound weight ratios of PHNP-2000 and ApoA1 were set at 50:1, 10:1, and 2:1, and PHNP-A1(SMR) and PHNP-A1(BT) were synthesized. The results of the quantified ApoA1 using ELISA are shown by yield in Fig. 19 and by recovery rate in Fig. 20.

[0098] As shown in Figs. 19 and 20, when synthesizing PHNP-A1(SMR) and PHNP-A1(BT) at the same weight ratio of the synthetic compound, it was confirmed that PHNP-A1(SMR) had higher numerical values in terms of the yield and recovery rate of ApoA1. Therefore, it can be seen that the synthetic method using a micro vortex device when incorporating apolipoprotein into nanoparticles is particularly excellent.

[0099] Also, human intrahepatic biliary epithelial cells (iHBEC) were treated with PHNP-2000, PHNP-A1(BT), and PHNP-A1(SMR) respectively. After 24 hours, confocal microscope images were taken and their fluorescence intensities were quantified. As shown in Fig. 21, as confirmed by the fluorescence images, it was confirmed that PHNP-A1(SMR) was delivered to the cells the most, and it was confirmed that it was delivered more than twice that of PHNP-A1(BT).

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

[0101] That is, as a synthetic method for adding protein to nanoparticles, it can be seen that the method using a micro vortex device is particularly excellent compared to conventional synthetic methods.

Example

[0102] Synthetic method of hybrid nanoparticles containing apolipoproteins (ApoE3 and ApoA1) Similar to Example 1, a scroll micro-vortex device was used to prepare PHNP-PEG2000 containing DPPC, DSPE-PEG2000, and PLGA, and then PHNP-E3 / A1 was synthesized 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) at the weight ratio (ApoE3 (mg) / ApoA1 (mg)) shown in Table 2 and prepared as a mixed aqueous solution.

[0104]

Table 2

[0105] The mixed aqueous solution of PHNP-PEG2000 and ApoE3 / ApoA1 was injected into each inlet of the micro-vortex device to synthesize PHNP-E3 / A1. Here, the Reynolds number was 250. The synthesized final PHNP-E3 / A1 was purified by centrifugation using an Amicon filter (MWCO 50 kDa).

[0106] The results of confirming the particle size and polydispersity index (PDI) of PHNP-E3 / A1 by DLS in a PBS (1% trehalose) buffer environment are shown in Fig. 22. As shown in Fig. 22, PHNP-E3(500 / 0) has a particle size of 85.55 ± 1.77 nm and a PDI of 0.23 ± 0.03, PHNP-E3 / A1(250 / 25) has a particle size of 70.38 ± 5.31 nm and a PDI of 0.08 ± 0.03, PHNP-E3 / A1(250 / 50) has a particle size of 68.74 ± 3.20 nm and a PDI of 0.09 ± 0.02, PHNP-E3 / A1(250 / 100) has a particle size of 96.58 ± 12.16 nm and a PDI of 0.26 ± 0.01, PHNP-E3 / A1(250 / 250) has a particle size of 73.03 ± 5.45 nm and a PDI of 0.23 ± 0.01, and PHNP-A1(0 / 500) has a particle size of 75.55 ± 2.88 nm and a PDI of 0.26 ± 0.03. Therefore, based on the particle size of less than 100 nm and the PDI result of less than 0.3, it can be seen that in the generated PHNP-E3 / A1, ApoE3 and ApoA1 are uniform and have high dispersibility.

[0107] The results of confirming the dispersion stability of PHNP-E3 / A1 synthesized with ApoE3 and ApoA1 at each synthetic compound weight ratio by DLS under the conditions of 4°C and a PBS (1% trehalose) solution are shown in Fig. 23. As shown in Fig. 23, it was confirmed that by 14 days, all PHNP-E3 / A1 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 over 14 days without change in particle size.

[0108] In addition, it was confirmed by fluorescence resonance energy transfer (FRET) that ApoE3 and ApoA1 were simultaneously introduced into the nanoparticles of PHNP-PEG2000. That is, in order to confirm the fluorescence intensity, Alexa Fluor 488 fluorescent molecules were bound to ApoE3, and Alexa Fluor 568 fluorescent molecules were bound to ApoA1 for confirmation. When the Alexa Fluor 488 bound to ApoE3 gave light energy at 470 nm that emitted fluorescence, if the Alexa Fluor 568 fluorescent molecules bound to ApoA1 emitted light energy at 600 nm, which was the fluorescence wavelength, it could be said that ApoE3 and ApoA1 coexisted in the nanoparticles at the same time.

[0109] To confirm this, the results of the fluorescence wavelengths emitted when PHNP-PEG2000, PHNP-E3, PHNP-A1, and PHNP-E3 / A1 were given energy at 470 nm are shown in Fig. 24. As shown in Fig. 24, in PHNP-PEG2000, which was the control group, there was almost no fluorescence intensity. In PHNP-E3, since Alexa Fluor 488 emitted fluorescence, it showed the highest intensity at 520 nm. In addition, in PHNP-A1, fluorescence was emitted at 600 nm, but it was confirmed that the intensity was weaker than that of the fluorescence 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, thick gray arrow), and the fluorescence intensity of Alexa Fluor 568 bound to ApoA1 increased compared to PHNP-A1 (600 nm, thin gray arrow). As a result of comparing the fluorescence spectrum of PHNP-E3 / A1 with the fluorescence spectra of PHNP-E3 and PHNP-A1 respectively, the phenomenon that the fluorescence intensity of Alexa Fluor 488 decreased and the Alexa Fluor 568 fluorescence increased proved the FRET effect, so it was confirmed that two kinds of proteins coexisted in PHNP-E3 / A1 at the same time.

Claims

1. In a scroll micro-vortex device having a first inlet, a second inlet, and an outlet, the steps of injecting hybrid nanoparticles into the first inlet and injecting a protein into the second inlet; and a step of adding the protein to the hybrid nanoparticles by a scroll micro-vortex, characterized by comprising: A method for producing protein-containing hybrid nanoparticles.

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

3. The 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 apolipoprotein A1, A2, E2, E3, J, and M.

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

1.

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

1.

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

8. The hybrid nanoparticles are produced by a production method including, in the scroll micro-vortex device, a step of injecting a phospholipid into the first inlet and injecting a polymer into the second inlet, and a step of mixing the phospholipid and the polymer by a scroll micro-vortex, characterized by: The method for producing protein-containing hybrid nanoparticles according to claim 1.

9. The phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyl oleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine (DSPE), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleoyloxy]-benzamide (VL-5), dioctadecylamidoglycyl spermine 4 trifluoroacetate (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3 dimethylhydroxyethylammonium bromide (DORIE), 1,2-Dimyristyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-Dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanammonium bromide (GAP-DLRIE), N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), Ethyl Phosphocholine (Ethyl 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, characterized in that it is at least one selected from the group consisting of, the method for producing a protein-containing hybrid nanoparticle according to claim 8.,

10. The method for producing protein-containing hybrid nanoparticles according to claim 9, characterized in that the phospholipid is DPPC and DSPE-PEG.

11. The method for producing protein-containing hybrid nanoparticles according to claim 10, characterized in that the molecular weight of PEG in the DSPE-PEG is 2000 to 5000.

12. The method for producing protein-containing hybrid nanoparticles according to claim 10, characterized in that the synthetic compounding weight ratio of the DPPC and the DSPE-PEG is 2.3:1 to 1:

1.

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

14. The method for producing protein-containing hybrid nanoparticles according to claim 8, characterized in that the hybrid nanoparticles are polymer-lipid hybrid nanoparticles (PHNP).

15. Protein-containing hybrid nanoparticles produced by the production method according to claims 1 to 14.

16. In a spiral micro-vortex device having a first inlet, a second inlet, and an outlet, A first step of producing hybrid nanoparticles, comprising the step of injecting a phospholipid into the first inlet and injecting a polymer into the second inlet, and the step of mixing the phospholipid and the polymer by spiral micro-vortices; A second step of producing protein-containing hybrid nanoparticles, comprising the step of injecting the hybrid nanoparticles obtained at the first inlet and injecting a protein into the second inlet. The method for producing protein-containing hybrid nanoparticles, characterized in that it comprises the above steps.

17. The method for producing protein-containing hybrid nanoparticles according to claim 16, characterized in that the protein is apolipoprotein or a polymer having amphoteric properties.

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

1.

19. The phospholipids are 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyl oleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine (DSPE), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleoyloxy]-benzamide (VL-5), dioctadecylamidoglycyl spermine 4 trifluoroacetate (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3 dimethylhydroxyethylammonium bromide (DORIE), 1,2-Dimyristyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanammonium bromide (GAP-DLRIE), N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl 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, wherein the method for producing the protein-containing hybrid nanoparticles according to claim 16 is characterized in that it is at least one selected from the group consisting of

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

21. The method for producing protein-containing hybrid nanoparticles according to claim 16, wherein the hybrid nanoparticles are polymer-lipid hybrid nanoparticles (PHNP).

Images