Novel reconstituted high density lipoprotein nanoparticle

Abstract

To provide a reconstituted high density lipoprotein (rHDL) that has a therapeutic effect and does not exhibit toxicity in vivo, and also to provide a composition for preventing or treating a neurodegenerative disease comprising the rHDL.SOLUTION: The present disclosure relates to a reconstituted high density lipoprotein (rHDL) nanoparticle and a composition for preventing or treating a neurodegenerative disease comprising the same. Specifically, the present disclosure relates to a rHDL nanoparticle prepared by mixing a phospholipid and an apolipoprotein, and the rHDL nanoparticle of the present disclosure has amyloid-beta (Aβ) aggregation inhibitory effect.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to reconstituted high density lipoprotein (rHDL). High density lipoprotein (HgDLP) nanoparticles and neurodegenerative diseases containing same More specifically, the present disclosure relates to a composition for preventing or treating a disease. spholipid and apolipoprotein E Regarding the rHDL nanoparticles containing it as a component, the rHDL nanoparticles of the present disclosure have excellent amyloid activity. It has the effect of inhibiting Abeta (Aβ) aggregation. [Background technology]

[0002] In recent years, with the rapid increase in the elderly population, various neurodegenerative diseases (Neurodegenerative The number of patients suffering from chronic pulmonary disease (COPD) is increasing, and there is a need for treatment and prevention. Neurodegenerative diseases are characterized by motor and memory disorders due to the decline or loss of function of nerve cells. It is a mental illness that causes various symptoms, including memory loss and cognitive impairment. Even in a normal adult brain, many nerve cells die every day, and the number of nerve cells that die with age is increasing. It increases exponentially.

[0003] The main neurodegenerative diseases are Alzheimer's disease, Parkinson's disease, and Rheumatism. These include Rheumatic disease and Huntington's disease, and the causes of these diseases have not yet been fully elucidated. No. Medications for treating Alzheimer's disease include acetylcholinesterase inhibitors or N MDA (N-methyl-D-aspartate) receptor antagonists are used, Medication for treating Parkinson's disease includes L-dopa, dopamine agonists, and MAO- B inhibitors or COMT inhibitors are used as treatments for Huntington's disease. The above-mentioned drugs are not fundamental treatments. It is known that these are merely methods to delay the onset of symptoms or to alleviate symptoms. Therefore, there is a continuing need for new drugs that can fundamentally prevent or treat these diseases. It is being done.

[0004] In particular, Alzheimer's disease (AD) is a type of dementia. It is the most common form of dementia and the leading neurodegenerative disease. 20% of people over 80 years of age It is estimated that the above are affected by Alzheimer's disease, and as the aging society progresses, Alzheimer's disease is caused by the amyloid precursor protein ( amyloid precursor protein (APP) binds to β-secretase Amyloid beta (Aβ) is produced by successive cleavage by γ-secretase and γ-secretase. Senile plaques, which are deposits of β-lactams in brain tissue, and microtubule-associated proteins Neurofibrillary tangles caused by hyperphosphorylation of certain Tau proteins Neurofibrillary tangles (NTFs) are the main pathological feature. In particular, when Aβ, a type of protein, accumulates in the brain, it can cause damage to the blood-brain barrier (BBB). d-brain-barrier) and impairs the delivery of essential nutrients from the cerebral blood vessels to nerve cells. Not only does this slow the progression of the disease, but it also causes persistent inflammation of the nerves, which in turn causes the death of microglial cells. It could disrupt the activity of immune cells and ultimately damage the function of synapses, the areas where nerve cells send and receive signals. Such a series of pathophysiological factors ultimately leads to deterioration of memory, judgment, and , which gradually reduces intellectual functions such as language ability and daily life functions, and personality and behavioral disorders It is known that it causes excessive Aβ production in the brain. , preventing the aggregation of Aβ produced or effectively reducing the aggregated Aβ structure. This is thought to be an important part of preventing and treating Alzheimer's disease.

[0005] On the other hand, high density lipoprotein (HDL) is a type of lipoprotein formed from apolipoproteins. It is the main cholesterol carrier in the blood and has a high density (>1.063 g / mL) and small Mature HDL particles are characterized by their size (stroke diameter = 5-17 nm). It exists in a globular form containing terpenes, phospholipids, and various apolipoproteins. Polar lipids, phospholipids, and free cholesterol are present in the outer layer of these particles, which is more hydrophobic. The lipids, e.g., esterified cholesterol and triglycerides, are present in the center of the particle. Newly formed or nascent HDL particles in the liver and intestine are lipid-poor and consist of discs (d The protein components are contained in the outer layer, and the major protein is Apolipoproteins, which are the components of fat, are the major proteins in the body. Starting with polyprotein A1 (Apo A1), Apo A2, Apo A3, and Apo B1 are classified according to their functions. o A4, Apo C3, Apo D, Apo E, Apo J, Apo M, etc. It is known that this can happen.

[0006] Apo A1 is synthesized in the liver and intestines and controls the physiological function of HDL in the blood. Remove cholesterol from the surrounding tissue and then deliver it to reverse cholesterol transport (RCT; The reverse cholesterol transport mechanism causes These Apo A1-based blood lipoproteins serve to transport the The physiological function of HDL in the blood can prevent and treat many risk factors that cause arteriosclerosis. It is known that reconstituted high density lipoproteins (HDLPs) are a type of protein that is involved in the synthesis of glycerol. Historically, this has been based on Apo A1.

[0007] In contrast, Apo E is synthesized in the liver and neuroglia, It is known to play a role in controlling cholesterol and lipid homeostasis in the brain. In humans, the types of isoform proteins are: These include Apo E2, Apo E3, and Apo E4, and their combinations are involved in aging. It is known that affecting brain homeostasis can alter the incidence of Alzheimer's disease. It has been reported that Apo E4 is the main cause of Alzheimer's disease, which is It is known to be involved in the aggregation of amyloid beta (Aβ) and ultimately damage the BBB. On the other hand, Apo E2 and Apo E3 have been reported to play a protective role. Apo E2, Apo E3, and Apo E4 are formed by two amino acid mutations These gene mutations have been shown to alter lipidation patterns and receptor binding affinities. It is known that Apo E mainly binds to LDLR (Low-Density Lipo The A protein Receptor group is widely distributed in the BBB. This can have a major impact on how po E enters and leaves the brain.

[0008] Patients with cognitive impairment and Alzheimer's disease have significantly lower HDL levels than normal individuals. HDL is known to be effective in preventing cognitive impairment, including Alzheimer's disease. However, HDL is a deficiency disorder that affects many different apoptotic cells in the body. Dynamically reacting with lipoproteins, they coexist with each other, and are abundant in HDL isolated from human plasma. The diversity of these factors makes analytical mechanistic studies difficult. Summary of the Invention [Problem to be solved by the invention]

[0009] The object of the present invention is to provide rHDL that has a therapeutic effect and is not toxic in vivo. do.

[0010] The present invention also provides a composition for preventing or treating a neurodegenerative disease, comprising rHDL. The purpose of this document is to:

[0011] Furthermore, the present invention relates to a method for treating a neurodegenerative disease comprising administering rHDL to a patient suffering from a neurodegenerative disease. The objective is to provide a method for treating neurodegenerative diseases. [Means for solving the problem]

[0012] The present disclosure relates to a method for the preparation of a phospholipid-containing ... and at least one of Apo E2 and / or Apo E3. Provides high density lipoprotein (rHDL).

[0013] The present disclosure includes a phospholipid and apolipoprotein E, Protein E in a weight ratio of 0.25:1 to 2.5:1, preferably 0.5:1 to 1.5:1. The present invention provides a reconstituted high density lipoprotein, characterized in that it is produced from a mixture containing

[0014] In one embodiment, the phospholipids and acetylcholine in the reconstituted high density lipoprotein (rHDL) are The weight ratio of polypoprotein E is 0.2:1 to 2.5:1, preferably 0.2:1 to 2. .5:1, and more preferably 0.2:1 to 0.5:1.

[0015] In one embodiment, the density of the reconstituted high density lipoprotein (rHDL) is 0.1 ~2.0g / mL, preferably 0.2-1.5g / mL, more preferably 0.3-1.2 g / mL.

[0016] In one embodiment, the apolipoprotein E is apolipoprotein E2, E3, or E2 and E3.

[0017] In one embodiment, the apolipoprotein further comprises apolipoprotein A1. nothing.

[0018] The present disclosure relates to a method for preventing or treating a neurodegenerative disease, comprising administering the reconstituted high density lipoprotein to a subject. In one embodiment, the reconstituted high density lipoprotein is amyloid. It inhibits the aggregation of Abeta (Aβ) and maintains the homeostasis of brain tissue.

[0019] The present disclosure includes administering the reconstituted high density lipoprotein to a patient suffering from a neurodegenerative disease. The present invention provides a method for treating neurodegenerative diseases.

[0020] The present disclosure relates to a method for feeding a phospholipid solution into a filter using three inlets and one outlet. ) into the second inlet located in the center of the microfluidic device, and apolipoprotein By injecting the solution into the first and third inlets located on both sides, phospholipids and apo Reconstitution with at least one of Apolipoprotein E The present invention provides a method for producing high density lipoprotein (rHDL). The microfluidic device may include micropillars.

[0021] In one embodiment, the synthetic blend weight ratio of the phospholipid to apolipoprotein E is 0. .25:1 to 2.5:1, preferably 0.5:1 to 1.5:1.

[0022] The present disclosure relates to a reconstituted high density lipoprotein (rHDL) produced by the above-mentioned production method. to provide. Effect of the Invention

[0023] The rHDL nanoparticles and compositions comprising the rHDL nanoparticles of the present disclosure are A) has the effect of inhibiting the aggregation of A). Therefore, the rHDL nanoparticles of the present disclosure are effective in preventing neurodegenerative diseases. It can be used for the prevention or treatment of diseases.

[0024] Furthermore, the rHDL of the present disclosure can be produced using a microfluidic device, Single-step continuous synthesis This ensures consistent properties (no batch-to-batch variability) It is possible to mass-produce rHDL with this function. Compared to conventional rHDL, it has no surfactants or other additives. Since it does not contain any additional ingredients, it does not show any toxicity in the body. [Brief description of the drawings]

[0025] [Figure 1]Schematic diagram of a microfluidic device for producing rHDL, which utilizes the effect of intense mixing of apolipoproteins dissolved in a hydrophilic solution and phospholipids dissolved in a lipophilic solution as they pass through a continuously propagating microvortex induced by micropillars. [Diagram 2] FIG. 2 is a graph showing the production yield and particle size uniformity with and without micropillars in the microfluidic device for producing rHDL nanoparticles shown in FIG. 1 , illustrating the effect of continuously propagating microvortexes on nanoparticle formation. [Diagram 3] FIG. 2 is a graph showing the production yield and particle size uniformity with and without micropillars in the microfluidic device for producing rHDL nanoparticles shown in FIG. 1 , illustrating the effect of continuously propagating microvortexes on nanoparticle formation. [Figure 4] FIG. 1 shows the size and distribution of nanoparticles according to the synthetic blend ratio of phospholipid (DMPC) and apolipoprotein E3, as well as the resulting amount (volume). [Diagram 5] FIG. 1 shows the size and distribution of nanoparticles according to the synthetic blend ratio of phospholipid (DMPC) and apolipoprotein E3, as well as the resulting amount (volume). [Figure 6] FIG. 1 shows the size and distribution of nanoparticles according to the synthetic blend ratio of phospholipid (DPPC) and apolipoprotein E3, and the resulting production amount (volume). [Figure 7] FIG. 1 shows the size and distribution of nanoparticles according to the synthetic blend ratio of phospholipid (DPPC) and apolipoprotein E3, and the resulting production amount (volume). [Figure 8] FIG. 1 shows the size and distribution of nanoparticles according to the synthetic blend ratio of phospholipid (POPC) and apolipoprotein E3, and the resulting production amount (volume). [Figure 9] FIG. 1 shows the size and distribution of nanoparticles according to the synthetic blend ratio of phospholipid (POPC) and apolipoprotein E3, and the resulting production amount (volume). [Figure 10]1 is a diagram showing the results of DLS measurements to compare the size distributions of apolipoproteins E2 and E3 with those of rHDL nanoparticles formed using them. There is no difference in size between apolipoproteins E2 and E3, and the rHDL nanoparticles formed by them form large particles of about 5 to 10 nm by binding with phospholipids, and there is also no difference due to the apolipoprotein. [Figure 11] TEM photograph of rHDL nanoparticles containing apolipoprotein E3 produced with a synthetic blend weight ratio of phospholipid and apolipoprotein of 0.75:1. [Figure 12] TEM photograph of rHDL nanoparticles containing apolipoprotein E3 produced with a synthetic blend weight ratio of phospholipid and apolipoprotein of 1.25:1. [Figure 13] TEM photograph of rHDL nanoparticles containing apolipoprotein E3 produced with a synthetic blend weight ratio of phospholipid and apolipoprotein of 2.5:1. [Figure 14] FIG. 13 is a DLS measurement result showing uniform distribution of hybrid rHDL nanoparticles containing apolipoproteins E2 and E3 simultaneously. [Figure 15] FIG. 1 shows results showing that rHDL nanoparticles containing apolipoprotein E3 contain relatively low and high amounts of phospholipids depending on the structural analysis of the molecular morphology. [Figure 16] This is a graph showing the results after synthesis relative to the size of rHDL nanoparticles containing apolipoprotein E3 after synthesis, when the nanoparticles contain relatively small and large amounts of phospholipids. [Figure 17] 1 is a graph showing the Aβ aggregation inhibitory effect over time for various concentrations of rHDL nanoparticles containing apolipoprotein E2. [Figure 18] 1 is a graph showing the Aβ aggregation inhibitory effect over time for various concentrations of rHDL nanoparticles containing apolipoprotein E3. [Figure 19] 1 is a graph showing the Aβ aggregation inhibitory effect over time for each concentration of hybrid rHDL nanoparticles simultaneously containing apolipoproteins E2 and E3. [Figure 20]1 is a graph showing the Aβ aggregation inhibitory effect over time for each concentration of rHDL nanoparticles depending on the type of apolipoprotein contained in the rHDL nanoparticles. [Figure 21] 1 is a graph comparing the Aβ aggregation inhibitory effect over time between pure apolipoprotein E3 and rHDL nanoparticles formed therewith. [Figure 22] This is a graph showing the results of an experiment on the intracellular absorption amount to represent transcytosis into brain tissue via brain microvascular endothelial cells (BMECs) for rHDL nanoparticles containing apolipoproteins E2 or E3, and hybrid rHDL nanoparticles containing apolipoproteins E2 and E3. [Figure 23] This is a graph showing the results of an experiment on the intracellular absorption amount for rHDL nanoparticles containing apolipoproteins E2 or E3, and hybrid rHDL nanoparticles containing apolipoproteins E2 and E3, to demonstrate transport through astrocytes at the blood-brain barrier. [Figure 24] This shows the results of administering saline to an animal model of Alzheimer's disease (5xFAD) for three months, and then photographing the distribution of amyloid in brain tissue. [Diagram 25] This is a diagram showing the results of administering rHDL nanoparticles containing apolipoprotein E3 to an Alzheimer's disease model animal (5xFAD) for three months, and then photographing the distribution of amyloid in brain tissue. [Figure 26] FIG. 1 shows the amyloid concentration in CSF after administration of rHDL nanoparticles containing apolipoprotein E3 to normal mice (wild type; WT) and Alzheimer's disease model animals (5xFAD) for three months. [Figure 27] FIG. 1 shows the results of administering rHDL nanoparticles containing apolipoprotein E3 to normal mice (wild type; WT) and Alzheimer's disease model animals (5xFAD) for three months, and showing the amyloid concentration in plasma thereafter. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Hereinafter, a person having ordinary skill in the art to which the present disclosure pertains will be described with reference to the accompanying drawings. The present disclosure will now be described in detail with reference to the embodiments and examples thereof so that it may be easily implemented. However, the present disclosure can be implemented in various forms, and the following embodiments and examples are provided. However, the present invention is not limited to the above.

[0027] Throughout this specification, when a part is said to "comprise" a certain element, this is specifically Unless otherwise specified, other elements may be included, rather than being excluded. This means that...

[0028] The term "apolipoprotein E" refers to a protein that is produced by the APOE gene or a functional variant thereof. In a preferred embodiment, apolipoprotein refers to a mammalian protein encoded by Protein E is a human protein encoded by the human APOE gene on chromosome 19. Apolipoprotein E is one of the isoforms of the APOE gene product. One example is apolipoprotein E2 ("APOE2"), apolipoprotein E3 ( "APOE3") and apolipoprotein E4 (APOE4). has three major alleles (epsilon 2, epsilon 3, and epsilon 4) Any allele may be used in the various embodiments of the present invention. A "functional variant" of APOE is one that has the same or similar biological function as the APOE gene product. The present invention relates to a mutant form of a mammalian protein encoded by the APOE gene that maintains its functional function. In some cases, functional variants are involved in the transcription factor agonist (TGA) expression in the protein encoded by the human APOE gene. Contains amino acid insertions, deletions, and / or substitutions compared to the protein. In some cases, functional The typical variant is a fragment of the protein encoded by the human APOE gene. do.

[0029] In some embodiments, apolipoprotein E2 is identified in GenBank under accession number AR a protein disclosed in Q79459 or at least 95% sequence identity thereto; Preferably, there is at least 98% or 99% sequence identity. Apolipoprotein E3 is disclosed in GenBank under the accession number ARQ79461.1. or a sequence identity of at least 95% thereto, preferably at least There is 98% or 99% sequence identity.

[0030] In some embodiments, apolipoprotein A in reconstituted high density lipoprotein (rHDL) is Protein E is a recombinant protein produced by genetic engineering or by chemical synthesis. It is a synthetic protein that has been produced.

[0031] The term "apolipoprotein A1" refers to the APOA1 gene or a functional variant thereof. In a preferred embodiment, the apoA protein is a mammalian protein encoded by Lipoprotein A1 is a human protein encoded by the APOA1 gene on chromosome 11. The "functional variant" is a protein that is identical to the APOA1 gene product. or mammalian genes encoded by the APOA1 gene that maintain similar biological functions. In some cases, the functional variant refers to a variant of the human APOA1 gene. Amino acid insertions, deletions, and / or deletions compared to the protein encoded by the gene In some cases, the functional variant is a variant encoded by the human APOA1 gene. It is a fragment of a protein.

[0032] In some embodiments, apolipoprotein A1 is identified in GenBank as accession number AA S68227.1 or a protein with at least 95% sequence identity thereto Preferably, the sequence identity is at least 98% or 99%.

[0033] In some embodiments, apolipoprotein A in reconstituted high density lipoprotein (rHDL) is Protein A1 is a recombinant protein produced by genetic engineering or chemically synthesized. It is a synthetic protein produced by

[0034] The present disclosure relates to phospholipids and apolipoproteins. and at least one of the following: (rHDL) is provided.

[0035] As used herein, "reconstituted high density lipoprotein (rHDL)" refers to The term "ed high density lipoprotein" refers to phospholipids and The term "HDL-like particles" refers to non-naturally occurring HDL-like particles that contain HDL and apolipoproteins.

[0036] In one embodiment, the apolipoprotein E is apolipoprotein E2 or E3. In another embodiment, the apolipoprotein is apolipoprotein E2 and E3 and may also include apolipoprotein A1. In this study, reconstituted high-density lipoprotein (rHDL) was synthesized by the method described above. In some embodiments, reconstituted high density lipoprotein (rHDL) contains apolipoproteins E2, E3 and A1.

[0037] In some embodiments, the reconstituted high density lipoprotein (rHDL) is apolipoprotein. In some embodiments, the protein E2 and / or E3 is present, but the other apolipoproteins are not present. In this state, reconstituted high density lipoprotein (rHDL) is composed of apolipoproteins A1, E 2 and / or E3, and does not include other apolipoproteins. In addition, reconstituted high density lipoprotein (rHDL) does not contain apolipoprotein E4. In some embodiments, the reconstituted high density lipoprotein (rHDL) is an esterified cholesterol-rich lipoprotein. In some embodiments, the reconstituted high density lipase does not contain sterols or triglycerides. The renal HDL is composed of apolipoproteins A1, A2, A4, C3, D, J, and does not contain M.

[0038] The term "phospholipid" as used herein specifically refers to 1,2-dioleoyl-sn-glycol. Lysero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), di Lauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glyceryl D-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) , distearoylphosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl 1-palmitoyl-2-myristoyl phosphatidylcholine (MPPC), Phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC) , 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2- Diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-dieicosapentaenoic acid Noyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatase Phatidylcholine (POPC), Lysophosphatidylcholine, Dilinoleoylphosphatidylcholine Dystearoylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristo Dipalmitoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DMPE), DPPE, palmitoyloleoylphosphatidylethanolamine ( POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[( 3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamate VL-5) , dioctadecylamidoglycylspermine 4-trifluoroacetate (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 (DOT AP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium DORIE, 1,2-Dimyristyloxy-propyl-3-dimethyl -Hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy- N-[2(Sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanediol N-(3-aminopropyl)-N,N-trifluoroacetate (DOSPA) -Dimethyl-2,3-bis(dodecyloxy)-1-propane ammonium bromide ( GAP-DLRIE), Nt-butyl-N′-tetradecyl-3-tetradecylamino Propionamidine (diC14-amidine), Ethylphosphocholine (Ethyl PC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesterol Lyl-spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane D-Lin-MC3-DMA (MC3, DLin-MC3-D MA), DLin-KC2-DMA, and DLin-DMA It may be, but is not limited to,

[0039] In some embodiments, the reconstituted high density lipoprotein is 1,2-dioleoyl-sn -Glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC) , dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glyceryl Lysero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPP C), distearoylphosphatidylcholine (DSPC), 1-myristoyl-2-palladium Myristoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine Phatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine Phosphorus (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPP C), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1, 2-Diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-diamino Cosanoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyl oleoyl Phosphatidylcholine (POPC), Lysophosphatidylcholine, Dilinoleoylphosphatidylcholine Phosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimer Dipalmitoyl phosphatidylethanolamine (DMPE), Dipalmitoyl phosphatidyl Ethanolamine (DPPE), Palmitoyloleoylphosphatidylethanolamine POPE, lysophosphatidylethanolamine, N1-[2-((1S)-1- [(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butyl Carboxyamide)ethyl]-3,4-di[oleyloxy]-benzamide) (VL- 5), dioctadecylamidoglycylspermine 4-trifluoroacetate (DOGS), 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol ( DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammonium proton Pan (DOTMA), 1,2-dioleyl-3-trimethylammonium-propane (D OTAP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethyl a Ammonium bromide (DORIE), 1,2-dimyristyloxy-propyl-3-dimethyl Diethyl-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy C-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanediol Aminotrifluoroacetate (DOSPA), N-(3-aminopropyl)-N ,N-Dimethyl-2,3-bis(dodecyloxy)-1-propane ammonium bromide GAP-DLRIE, Nt-butyl-N'-tetradecyl-3-tetradecyl diC14-amidine, ethylphosphocholine (Eth yl PC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesterol Steryl-spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane Dopan (DODMA), or D-Lin-MC3-DMA (MC3, DLin-MC3 -DMA), DLin-KC2-DMA, and DLin-DMA were selected from 1, 2, and 3 or four phospholipids.

[0040] In some embodiments, the reconstituted high density lipoprotein comprises DMPC. In some embodiments, the reconstituted high density lipoprotein comprises DPPC. , reconstituted high density lipoprotein contains POPC.

[0041] In some embodiments, the reconstituted high density lipoprotein comprises DMPC and other phosphoryl groups. In some embodiments, the reconstituted high density lipoprotein does not contain DPPC. In some embodiments, the reconstituted high density lipoprotein comprises a phospholipid and no other phospholipids. contains POPC and no other phospholipids.

[0042] The present disclosure relates to a method for the preparation of a phospholipid-containing composition comprising: In some embodiments, a reconstituted high density lipoprotein is provided, comprising: In this study, a synthetic formulation for reconstituted high density lipoprotein (HDLP) was developed that contains phospholipids and apolipoproteins. The weight ratio of the material E is 0.25:1 to 2.5:1, preferably 0.5:1 to 1.5:1. In one embodiment, the apolipoprotein E is apolipoprotein E2, E3, or E2 and E3.

[0043] In some embodiments, the reconstituted high density lipoprotein (rHDL) comprises phospholipids and produced from a mixture containing apolipoprotein E in weight ratios of 0.25:1 to 2.5:1 In some embodiments, the reconstituted high density lipoprotein (rHDL) comprises phospholipids and It was produced from a mixture containing apolipoprotein E in a weight ratio of 0.5:1 to 1.5:1. In some embodiments, reconstituted high density lipoprotein (rHDL) is a lipid-rich lipid comprising phospholipids and acetylcholine. The antibodies were produced from mixtures containing polypoprotein E in weight ratios ranging from 0.1:1 to 5:1.

[0044] The present disclosure relates to a method for the preparation of a phospholipid-containing medicament comprising administering to a patient a small amount of apolipoprotein E. In some embodiments, a reconstituted high density lipoprotein is provided, the reconstituted high density lipoprotein comprising at least one of: Weight distribution of phospholipids and apolipoprotein E in reconstituted high density lipoprotein (rHDL) The ratio is 0.1:1 to 5:1. In some embodiments, the reconstituted high density lipoprotein The weight ratio of phospholipids to apolipoprotein E in rHDL is 0.1:1 to 3:1. In some embodiments, the phospholipids in the reconstituted high density lipoprotein (rHDL) are The weight ratio of the protein to apolipoprotein E is 0.2:1 to 1.5:1. In the endothelial cell, phospholipids and apolipoproteins in reconstituted high-density lipoprotein (rHDL) were The weight ratio to quality E is 0.2:1 to 0.5:1.

[0045] In some embodiments, the phospholipids in reconstituted high density lipoprotein (rHDL) are The weight ratio of apolipoprotein E2 and apolipoprotein E3 is 1:5:5 to 30:5: In some embodiments, the phosphorus in reconstituted high density lipoprotein (rHDL) is 5. The weight ratio of lipids, apolipoprotein E2 and apolipoprotein E3 is 2:5:5 to 1. In some embodiments, reconstituted high density lipoprotein (rHDL) The weight ratio of phospholipids, apolipoprotein E2 and apolipoprotein E3 in :5~5:5:5.

[0046] In some embodiments, apolipoprotein A in reconstituted high density lipoprotein (rHDL) is The weight ratio of protein E2 and apolipoprotein E3 is 1:10 to 10:1. In an embodiment, apolipoprotein E in reconstituted high density lipoprotein (rHDL) In some embodiments, the weight ratio of 2 to apolipoprotein E3 is 1:5 to 5:1. In the present study, apolipoprotein E2 and apolipoprotein E3 in reconstituted high density lipoprotein (rHDL) were In some embodiments, the weight ratio of the reconstituted polyprotein E3 to the reconstituted polyprotein E4 is 1:2 to 2:1. Apolipoprotein E2 and apolipoproteins in high density lipoprotein (rHDL) The weight ratio of E3 is 1:1.

[0047] In some embodiments, the density of the reconstituted high density lipoprotein (rHDL) is 0.1 In some embodiments, the reconstituted high density lipoprotein (rH In some embodiments, the density of the reconstituted high-density collagen (DL) is 0.2 to 1.5 g / mL. The density of renal HDL is 0.3 to 1.2 g / mL.

[0048] In some embodiments, the reconstituted high density lipoprotein (rHDL) is less than 50 nm. , nanoparticles having a major axis length of less than 25 nm, or less than 20 nm. In the study, reconstituted high density lipoprotein (rHDL) was nanoparticles with a long axis length of more than 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm In some embodiments, the reconstituted high density lipoprotein (rHDL) is Nanoparticles having a major axis length of 1 to 100 nm, 5 to 50 nm, 5 to 25 nm, or 5 to 20 nm. It is a particle.

[0049] In some embodiments, the reconstituted high density lipoprotein (rHDL) is 5 to 200 kDa. Da, 5-150 kDa, 5-100 kDa, or 5-60 kDa nanoparticles.

[0050] In one aspect, the present disclosure provides a method for the preparation of a reconstituted high density lipoprotein as described herein and a pharma- ceutical composition comprising the reconstituted high density lipoprotein ... The present invention provides a pharmaceutical composition comprising an acceptable excipient.

[0051] In some embodiments, the pharmaceutical composition comprises a reconstituted high density apolipoprotein E2. A first set of high density lipoproteins, and a reconstituted high density lipoproteins containing apolipoprotein E3. In some embodiments, the pharmaceutical composition comprises a second set of proteins. Reconstituted high density lipoprotein containing both protein E2 and apolipoprotein E3 Contains gypsum.

[0052] In some embodiments, the pharmaceutical composition comprises a reconstituted high density apolipoprotein E2. The first set of high-density lipoproteins, reconstituted high-density lipoproteins containing apolipoprotein E3 A second set of proteins, and a reconstituted high density lipoprotein containing apolipoprotein A1. In some embodiments, the pharmaceutical composition comprises a third set of apolipoprotein A. Contains reconstituted high density lipoproteins containing E2, E3 and A1.

[0053] In some embodiments, the reconstituted high density lipoprotein (rHDL) in the pharmaceutical composition At least 80% of the nanoparticles have a major axis length of 10 to 20 nm. In one embodiment, at least 7% of the reconstituted high density lipoprotein (rHDL) in the pharmaceutical composition is 0% are nanoparticles having a major axis length of 10 to 20 nm. At least 60% of the reconstituted high density lipoprotein (rHDL) in the biological composition is The nanoparticles have a major axis length of 20 nm. In some embodiments, the pharmaceutical composition At least 50% of reconstituted high-density lipoprotein (rHDL) has a long axis of 10–20 nm. In some embodiments, the reconstituted high density linker in the pharmaceutical composition is a nanoparticle having a length of 100 nm. At least 40% of lipoproteins (rHDL) are nanoparticles with a long axis length of 10 to 20 nm. It is a particle.

[0054] In some embodiments, the reconstituted high density lipoprotein (rHDL) in the pharmaceutical composition At least 80% of the nanoparticles have a major axis length of 5 to 50 nm. At least 70% of the reconstituted high density lipoprotein (rHDL) in the pharmaceutical composition % are nanoparticles having a major axis length of 5 to 50 nm. At least 60% of the reconstituted high density lipoprotein (rHDL) in the composition is 5-50 nM. In some embodiments, the nanoparticles have a major axis length of about 100 nm. At least 50% of rHDL has a long axis length between 5 and 50 nm. In some embodiments, the reconstituted high density lipoprotein in the pharmaceutical composition is a nanoparticle. At least 40% of the protein (rHDL) is a nanoparticle with a long axis length of 5-50 nm. .

[0055] In another aspect, the present disclosure provides a method for treating a neurodegenerative disease comprising administering to a patient the reconstituted high density lipoprotein. In one embodiment, a composition for preventing or treating reconstituted high density lipoprotein (HDLP) is provided. The substance may inhibit the aggregation of amyloid beta (Aβ). Constitutive high density lipoproteins can maintain brain tissue homeostasis.

[0056] The term "prevention" as used herein means suppressing or delaying the onset of a disease by administering a composition. The term "treatment" refers to any action that extends the lifespan of a disease by administering a composition to a suspected disease. means any action that improves or beneficially alters the condition of an affected individual. do.

[0057] In one embodiment, the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, Pick's disease , Huntington's disease, Creutzfeldt-Jakob disease, Lou Gehrig's disease, spinocerebellar degeneration, Spinocerebellar ataxia, prion disease, cognitive impairment, senile dementia, Lewy body dementia, frontal lobe Temporal dementia, vascular dementia, alcoholic dementia, presenile dementia, Machado Joseph Disease, dystonia, multiple system atrophy, progressive supranuclear palsy, Friedreich's ataxia, temporal lobe These include, but are not limited to, epilepsy, or stroke.

[0058] The present disclosure relates to a method for the preparation of phospholipids and apolipoprotein E cysts. and at least one of: The method includes: dissolving a hydrophilic solution containing apolipoprotein E in a first solution of a microfluidic device; and injecting a phospholipid solution into a second inlet of the microfluidic device. Step 3: Collect reconstituted high density lipoprotein (rHDL) from the outlet of the microfluidic device. The method may further comprise a step of:

[0059] The present disclosure also provides a method for introducing a first hydrophilic solution containing a first apolipoprotein into three inlets and injecting into a first inlet of a microfluidic device having one outlet; into a second inlet of the microfluidic device; and and injecting a second hydrophilic solution containing the second hydrophilic solution into a third inlet of the microfluidic device. A reconstituted high density lipoprotein comprising at least one of a phospholipid and an apolipoprotein. The present invention provides a method for producing rHDL.

[0060] In some embodiments, the microfluidic device comprises a first fluidic device located at the center of the microfluidic device. In some embodiments, the second inlet is located on one of the two sides, and the first and third inlets are located on the other side. wherein the microfluidic device is configured to mix a phospholipid solution and a hydrophilic solution. It further comprises micropillars.

[0061] In some embodiments, the first apolipoprotein and the second apolipoprotein In some embodiments, the first apolipoprotein is Both the protein and the second apolipoprotein are apolipoprotein E3. In one embodiment, the first apolipoprotein is apolipoprotein E2 and the second apolipoprotein is In some embodiments, the lipoprotein is apolipoprotein E3. The lipoprotein is apolipoprotein E2 or E3, and the second apolipoprotein is apolipoprotein A1.

[0062] In some embodiments, the first hydrophilic solution, the phospholipid solution, and the second hydrophilic solution are injected simultaneously.

[0063] In some embodiments, the method includes producing reconstituted high density lipoprotein (rHDL). The method further comprises the step of collecting via the mouth.

[0064] In some embodiments, a phospholipid is injected into an inlet of a microfluidic device, and a first and The weight ratio of the second apolipoprotein is 0.25:1 to 2.5:1. In the embodiment, a phospholipid is injected into an inlet of a microfluidic device, and a first and a second apolipoprotein are injected into the inlet of the microfluidic device. In some embodiments, the weight ratio of the polysaccharide to the protein is 0.5:1 to 1.5:1. A phospholipid and a first and a second apolipoprotein are injected into an inlet of a microfluidic device. The weight ratio is 0.1:1 to 5:1.

[0065] In some embodiments, a phospholipid is injected into the inlet of the microfluidic device and a first alpha The weight ratio of the first apolipoprotein to the second apolipoprotein is 1:2:2 to 10:2: 2. In some embodiments, a phospholipid is injected into an inlet of a microfluidic device; The weight ratio of the first apolipoprotein to the second apolipoprotein is 1:1:1 to 3. In some embodiments, the phospholipid injected into the inlet of the microfluidic device is The weight ratio of the protein to the first apolipoprotein to the second apolipoprotein is 1:5: In some embodiments, the ratio of the mixture injected into the inlet of the microfluidic device is 5 to 30:5:5. The weight ratio of the phospholipid to the first apolipoprotein to the second apolipoprotein is In some embodiments, the ratio of the inlet of the microfluidic device to the The weight ratio of the injected phospholipid, the first apolipoprotein, and the second apolipoprotein The ratio is 2:5:5 to 5:5:5.

[0066] In some embodiments, the method further comprises: feeding the phospholipid solution into three inlets and and a second inlet located in the center of a microfluidic device having one outlet. Injecting the apolipoprotein solution into the first and third inlets located on both sides. In one embodiment, the microfluidic device comprises a microfluidic device. It may include pillars.

[0067] As used herein, the term "microfluidic device" refers to a plastic device that includes organic polymeric materials. Fluids are applied to substrates made from a variety of materials, including silicon, glass, metal, or ceramic. By this term is meant a device containing microchannels configured to allow for the passage of liquid.

[0068] The term "micropillar" or "micropillar structure" as used herein means a micropillar structure. By creating vortexes in the microfluidic device, different types of flow injected into the microfluidic device can be It means a columnar structure for efficient mixing of the body.

[0069] In some embodiments, the method further comprises: The method further comprises the step of purifying by centrifugation.

[0070] In one aspect, the present disclosure provides a reconfigurable high density readout device manufactured by the methods described herein. Provides renal HDL (rHDL).

[0071] In another aspect, the present disclosure provides a reconstituted high density lipoprotein as described herein or Preventing or treating a neurodegenerative disease in a subject, comprising administering to the subject a pharmaceutical composition as described above. In some embodiments, the disease is Parkinson's disease, Alzheimer's disease, or a combination of both. Zheimer's disease, Pick's disease, Huntington's disease, Creutzfeldt-Jakob disease, Lou Gehri Chung's disease, spinocerebellar degeneration, spinocerebellar ataxia, prion disease, cognitive impairment, senile dementia, Dementia with Bee bodies, frontotemporal dementia, vascular dementia, alcoholic dementia, presenile cognition Machado-Joseph disease, dystonia, multiple system atrophy, progressive supranuclear palsy, Friedrich- The condition is selected from the group consisting of Ihi ataxia, temporal lobe epilepsy, and stroke.

[0072] In some embodiments, the reconstituted high density lipoprotein or pharmaceutical composition is amylo. In an amount sufficient to inhibit the aggregation of Aβ or maintain brain tissue homeostasis It is administered at .

[0073] The present disclosure will now be described in more detail with reference to the following examples, which are provided for illustrative purposes only. It is merely intended to be illustrative and not to limit the scope of the present disclosure. EXAMPLES

[0074] Fabrication of rHDL nanoparticles using a microfluidic device 1-1. Design of a microfluidic device for producing rHDL nanoparticles Reconstituted high density lipoprotein by mixing phospholipids and apolipoproteins (rHDL; reconstituted high density lipopro Microfluidic device for producing tein nanoparticles We designed a microfluidic device (Figure 1). The microfluidic device has three inlets and It has one outlet, and the central inlet of the three inlets is hydrophobic. Hydrophobic phospholipids and hydrophobic drugs are inserted into the two inlets on either side of the Inject hydrophilic apolipoproteins and hydrophilic drugs from In addition, a microfluidic device was used to effectively mix lipids and apolipoproteins. A micropillar structure was introduced inside the device.

[0075] 1-2. Preparation of rHDL nanoparticles 1 Using a microfluidic device, phospholipids (DMPC) and apolipoproteins were synthesized in the following manner. Nanoparticles containing rHDL were prepared using rHDL-containing glycerol.

[0076] DMPC (1,2-dimyristoyl-sn-glyceryl) in absolute ethanol o-3-phosphocholine solution and apolipoprotein solution in PBS Then, one syringe was filled with 0.83 ml of anhydrous ethanol. Fill the other two syringes with DMPC solution at 100 µg / mL and 0.5 µL of PBS. Add approximately 1.25 mL of apolipoprotein solution (total approximately 2.5 mL) at a concentration of 0.2 mg / mL. All syringes were debubbled after each filling.

[0077] Connect each injection needle to the inlet of the microfluidic device with a tube and wash the microfluidic device. To achieve this, PBS was injected at a flow rate of 1 mL / min. Then, D The infusion flow rate of the MPC solution was set to 0.8 mL / min, and the infusion flow rate of the apolipoprotein solution was set to 2 The flow rate was set at 0.2 mL / min. rHDL nanoparticles produced from the outlet of the microfluidic device. The nanoparticles were collected, mixed with PBS, and then centrifuged using a 10K filter. The purification was carried out three times for 20 minutes at 4°C with the rotor at maximum speed. After the last purification, approximately 250 μL of the residue was The remaining nanoparticle solution was stored at 4°C.

[0078] 1-3. Preparation of rHDL nanoparticles 2 In the same manner as in 1-2, rHDL containing phospholipids (DPPC) and apolipoproteins was Nanoparticles were prepared.

[0079] 1-4. Preparation of rHDL nanoparticles 3 In the same manner as in 1-2, rHDL containing phospholipids (POPC) and apolipoproteins was Nanoparticles were prepared.

[0080] 1-5. Effects of micropillar structure To confirm the mixing efficiency of lipids and apolipoproteins with and without micropillar structures Simulation and red ink used to observe the distribution of phospholipids in a microfluidic device As a result, as shown in Figure 1, the micropillar structure created a microvortex ( Microvortexes are generated, allowing phospholipids and apolipoproteins to mix more efficiently. In addition, the production of rHDL nanoparticles was confirmed by quantification of BCA protein. When we checked the yield, as shown in Figure 2, the yield was higher than that without micropillars. It was confirmed that the production yield improved when clopylar was present.

[0081] In addition, as shown in Figure 3, rHDL nanoparticles produced by two different microfluidic devices were DLS data was measured after dissolving in BS, and the results were compared with the case without micropillars. In addition, the size of rHDL nanoparticles produced in a microfluidic device with micropillars was The particle size distribution is much more uniform and the degree of aggregation is reduced, It was confirmed that homogeneity was improved. EXAMPLES

[0082] Optimization of the ratio of phospholipids and apolipoprotein E3 The synthetic ratio of phospholipids and apolipoproteins used in the production of rHDL nanoparticles was To optimize the average size and size of rHDL nanoparticles, we synthesized them in the following manner: The size distributions were compared.

[0083] 2-1. rHDL nanoparticles containing phospholipids (DMPC) and apolipoproteins The same method as in Example 1-2 was used to determine the ratio of phospholipid (DMPC) and apolipoprotein. The rHDL nanoparticles produced at different ratios were dissolved in PBS and analyzed by Zetasizer N. Dynamic Light Scattering (DLS) was performed using the ano ZS. The change in particle size distribution due to the aggregation phenomenon of nanoparticles caused by tering was measured. Results are displayed as intensity, volume, and number. was plotted by

[0084] To artificially produce stable HDL, it is necessary to make rHDL dense and small. Therefore, for use as a pharmaceutical, stable rHDL needs to be produced in large quantities.

[0085] As shown in FIG. 4, apolipoprotein E3 (GenB ank:ARQ79461.1) synthetic blend weight ratio of 0.75:1 to 1.25:1 When the synthetic blend weight ratio was 0.75:1, the nanoparticles were It was confirmed that the final product, rHDL, was obtained with small and uniform size.

[0086] The HDL naturally produced in the human body has a diameter of 20 nm or less, but the particle size of the rHDL disclosed herein is It can be seen that most of the particles have a size of 20 nm or less. As a result, the distribution of HDL with a stable particle size of 10 to 20 nm was calculated. When the composition weight ratio is 0.75:1, it is 70% or more. When the composition weight ratio is 1.25:1, it is 70% or more. In the case of the synthetic blend weight ratio, the content was 20% or more, but in the case of the synthetic blend weight ratio of 2.5:1, it was less than 5%. It was confirmed that.

[0087] The synthetic blend weight ratio of apolipoprotein E3 to phospholipid (DMPC) is 2.5:1 Above this, more phospholipids than are required for nanoparticle formation are present, and the remaining phospholipids Stably formed rHDL binds to each other to form clusters. or further aggregate to form phospholipid aggregates. It can be seen that...

[0088] In addition, the synthetic weight ratio of apolipoprotein E3 to phospholipid (DMPC) was 0. If the ratio is less than 25:1, the amount of apolipoprotein contained is greater than that required for nanoparticle formation. In rare cases, apolipoproteins that were not formed into nanoparticles were observed to aggregate.

[0089] In general, apolipoproteins are relatively expensive compared to phospholipids, so apolipoproteins are synthesized in large amounts. It is more preferable from the viewpoint of mass production not to include more polypoproteins than necessary. For efficient mass production, relatively inexpensive phospholipids are synthesized in excess of the required amount. It is preferable to subsequently remove the phospholipids together with the organic solvent.

[0090] Therefore, after synthesizing the nanoparticles, the organic solvent is removed, and the nanoparticles are synthesized. By removing as much of the remaining phospholipids as possible, a uniform and stable rHDL is produced. Therefore, the phospholipids and apolipoproteins contained in the final product, rHDL nanoparticles, The weight ratio is even smaller than the synthetic blend weight ratio.

[0091] 2-2. rHDL nanoparticles containing phospholipids (DPPC) and apolipoproteins The same method as in Examples 1-3 was used to determine the ratio of phospholipid (DPPC) and apolipoprotein. The rHDL nanoparticles produced at different ratios were dissolved in PBS and analyzed by Zetasizer N. Dynamic Light Scattering (DLS) was performed using the ano ZS. The change in particle size distribution due to the aggregation phenomenon of nanoparticles caused by tering was measured.

[0092] As shown in Figure 6, the synthetic formulation of apolipoprotein E3 to phospholipid (DPPC) At a weight ratio of 1.1:1, the final product, rHDL, had small nanoparticle size and uniformity. It was confirmed that it can be obtained.

[0093] Specifically, as shown in FIG. 7, the distribution of HDL with a particle size of 40 nm or less was measured. The calculation confirmed that the ratio was 50% or more when the synthetic blend weight ratio was 1.1:1.

[0094] The synthetic blend weight ratio of apolipoprotein E3 to phospholipid (DPPC) is 2.5:1 Above this, more phospholipids than are required for nanoparticle formation are present, and the remaining phospholipids Stably formed rHDL binds to each other to form clusters. or further aggregate to form phospholipid aggregates. It was confirmed that.

[0095] In addition, the synthetic weight ratio of apolipoprotein E3 to phospholipid (DPPC) was 0. If the ratio is less than 5:1, the amount of phospholipid contained will be less than the amount required for nanoparticle formation. It was confirmed that the instability of the constituent materials increased and the size distribution of the aggregates became non-uniform.

[0096] 2-3. rHDL nanoparticles containing phospholipids (POPC) and apolipoproteins The same method as in Examples 1-4 was used to determine the ratio of phospholipids (POPC) and apolipoproteins. The rHDL nanoparticles produced at different ratios were dissolved in PBS and analyzed by Zetasizer N. Dynamic Light Scattering (DLS) was performed using the ano ZS. The change in particle size distribution due to the aggregation phenomenon of nanoparticles caused by tering was measured.

[0097] As shown in Figure 8, synthetic formulation of apolipoprotein E3 to phospholipids (POPC) When comparing the weight ratio of 1.2:1 with that of 1.35:1, the composite weight ratio of 1.2 In the case of 1, the final product, rHDL, was obtained with small nanoparticle size and uniformity. confirmed.

[0098] Specifically, as shown in FIG. 9, the distribution of HDL with a particle size of 40 nm or less was measured. The calculation results showed that the ratio was 60% or more when the synthetic blend weight ratio was 1.2:1, and It was confirmed that the ratio was 20% or more when the ratio was 1.35:1.

[0099] A synthetic blend of apolipoprotein E3 to phospholipid (POPC) at a weight ratio of 2.5:1 Above this, more phospholipids than are required for nanoparticle formation are present, and the remaining phospholipids Stably formed rHDL binds to each other to form clusters. or further aggregate to form phospholipid aggregates. It was confirmed that.

[0100] In addition, the synthetic blend weight ratio of apolipoprotein E3 to phospholipid (POPC) was 0. If the ratio is less than 5:1, the amount of phospholipid contained will be less than the amount required for nanoparticle formation. It was confirmed that the instability of the constituent materials increased and the size distribution of the aggregates became non-uniform. EXAMPLES

[0101] Preparation of rHDL nanoparticles containing apolipoproteins E2 or E3 The method for producing rHDL nanoparticles containing apolipoprotein E3 in the same manner as in Examples 1 and 2 was carried out. In addition, rHDL containing apolipoprotein E2 (GeneBank: ARQ79459) Nanoparticles were produced. The particle size distribution was then measured using DLS in the same manner as in Example 2. was measured and compared with rHDL nanoparticles containing apolipoprotein E3.

[0102] As shown in FIG. 10, rHDL nanoparticles containing apolipoprotein E3 and apolipoprotein There was little difference in shape between the rHDL nanoparticles containing protein E2 and the nanoparticles containing protein E2 under the same conditions. It was confirmed that they could produce offspring. EXAMPLES

[0103] Morphology of rHDL nanoparticles containing apolipoprotein E3 According to the manufacturing method of Example 1, apolipoprotein E3 to phospholipid (DMPC) The rHDL nanoparticles were prepared with a weight ratio of 0.75:1 and analyzed by transmission electron microscopy (TEM). Transmission electron microscopy (TEM) was used to The morphological characteristics were observed. rHDL nanoparticles were dissolved in PBS and then plated on nickel grids. The plate was placed on top, negatively stained, and allowed to dry thoroughly before photographing.

[0104] As shown in Figure 11, rHDL nanoparticles containing apolipoprotein E3 were formed by forming two layers of apolipoprotein E3. Lipoproteins are wrapped around phospholipids in a discoid shape. ) was confirmed.

[0105] On the other hand, as shown in Figs. 12 and 13, when the weight ratio is 1.25:1, a stable disk-shaped structure is formed. It was found that the amount of rHDL decreased, and when the weight ratio exceeded 2.5:1, stable rHDL was observed. It can be seen that L is hardly observed.

[0106] When the weight ratio exceeds 2.5:1, the remaining phospholipids will dissolve the already formed stable rHDL. It binds like a cloud and increases the particle size of rHDL, but in this case, rHDL becomes anxious. Because rHDL has a fixed morphology, it is not possible to maintain a constant concentration of rHDL in the body. Apolipoprotein E (ApoE) acts on the LDLR (low-density receptor) in the body. They cross the BBB via the lipoprotein receptor or They transport myloid beta out of the brain, but phospholipids bind to these apolipoproteins. These proteins form a cloud around the protein, potentially making it difficult for rHDL to perform its functions. be. EXAMPLES

[0107] Preparation of hybrid rHDL nanoparticles containing multiple apolipoproteins The rHDL nanoparticles of the present disclosure are characterized by the type and composition of apolipoproteins surrounding phospholipids. Therefore, in this embodiment, the phospholipid-enclosing acetylcholinesterase has different functions and effects depending on the acetylcholinesterase. Hybrid rHDL nanoparticles composed of multiple types of polypoproteins were prepared.

[0108] Apolipoprotein A1 (GenBank: AAS68227.1) is a highly biocompatible To prepare rHDL nanoparticles containing both rHDL and apolipoprotein E3, 0.8mg / mL DMPC solution, 0.2mg / mL apolipoprotein A1 solution, and rHDL nanoparticles were prepared according to Example 1 using both rHDL and apolipoprotein E3 solutions. did.

[0109] In addition, when apolipoprotein E2 solution and apolipoprotein E3 solution are used together, We have demonstrated that hybrid rHDL nanoparticles containing polypoproteins E2 and E3 can be produced. In order to prepare the hybrid nanoparticles of the present disclosure, Prepare 1.25 mL each of ApoE2 and ApoE3 solutions with concentrations of 0.83 m and mix them together. Prepare 0.8 mL of DMPC solution with a concentration of 100 µg / mL and prepare nanoparticles according to Example 1. As a result, as shown in FIG. 10, a hybrid containing apolipoproteins E2 and E3 was It was confirmed that red rHDL nanoparticles could be produced as uniform nanomaterials.

[0110] When apolipoprotein A1 solution and apolipoprotein E3 solution were used together, rHDL nanoparticles containing apolipoprotein A1 and rH rHDL nanoparticles and hybrid rHDL nanoparticles containing apolipoproteins A1 and E3 Both particles can be present. In addition, ELISA confirmed that the hybrid It was confirmed that rHDL nanoparticles were produced at a ratio of approximately 10% of the total nanoparticles. It was done.

[0111] In conclusion, the conventional incubation method The protein is mixed with a phospholipid carrier and left to stand, but the apolipoproteins remain aggregated together. Therefore, even if apolipoproteins E2 and E3 were added to a culture dish at the same time, the E2 and E3 proteins Proteins do not spontaneously fuse with each other to produce hybrid rHDL. It is understood that the disclosed manufacturing methods result in the production of hybrid rHDL nanoparticles. EXAMPLES

[0112] Phospholipids and apolipoproteins in rHDL nanoparticles containing apolipoprotein E3. Quality composition The final phospholipids and apolipoproteins of the rHDL nanoparticles prepared according to Examples 1 and 2 To confirm the protein composition ratio, BCA assay and Lipid quantification were performed. The results are shown in Table 1.

[0113] [Table 1]

[0114] As shown in Table 1, the phospholipids of rHDL nanoparticles produced at different times were determined in a total of six groups. The ratio of protein to apolipoprotein was 0.2:1 to 0.5:1. The rHDL was produced with a synthetic phospholipid:apolipoprotein weight ratio of 0.75:1. In the case of Therefore, excess phospholipids are removed together with the organic solvent during the rHDL production process, It can be seen that the phospholipid content in the final product, rHDL, is reduced.

[0115] The rHDL of the present disclosure contains phospholipids in an apolipoprotein structure that forms two layers. The phospholipids after synthesis of rHDL are produced in a synthetic mixture ratio optimized for mass production: Apolipoproteins were determined to be between 0.2:1 and 0.5:1.

[0116] However, if excess phospholipids remain with the organic solvent during the rHDL production process, After synthesis of rHDL, the phospholipid:apolipoprotein ratio was 0.2:1 to 2.5:1. If excess phospholipids are not completely removed together with the organic solvent and remain, the ratio of the excess phospholipids is It may be 0.2:1 to 1.5:1. EXAMPLES

[0117] Measurement and calculation of the density of rHDL nanoparticles The molecular weight of the apolipoprotein E3 used in the synthesis and the length of the material used in the synthesis Based on the volume range of the final synthesis product from DLS measurements, the density of the rHDL nanoparticles was calculated as I calculated it.

[0118] The mass information of the rHDL nanoparticles is as follows: Molecular weight of apolipoprotein E3 The molecular weight of the final synthesis product is 35.20 kDa, and apolipoprotein E3 forms two layers. The total mass of the constituent proteins is calculated to be 70.40 kDa. Preferred final lipid for rHDL nanoparticles prepared according to 1 and 2: apolipoprotein The ratio of the proteins was confirmed to be 0.2:1 to 0.5:1. The mass of the lipids contained was calculated to be 14.08 to 35.20 kDa. The molecular weight of the rHDL nanoparticles is 84.48–10, which is the sum of the constituent proteins and lipids. 5.60 kDa (140.24 × 10 -21 ~175.30×10 -21 g) is calculated as It was done.

[0119] The volume information of rHDL nanoparticles is as follows. The morphology of the L nanoparticles was estimated. The results are shown in Figures 14 to 16. The diameter of the rHDL nanoparticles containing apolipoprotein E3 was It was confirmed that the size distribution was 8 to 15 nm. Assuming that the short axis (height) of the rHDL nanoparticle in e) is 2.5 nm, the nanoparticle will be spherical or The volume of the ellipsoid is 125.6 to 441.6 nm 3 It was calculated that: Therefore, the density of the rHDL nanoparticles is estimated to be 0.3–1.0 times the mass and volume estimated from the experimental data. Calculated as a density distribution of 1.2 g / mL. [Test Example 1] Confirmation of Aβ aggregation inhibitory effect of rHDL nanoparticles To confirm the amyloid beta (Aβ) aggregation inhibitory effect of the rHDL nanoparticles produced in the examples, In order to do so, the following experiment was carried out.

[0120] 1 μM Aβ1-42 labeled with 488 fluorescence and three concentrations (0.01, 0.1, 0 rHDL nanoparticles (0.5 μM) were prepared and simultaneously treated in a 96-well plate. , 10, 20, 30, 40, 50, 60, 120, 150, 240, 300, 360, 7 The fluorescence values ​​were photographed after 20 and 1440 minutes, respectively. The fluorescence values ​​of the plates treated separately were also confirmed. The fluorescence intensity decreases due to self-quenching, so the rHDL nanoparticles If the fluorescence value when treated with 488-Aβ is higher than that when treated with 488-Aβ alone, it is considered that Aβ This can be interpreted as having an aggregation inhibitory effect.

[0121] 1-1. rHDL nanoparticles containing apolipoprotein E2 or E3 As shown in Figures 17 and 18, when 488-Aβ was treated alone, 488-Aβ increased over time. It was confirmed that the fluorescence intensity decreased due to self-quenching caused by aggregation. The combination of rHDL nanoparticles containing apolipoprotein E2 or E3 was more effective than the combination of rHDL nanoparticles containing apolipoprotein E2 or E3. It was confirmed that the fluorescence value was high when treated with rHDL nanoparticles. It was confirmed that the fluorescence value was higher as the concentration increased.

[0122] Therefore, it is clear that the rHDL nanoparticles of the present disclosure have an excellent Aβ aggregation inhibitory effect. .

[0123] 1-2. Hybrid rHDL nanoparticles containing apolipoproteins E2 and E3 As shown in FIG. 19, apolipoprotein E was significantly increased in the 488-Aβ-treated group compared with the 488-Aβ-treated group alone. The fluorescence intensity was higher when the hybrid rHDL nanoparticles containing E2 and E3 were treated together. As the concentration of hybrid rHDL nanoparticles increased, the fluorescence intensity increased. In particular, when treated with 0.5 μM hybrid rHDL nanoparticles, the fluorescence value There was almost no decrease in Aβ aggregation, confirming that the compound has a remarkable effect in inhibiting Aβ aggregation. [Test Example 2] Comparison of Aβ aggregation inhibition effects depending on the type of apolipoprotein contained in rHDL nanoparticles To compare the effects of different types of apolipoproteins contained in rHDL nanoparticles, To achieve this, we used rHDL nanoparticles containing the same concentrations of apolipoproteins E2 and E3 (0.5 μM). , amyloid of hybrid rHDL nanoparticles containing apolipoprotein E2 or E3 The beta (Aβ) aggregation inhibitory effect was compared in the same manner as in Test Example 1.

[0124] As shown in FIG. 20, rHDL nanoparticles containing apolipoproteins E2 or E3 and apolipoprotein E Both hybrid rHDL nanoparticles containing polypoproteins E2 and E3 exhibited excellent A The hybrid rHDL nanoparticles showed a β-aggregation inhibitory effect, especially after 10 hours. The most effective treatment was observed within the first 20 minutes. The phenomenon of deaggregation of β and a temporary increase in fluorescence intensity was observed. It was. [Test Example 3] Comparison of the Aβ aggregation inhibitory effects of pure apolipoproteins and rHDL nanoparticles Treatment with apolipoprotein alone, rather than with the rHDL nanoparticles of the present disclosure, also inhibited Aβ To confirm whether the aggregation inhibitory effect was exerted, we treated the cells with Aβ and apolipoprotein E3. or rHDL nanoparticles containing Aβ and apolipoprotein E3 were mixed in a 1:0.1 ratio. The change in fluorescence value was confirmed in the same manner as in Test Example 1.

[0125] As shown in FIG. 21, treatment with apolipoprotein E3 did not significantly affect Aβ expression compared with treatment with Aβ alone. The fluorescence intensity was higher than that of the rHDL nanoparticles containing apolipoprotein E3. The fluorescence value was significantly higher when treated with , indicating the Aβ aggregation inhibitory effect of the rHDL nanoparticles of the present disclosure. I found it to be extremely excellent. [Test Example 4] Comparison of transport of rHDL nanoparticles through blood-brain barrier endothelial cells rHDL nanoparticles containing apolipoprotein E2 or E3, and Hybrid rHDL nanoparticles containing proteins E2 and E3 were used to stimulate the expression of cerebral microvascular endothelial cells (b rain microvascular endothelial cell;BMEC We performed an experiment to examine the extent of transcytosis into brain tissue via The results are shown in Figure 22.

[0126] As shown in FIG. 22, a hybrid rHDL nucleic acid containing apolipoproteins E2 and E3 was In the case of nanoparticles, the brain microvascular endothelial cells of the blood-brain barrier Transport into brain tissue via tr endothelial cells (BMEC) anscytosis) is a mechanism by which rHDL nucleic acids containing a single apolipoprotein E2 or E3 It was confirmed that E2 and E3 are superior to other proteins. When the same amount of nanomaterial was delivered to the brain microvascular endothelial cell receptors that react with the single Hybrid rHDL is more effective than apolipoprotein (E2 or E3) rHDL. This means that the compound can be effectively absorbed into cells.

[0127] Therefore, if rHDL is used as a therapeutic or delivery agent in the future, the lowest doses may be sufficient. This allows them to cross the brain microvascular endothelial cell barrier more efficiently, resulting in more effective drug delivery. It is clear that efficacy and drug delivery are possible. [Test Example 5] Comparison of transport of rHDL nanoparticles in astrocytes at the blood-brain barrier rHDL nanoparticles containing apolipoprotein E2 or E3, and Hybrid rHDL nanoparticles containing substrates E2 and E3 bind to astrocytes at the blood-brain barrier. The results are shown in Figure 23. As shown in.

[0128] As shown in FIG. 23, a hybrid rHDL nucleic acid containing apolipoproteins E2 and E3 was In the case of rHDL, blood-brain It is more efficient at transporting through the astrocytes of the barrier and is more effective in transporting It can be seen that it has a more excellent effect on maintaining homeostasis in tissues.

[0129] This is because the same amount of nanomaterials was attached to the receptors of astrocytes to which E2 and E3 react, respectively. When delivered to the brain, hybrid rHDL nanoparticles containing apolipoproteins E2 and E3 This means that the receptors can react to the maximum extent possible, so the smallest dose will have the greatest effect. I know what I can do. [Test Example 6] Comparison of amyloid beta removal from brain tissue in animal models of Alzheimer's disease Apolipoprotein E3-containing rHDL nanoparticles were used in animal models of Alzheimer's disease. The results are shown in Figure 24-2. Shown in Figure 7.

[0130] Equal numbers of male and female normal mice and Alzheimer's disease animal model (5xFAD) were used. The subjects were divided into a control group and a rHDL nanoparticle-treated group, and the control group was given saline intravenously. The group administered rHDL nanoparticles was administered with rHDL nanoparticles of the present disclosure containing apolipoprotein E3. Nanoparticles (2.5 mg / kg) were administered once every 3 days for a total of 33 doses. After 1 h, the brain was removed and the left brain was fixed in 4% PFA (paraformaldehyde) solution. The fixed brain tissue was frozen in OCT compound and then microtomed. The tissue sections were cut to a thickness of 4 μm using a microtome. The Aβ-specific antibody and DAB (3,3'-diaminobenzidine tetraacetate) were used. The reaction was performed using a color developer (tetrahydrochloride salt). The stained slides were photographed using a mirror, and the results are shown in Figures 24 and 25.

[0131] As shown in FIG. 24, the effect of saline on the development of Alzheimer's disease was improved by administering saline for three months. In the disease animal model (5xFAD), excessive Aβ deposition was observed in the brain. As shown in FIG. 25, administration of rHDL nanoparticles containing apolipoprotein E3 increased A Therefore, it is possible that rHDL nanoparticles containing E3 can be used to treat Alzheimer's disease. It has been found that the Aβ protein in brain tissue that causes AIMS can be effectively removed.

[0132] In addition, to compare the Aβ concentration in the cerebrospinal fluid of each experimental group, mice were The rat was anesthetized, and then the cerebellum and medulla oblongata were removed. The cerebrospinal fluid (cerebrospinal fluid) is transferred to the brain via the cisterna magna (cisterna magna) between the gingata. To compare the Aβ concentration in plasma, To obtain the cerebrospinal fluid, blood was taken from the jugular vein and treated with heparin (5 IU / mL). Approximately 0.13 mL of whole blood was collected in the tube. The collected blood was centrifuged to separate the plasma. Ta.

[0133] As a result, as shown in Figures 26 and 27, rHDL nanoparticles containing apolipoprotein E3 were obtained. Aβ in cerebrospinal fluid and plasma of an animal model of Alzheimer's disease (5xFAD) administered with nanoparticles It was confirmed that the concentration was significantly decreased compared to the group administered physiological saline.

Claims

1. Contains phospholipids and apolipoprotein E, Reconstituted high density lipoprotein (rHDL) produced from a mixture of phospholipids and apolipoprotein E in a weight ratio of 0.5:1 to 1.5:

1.

2. The reconstituted high density lipoprotein (rHDL) according to claim 1, characterized in that the weight ratio of phospholipids to apolipoprotein E in the reconstituted high density lipoprotein (rHDL) is 0.2:1 to 2.5:

1.

3. The reconstituted high density lipoprotein (rHDL) according to claim 2, characterized in that the weight ratio of phospholipids to apolipoprotein E in the reconstituted high density lipoprotein (rHDL) is 0.2:1 to 1.5:

1.

4. The reconstituted high density lipoprotein (rHDL) according to claim 3, characterized in that the weight ratio of phospholipids to apolipoprotein E in the reconstituted high density lipoprotein (rHDL) is 0.2:1 to 0.5:

1.

5. The reconstituted high density lipoprotein (rHDL) according to claim 1, characterized in that the density of the reconstituted high density lipoprotein (rHDL) is 0.1-2.0 g / mL.

6. The reconstituted high density lipoprotein (rHDL) according to claim 5, characterized in that the density of the reconstituted high density lipoprotein (rHDL) is 0.2 to 1.5 g / mL.

7. The reconstituted high density lipoprotein (rHDL) according to claim 6, characterized in that the density of the reconstituted high density lipoprotein (rHDL) is 0.3 to 1.2 g / mL.

8. 2. The reconstituted high density lipoprotein (rHDL) of claim 1, characterized in that it contains apolipoproteins E2, E3, or both E2 and E3.

9. The reconstituted high density lipoprotein (rHDL) of claim 1, further comprising apolipoprotein A1.

10. 2. The reconstituted high density lipoprotein (rHDL) of claim 1, which is characterized in that it does not contain any apolipoproteins other than apolipoproteins E2, E3 or A1.

11. 2. The reconstituted high density lipoprotein (rHDL) according to claim 1, characterized in that it does not contain apolipoprotein E4.

12. The phospholipid may be 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-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachido ...DAPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC Phosphocholine (DBPC), 1,2-dieicosanoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide) (VL-5), dioctadecylaminoglycylspermine 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-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl The reconstituted high density lipoprotein (rHDL) according to claim 1, characterized in that at least one of the following is selected from the group consisting of dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl-spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), and D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA.

13. A pharmaceutical composition comprising the reconstituted high density lipoprotein of any one of claims 1 to 12 and a pharma- ceutically acceptable excipient.

14. 14. The pharmaceutical composition of claim 13, comprising a first set of reconstituted high density lipoproteins comprising apolipoprotein E2 and a second set of reconstituted high density lipoproteins comprising apolipoprotein E3.

15. The pharmaceutical composition of claim 13, comprising a reconstituted high density lipoprotein that includes both apolipoprotein E2 and apolipoprotein E3.

16. A pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising the reconstituted high density lipoprotein according to any one of claims 1 to 12.

17. The pharmaceutical composition for preventing or treating a neurodegenerative disease according to claim 16, characterized in that the reconstituted high density lipoprotein can inhibit amyloid beta (Aβ) aggregation and maintain brain tissue homeostasis.

18. 17. The pharmaceutical composition for preventing or treating a neurodegenerative disease according to claim 16, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, Creutzfeldt-Jakob disease, Lou Gehrig's disease, spinocerebellar degeneration, spinocerebellar ataxia, prion disease, cognitive dysfunction, senile dementia, dementia with Lewy bodies, frontotemporal dementia, vascular dementia, alcoholic dementia, presenile dementia, Machado-Joseph disease, dystonia, multiple system atrophy, progressive supranuclear palsy, Friedreich's ataxia, temporal lobe epilepsy, and stroke.

19. 1. A method for producing a reconstituted high density lipoprotein (rHDL) comprising phospholipids and apolipoprotein E, comprising the steps of: Injecting a first hydrophilic solution containing a first apolipoprotein into a first inlet of a microfluidic device having three inlets and one outlet; injecting a phospholipid solution into a second inlet of the microfluidic device; Injecting a second hydrophilic solution containing a second apolipoprotein into a third inlet of the microfluidic device.

20. 20. The method for producing reconstituted high density lipoprotein (rHDL) according to claim 19, characterized in that the microfluidic device comprises a second inlet located in the center of the microfluidic device and first and third inlets located on either side of the device.

21. 21. The method for producing reconstituted high density lipoprotein (rHDL) according to claim 19 or 20, characterized in that both the first apolipoprotein and the second apolipoprotein are apolipoprotein E2.

22. 21. The method for producing reconstituted high density lipoprotein (rHDL) according to claim 19 or 20, characterized in that both the first apolipoprotein and the second apolipoprotein are apolipoprotein E3.

23. 21. A method for producing reconstituted high density lipoprotein (rHDL) according to claim 19 or 20, characterized in that the first apolipoprotein is apolipoprotein E2 and the second apolipoprotein is apolipoprotein E3.

24. 21. A method for producing reconstituted high density lipoprotein (rHDL) according to claim 19 or 20, characterized in that the first apolipoprotein is apolipoprotein E2 or E3 and the second apolipoprotein is apolipoprotein A1.

25. 21. The method for producing reconstituted high density lipoprotein (rHDL) according to claim 19 or 20, characterized in that the first hydrophilic solution, the phospholipid solution and the second hydrophilic solution are injected simultaneously.

26. 21. The method for producing reconstituted high density lipoprotein (rHDL) according to claim 19 or 20, further comprising the step of collecting the reconstituted high density lipoprotein (rHDL) from an outlet.

27. The method for producing reconstituted high density lipoprotein (rHDL) according to claim 19 or 20, characterized in that the weight ratio of phospholipids and apolipoprotein E injected into the inlet of the microfluidic device is 0.25:1 to 2.5:

1.

28. The method for producing reconstituted high density lipoprotein (rHDL) according to claim 27, characterized in that the weight ratio of phospholipids and apolipoprotein E injected into the inlet of the microfluidic device is 0.5:1 to 1.5:1.

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