PEGylated reconstituted high-density lipoprotein nanoparticles

Abstract

This invention relates to pegylated reconstituted high-density lipoprotein nanoparticles that have preventive or therapeutic effects on neurodegenerative diseases. Specifically, the present invention relates to nanoparticles and a method for producing the same, characterized in that the phospholipid layer of the produced nanoparticles is protected by PEG by including PEG-lipid or a derivative thereof in a step of preparing a fluid containing a hydrophobic substance and a fluid containing a hydrophilic substance. Pegylated nanoparticles possess excellent stability because they maintain existing blood-brain barrier transport capabilities while evading removal mechanisms by the body's immune response. Their high circulating capacity in the body allows them to exert long-term pharmacological effects, making them effective as drugs or drug carriers.

Description

Technical Field

[0001] The present invention relates to pegylated reconstituted high-density lipoprotein nanoparticles and a method for producing the same. The pegylated reconstituted high-density lipoprotein nanoparticles of the present invention exhibit a therapeutic and preventive effect on neurodegenerative diseases based on the characteristic of excellent in vivo stability.

Background Art

[0002] In recent years, with the rapid increase in the elderly population, the number of patients suffering from various neurodegenerative diseases has been increasing, and the interest in their treatment and prevention has been growing. Neurodegenerative diseases are qualitative diseases that cause various symptoms such as movement disorders, memory disorders, and cognitive disorders due to the decline or loss of nerve cell functions. Not only in nervous system diseases, but also in the normal adult brain, a large number of nerve cells die every day, and the number of nerve cells that die with aging increases exponentially.

[0003] In particular, Alzheimer's disease (AD) is the most common form of dementia and a representative neurodegenerative disease. Alzheimer's disease, the incidence of which is rapidly increasing in an aging society, is characterized by senile plaques in which amyloid-β (Aβ) generated by successive cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase deposits in brain tissue, and neurofibrillary tangles due to hyperphosphorylation of Tau protein, a microtubule-associated protein.

[0004] In recent years, the development of therapeutic drugs targeting these pathological features of Alzheimer's disease has been actively promoted. The most common form of therapeutic drug is antibody therapy, and active research is being conducted on the development of effective antibodies against the aforementioned target substances and the optimization of their delivery to diseased tissues. However, there have been continuous reports that these antibody-based therapeutic drugs can cause cerebral edema and intracerebral microbleeds, leading to a fatal outcome for the patient.

[0005] On the other hand, reconstituted high-density lipoprotein nanoparticles (rHDL) have the advantage of being very similar to substances present in the body, thus avoiding the side effects always observed with antibody-based therapies. However, when administered into the body, they form a protein corona with various proteins in the blood and are taken up by immune cells residing in normal organs other than the target organ (brain) (liver, spleen, lungs, etc.). As a result, problems arise such as rapid removal from the bloodstream and decreased circulatory and targeting efficiency. Furthermore, when administered into the body, inflammatory cytokines (IL-1, IL-6, TNF-α, etc.), which are the cause of inflammatory responses, are adsorbed among various blood proteins, so there is a potential risk of secondary inflammatory side effects occurring when taken up by the brain or other normal organs.

[0006] Therefore, in this invention, we have developed a composition and production method for pegylated reconstituted high-density lipoprotein nanoparticles (P-rHDL) that exhibit excellent therapeutic effects against neurodegenerative diseases and superior in vivo stability. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Korean Registered Patent Publication No. 10-2531293 [Patent Document 2] Korean Registered Patent Publication No. 10-2631907 [Non-patent literature]

[0008] [Non-Patent Document 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). [Non-Patent Document 2] Kim, Y. et al.Single step reconstitution of multifunctional high-density lipoprotein-derived nanomaterials using microfluidics.ACS Nano 7,11,9975-9983(2013). [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention aims to provide PEGylated reconstituted high-density lipoprotein nanoparticles (P-rHDL) that have excellent stability in the body.

[0010] Furthermore, the present invention provides a method for producing pegylated reconstituted high-density lipoprotein nanoparticles (P-rHDL).

[0011] Furthermore, the present invention aims to provide a composition for the prevention or treatment of neurodegenerative diseases comprising pegylated reconstituted high-density lipoprotein nanoparticles (P-rHDL).

[0012] The present invention aims to provide a method for treating neurodegenerative diseases, comprising administering pegylated reconstituted high-density lipoprotein nanoparticles (P-rHDL) or reconstituted high-density lipoprotein nanoparticles (rHDL) to patients with neurodegenerative diseases. [Means for solving the problem]

[0013] The present invention provides reconstituted high-density lipoprotein (rHDL) nanoparticles containing PEG-lipid, phospholipid, and apolipoprotein E.

[0014] In one embodiment, the combined molar ratio of the apolipoprotein to the pegylated lipid is 1:0.5 to 1:50, preferably 1:1 to 1:10, and more preferably 1:1 to 1:5.

[0015] In one embodiment, the average molecular weight of the PEG is 550 (0.5k) to 5,000 (5k), preferably 1,000 (1k) to 2,000 (2k).

[0016] In one embodiment, the size of the reconstituted high-density lipoprotein (rHDL) nanoparticles may be 5 to 50 nm, and preferably 10 to 35 nm.

[0017] In one embodiment, the PEG in the PEGylated lipid may be PEG or a derivative thereof.

[0018] In one embodiment, the apolipoprotein may be apolipoprotein E2 or apolipoprotein E3.

[0019] In one embodiment, the reconstituted high-density lipoprotein nanoparticles (rHDL) may further contain apolipoprotein A1.

[0020] In one embodiment, the phospholipids are 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimryristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), and 1,2-diarachidoyl-sn-glycero- Cello-3-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]butylcarboxyamide)ethyl]-3,4-di[oleyloxy]-benzamide)(VL-5), dioctadecylamideglycylspermine 4-Trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyl-3-trimethylammoniumpropane (DOTAP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-Dioleyloxy-N-[2(speminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanammonium bromide (GAP-DLRIE), N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl-spemine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA may be selected from at least one of the group consisting of, but not limited to, these.,

[0021] The present invention provides a composition for preventing or treating neurodegenerative diseases with improved in vivo safety and stability, which contains the reconstituted high-density lipoprotein nanoparticles (rHDL).

[0022] 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, prion disease, cognitive impairment, senile dementia, dementia with Lewy bodies, frontotemporal dementia, vascular dementia, alcohol dementia, early-onset dementia, Machado-Joseph disease, dystonia, multiple system atrophy, progressive supranuclear palsy, Friedreich's ataxia, temporal lobe epilepsy, and stroke, but is not limited thereto, and preferably, it may be Alzheimer's disease.

[0023] The reconstituted high-density lipoprotein nanoparticles (rHDL) can avoid the phagocytosis of immune cells while maintaining the specific ligand activity against the blood-brain barrier tissue.

[0024] The present invention provides an improved method for treating neurodegenerative diseases, comprising administering reconstituted high-density lipoprotein nanoparticles with improved in vivo safety and stability to patients with neurodegenerative diseases.

[0025] The present invention provides a method for producing pegylated reconstituted high-density lipoprotein (P-rHDL) by injecting a pegylated lipid solution into a single channel inlet located in the center of a microfluidic device having three inlets and one outlet, and injecting high-density lipoprotein (HDL) or reconstituted high-density lipoprotein (rHDL) containing apolipoproteins and phospholipids into the two channel inlets on either side.

[0026] The present invention provides reconstituted high-density lipoprotein nanoparticles (rHDL) produced by the above-described manufacturing method. [Effects of the Invention]

[0027] The pegylated reconstituted high-density lipoprotein nanoparticles (P-rHDL) and compositions containing the same of the present invention can be used for the prevention or treatment of neurodegenerative diseases.

[0028] Furthermore, the pegylated reconstituted high-density lipoprotein nanoparticles (P-rHDL) of the present invention have the effect of avoiding loss of efficacy of therapeutic drugs due to the body's immune response through pegylation, thereby exhibiting excellent efficacy even at low doses. [Brief explanation of the drawing]

[0029] [Figure 1] This is a schematic diagram illustrating a method for synthesizing pegylated high-density lipoprotein nanoparticles (P-rHDL) using the reconstituted high-density lipoprotein nanoparticles (rHDL) of the present invention. [Figure 2] This is a diagram showing the DLS measurement results for P-rHDL, in which the average molecular weight of polyethylene glycol (PEG) in the PEGylated lipid is 0.5k and the synthesis molar ratio with apolipoprotein is 1:1. [Figure 3]This is a diagram showing the DLS measurement results for P-rHDL, in which the average molecular weight of PEG in the PEGylated lipid is 1k and the synthesis molar ratio with apolipoprotein is 1:1. [Figure 4] This is a diagram showing the DLS measurement results for P-rHDL, in which the average molecular weight of PEG in the PEGylated lipid is 2k and the synthesis molar ratio with apolipoprotein is 1:1. [Figure 5] This is a diagram showing the DLS measurement results for P-rHDL, in which the average molecular weight of PEG in the PEGylated lipid is 3k and the synthesis molar ratio with apolipoprotein is 1:1. [Figure 6] This is a diagram showing the DLS measurement results for P-rHDL, in which the average molecular weight of PEG in the PEGylated lipid is 5k and the synthesis molar ratio with apolipoprotein is 1:1. [Figure 7] This is a diagram showing the DLS measurement results for non-pegged rHDL. [Figure 8] This graph shows the DLS measurement results for P-rHDL with synthetic molar ratios of apolipoprotein and PEG2k-lipid (PEG2k-lipid) of 1:0.5 and 1:1. [Figure 9] This graph shows the DLS measurement results for P-rHDL with a synthetic molar ratio of apolipoprotein to PEG-2k-lipid of 1:5 and 1:10. [Figure 10] This graph shows the DLS measurement results for P-rHDL with synthetic molar ratios of apolipoprotein to PEG-2k-lipid of 1:20 and 1:50. [Figure 11] This figure shows the results of comparing the apolipoprotein uptake efficiency of P-rHDL with molar ratios of rHDL to PEG-2k-lipid of 1:5, 1:10, and 1:20, respectively. [Figure 12] This figure shows the results of comparing the surface charge using the zeta potential values ​​of rHDL and P-rHDL. [Figure 13] This figure shows the results of comparing the dispersion stability and storage stability of P-rHDL using DLS at day 0 and day 21. [Figure 14] This figure shows the results of confirming the amyloid-beta aggregation suppression effect when amyloid-beta and P-rHDL are treated simultaneously. [Figure 15] This figure shows the results of examining the decomposition effect of aggregated amyloid-beta when treated with P-rHDL after amyloid-beta aggregation induction. [Figure 16] This is a diagram showing the absorption spectrum measurement results of P-rHDL incorporating fluorescent PEG-pegylated lipids with an average molecular weight of 1k. [Figure 17] This is a diagram showing the absorption spectrum measurement results of P-rHDL incorporating fluorescent PEG-pegylated lipids with an average molecular weight of 2k. [Figure 18] This is a diagram showing the absorption spectrum measurement results of P-rHDL incorporating fluorescent PEG-pegylated lipids with an average molecular weight of 3k. [Figure 19] This figure shows the results of confirming the phagocytic evasion function of immune cells against fluorescently labeled rHDL and fluorescently labeled P-rHDL using a confocal laser microscope. [Figure 20] This figure shows the results of confocal laser microscopy to confirm the intracellular delivery efficiency of fluorescently labeled rHDL in human brain microvascular endothelial cells (hBMECs). [Figure 21] This figure shows the results of confocal laser microscopy to confirm the intracellular delivery efficiency of fluorescently labeled P-rHDL in hBMEC. [Figure 22] This figure shows the results of confocal laser microscopy confirmation of the intracellular delivery efficiency of fluorescently labeled rHDL·P-rHDL in hBMEC monolayers. [Figure 23] This figure shows the results of a quantitative analysis of the intracellular delivery efficiency of fluorescently labeled rHDL·P-rHDL in hBMEC monolayers, based on fluorescence imaging. [Figure 24] This figure shows the results of confocal laser microscopy to confirm the intracellular delivery efficiency of fluorescently labeled rHDL·P-rHDL in a monolayer of stem cell-derived brain microvascular endothelial cells (iBMEC). [Figure 25] This figure shows the results of a quantitative analysis of the intracellular delivery efficiency of fluorescently labeled rHDL·P-rHDL in iBMEC monolayers, based on fluorescence imaging. [Figure 26]This graph compares the blood-brain barrier (BBB) ​​permeability efficiency of rHDL and P-rHDL up to 4 hours in the hBMEC monolayer within Transwell. [Figure 27] This graph compares the blood-brain barrier (BBB) ​​permeability of rHDL and P-rHDL up to 24 hours in the hBMEC monolayer within the Transwell. [Modes for carrying out the invention]

[0030] The embodiments and examples of the present invention will be described in detail below so that those with ordinary skill in the art to which the present invention pertains can easily implement it. However, the present invention can be implemented in various forms and is not limited to the embodiments and examples described below.

[0031] In the entirety of this specification, when a part "includes" a certain component, unless otherwise specified, this does not mean that other components are excluded, but rather that other components may be included.

[0032] The present invention provides a reconstituted high-density lipoprotein (rHDL) comprising phospholipids, PEG-lipids, and apolipoproteins.

[0033] As used herein, the term “reconstituted high-density lipoprotein (rHDL)” refers to HDL pseudoparticles artificially created to mimic the biological effects of naturally occurring high-density lipoprotein (HDL).

[0034] As used herein, the term "PEGylated" means that the surface of a substance has been modified with PEG or a derivative thereof.

[0035] As used herein, the terms "PEG" or "polyethylene glycol" refer to a linear polymer having a hydroxyl group at its terminus, H-(OCH2CH2) n It has an -OH structure, and its terminal end may be substituted with a functional group such as a carboxylic acid, amine, azide, cyanine, or NHS ester.

[0036] The PEG used herein may have an average molecular weight of 550 (0.5k) to 5,000 (5k), preferably 1,000 (1k) to 2,000 (2k).

[0037] As used herein, the term “neurodegenerative disease” means a disease in which the function of the nervous system is reduced or lost due to degenerative changes in nerve cells, and may preferably be at least one 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 impairment, senile dementia, Lewy body dementia, frontotemporal dementia, vascular dementia, alcoholic dementia, presenile dementia, Machado-Joseph disease, myodystonia, multiple system atrophy, progressive supranuclear palsy, Friedreich’s ataxia, temporal lobe epilepsy, and stroke, and more preferably Alzheimer’s disease.

[0038] 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 may be any one of the isoforms of the APOE gene product, e.g., apolipoprotein E2 ("APOE2"), apolipoprotein E3 ("APOE3"), and apolipoprotein E4 (APOE4). The “functional variant” refers to a variant of a mammalian protein encoded by the APOE gene that maintains the same or similar biological function as the APOE gene product. In some cases, the functional variant involves amino acid insertions, deletions, and / or substitutions compared to the protein encoded by the human APOE gene. In some cases, the functional variant is a fragment of the protein encoded by the human APOE gene.

[0039] In some embodiments, apolipoprotein E2 is a protein registered in GenBank with accession number ARQ79459, or has at least 95% sequence identity with it, preferably at least 98% or 99% sequence identity. In one embodiment, apolipoprotein E3 is a protein registered in GenBank with accession number ARQ79461.1, or has at least 95% sequence identity with it, preferably at least 98% or 99% sequence identity.

[0040] In some embodiments, the apolipoprotein E in the reconstituted high-density lipoprotein (rHDL) is a recombinant protein produced by genetic engineering or a synthetic protein produced by chemical synthesis.

[0041] 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 the mammalian protein encoded by the APOA1 gene that maintains the same or similar biological function as the APOA1 gene product. In some cases, a functional variant involves amino acid insertions, deletions, and / or substitutions compared to the protein encoded by the human APOA1 gene. In some cases, a functional variant is a fragment of the protein encoded by the human APOA1 gene.

[0042] The term "phospholipid" as used herein specifically refers to 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (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-Diarachidoyl-sn -Glycero-3-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]butylcarboxyamide)ethyl]-3,4-di[oleyloxy]-benzamide)(VL-5), dioctadecylamideglycylspermine 4-Trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyl-3-trimethylammoniumpropane (DOTAP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,At least one of the following may be selected, but is not limited to, the group consisting of 3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneammonium bromide (GAP-DLRIE), Nt-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl-spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA.

[0043] As used herein, "PEG-lipid" may be PEG or its derivatives, specifically including 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (PEG PE), 1,2-dimyristoyl-rac-glycerol-methoxypolyethylene glycol (DMG-PEG), distearoyl-rac-glycerol-methoxypolyethylene 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-amine (Cholesterol-PEG-Amine), cholesterol-polyethylene glycol-azide (Cholesterol-PEG-Azide), and cholesterol-polyethylene glycol-dibenzocyclooctin (Chole Cholesterol-PEG-DBCO), Cholesterol-polyethylene glycol-fluorescein isothiocyanate (Cholesterol-PEG-FITC), Cholesterol-polyethylene glycol-maleimide (Cholesterol-PEG-Maleimide), Cholesterol-polyethylene glycol-N-hydroxysuccinimide (Cholesterol-PEG-NHS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)] (DSPE-PEG-Biotin), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)] (DSPE-PEG-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[fluorescein isothiocyanate(polyethylene glycol)] (DSPE-PEG-FITC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)] (DSPE-PEG-Maleimide), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[azide (polyethylene glycol)] (DSPE-PEG-Azide), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP (polyethylene glycol)] (DSPE-PEG-PDP), 1,2-Distearoyl-sn-glycero It may be at least one selected from the group consisting of -3-phosphoethanolamine-N-[amino(polyethylene glycol)](DSPE-PEG-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl(polyethylene glycol)](DSPE-PEG-NHS), and N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]}(C16-PEG-Ceramide), but is not limited to these. [Examples]

[0044] Method for producing pegylated reconstituted high-density lipoprotein nanoparticles (P-rHDL) To produce PEGylated recombinant high-density lipoprotein (P-rHDL) nanoparticles, high-density lipoprotein (rHDL) nanoparticles were produced using a microfluidic device equipped with three inlets and one outlet, and having micropillars inside the apparatus, as shown in Figure 1. Subsequently, PEGylated lipids were added to the rHDL to produce P-rHDL.

[0045] 1-1. Method for manufacturing P-rHDL using a microfluidic device 1 First, to produce rHDL, the phospholipid DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was prepared in anhydrous ethanol to a concentration of 0.83 mg / mL, and the apolipoprotein was prepared in PBS or physiological saline to a concentration of 0.2 mg / mL.

[0046] Approximately 1 mL of DMPC solution was injected through the central inlet of the microfluidic device, and 5 mL of apolipoprotein solution was injected through the two channels located on either side. The injection rate of the DMPC solution was set to 0.8 mL / min, and the injection rate of the apolipoprotein was set to 4.4 mL / min. The two solutions were efficiently mixed by the micropillars inside the device, and rHDL was finally obtained from the efflux channel of the micromixing channel device. The obtained rHDL was purified using a 10K Amicon filter at 3900 rpm for 20 minutes and prepared to a volume of 2.5 mL in PBS or physiological saline for the next step.

[0047] Subsequently, 500 μL of PEGylated lipid PEG2k-lipid (18:0PEG2000PE) at a concentration of 0.82 mg / mL was prepared to pegyrate rHDL. The injection rate of the PEGylated lipid PEG2k-lipid solution was set to 0.8 mL / min, and the injection rate of the rHDL solution was set to 4.4 mL / min. Then, P-rHDL was synthesized by connecting to a new microfluidic device. The P-rHDL obtained from the efflux channel of the microfluidic device was mixed with PBS or physiological saline, and then purified three times using a new 10K filter at 25°C and maximum centrifuge speed: once for 20 minutes and twice for 15 minutes. The residue after final purification was diluted with physiological saline to a total of 1 mL, and then passed through a 0.2 μm syringe filter to obtain the final purified P-rHDL. The prepared P-rHDL was stored at 4°C until use in the next experiment.

[0048] 1-2. Method for manufacturing P-rHDL using a microfluidic device 2 Another method for producing P-rHDL was prepared as follows: The phospholipid DMPC and pegylated lipid to be used to produce rHDL were prepared as a single solution and injected into the central inlet of a microfluidic device, and the apolipoprotein solution was injected through two channels located on either side.

[0049] In detail, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and PEG solutions were prepared in anhydrous ethanol. First, an 8.3 mg / mL DMPC solution was prepared in anhydrous ethanol, and then a 0.82 mg / mL PEG2k-lipid (18:0 PEG2000 PE, molar ratio of 1 to apolipoprotein) solution was prepared in anhydrous ethanol. The PEG2k-lipid solution was completely dispersed in a bath sonicator for approximately 30 minutes and then visually confirmed.

[0050] 100 μL of the DMPC solution, 100 μL of the PEG2 k-lipid solution, and 800 μL of anhydrous ethanol were mixed, and then homogenized using a digital locker for approximately 10 minutes to produce the DMPC / PEG2 k-lipid solution.

[0051] Subsequently, P-rHDL containing phospholipid (DMPC), PEG, and apolipoprotein was produced using the same method as in Example 1-1.

[0052] 1-3. Optimization of PEG molecular weight of PEGylated lipids during P-rHDL production. To investigate the influence of the average molecular weight of PEG in pegylated lipids on the production of P-rHDL, we examined whether P-rHDL synthesis is possible depending on the molecular weight of PEG in the pegylated lipids. The P-rHDL synthesis method was the same as in Examples 1-2, using pegylated lipids with PEG molecular weights of 0.5, 1, 2, 3, and 5k. The molar ratio of apolipoprotein to pegylated lipid was set to 1:1.

[0053] The P-rHDL synthesized under the above conditions was diluted 10-fold with PBS, and then its size distribution was measured using dynamic light scattering (DLS), as shown in Figures 2-6. This confirmed that P-rHDL is synthesized regardless of the PEG molecular weight. In particular, it was confirmed that the PEG molecular weight that showed a graph shape similar to that of rHDL in Figure 7, i.e., a size similar to that of rHDL, was 1k or 2k, as shown in Figures 3 and 4.

[0054] 1-4. Optimization of the pegylated lipid blending ratio during P-rHDL production. As shown in Figures 8-10, in order to optimize the pegylated lipid blending ratio of P-rHDL, P-rHDL was produced using the methods of Examples 1-2 and 1-3, according to the blending ratio of PEG2k-lipid to apolipoprotein ApoE3 (1:0, 1:0.5, 1:1, 1:5, 1:10, 1:20, and 1:50), and the size of the produced P-rHDL was compared using DLS.

[0055] [Table 1]

[0056] As shown in Table 1, the PEG2k-lipid was prepared by adjusting the blending ratio based on apolipoprotein, and P-rHDL was produced using the method of Example 1-1.

[0057] [Table 2]

[0058] As shown in Table 2 and Figures 8-10, the size and uniformity of P-rHDL synthesized for each mixing ratio were confirmed by DLS measurement. In the case of 1:0.5, the size of the synthesized P-rHDL was 70 nm and the PDI was 0.4, which differed significantly from rHDL. For all mixing ratios except 1:0.5 (1:1, 1:5, 1:10, 1:20, 1:50), the PDI was confirmed to be similar to that of rHDL. The size of P-rHDL at mixing ratios of 1:1 and 1:50 was approximately twice as large as that of unpegylated rHDL, with particle sizes of 31.44 nm and 32.71 nm, respectively. Furthermore, as shown in Figures 8 and 10, from the number-precent graph (number distribution basis), which is the DLS result for P-rDHL, it was confirmed that P-rDHL, unlike rHDL, generates two types of particles and is not uniform. At mixing ratios of 1:5, 1:10, and 1:20, the particle sizes were 16.39, 18.19, and 11.32 nm, respectively, which are similar in size to rHDL. We confirmed that the DLS distribution in Figures 9 and 10 and the PDI values ​​in Table 2 were similar. In other words, we confirmed that the most ideal conditions for P-rHDL according to the PEG2k-lipid mixing ratio are 1:5, 1:10, and 1:20.

[0059] To confirm the incorporation efficiency of apolipoprotein E3 (ApoE3) in P-rHDL synthesized under various mixing ratio conditions (1:5, 1:10, 1:20), ApoE3 was quantitatively analyzed using a BCA (Bicinchoninic acid) assay.

[0060] [Table 3]

[0061] As shown in Table 3 and Figure 11, we confirmed that the uptake efficiency of unpegylated rHDL and ApoE3 was similar at all PEG2k-lipid formulation ratios of 1:5, 1:10, and 1:20.

[0062] Of the three mixing ratios (1:5, 1:10, and 1:20) of ApoE3 and PEG2k-lipid used to synthesize uniformly sized P-rHDL, the 1:5 ratio, which resulted in the least uptake of the extracorporeal substance PEG, was selected as the optimal condition considering long-term repeated in vivo administration and pharmaceutical production. Therefore, in subsequent validation experiments, P-rHDL was produced using the 1:5 condition.

[0063] [Table 4]

[0064] As shown in Table 4 and Figure 12, the surface charges of P-rHDL and rHDL were confirmed to be similar. In other words, the presence or absence of PEG incorporation did not affect the surface charge. This suggests that PEG does not affect the efficacy of P-rHDL and may exhibit similar efficacy to rHDL. [Examples]

[0065] Verification of P-rHDL stability As shown in Table 5 and Figure 13 below, when P-rHDL synthesized under optimal conditions was measured by DLS immediately after synthesis (0d), its size was 16.39 nm and its PDI was 0.24, confirming that it exhibited similar size and PDI values ​​to rHDL. Furthermore, P-rHDL stored for 21 days at 2-8°C to maintain the stability of apolipoprotein and P-rHDL was also confirmed to have a size of 20.06 nm and a PDI of 0.31. This suggests that uniformly synthesized P-rHDL has storage stability that allows it to maintain the same size as immediately after synthesis for at least 21 days without aggregation between nanoparticles during storage, i.e., without non-uniform nanoparticles of 50 nm or larger.

[0066] [Table 5] [Examples]

[0067] Verification of amyloid-beta (Aβ) resolution with and without pegylation of P-rHDL Fluorescently labeled amyloid-beta (Aβ; Beta-Amyloid(1-42), HiLyte TM To confirm the potential for Aβ removal effects using Fluor 488-labeled rHDL, the Aβ aggregation inhibition and resolution capabilities of rHDL and P-rHDL were compared. Aβ has the property of aggregating with each other to form plaques, which are the main cause of the progression of Alzheimer's dementia. When Aβ aggregates, the distance between the fluorescent particles labeled with Aβ shortens, causing a self-quenching effect and a decrease in fluorescence intensity. Based on this, the aggregation inhibition or resolution capability of P-rHDL was confirmed.

[0068] To confirm the inhibitory effect on Aβ aggregation, P-rHDL and control group rHDL were treated simultaneously with Aβ, and the fluorescence intensity was measured at time intervals (0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 8, 24, 48, 72 hours) using a microplate reader. As shown in Figure 14, 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 that it exerts an inhibitory effect on agglutinating Aβ. Furthermore, when comparing the degree of fluorescence reduction of the control group rHDL and Aβ, they were similar, confirming that the two nanoparticles exert an inhibitory effect on the aggregation of almost the same pathogen.

[0069] Furthermore, as shown in Figure 15, to confirm the degrading effect of aggregated Aβ, Aβ aggregation was induced for up to 24 hours. Then, at the 24-hour aggregation induction point, P-rHDL and rHDL were treated, respectively, and the fluorescence intensity was measured at each time point (24, 24.33, 24.67, 25, 26, 7, 28, 30, 48, and 72 hours) using a microplate reader. The fluorescence intensity of Aβ increased immediately after treatment with P-rHDL, confirming that the aggregated Aβ was degraded into peptides by P-rHDL. In addition, a comparison of the degree of fluorescence increase of Aβ after treatment with the control group (rHDL) showed similar results, confirming that P-rHDL exhibits almost the same Aβ degrading effect. [Examples]

[0070] Verification of the pegylation effect of P-rHDL by avoiding phagocytosis by immune cells. 4-1. Production of fluorescently labeled P-rHDL (fluorescein isothiocyanate (FITC)) To verify the pegylation effect using fluorescence, fluorescently labeled P-rHDL was prepared using fluorescein isothiocyanate (FITC) labeled PEG-lipid.

[0071] The synthesis method for FITC-labeled P-rHDL was the same as in Examples 1-2, except that FITC-PEG-lipids with molecular weights of PEG of 1k, 2k, and 3k were used as the pegylated lipids, and a molar ratio of apolipoprotein to pegylated lipid of 1:5 was applied.

[0072] As shown in Figures 16 and 17, when P-rHDL was produced using FITC-PEG with average PEG molecular weights of 1k and 2k, a similar spectral peak was observed at 490 nm, which is known as the absorption spectral peak of the fluorescent substance FITC. As shown in Figure 18, when P-rHDL was produced using FITC-PEG with an average PEG molecular weight of 3k, the absorption spectral peak of FITC was not observed at 490 nm.

[0073] In other words, while P-rHDL can be synthesized when the average molecular weight of PEG is 0.5 to 5 k, the preferred range is 1 to 2 k.

[0074] 4-2. Verification of the pegylation effect of P-rHDL by avoiding phagocytosis by immune cells. To verify the immunosuppressive evasion ability of P-rHDL, fluorescently labeled P-rHDL was synthesized using the method of Example 1-2, labeled with cyanine 5.5 N-hydroxysuccinimide ester (Cy5.5-NHS). To confirm the synthesis of fluorescently labeled P-rHDL, the absorbance spectrum at 665 nm was measured using a microplate reader, and the Cy5.5 absorbance was analyzed.

[0075] Subsequently, the degree of phagocytosis by immune cells was experimentally investigated using synthesized fluorescently labeled P-rHDL to verify the immune response evasion ability of P-rHDL. THP-1 cells used as immune cells were prepared by treating them with phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng / mL for 24 hours to differentiate them into macrophages. Differentiated THP-1 cells were treated with the same concentrations of fluorescently labeled rHDL and fluorescently labeled P-rHDL, and then the rHDL and P-rHDL that entered the cells by phagocytosis were observed using a confocal laser scanning microscope (CLSM).

[0076] As shown in Figure 19, we confirmed that P-rHDL exhibited less phagocytosis by THP-1 cells compared to non-pegylated rHDL. This means that, as described in the references, P-rHDL can evade phagocytosis by immune cells by incorporating PEG functional groups on its particle surface. [Examples]

[0077] Confirmation of the delivery efficiency of P-rHDL to brain microvascular endothelial cells. The intracellular delivery efficiency of P-rHDL was confirmed using brain microvascural endethelial cells (BMECs), which are the main cells that form 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, 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, and 4-Chlorobenzenesulfonate Salt (DiD) fluorescent molecules, respectively. In the control group, rHDL was labeled with Alexa Fluor 568 and DiD fluorescent molecules for ApoE3 and DMPC, respectively. To confirm the extent of delivery to human brain microvascular endothelial cells (human BMECs; hBMECs), cells were treated with P-rHDL and rHDL at 37°C and 5% CO2 for 4 hours. The cells were then fixed, the nucleus and cytoplasm were stained, and observed using a confocal laser scanning microscope (CLSM). As shown in Figures 20 and 21, it was confirmed that P-rHDL was delivered to the area around the cell nucleus, similar to rHDL in the control group.

[0078] Furthermore, to confirm that P-rHDL was delivered into cells as single nanoparticles without degradation, z-stack images of P-rHDL and rHDL incorporated into hBMEC were obtained using CLSM. Examination of two different cross-sectional images of hBMEC using z-stack imaging revealed, as shown in Figures 20 and 21, that all components of P-rHDL delivered into cells—PEG2k-lipid, ApoE3, and DMPC—were incorporated around the cell nucleus and overlapped in one location. The degree of delivery to the cell nucleus and the overlap of nanoparticle components were similar to that of the control group, rHDL. This suggests that, despite being pegylated, P-rHDL can exert functional effects equivalent to rHDL in terms of intracellular delivery efficiency.

[0079] Next, as shown in Figures 22 and 24, human-derived brain microvascular endothelial cells (hBMECs) and induced pluripotent stem cell-derived brain microvascular endothelial cells (iBMECs) formed a monolayer similar to the blood-brain barrier in vivo, and the intracellular delivery efficiency of P-rHDL was compared with that of rHDL in the control group. As shown in Figure 22, in hBMECs, P-rHDL showed a similar intracellular delivery efficiency to rHDL, and as shown in Figure 23, no significant difference was confirmed in quantitative analysis based on fluorescence imaging. As shown in Figure 24, in iBMECs, P-rHDL showed a similar intracellular delivery efficiency to rHDL, and as shown in Figure 25, no significant difference was confirmed in quantitative analysis based on fluorescence imaging. Therefore, since P-rHDL shows a similar delivery efficiency to rHDL in BMECs, it can be seen that P-rHDL is not affected by pegylation and functionally exhibits a delivery effect equivalent to rHDL. [Examples]

[0080] Confirmation of P-rHDL's blood-brain barrier permeability efficiency. To confirm the blood-brain barrier permeability of P-rHDL, a monolayer of hBMEC was formed in the insert portion of a Ranswell plate. Each plate was treated with DiD-labeled P-rHDL and rHDL, respectively, and the permeability efficiency at different time intervals (4 and 24 hours) was compared with that of a control group (fluorescein, rhodamine B, and FITC-dextran (4, 70, and 150 kDa)). The permeated control group, rHDL, and P-rHDL were collected from the bottom well of the Ranswell plate, and fluorescence measurements were performed using a microplate reader to compare the permeability.

[0081] As shown in Figure 26, at the initial permeation point of 4 hours, P-rHDL showed no significant difference compared to rHDL, exhibiting similar blood-brain barrier permeability. As shown in Figure 27, at 24 hours of blood-brain barrier permeability, P-rHDL showed a statistically significant difference (p=0.0053) compared to rHDL based on statistical analysis. However, since P-rHDL and rHDL showed similar cell delivery efficiencies, it is unlikely that pegylation contributed to improved permeability, and therefore, this difference can be excluded from the statistical significance. Thus, it can be said that P-rHDL showed similar blood-brain barrier permeability to rHDL up to 24 hours. In comparison with the control group, both rHDL and P-rHDL showed more than twice the permeability, confirming a statistically significant difference.

Claims

1. In pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles containing pegylated lipids, phospholipids, and apolipoprotein E, Peginated reconstituted high-density lipoprotein nanoparticles (P-rHDL) characterized in that the synthetic molar ratio of apolipoprotein E to pegylated lipid is 1:0.5 to 1:

50.

2. The pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles according to claim 1, characterized in that the synthetic blending molar ratio of apolipoprotein E to pegylated lipid is 1:1 to 1:

10.

3. The pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles according to claim 1, characterized in that the synthetic blending molar ratio of apolipoprotein E to pegylated lipid is 1:1 to 1:

5.

4. The pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles according to claim 1, characterized in that the average molecular weight of the polyethylene glycol in the pegylated lipid is 550 to 5,000.

5. The pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles according to claim 1, characterized in that the average molecular weight of the polyethylene glycol in the pegylated lipid is 1,000 to 2,000.

6. The pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles according to claim 1, characterized in that the size of the pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles is 5 to 50 nm.

7. The pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles according to claim 1, characterized in that the size of the pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles is 10 to 35 nm.

8. The pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles according to claim 1, characterized in that the apolipoprotein E is apolipoprotein E2, E3, or E2 and E3.

9. The pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles according to claim 1, which may further contain apolipoprotein A1.

10. The phospholipids mentioned above are 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1,2-dimryristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachidoyl-sn-glycero-3- 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]butylcarboxyamide)ethyl]-3,4-di[oleyloxy]-benzamide)(VL-5), dioctadecylamideglycylspermine 4-Trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyl-3-trimethylammoniumpropane (DOTAP), (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-Dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneammonium bromide (GAP-DLRIE), N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl The pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles according to claim 1, characterized in that at least one is selected from the group consisting of PC, dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl-spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), D-Lin-MC3-DMA (MC3,DLin-MC3-DMA), DLin-KC2-DMA, and DLin-DMA.

11. A composition for the prevention or treatment of neurodegenerative diseases, comprising pegylated reconstituted high-density lipoprotein nanoparticles according to any one of claims 1 to 10.

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

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

14. The steps include injecting a phospholipid and pegylated lipid solution into a second inlet located in the center of a microfluidic device having three inlets and one outlet, The steps include injecting high-density lipoprotein (HDL) or reconstituted high-density lipoprotein (rHDL) containing apolipoproteins and phospholipids into the first and third inlets located on both sides, A method for producing pegylated reconstituted high-density lipoprotein (rHDL) nanoparticles comprising pegylated lipids, phospholipids, and apolipoprotein E.

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