Pegylated recombinant high-density lipoprotein nanoparticles

Abstract

The invention relates to pegylated recombinant high-density lipoprotein nanoparticles, which have the effect of preventing or treating neurodegenerative diseases. Specifically, the present invention relates to a nanoparticle and a method for preparing the same, in which a polyethylene glycol-lipid (PEG-lipid) or a derivative thereof is added during the preparation of a fluid containing a hydrophobic substance and a fluid containing a hydrophilic substance, thereby protecting a phospholipid layer of the prepared nanoparticle by PEG, and to a method for preparing the same. The pegylated nanoparticles not only maintain the original blood-brain barrier-crossing delivery ability, but also have the ability to avoid a clearance mechanism caused by in-vivo immune action, thus having excellent stability, and exhibiting a long-term pharmacological effect on the basis of the high in-vivo circulation ability thereof. Therefore, the compound can be effectively used as a drug or a drug delivery vehicle.

Description

Technical Field

[0001] This invention relates to polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles and their preparation method. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles of this invention exhibit therapeutic and preventative effects against neurodegenerative diseases due to their excellent in vivo stability. Background Technology

[0002] In recent years, with the surge in the aging population and the increasing number of patients with various neurodegenerative diseases, interest in their treatment and prevention has grown significantly. Neurodegenerative diseases are characterized by a reduction or loss of nerve cell function, leading to various symptoms such as motor impairment, memory impairment, and cognitive impairment. Nerve cells die in large numbers every day not only in neurological diseases but also in the brains of normal adults, and the number of dying nerve cells increases exponentially with age.

[0003] In particular, Alzheimer's disease (AD) is the most common form of dementia and a representative neurodegenerative disease. With the increasing aging of society, the number of Alzheimer's patients is rapidly increasing. Its main pathological features are senile plaques and neurofibrillary tangles. Senile plaques are produced by the deposition of β-amyloid protein (Aβ), which is generated by the sequential cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase, in brain tissue. Neurofibrillary tangles are caused by the hyperphosphorylation of Tau protein, a microtubule-associated protein.

[0004] In recent years, therapeutics targeting the pathological features of Alzheimer's disease have been actively developed. The most common form of treatment utilizes antibodies, and research is underway to develop effective antibodies against the aforementioned targets and optimize their delivery to diseased tissues. However, there are ongoing reports that antibody-based treatments may cause cerebral edema or intracranial microvascular hemorrhage, potentially plunging patients into life-threatening situations.

[0005] On the other hand, recombinant high-density lipoprotein nanoparticles (rHDL) have the advantage of not producing the side effects commonly seen in antibody therapies because they are almost identical to substances present in the body. However, when injected into the body, they are absorbed by immune cells in normal organs (liver, spleen, lungs, etc.) other than the target organ (brain) due to the formation of a protein corona by various proteins in the blood. This results in their rapid disappearance from the bloodstream, leading to problems with low circulation capacity and targeting efficiency. Furthermore, when injected into the body, they adsorb various pro-inflammatory cytokines (IL-1, IL-6, TNF-α, etc.) from various proteins in the blood that trigger inflammatory responses, posing a potential problem of side effects caused by secondary inflammatory reactions when absorbed by the brain or other normal organs.

[0006] Therefore, this invention develops the composition and preparation method of polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles (P-rHDL), which can have excellent therapeutic effects on neurodegenerative diseases and have excellent in vivo stability.

[0007] Existing technical documents

[0008] Patent Document 1: Korean Patent No. 10-2531293

[0009] Patent Document 2: Korean Patent No. 10-2631907

[0010] Non-patent literature 1: Rumiana, T. et al. Lipid nanoparticles-from liposomes to mRNA vaccine delivery, a landscape of research diversity and advancement. ACS Nano 15, 11, 16982-17015 (2021).

[0011] Non-patent literature 2: Kim, Y. et al. Single step reconstitution of multifunctional high-density lipoprotein-derived nanomaterials using microfluidics. ACS Nano 7, 11, 9975-9983 (2013). Summary of the Invention

[0012] Technical issues

[0013] The purpose of this invention is to provide PEGylated recombinant high-density lipoprotein nanoparticles (P-rHDL) that exhibit excellent stability in vivo.

[0014] Furthermore, the present invention provides a method for preparing polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles (P-rHDL).

[0015] Furthermore, the object of the present invention is to provide a composition for the prevention or treatment of neurodegenerative diseases, comprising polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles (P-rHDL).

[0016] The purpose of this invention is to provide a method for treating neurodegenerative diseases, the method comprising the step of administering polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles (P-rHDL) or recombinant high-density lipoprotein nanoparticles (rHDL) to a patient with a neurodegenerative disease.

[0017] Technical solution

[0018] The present invention provides recombinant high-density lipoprotein (rHDL) nanoparticles comprising polyethylene glycol-modified lipids (PEG-lipid), phospholipids and apolipoprotein E.

[0019] In one embodiment, the molar ratio of the apolipoprotein to the polyethylene glycolated lipid is 1:0.5 to 1:50, preferably 1:1 to 1:10, and more preferably 1:1 to 1:5.

[0020] In one embodiment, the average molecular weight of the PEG is from 550 (0.5k) to 5000 (5k), preferably from 1000 (1k) to 2000 (2k).

[0021] In one embodiment, the recombinant high-density lipoprotein (rHDL) nanoparticles may be 5 nm to 50 nm in size, preferably 10 nm to 35 nm.

[0022] In one embodiment, the PEG in the polyethylene glycol-modified lipid can be PEG or a derivative thereof.

[0023] In one embodiment, the apolipoprotein may be apolipoprotein E2 or apolipoprotein E3.

[0024] In one embodiment, the recombinant high-density lipoprotein nanoparticles (rHDL) may further comprise apolipoprotein A1.

[0025] In one embodiment, the phospholipid may be selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), lecithinylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl 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 (PSPC), etc. Palmitoylphosphatidylcholine (SPPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), 1,2-dianarachidoyl-sn-glycerol-3-phosphocholine (DBPC), 1,2-diecosanoyl-sn-glycerol-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1- [2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamoyl)ethyl]-3,4-di[oleoyloxy]-benzamide)(VL-5), N1-[2-((1S)-1-[(3-aminopropyl)bis(octadecylamidoglycerin)glycerylamine-4-trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), ( 1,2-Dioleopropyl(3-dimethylhydroxyethylammonium bromide)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-Dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 2,3-Dioleopropyloxy-N-[2-(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propanediamine trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanediamine bromide (GAP-DLRIE), N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropanediamine (diCl4-amidine), Ethylphosphocholine (Ethyl... The invention comprises one or more of the following groups: PC, dimethyl dioctadecyl ammonium bromide (DDAB), N4-cholesterol spermine (GL67), 1,2-dioleoxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA, but is not limited thereto.

[0026] The present invention provides a composition for the prevention or treatment of neurodegenerative diseases with improved in vivo safety and stability, comprising the recombinant high-density lipoprotein nanoparticles (rHDL).

[0027] In one embodiment, the neurodegenerative disease may be 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, prions, cognitive impairment, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, vascular dementia, alcoholic dementia, early-onset Alzheimer's disease, Machado-Joseph disease, dystonia, multiple system atrophy, progressive supranuclear palsy, Friedreich ataxia, temporal lobe epilepsy, and stroke, but is not limited thereto. Preferably, it may be Alzheimer's disease.

[0028] The recombinant high-density lipoprotein nanoparticles (rHDL) can maintain specific ligand activity against the blood-brain barrier tissue while avoiding phagocytosis by immune cells.

[0029] The present invention provides an improved method for treating neurodegenerative diseases, the method comprising the step of administering recombinant high-density lipoprotein nanoparticles having improved in vivo safety and stability to patients with neurodegenerative diseases.

[0030] The present invention provides a method for preparing polyethylene glycol-modified recombinant high-density lipoprotein (P-rHDL), the method comprising: injecting a polyethylene glycol-modified lipid solution into a central channel inlet in a microfluidic device comprising three inlets and one outlet, and injecting high-density lipoprotein (HDL) or recombinant high-density lipoprotein (rHDL) containing apolipoproteins and phospholipids into two channel inlets on both sides.

[0031] This invention provides a recombinant high-density lipoprotein nanoparticle (rHDL) prepared by the aforementioned preparation method.

[0032] The effects of the invention

[0033] The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles (P-rHDL) and compositions containing them of the present invention can be used to prevent or treat neurodegenerative diseases.

[0034] Furthermore, the polyethylene glycolated recombinant high-density lipoprotein nanoparticles (P-rHDL) of the present invention have the effect of avoiding the loss of efficacy of therapeutic agents due to in vivo immune response through polyethylene glycolation, thereby achieving excellent effects even at low doses. Attached Figure Description

[0035] Figure 1This is a schematic diagram illustrating the method of synthesizing polyethylene glycol-modified high-density lipoprotein nanoparticles (P-rHDL) using recombinant high-density lipoprotein nanoparticles (rHDL) according to the present invention.

[0036] Figure 2 The results of DLS determination of P-rHDL with an average molecular weight of polyethylene glycol (PEG) of 0.5k and a synthetic molar ratio of PEG to apolipoprotein of 1:1 are shown.

[0037] Figure 3 The results of DLS determination of P-rHDL with an average molecular weight of 1k and a synthetic molar ratio of PEG to apolipoprotein of 1:1 are shown.

[0038] Figure 4 The results of DLS determination for P-rHDL with an average molecular weight of 2k and a synthetic molar ratio of PEG to apolipoprotein of 1:1 are shown.

[0039] Figure 5 The results of DLS determination for P-rHDL with an average molecular weight of 3k and a synthetic molar ratio of PEG to apolipoprotein of 1:1 are shown.

[0040] Figure 6 The results of DLS determination for P-rHDL with an average molecular weight of 5k and a synthetic molar ratio of PEG to apolipoprotein of 1:1 are shown.

[0041] Figure 7 The results of DLS determination for unpolyglycolated rHDL are shown.

[0042] Figure 8 The results of DLS assays for P-rHDL with synthetic molar ratios of apolipoprotein to polyethylene glycol-modified lipid (PEG2k-lipid) of 1:0.5 and 1:1 are shown.

[0043] Figure 9 The results of DLS assays for P-rHDL with apolipoprotein and polyethylene glycol-modified lipid (PEG2k-lipid) synthesized in molar ratios of 1:5 and 1:10 are shown.

[0044] Figure 10 The results of DLS assays for P-rHDL with a synthetic molar ratio of apolipoprotein to polyethylene glycol-modified lipid (PEG2k-lipid) of 1:20 and 1:50 are shown.

[0045] Figure 11 The results show the apolipoprotein introduction efficiency of P-rHDL when the mixing molar ratios of rHDL and PEG-2k-lipid are 1:5, 1:10, and 1:20.

[0046] Figure 12 The results of comparing surface charge using the ZETA potential values ​​of rHDL and P-rHDL are shown.

[0047] Figure 13 The results show the dispersibility and storage stability of P-rHDL compared by DLS on day 0 and day 21.

[0048] Figure 14 The results show the inhibitory effect of β-amyloid aggregation when treated with β-amyloid and P-rHDL simultaneously.

[0049] Figure 15 The results show the effect of P-rHDL treatment on the decomposition of aggregated β-amyloid protein after inducing β-amyloid protein aggregation.

[0050] Figure 16 The absorbance spectra of P-rHDL, a fluorescent PEGylated lipid with an average molecular weight of 1 kJ, are shown.

[0051] Figure 17 The absorbance spectra of P-rHDL, a fluorescent PEGylated lipid with an average molecular weight of 2k introduced with PEG, are shown.

[0052] Figure 18 The absorbance spectra of P-rHDL, a fluorescent PEGylated lipid with an average molecular weight of 3k introduced with PEG, are shown.

[0053] Figure 19 The results show the evasion function of fluorescently labeled rHDL and fluorescently labeled P-rHDL against phagocytosis by immune cells, as confirmed by laser confocal scanning microscopy.

[0054] Figure 20 The results show the results of confirming the intracellular delivery efficiency of fluorescently labeled rHDL in human brain microvascular endothelial cells (hBMECs) using laser confocal scanning microscopy.

[0055] Figure 21 The results show the results of confirming the intracellular delivery efficiency of fluorescently labeled P-rHDL in hBMECs using laser confocal scanning microscopy.

[0056] Figure 22 The results show the intracellular delivery efficiency of fluorescently labeled rHDL and P-rHDL in hBMEC monolayers, confirmed by laser confocal scanning microscopy.

[0057] Figure 23The results show the results of a quantitative analysis of the intracellular delivery efficiency of fluorescently labeled rHDL and P-rHDL in hBMEC monolayers based on fluorescence images.

[0058] Figure 24 The results show the results of intracellular delivery efficiency of fluorescently labeled rHDL and P-rHDL in a stem cell-derived brain microvascular endothelial cell (iBMEC) monolayer, confirmed by laser confocal scanning microscopy.

[0059] Figure 25 The results show the results of a quantitative analysis of the intracellular delivery efficiency of fluorescently labeled rHDL and P-rHDL in iBMEC monolayers based on fluorescence images.

[0060] Figure 26 A graph comparing the BBB penetration efficiency of rHDL and P-rHDL in Transwell via the hBMEC monolayer up to 4 hours.

[0061] Figure 27 A graph comparing the BBB penetration efficiency of rHDL and P-rHDL in Transwell through the blood-brain barrier (BBB) ​​formed by the hBMEC monolayer up to 24 hours. Detailed Implementation

[0062] The following will describe in detail the embodiments and examples of the present invention, enabling those skilled in the art to readily implement it. However, the present invention can be implemented in various forms and is not limited to the embodiments and examples described herein.

[0063] Throughout this specification, when a part “includes” a structural element, unless otherwise stated otherwise, it means that other structural elements may also be included rather than excluded.

[0064] This invention provides a reconstituted high-density lipoprotein (rHDL) comprising phospholipids, PEG-lipids, and apolipoproteins.

[0065] The term "recombinant high-density lipoprotein (rHDL)" as used in this invention refers to artificially manufactured HDL mimic particles designed to mimic the biological effects of naturally occurring high-density lipoprotein (HDL).

[0066] The term "PEGylated" as used in this invention refers to the modification of the surface of a substance by PEG or its derivatives.

[0067] As used in this invention, the term "PEG" or "polyethylene glycol" refers to a linear polymer with terminal hydroxyl groups and the structure H-(OCH2CH2). n The -OH group at the end can be replaced by functional groups such as carboxylic acid, amine, azide, cyanine, and ester.

[0068] The average molecular weight of PEG used in this invention can be from 550 (0.5k) to 5000 (5k), preferably from 1000 (1k) to 2000 (2k).

[0069] The term "neurodegenerative disease" as used in this invention refers to a disease in which the function of the nervous system is reduced or lost due to degenerative changes in nerve cells. Preferably, it can be one or more of the following: Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, Creutzfeldt-Jakob disease, Lou Gehrig's disease, spinocerebellar degeneration, spinocerebellar ataxia, prions, cognitive impairment, senile dementia, Lewy body dementia, frontotemporal dementia, vascular dementia, alcoholic dementia, early-onset dementia, Machado-Joseph disease, dystonia, multiple system atrophy, progressive supranuclear palsy, Friedreich ataxia, temporal lobe epilepsy, and stroke. More preferably, it can be Alzheimer's disease.

[0070] The term "apolipoprotein E" refers to 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 can be any isoform of the APOE gene product, for example, apolipoprotein E2 ("APOE2"), apolipoprotein E3 ("APOE3"), and apolipoprotein E4 ("APOE4"). A "functional variant" refers to a variant of a mammalian protein encoded by an APOE gene that retains the same or similar biological function as the APOE gene product. In some cases, functional variants include amino acid insertions, deletions, and / or substitutions compared to proteins encoded by the human APOE gene. In some cases, functional variants are fragments of proteins encoded by the human APOE gene.

[0071] In some embodiments, apolipoprotein E2 is the protein with GenBank accession number ARQ79459 or has at least 95% sequence identity with it, preferably at least 98% or 99% sequence identity. In some embodiments, apolipoprotein E3 is the protein with GenBank accession number ARQ79461.1 or has at least 95% sequence identity with it, preferably at least 98% or 99% sequence identity.

[0072] In some embodiments, the apolipoprotein E in recombinant high-density lipoprotein (rHDL) is a recombinant protein produced by genetic engineering or a synthetic protein produced by chemical synthesis.

[0073] The term "apolipoprotein A1" refers to a mammalian protein encoded by the APOA1 gene or a functional variant thereof. In a preferred embodiment, apolipoprotein A1 is a human protein encoded by the human APOA1 gene located on chromosome 11. A "functional variant" refers to a variant of a mammalian protein encoded by the APOA1 gene that retains the same or similar biological function as the APOA1 gene product. In some cases, functional variants include amino acid insertions, deletions, and / or substitutions compared to the protein encoded by the human APOA1 gene. In some cases, functional variants are fragments of the protein encoded by the human APOA1 gene.

[0074] Specifically, the "phospholipid" used in this invention can be selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), lecithinylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-phosphatidylcholine (PSPC), etc. Palmitoylphosphatidylcholine (SPPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), 1,2-dianarachidoyl-sn-glycerol-3-phosphocholine (DBPC), 1,2-dieanoyl-sn-glycerol-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N 1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamoyl)ethyl]-3,4-di[oleoyloxy]-benzamide)(VL-5), N1-[2-((1S)-1-[(3-aminopropyl)bis(octadecylamidoglycerin)glycerylamine-4-trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), (1,2-Dioleopropyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 2,3-dioleopropyloxy-N-[2-(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propanediamine trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanediamine bromide (GAP-DLRIE), N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropanediamine (diCl4-amidine), ethylphosphocholine (Ethyl) The invention comprises one or more of the following, but is not limited to: PC, dimethyl dioctadecyl ammonium bromide (DDAB), N4-cholesterol spermine (GL67), 1,2-dioleoxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA.

[0075] The "polyethylene glycol-lipid" used in this invention can be PEG or its derivatives. Specifically, it can be selected from 1,2-dimyristoyl-racemic-glycerol methoxy polyethylene glycol (DMG-PEG), including 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy polyethylene glycol] (PEG PE), distearyl-racemic-glycerol methoxy polyethylene glycol (DSG-PEG), polyethylene glycol ceramide (PEG-Ceramide), polyethylene glycol-phosphatidylethanolamine (PEG-PE), dodecyl-polyethylene glycol-carboxylic acid (C12-PEG-COOH), octadecyl-polyethylene glycol-carboxylic acid (C18-PEG-COOH), cholesterol-polyethylene glycol-acid (Cholesterol-PEG-Acid), cholesterol-polyethylene glycol-carboxylic acid (Cholesterol-polyethylene glycol-carboxylic acid), etc. Cholesterol-PEG-Amine, Cholesterol-PEG-Azide, Cholesterol-PEG-DBCO, Cholesterol-PEG-FITC, Cholesterol-PEG-Maleimide, Cholesterol-N-hydroxysuccinimide (Cholesterol-PEG-NHS), 1,2-stilbene-sn-glycerol-3-phosphate ethanolamine-N-[Biotinyl (polyethylene glycol)](DSPE-PEG-Biotin), 1,2-stilbene-sn-glycerol-3-phosphate ethanolamine-N-[carboxyl (polyethylene glycol)](DSPE-PEG-COOH), 1,2-stilbene-sn-glycerol-3-phosphate ethanolamine-N-[fluorescein isothiocyanate (polyethylene glycol)](DSPE-PEG-FITC), 1,2-stilbene-sn-glycerol- 3-Phosphoethanolamine-N-[maleimide (polyethylene glycol)] (DSPE-PEG-Maleimide), 1,2-stilbene-sn-glycerol-3-phosphate ethanolamine-N-[azido (polyethylene glycol)] (DSPE-PEG-Azide), 1,2-stilbene-sn-glycerol-3-phosphate ethanolamine-N-[PDP (polyethylene glycol)] (DSPE-PEG-PDP), 1,2-stilbene-sn-glycerol-3-phosphate ethanolamine-N-[amino (polyethylene glycol)] (DSPE-PEG-NH2), 1,The PEG-N-[succinyl-sn-glycerol-3-phosphate ethanolamine] (DSPE-PEG-NHS), N-palmitoylsphingosine-1-{succinyl[methoxy(polyethylene glycol)]} (C16-PEG-Ceramide), and others, but not limited thereto.

[0076] Example 1

[0077] Preparation method of polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles (P-rHDL)

[0078] To prepare PEGylated recombinant highdensity lipoprotein (P-rHDL) nanoparticles, such as... Figure 1 As shown, a microfluidic device with three inlets and one outlet and microcolumns inside the device is used to prepare high-density lipoprotein nanoparticles (rHDL), and then polyethylene glycol-modified lipids are added to the rHDL to prepare P-rHDL.

[0079] 1-1. Preparation method of P-rHDL using microfluidic devices

[0080] First, in order to prepare rHDL, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at a concentration of 0.83 mg / mL was prepared as a phospholipid in anhydrous ethanol, and apolipoprotein at a concentration of 0.2 mg / mL was prepared in PBS (phosphate buffer) or physiological saline.

[0081] Approximately 1 mL of DMPC solution was injected into the central inlet of the microfluidic device, and 5 mL of apolipoprotein solution was injected into the two channels on either side. The injection flow rate of the DMPC solution was set at 0.8 mL / min, and the injection flow rate of the apolipoprotein solution was set at 4.4 mL / min. The two solutions were effectively mixed through the microcolumn inside the device, and rHDL was obtained by flowing out through the effluent channel of the micromixing channel device. The obtained rHDL was purified using a 10K Amicon filter at 3900 rpm for 20 minutes and added to PBS or physiological saline to make 2.5 mL for the next step.

[0082] Next, to PEGylate rHDL, 500 μL of PEG2k-lipid (18:0PEG2000 PE) at a concentration of 0.82 mg / mL was prepared. The injection flow rate of the PEG2k-lipid solution was set to 0.8 mL / min, and the injection flow rate of the rHDL solution was set to 4.4 mL / min. The solution was then connected to a new microfluidic device to synthesize P-rHDL. The P-rHDL obtained through the effluent channel of the microfluidic device was mixed with PBS or physiological saline and purified three times at maximum centrifugation speed at 25 °C using a new 10K filter, once for 20 min and twice for 15 min. The final purified residue was diluted with physiological saline to a total volume of 1 mL and then filtered through a 0.2 μm syringe filter to obtain the finally purified P-rHDL. The prepared P-rHDL was stored at 4 °C until used in the next experiment.

[0083] 1-2. Preparation method of P-rHDL using microfluidic devices 2

[0084] Another method for preparing P-rHDL is as follows: A single solution of phospholipid DMPC and polyethylene glycol-modified lipids used to prepare rHDL is prepared and injected into the central inlet of a microfluidic device, while apolipoprotein solution is injected into two channels located on either side.

[0085] Specifically, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and PEG solutions were added to anhydrous ethanol. First, an 8.3 mg / mL DMPC solution was prepared in anhydrous ethanol. Then, a 0.82 mg / mL PEG2k-lipid solution (18:0 PEG2000 PE, molar ratio to apolipoprotein 1) was prepared in anhydrous ethanol. The PEG2k-lipid solution was completely dispersed by sonication in a water bath for approximately 30 minutes, and the dispersion was confirmed visually.

[0086] Mix 100 μL of the DMPC solution, 100 μL of the PEG2k-lipid solution and 800 μL of anhydrous ethanol, and then homogenize using a digital shaker for about 10 minutes to prepare the DMPC / PEG2k-lipid solution.

[0087] Then, P-rHDL containing phospholipids (DMPC), PEG and apolipoproteins was prepared using the same method as in Examples 1-1.

[0088] 1-3. Optimization of the PEG molecular weight of PEGylated lipids during P-rHDL preparation

[0089] To confirm the effect of the average molecular weight of PEG in the polyethylene glycol-modified lipids on the preparation of P-rHDL, the synthesis of P-rHDL was determined based on the molecular weight of PEG in the polyethylene glycol-modified lipids. The synthesis method for P-rHDL was the same as in Examples 1-2, using polyethylene glycol-modified lipids with PEG molecular weights of 0.5, 1, 2, 3, and 5 kJ. In this case, the molar ratio of apolipoprotein to polyethylene glycol-modified lipids was 1:1.

[0090] The P-rHDL synthesized under the described conditions was diluted 10-fold with PBS, and its size distribution was then measured using dynamic light scattering (DLS) and plotted on the graph. Figures 2-6 This confirms that P-rHDL can be synthesized regardless of the molecular weight of PEG. In particular, through... Figure 3 , Figure 4 Confirmation and Figure 7 The graph shape is similar to that of the DLS results for rHDL, showing PEG molecules of similar size to rHDL, which are 1k or 2k.

[0091] 1-4. Optimization of the PEGylated lipid mixing ratio during P-rHDL preparation

[0092] like Figure 8 , Figure 9 , Figure 10 As shown, in order to optimize the mixing ratio of PEGylated lipids in P-rHDL, P-rHDL was prepared according to the methods of Examples 1-2 and 1-3. In this case, the preparation was carried out according to the mixing ratio of apolipoprotein ApoE3 and PEG2k-lipid (1:0, 1:0.5, 1:1, 1:5, 1:10, 1:20, 1:50), and the size of the prepared P-rHDL was compared using DLS.

[0093] Table 1

[0094]

[0095] As shown in Table 1, P-rHDL was prepared according to the mixing ratios of PEG2k-lipid based on apolipoproteins using the method of Example 1-1.

[0096] Table 2

[0097]

[0098]

[0099] As shown in Table 2 and Figure 8 , Figure 9, Figure 10 As shown, the size and uniformity of P-rHDL synthesized at various mixing ratios were confirmed by DLS measurement. At a 1:0.5 ratio, the synthesized P-rHDL had a size of 70 nm and a PDI of 0.4, exhibiting a significantly different pattern from rHDL. It was confirmed that the PDI was similar to rHDL at all mixing ratios other than 1:0.5 (1:1, 1:5, 1:10, 1:20, 1:50). The particle sizes of P-rHDL at mixing ratios of 1:1 and 1:50 were confirmed to be 31.44 nm and 32.71 nm, respectively, approximately twice that of unpolyethylene glycol-modified rHDL. Furthermore, as... Figure 8 , Figure 10 As shown, in the numerical percentile (numerical dispersion standard) of the DLS results for P-rDHL, it was confirmed that, unlike rHDL, P-rDHL produces two different types of particles, leading to inhomogeneity. It was confirmed that at mixing ratios of 1:5, 1:10, and 1:20, the particle sizes were 16.39 nm, 18.19 nm, and 11.32 nm, respectively, similar to the size of rHDL. Figure 9 , Figure 10 The DLS distribution map or the PDI values ​​in Table 2 are similar. That is, the most ideal conditions for P-rHDL based on the mixing ratio of PEG2k-lipid were confirmed to be a mixing ratio of 1:5, 1:10, and 1:20.

[0100] To confirm the incorporation efficiency of apolipoprotein E3 (ApoE3) in P-rHDL synthesized at various mixing ratios (1:5, 1:10, 1:20), ApoE3 was quantitatively analyzed using a bicinchoninic acid (BCA) assay.

[0101] Table 3

[0102]

[0103]

[0104] As shown in Table 3 and Figure 11 As shown, it was confirmed that when the mixing ratio of PEG2k-lipid was 1:5, 1:10, and 1:20, the introduction efficiency of unpolyethylene glycolated rHDL was similar to that of ApoE3.

[0105] Among the three mixing ratios (1:5, 1:10, and 1:20) of ApoE3 and PEG2k-lipid to synthesize uniformly sized P-rHDL, considering long-term repeated in vivo administration and drug production, the 1:5 mixing ratio, which introduces the least amount of PEG as an in vitro substance, was selected as the most suitable condition. In subsequent verification experiments, P-rHDL was prepared using the 1:5 condition.

[0106] Table 4

[0107]

[0108] As shown in Table 4 and Figure 12 As shown, the surface charge of P-rHDL is confirmed to be at a similar level to that of rHDL. That is, it can be seen that the introduction of PEG does not affect the surface charge. This may mean that PEG does not affect the efficacy of P-rHDL and can exhibit similar efficacy to rHDL.

[0109] Example 2

[0110] Stability verification of P-rHDL

[0111] As shown in Table 5 below and Figure 13 As shown, the DLS results of P-rHDL after confirming optimal synthesis conditions (day 0) showed a size of 16.39 nm and a PDI of 0.24, exhibiting similar size and PDI level to rHDL. Furthermore, P-rHDL stored at 2–8°C for 21 days to maintain the stability of both apolipoprotein and P-rHDL showed a size of 20.06 nm and a PDI of 0.31. This indicates that uniformly mixed P-rHDL exhibits storage stability, with no aggregation between nanoparticles during storage; that is, it can maintain a size similar to that of freshly synthesized P-rHDL for at least 21 days without the presence of non-uniform nanoparticles larger than 50 nm.

[0112] Table 5

[0113]

[0114] Example 3

[0115] Verification based on the ability of P-rHDL to degrade β-amyloid (Aβ) whether or not it is PEGylated.

[0116] To confirm the potential effectiveness of removing the pathogenic substance Aβ, fluorescently labeled β-amyloid (Aβ, Beta-Amyloid(1-42), HiLyte) was used. TMA comparison of the abilities of rHDL and P-rHDL to inhibit Aβ aggregation and degradation was conducted using Fluor 488-labeled rHDL. Since Aβ has the property of aggregating to form plaques, and plaques are a major cause of Alzheimer's disease, when Aβ aggregates, the fluorescent molecules labeled with Aβ move closer together, resulting in a self-quenching effect and a decrease in fluorescence intensity. This confirmed the ability of P-rHDL to inhibit aggregation and degradation.

[0117] To confirm the effect of inhibiting Aβ aggregation, P-rHDL and rHDL (as a control) were simultaneously used to treat Aβ, and the fluorescence intensity was measured at different time points (0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 8, 24, 48, 72 hours) using a microplate reader. Figure 14 As shown, the fluorescence reduction of Aβ treated simultaneously with P-rHDL was approximately 7 times lower than that of the group treated with Aβ alone, confirming an inhibitory effect on Aβ aggregation. Furthermore, when comparing the fluorescence reduction of rHDL and Aβ as a control group, the similar fluorescence reduction levels confirmed that both nanoparticles exhibited almost identical effects in inhibiting pathogenic substance aggregation.

[0118] And, as Figure 15 As shown, to confirm the degradation effect on aggregated Aβ, Aβ aggregation was induced for 24 hours. Then, at the 24-hour time point after aggregation induction, P-rHDL and rHDL were applied, and the fluorescence intensity was measured at different time points (24, 24.33, 24.67, 25, 26, 28, 30, 48, and 72 hours) using a microplate reader. After treatment with P-rHDL, the fluorescence intensity of aggregated Aβ immediately increased, thus confirming that aggregated Aβ was degraded into peptide form by P-rHDL. Furthermore, when comparing the fluorescence increase of rHDL (as a control group) with that of treated Aβ, the similar fluorescence increase confirmed that they exhibited almost identical Aβ degradation effects.

[0119] Example 4

[0120] The efficacy of P-rHDL PEGylation was verified by phagocytosis by immune cells.

[0121] 4-1. Preparation of fluorescent (FITC) labeled P-rHDL

[0122] To verify the PEGylation effect via fluorescence, fluorescently labeled P-rHDL was prepared using PEG-lipid labeled with fluorescein isothiocyanate (FITC).

[0123] The synthesis method of FITC-labeled P-rHDL is the same as that in Examples 1-2, but FITC-PEG-lipids with molecular weights of 1k, 2k, and 3k are used as PEGylated lipids, and the molar ratio of apolipoprotein to PEGylated lipids is 1:5.

[0124] like Figures 16 to 17 As shown, when P-rHDL was prepared using FITC-PEG with average molecular weights of 1k and 2k, a similar spectral peak was identified at 490 nm, which is the absorbance peak of the known fluorescent substance FITC. Figure 18 As shown, when P-rHDL was prepared using FITC-PEG with an average molecular weight of 3k, no absorbance peak of FITC was observed at 490nm.

[0125] That is, it is known that P-rHDL can be synthesized when the average molecular weight of PEG is 0.5k to 5k, but the preferred range is 1k to 2k.

[0126] 4-2. Verification of the PEGylation effect of P-rHDL by phagocytosis by immune cells.

[0127] To verify the immune evasion ability of P-rHDL, fluorescently labeled P-rHDL with 5,5-N-hydroxysuccinimide ester (Cy5.5-NHS) was synthesized using the methods described in Examples 1-2. To confirm the synthesis of the fluorescently labeled P-rHDL, the absorbance spectrum at 665 nm was measured using a microplate reader, and the absorbance of Cy5.5 was analyzed.

[0128] Then, the immune evasion ability of P-rHDL was verified by using the synthetic fluorescently labeled P-rHDL to test the degree of phagocytosis induced by immune cells. THP-1 cells, used as immune cells, were treated with phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng / mL for 24 hours to differentiate into macrophages. Differentiated THP-1 cells were then treated with the same concentrations of fluorescently labeled rHDL and fluorescently labeled P-rHDL, and the amount of rHDL and P-rHDL entering the cells due to phagocytosis was observed using a confocal laser scanning microscope (CLSM).

[0129] like Figure 19As shown, it was confirmed that in the case of P-rHDL, less phagocytosis induced by THP-1 cells occurred compared to unpolyethylene glycol-modified rHDL. As described in the references, this suggests that P-rHDL can evade phagocytosis by immune cells by introducing PEG functional groups on the particle surface.

[0130] Example 5

[0131] Confirm the delivery efficiency of P-rHDL into brain microvascular endothelial cells

[0132] The intracellular delivery efficiency of P-rHDL was confirmed using brain microvascular endothelial cells (BMECs), the main cells constituting the blood-brain barrier (BBB). To confirm the cellular delivery efficiency, PEG2k-lipid, ApoE3, and rHDL components of P-rHDL were labeled with FITC, Alexa Fluor 568, and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DiD), respectively. ApoE3 and DMPC in rHDL, which served as the control group, were labeled with Alexa Fluor 568 and DiD, respectively. To confirm the extent of delivery to human brain microvascular endothelial cells (hBMECs), cells were treated with P-rHDL and rHDL for 4 hours at 37°C and 5% CO2, respectively. Cells were then fixed, and the nucleus and cytoplasm were stained. Observation was performed using a confocal laser scanning microscope (CLSM). Figure 20 , Figure 21 As shown, it was confirmed that P-rHDL was delivered to the perinuclear region in a similar manner to rHDL in the control group.

[0133] Furthermore, to confirm whether P-rHDL was delivered into cells as individual nanoparticles without degradation, z-axis (z-stack) images of P-rHDL and rHDL absorbed in hBMECs were obtained using CLSM. Figure 20 , Figure 21As shown, when two different cross-sectional images of hBMEC were confirmed using Z-stack imaging, it was confirmed that PEG2k-lipid, ApoE3, and DMPC, components of P-rHDL, delivered into the cells, were all absorbed around the cell nucleus and overlapped in one location. The degree of overlap between the components delivered to the cell nucleus and the nanoparticles was similar to that of rHDL, which served as the control group. This could mean that even after P-rHDL is PEGylated, it can achieve the same functional effect as rHDL in terms of intracellular delivery efficiency.

[0134] Next, as Figure 22 , Figure 24 As shown, human brain microvascular endothelial cells (hBMEC) and induced pluripotent stem cell-derived brain microvascular endothelial cells (iBMEC) were used to form a monolayer similar to the blood-brain barrier in vivo, and the intracellular delivery efficiency of P-rHDL was compared with that of rHDL as a control group. Figure 22 As shown, P-rHDL exhibited intracellular delivery efficiency in hBMECs similar to that of rHDL, and as Figure 23 As shown, no significant differences were confirmed in the quantitative analysis comparison based on fluorescence images. Figure 24 As shown, P-rHDL exhibited intracellular delivery efficiency similar to that of rHDL in iBMECs, and as... Figure 25 As shown, no significant differences were confirmed in the quantitative analysis comparison based on fluorescence images. Therefore, since P-rHDL exhibits similar delivery efficiency to rHDL in BMEC, it can be seen that P-rHDL can functionally achieve the same level of delivery effect as rHDL without being affected by polyethylene glycol conversion.

[0135] Example 6

[0136] Confirming the blood-brain barrier penetration efficiency of P-rHDL

[0137] To confirm the blood-brain barrier penetration ability of P-rHDL, a monolayer of hBMEC was formed in the upper chamber (insert) of the transwell and treated with DiD fluorescently labeled P-rHDL and rHDL, respectively. The penetration efficiency at different time points (4 hours and 24 hours) was compared with that of the control group (fluorescein, rhodamine B, and FITC-dextran (4, 70, and 150 kDa)). The penetrated control group, rHDL, and P-rHDL were retrieved from the lower chamber (bottom well) of the transwell, and their fluorescence was measured using a microplate reader to compare their penetration.

[0138] like Figure 26 As shown, within the initial 4-hour penetration time point, P-rHDL exhibited similar blood-brain barrier penetration efficiency to rHDL without significant difference. Figure 27 As shown, regarding the blood-brain barrier penetration efficiency at 24 hours, statistical analysis revealed a significant difference between P-rHDL and rHDL (p = 0.0053). However, since P-rHDL and rHDL exhibited similar cell delivery efficiencies and there was no evidence that polyethylene glycol (PEG) could improve penetration efficiency, this significance was excluded. Therefore, it can be concluded that, up to 24 hours, P-rHDL demonstrated a similar level of blood-brain barrier penetration efficiency to rHDL. Compared to the control group, both rHDL and P-rHDL showed more than a 2-fold increase in penetration efficiency, thus indicating a significant difference.

Claims

1. A polyethylene glycol-modified recombinant high-density lipoprotein nanoparticle, comprising polyethylene glycol-modified lipids, phospholipids, and apolipoprotein E, characterized in that, The molar ratio of apolipoprotein E to polyethylene glycol-modified lipids is 1:0.5 to 1:

50.

2. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to claim 1, characterized in that, The molar ratio of apolipoprotein E to polyethylene glycolated lipids is 1:1 to 1:

10.

3. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to claim 1, characterized in that, The molar ratio of apolipoprotein E to polyethylene glycolated lipids is 1:1 to 1:

5.

4. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to claim 1, characterized in that, The polyethylene glycol in the PEGylated lipid has an average molecular weight of 550 to 5000.

5. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to claim 1, characterized in that, The polyethylene glycol in the PEGylated lipid has an average molecular weight of 1000 to 2000.

6. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to claim 1, characterized in that, The size of the polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles ranges from 5 nm to 50 nm.

7. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to claim 1, characterized in that, The size of the polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles ranges from 10 nm to 35 nm.

8. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to claim 1, characterized in that, The apolipoprotein E is apolipoprotein E2 or apolipoprotein E3, or includes both apolipoprotein E2 and apolipoprotein E3.

9. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to claim 1, characterized in that, It may also contain apolipoprotein A1.

10. The polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to claim 1, characterized in that, The phospholipid is selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine, lecithinylcholine, dilauroyl phosphatidylcholine, 1,2-dimyristoyl-sn-glycerol-3-phosphocholine, dipalmitoyl phosphatidylcholine, distearyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1,2-distearyl-sn-glycerol-3-phosphocholine, 1,2- Arachioyl-sn-glycerol-3-phosphate choline, 1,2-dieanoyl-sn-glycerol-3-phosphate choline, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, distearyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, palmitoyl oleoyl phosphatidylethanolamine, lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamoyl)ethyl]-3,4-di[oleoyloxy] [3-aminopropyl]-benzamide), N1-[2-((1S)-1-[(3-aminopropyl)bis(octadecylamidoglycerin)glycerylspermine, 4-trifluoroacetic acid, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane, (1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, 2,3-dioleoyloxy-N-[2( One or more of the following groups: [[(3-aminopropyl)-N,N-dimethyl-1-propanediamine trifluoroacetate], [[(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanediamine bromide], [[N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropanediamine], [[ethylphosphocholine], [[dimethylbisoctadecylammonium bromide], [[N4-cholesterol spermine], [[1,2-dioleoxy-3-dimethylaminopropane], [[D-Lin-MC3-DMA], [[DLin-KC2-DMA], and [[DLin-DMA]]] 11. A composition for the prevention or treatment of neurodegenerative diseases, characterized in that, It comprises polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles according to any one of claims 1 to 10.

12. The composition for the prevention or treatment of neurodegenerative diseases according to claim 11, characterized in that, The neurodegenerative diseases mentioned are 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, prions, cognitive impairment, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, vascular dementia, alcoholic dementia, Alzheimer's disease, Machado-Joseph disease, dystonia, multiple system atrophy, progressive supranuclear palsy, Friedreich ataxia, and temporal lobe epilepsy.

13. The composition for the prevention or treatment of neurodegenerative diseases according to claim 11, characterized in that, The neurodegenerative disease mentioned is Alzheimer's disease.

14. A method for preparing polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles, wherein the polyethylene glycol-modified recombinant high-density lipoprotein nanoparticles comprise polyethylene glycol-modified lipids, phospholipids, and apolipoprotein E, characterized in that, Includes the following steps: In a microfluidic device comprising three inlets and one outlet, a solution of phospholipids and polyethylene glycol-modified lipids is injected into the second inlet located in the middle; and High-density lipoprotein or recombinant high-density lipoprotein containing apolipoproteins and phospholipids is injected into the first and third inlets located on both sides.

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