KR20230145791A - Cationic lipid nanoparticles for mRNA vaccine - Google Patents
Cationic lipid nanoparticles for mRNA vaccine Download PDFInfo
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- KR20230145791A KR20230145791A KR1020220044633A KR20220044633A KR20230145791A KR 20230145791 A KR20230145791 A KR 20230145791A KR 1020220044633 A KR1020220044633 A KR 1020220044633A KR 20220044633 A KR20220044633 A KR 20220044633A KR 20230145791 A KR20230145791 A KR 20230145791A
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- phosphoethanolamine
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Classifications
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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- A—HUMAN NECESSITIES
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
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Abstract
본 발명은 a) DMKD(O,O′-dimyristyl-N-lysyl aspartate); b) 인지질; c) 스테롤계 지질; 및 d) 세포사멸 유도 물질을 포함하는 지질 나노입자; 또는 e) 핵산을 더 포함하는 지질 나노입자 복합체에 관한 것으로, 전달체로 사용이 가능하고, 비면역세포와 면역세포의 전달에 차별성을 나타내어 효율적으로 면역 획득이 가능한 발명에 관한 것이다.The present invention relates to a) DMKD (O,O'-dimyristyl-N-lysyl aspartate); b) phospholipids; c) sterol lipids; and d) lipid nanoparticles containing an apoptosis-inducing substance; or e) relates to a lipid nanoparticle complex further containing nucleic acid, which can be used as a delivery vehicle and is capable of efficiently acquiring immunity by showing differentiation in the delivery of non-immune cells and immune cells.
Description
본 발명은 mRNA 백신을 위한 양이온성 지질 나노입자, 상세하게는 a) DMKD(O,O′-dimyristyl-N-lysyl aspartate); b) 인지질; c) 스테롤계 지질; 및 d) 세포사멸 유도 물질을 포함하는 지질 나노입자; 또는 e) 핵산을 더 포함하는 지질 나노입자 복합체에 관한 것이다.The present invention relates to cationic lipid nanoparticles for mRNA vaccines, specifically a) DMKD (O,O′-dimyristyl-N-lysyl aspartate); b) phospholipids; c) sterol lipids; and d) lipid nanoparticles containing an apoptosis-inducing substance; or e) a lipid nanoparticle complex further comprising a nucleic acid.
지질 나노입자(lipid nanoparticle)는 난용성 물질을 봉입할 수 있는 지질로 이루어진 입자성 약물전달체를 의미한다. 지질 나노입자는 일반적으로 인지질, 콜레스테롤, 음이온성 지질, PEG 함유 지질을 사용하여 제조되고, 크기가 약 60 내지 150 나노미터(nm)에 해당한다. 지질 나노입자는 내부 구조에 따라 분류될 수 있고, 그 분류에 따라 입자 안정성, 전달 효과 등이 다르게 나타난다. Lipid nanoparticle refers to a particulate drug carrier made of lipid that can encapsulate poorly soluble substances. Lipid nanoparticles are generally prepared using phospholipids, cholesterol, anionic lipids, and PEG-containing lipids, and have a size of about 60 to 150 nanometers (nm). Lipid nanoparticles can be classified according to their internal structure, and depending on the classification, particle stability, delivery effect, etc. appear differently.
종래는 약물의 전달을 위하여 사용되는 지질의 종류, 사용량, 약물의 종류 등을 변화시켜가며 제조하려는 시도가 있어 왔고, 이와 관련하여, 지질의 특성에 따라 물리적 특성이 달라질 수 있고, 이온성 지질이 포함되지 않은 중성 나노입자의 경우 마이크로 크기의 거대 입자가 형성되거나 소수성 결합에 의해 침전이 발생하며, 음이온성 지질의 경우에도 표면전하 등에 따라 봉입 효율이 달라진다는 것이 알려진 바 있다(비특허문헌 1). 또한, 약물로써 RNA분자를 사용하고, 지질 나노입자를 약물전달체로 하는 복합체로 약물 전달효율을 높이려는 시도가 있어왔으나, RNA의 불투과성, 취약성, 면역원성, 큰 크기 등에 의한 문제가 있다. 다만, 지질 나노입자를 통한 siRNA 전달방법이 2018년 FDA 허가를 받으면서 가능하게 되었고, 이를 바탕으로 mRNA 백신 전달체로 지질 나노입자를 사용하는 많은 연구가 진행되었다.Conventionally, there have been attempts to manufacture drugs by changing the type of lipid used for drug delivery, the amount used, the type of drug, etc. In this regard, physical properties may vary depending on the characteristics of the lipid, and ionic lipids In the case of neutral nanoparticles that are not included, micro-sized large particles are formed or precipitation occurs due to hydrophobic bonds, and even in the case of anionic lipids, it is known that the encapsulation efficiency varies depending on the surface charge, etc. (Non-patent Document 1) . In addition, there have been attempts to increase drug delivery efficiency by using RNA molecules as drugs and complexes with lipid nanoparticles as drug delivery vehicles, but there are problems due to RNA's impermeability, fragility, immunogenicity, and large size. However, the siRNA delivery method through lipid nanoparticles became possible with FDA approval in 2018, and based on this, many studies were conducted on using lipid nanoparticles as an mRNA vaccine delivery vehicle.
이와 관련하여, 대한민국공개특허 제10-2021-0105889호에 특정 양이온성 지질을 사용함과 동시에 핵산 분자, 콜레스테롤, 인지질, PEG 포함 지질이 특정 비율로 인해 제조된 지질 나노입자가 개시되어 있고, 대한민국공개특허 제10-2021-0135494호에는 핵산과 결합된 지질 나노입자의 제조방법이 개시되어 있으며, 대한민국공개특허 제10-2021-0091120호에 특정 농도로 캡슐화하여 mRNA 로딩 지질 나노입자의 제조방법이 개시되어 있다.In this regard, Korea Patent Publication No. 10-2021-0105889 discloses lipid nanoparticles manufactured by using specific cationic lipids and at the same time containing nucleic acid molecules, cholesterol, phospholipids, and PEG-containing lipids in a specific ratio. Patent No. 10-2021-0135494 discloses a method for producing lipid nanoparticles bound to nucleic acids, and Korean Patent Publication No. 10-2021-0091120 discloses a method for producing mRNA-loaded lipid nanoparticles by encapsulating them at a specific concentration. It is done.
다만, 지질 나노입자는 종류에 따라 mRNA, 약물 등의 전달 효율, 안정성 등 특성에 많은 차이를 나타낸다. 이에, 본 발명자는 지질 나노입자 전달 특성의 개선을 위해 많은 연구를 거쳐 본 발명을 완성하였다.However, depending on the type, lipid nanoparticles show many differences in characteristics such as delivery efficiency and stability of mRNA and drugs. Accordingly, the present inventor completed the present invention through much research to improve the delivery characteristics of lipid nanoparticles.
본 발명은 종래 지질 나노입자가 체내 오래 순환할 수 있다는 면역회피성 입자와 달리, 면역세포의 포식작용을 유발하는 방식에 의해 mRNA 백신을 전달하고자 한다. 또한, 종래 mRNA 백신 전달체는 면역세포와 비면역세포를 구분하지 않고 전달하여 항원의 과발현에 따른 부작용 가능성을 내포하나, 본 발명은 비면역세포에 핵산을 전달하면서도 면역세포에 효과적으로 전달하여 면역세포가 직접 항원을 생산하여 효율적으로 면역 획득이 가능할 수 있는 전달체를 제공하고자 한다.The present invention seeks to deliver mRNA vaccines by inducing phagocytosis of immune cells, unlike conventional lipid nanoparticles, which are immune-evading particles that can circulate in the body for a long time. In addition, conventional mRNA vaccine delivery vehicles carry the possibility of side effects due to overexpression of antigens as they are delivered without distinguishing between immune cells and non-immune cells, but the present invention delivers nucleic acids to non-immune cells while effectively delivering them to immune cells, We aim to provide a delivery vehicle that can efficiently acquire immunity by directly producing antigens.
상기 목적을 달성하기 위하여, 본 발명은 a) DMKD(O,O′-dimyristyl-N-lysyl aspartate); b) 인지질; c) 스테롤계 지질; 및 d) 세포사멸 유도 물질을 포함하는 지질 나노입자를 제공한다.In order to achieve the above object, the present invention provides a) DMKD (O,O′-dimyristyl-N-lysyl aspartate); b) phospholipids; c) sterol lipids; and d) lipid nanoparticles containing an apoptosis-inducing substance.
본 발명의 일 양태에서, 상기 인지질은 In one aspect of the invention, the phospholipid is
1,2-디팔미토일-sn-글리세로-3-포스포에탄올아민(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
디스테아로일포스파티딜에탄올아민-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine), Distearoylphosphatidylethanolamine-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine),
디스테아로일포스파티딜에탄올말레이미드-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide), Distearoylphosphatidylethanol maleimide-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide),
1,2-디스테아로일-sn-글리세로-3-포스포콜린(1,2-Distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-디팔미토일-sn-글리세로-3-포스포콜린(1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-디라우로일-sn-글리세로-3-포스포콜린(1,2-dilauroylsn-glycero-3-phosphocholine, DLPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-디미리스토일-sn-글리세로-3-포스포콜린(1,2-dimyristoyl-snglycero-3-phosphocholine, DMPC), 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC),
1,2-디올레오일-sn-글리세로-3-포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-디미리스토일-sn-글리세로-3-포스포에탄올아민(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPE), 및1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and
1,2-디올레오일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE)로 구성된 군으로부터 선택된 1종 이상이다.At least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE).
본 발명의 일 양태에서, 상기 인지질은 In one aspect of the invention, the phospholipid is
1,2-디팔미토일-sn-글리세로-3-포스포에탄올아민(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
디스테아로일포스파티딜에탄올아민-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine) 및Distearoylphosphatidylethanolamine-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine) and
디스테아로일포스파티딜에탄올말레이미드-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide)Distearoylphosphatidylethanol maleimide-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide)
로 구성된 군으로부터 선택된 1종 이상이다.It is one or more types selected from the group consisting of.
구체적인 본 발명의 일 양태에서, 상기 인지질은 디스테아로일포스파티딜에탄올아민-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine)이다.In one specific embodiment of the present invention, the phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000- amine).
본 발명의 일 양태에서, 상기 스테롤계 지질은 콜레스테롤(cholesterol), 시토스테롤(sitosterol), 스티그마스테롤(stigmasterol), 캄페스테롤(campesterol), 및 에르고스테롤(ergosterol)로 구성된 군으로부터 선택된 1종 이상이다.In one aspect of the present invention, the sterol lipid is one or more selected from the group consisting of cholesterol, sitosterol, stigmasterol, campesterol, and ergosterol.
본 발명의 일 양태에서, 상기 스테롤계 지질은 콜레스테롤(cholesterol)이다.In one aspect of the present invention, the sterol-based lipid is cholesterol.
본 발명의 일 양태에서, 상기 세포사멸 유도 물질은 포스파티딜 세린(phosphatidylserine)이다.In one aspect of the present invention, the apoptosis-inducing substance is phosphatidylserine.
본 발명의 일 양태에서, 상기 DMKD(O,O′-dimyristyl-N-lysyl aspartate)는 나노입자 대비 몰백분율 45 내지 65 mol% 이다.In one aspect of the present invention, the mole percentage of DMKD (O,O'-dimyristyl-N-lysyl aspartate) is 45 to 65 mol% based on the nanoparticles.
본 발명의 일 양태에서, 상기 인지질은 나노입자 대비 몰백분율 14 내지 24 mol%이다..In one aspect of the present invention, the phospholipids have a molar percentage of 14 to 24 mol% relative to the nanoparticles.
본 발명의 일 양태에서, 상기 스테롤계 지질은 나노입자 대비 몰백분율 18 내지 30 mol% 이다.In one aspect of the present invention, the sterol-based lipid has a molar percentage of 18 to 30 mol% based on the nanoparticles.
본 발명의 일 양태에서, 상기 세포사멸 유도 물질은 나노입자 대비 몰백분율 1 내지 4 mol%이다.In one aspect of the present invention, the apoptosis-inducing substance has a molar percentage of 1 to 4 mol% based on the nanoparticles.
본 발명의 일 양태에서, 상기 지질 나노입자는 크기가 1 내지 500 nm이다.In one aspect of the invention, the lipid nanoparticles have a size of 1 to 500 nm.
또한, 본 발명은 a) DMKD(O,O′-dimyristyl-N-lysyl aspartate); b) 인지질; c) 스테롤계 지질; d) 세포사멸 유도 물질; 및 e) 핵산을 포함하는 지질 나노입자 복합체를 제공한다.In addition, the present invention provides a) DMKD (O,O'-dimyristyl-N-lysyl aspartate); b) phospholipids; c) sterol lipids; d) Apoptosis-inducing substances; and e) providing a lipid nanoparticle complex comprising nucleic acid.
본 발명의 일 양태에서, 상기 핵산은 DNA, siRNA, mRNA, 안티센스 RNA, 단일가닥 RNA, 또는 마이크로 RNA이다.In one aspect of the invention, the nucleic acid is DNA, siRNA, mRNA, antisense RNA, single-stranded RNA, or micro RNA.
본 발명의 일 양태에서, 상기 핵산은 mRNA이다.In one aspect of the invention, the nucleic acid is mRNA.
또한, 본 발명은 상기 지질 나노입자 복합체를 포함하는 약학적 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition containing the lipid nanoparticle complex.
또한, 본 발명은 상기 지질 나노입자 복합체를 포함하는 백신을 제공한다.Additionally, the present invention provides a vaccine comprising the lipid nanoparticle complex.
본 발명은 mRNA 백신 전달체로 사용이 가능하고, 비면역세포와 면역세포의 전달에 차별성을 나타내어 효율적으로 면역 획득이 가능하다는 이점이 있다.The present invention can be used as an mRNA vaccine delivery vehicle and has the advantage of being able to efficiently acquire immunity by showing differentiation in the delivery of non-immune cells and immune cells.
도 1은 본 발명 지질 나노입자의 모식도이다.
도 2는 본 발명의 일 실시형태에 따른 지질 나노입자의 안정성을 확인한 도이다.
도 3은 본 발명의 일 실시형태에 따른 지질 나노입자와 비교군의 단백질 발현을 공초점 현미경으로 확인한 도이다.
도 4 및 5는 본 발명의 일 실시형태에 따른 지질 나노입자와 비교군의 비면역세포에서 단백질 발현을 유세포 분석으로 확인한 도이다.
도 6 및 7는 본 발명의 일 실시형태에 따른 지질 나노입자와 비교군의 면역세포에서 단백질 발현을 유세포 분석으로 확인한 도이다.Figure 1 is a schematic diagram of the lipid nanoparticles of the present invention.
Figure 2 is a diagram confirming the stability of lipid nanoparticles according to an embodiment of the present invention.
Figure 3 is a diagram confirming protein expression of lipid nanoparticles according to an embodiment of the present invention and a comparison group using a confocal microscope.
Figures 4 and 5 are diagrams confirming protein expression in lipid nanoparticles according to an embodiment of the present invention and non-immune cells of a comparison group by flow cytometry.
Figures 6 and 7 are diagrams confirming protein expression in lipid nanoparticles according to an embodiment of the present invention and immune cells of a comparison group by flow cytometry.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 a) DMKD(O,O′-dimyristyl-N-lysyl aspartate); b) 인지질; c) 스테롤계 지질; 및 d) 세포사멸 유도 물질을 포함하는 지질 나노입자에 관한 것이다. The present invention relates to a) DMKD (O,O'-dimyristyl-N-lysyl aspartate); b) phospholipids; c) sterol lipids; and d) lipid nanoparticles containing an apoptosis-inducing substance.
본 발명에서 DMKD는 O,O′-디미리스틸-N-라이실 아스파테이트로, 하기 화학식의 화합물을 의미한다.In the present invention, DMKD refers to O,O'-dimyristyl-N-lysyl aspartate, a compound of the following formula.
본 발명에서, 상기 DMKD는 자기조립을 통해 나노크기의 입자 형성을 촉진할 수 있고, 그 구조에 의해 세포질로 mRNA 등의 방출, 특히 엔도솜 방출이 가능하다.In the present invention, the DMKD can promote the formation of nano-sized particles through self-assembly, and its structure allows the release of mRNA, etc. into the cytoplasm, especially endosome release.
본 발명의 일 양태에서, 상기 인지질은 In one aspect of the invention, the phospholipid is
1,2-디팔미토일-sn-글리세로-3-포스포에탄올아민(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
디스테아로일포스파티딜에탄올아민-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine), Distearoylphosphatidylethanolamine-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine),
디스테아로일포스파티딜에탄올말레이미드-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide), Distearoylphosphatidylethanol maleimide-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide),
1,2-디스테아로일-sn-글리세로-3-포스포콜린(1,2-Distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-디팔미토일-sn-글리세로-3-포스포콜린(1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-디라우로일-sn-글리세로-3-포스포콜린(1,2-dilauroylsn-glycero-3-phosphocholine, DLPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-디미리스토일-sn-글리세로-3-포스포콜린(1,2-dimyristoyl-snglycero-3-phosphocholine, DMPC), 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC),
1,2-디올레오일-sn-글리세로-3-포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-디미리스토일-sn-글리세로-3-포스포에탄올아민(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPE), 및1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and
1,2-디올레오일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE)로 구성된 군으로부터 선택된 1종 이상이다.At least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE).
본 발명의 일 양태에서, 상기 인지질은 1,2-디팔미토일-sn-글리세로-3-포스포에탄올아민(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPE), 디스테아로일포스파티딜에탄올아민-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine) 및 디스테아로일포스파티딜에탄올말레이미드-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide)로 구성된 군으로부터 선택된 1종 이상이다.In one aspect of the present invention, the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), distearo Ilphosphatidylethanolamine-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine) and distearoylphosphatidylethanol maleimide-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide).
구체적인 본 발명의 일 양태에서, 상기 인지질은 디스테아로일포스파티딜에탄올아민-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine)이다.In one specific embodiment of the present invention, the phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000- amine).
본 발명의 일 양태에서, 상기 인지질은 폴리에틸렌글리콜 변형을 포함할 수 있다. 구체적인 본 발명의 일 양태에서, 상기 인지질은 폴리에틸렌글리콜 변형을 포함할 수 있고, 인지질에 1 kDa 이상의 폴리에틸렌글리콜이 공유 결합될 수 있다. 구체적으로, 폴리에틸렌글리콜은 1 kDa 이상, 1.5 kDa 이상, 2 kDa 이상, 2.5 kDa 이상일 수 있다.In one aspect of the present invention, the phospholipid may include polyethylene glycol modification. In one specific aspect of the present invention, the phospholipid may include polyethylene glycol modification, and polyethylene glycol of 1 kDa or more may be covalently bonded to the phospholipid. Specifically, polyethylene glycol may be 1 kDa or more, 1.5 kDa or more, 2 kDa or more, and 2.5 kDa or more.
본 발명에서, 인지질은 지질 나노입자의 반감기와 안정성을 증가시킬 수 있다.In the present invention, phospholipids can increase the half-life and stability of lipid nanoparticles.
본 발명의 일 양태에서, 상기 스테롤계 지질은 콜레스테롤(cholesterol), 시토스테롤(sitosterol), 스티그마스테롤(stigmasterol), 캄페스테롤(campesterol), 및 에르고스테롤(ergosterol)로 구성된 군으로부터 선택된 1종 이상이다.In one aspect of the present invention, the sterol lipid is one or more selected from the group consisting of cholesterol, sitosterol, stigmasterol, campesterol, and ergosterol.
구체적인 본 발명의 일 양태에서, 상기 스테롤계 지질은 콜레스테롤(cholesterol)이다.In one specific aspect of the present invention, the sterol-based lipid is cholesterol.
본 발명에서, 스테롤계 지질은 이중층 구조를 지지할 수 있고, 안정성을 부여할 수 있다.In the present invention, sterol-based lipids can support the bilayer structure and provide stability.
본 발명의 일 양태에서, 상기 세포사멸 유도 물질은 포스파티딜 세린(phosphatidylserine)이다.In one aspect of the present invention, the apoptosis-inducing substance is phosphatidylserine.
본 발명에서, 세포사멸 유도 물질이 세포막 표면에 노출되는 경우, 세포사멸 신호(apoptosis signal)를 모방하여 식작용(phagocytosis)을 촉진할 수 있다. 따라서, 면역세포에서의 식작용을 촉진하여 전달체로써 유용하다.In the present invention, when an apoptosis-inducing substance is exposed to the cell membrane surface, it can promote phagocytosis by mimicking an apoptosis signal. Therefore, it is useful as a delivery vehicle by promoting phagocytosis in immune cells.
본 발명의 일 양태에서, 상기 DMKD(O,O′-dimyristyl-N-lysyl aspartate)는 나노입자 대비 몰백분율 45 내지 65 mol% 이다.In one aspect of the present invention, the mole percentage of DMKD (O,O'-dimyristyl-N-lysyl aspartate) is 45 to 65 mol% based on the nanoparticles.
구체적인 본 발명의 일 양태에서, 상기 DMKD는 나노입자 대비 몰백분율 47 내지 65 mol %, 구체적으로, 48 내지 65 mol%, 49 내지 65 mol%, 50 내지 65 mol%, 50 내지 63 mol%, 50 내지 62 mol%, 50 내지 61 mol%, 더 구체적으로, 50 내지 60 mol%이다.In one specific embodiment of the present invention, the DMKD is present at a molar percentage of 47 to 65 mol %, specifically, 48 to 65 mol %, 49 to 65 mol %, 50 to 65 mol %, 50 to 63 mol %, 50 mol %, relative to the nanoparticles. to 62 mol%, 50 to 61 mol%, more specifically, 50 to 60 mol%.
본 발명의 일 양태에서, 상기 인지질은 나노입자 대비 몰백분율 14 내지 24 mol%이다.In one aspect of the present invention, the phospholipid has a molar percentage of 14 to 24 mol% relative to the nanoparticles.
구체적인 본 발명의 일 양태에서, 상기 인지질은 나노입자 대비 몰백분율 14.5 내지 24 mol%, 15 내지 24 mol%, 15.5 내지 24 mol%, 16 내지 24 mol%, 16 내지 22 mol%, 16 내지 21.5 mol%, 16 내지 21 mol%, 16 내지 20.5 mol%, 더 구체적으로, 16 내지 20 mol%이다.In one specific embodiment of the present invention, the phospholipid is present in a molar percentage of 14.5 to 24 mol%, 15 to 24 mol%, 15.5 to 24 mol%, 16 to 24 mol%, 16 to 22 mol%, 16 to 21.5 mol relative to the nanoparticles. %, 16 to 21 mol%, 16 to 20.5 mol%, more specifically, 16 to 20 mol%.
본 발명의 일 양태에서, 상기 스테롤계 지질은 나노입자 대비 몰백분율 18 내지 30 mol%이다.In one aspect of the present invention, the sterol-based lipid has a molar percentage of 18 to 30 mol% based on the nanoparticles.
구체적인 본 발명의 일 양태에서, 상기 스테롤계 지질은 나노입자 대비 몰백분율 19 내지 30 mol%, 20 내지 30 mol%, 20.5 내지 30 mol%, 21 내지 30 mol%, 21 내지 29 mol%, 21 내지 28.5 mol%, 21 내지 28 mol%, 21 내지 27.5 mol%, 더 구체적으로, 21 내지 27 mol%이다.In a specific embodiment of the present invention, the sterol-based lipid is present in a molar percentage of 19 to 30 mol%, 20 to 30 mol%, 20.5 to 30 mol%, 21 to 30 mol%, 21 to 29 mol%, 21 to 30 mol%, relative to the nanoparticles. 28.5 mol%, 21 to 28 mol%, 21 to 27.5 mol%, more specifically, 21 to 27 mol%.
본 발명의 일 양태에서, 상기 세포사멸 유도 물질은 나노입자 대비 몰백분율 1 내지 4 mol%이다.In one aspect of the present invention, the apoptosis-inducing substance has a molar percentage of 1 to 4 mol% based on the nanoparticles.
구체적인 본 발명의 일 양태에서, 상기 세포사멸 유도 물질은 나노입자 대비 몰백분율 1.5 내지 4 mol%, 1.6 내지 4 mol%, 1.7 내지 4 mol%, 1.75 내지 4 mol%, 1.8 내지 4 mol%, 1.85 내지 4 mol%, 1.9 내지 4 mol%, 2 내지 4 mol%, 2 내지 3.8 mol%, 2 내지 3.6 mol%, 2 내지 3.5 mol%, 2 내지 3.4 mol%, 2 내지 3.3 mol%, 2 내지 3.25 mol%, 2 내지 3.2 mol%, 2 내지 3.15 mol%, 2 내지 3.1 mol%, 2 내지 3.1 mol%, 2 내지 3.05 mol%, 더 구체적으로, 2 내지 3 mol%이다.In a specific embodiment of the present invention, the apoptosis-inducing substance is used at a molar percentage of 1.5 to 4 mol%, 1.6 to 4 mol%, 1.7 to 4 mol%, 1.75 to 4 mol%, 1.8 to 4 mol%, 1.85 mol% relative to nanoparticles. to 4 mol%, 1.9 to 4 mol%, 2 to 4 mol%, 2 to 3.8 mol%, 2 to 3.6 mol%, 2 to 3.5 mol%, 2 to 3.4 mol%, 2 to 3.3 mol%, 2 to 3.25 mol%, 2 to 3.2 mol%, 2 to 3.15 mol%, 2 to 3.1 mol%, 2 to 3.1 mol%, 2 to 3.05 mol%, more specifically, 2 to 3 mol%.
본 발명의 일 양태에서, 상기 지질 나노입자는 크기가 1 내지 500 nm이다. 구체적인 본 발명의 일 양태에서, 상기 지질 나노입자는 크기가 2 내지 500 nm, 더 구체적으로 5 내지 500 nm, 5 내지 450 nm, 5 내지 400 nm, 5 내지 350 nm, 5 내지 300 nm, 5 내지 250 nm, 5 내지 240 nm, 5 내지 230 nm, 5 내지 220 nm, 5 내지 210 nm, 5 내지 200 nm, 10 내지 200 nm이다.In one aspect of the invention, the lipid nanoparticles have a size of 1 to 500 nm. In one specific aspect of the present invention, the lipid nanoparticles have a size of 2 to 500 nm, more specifically 5 to 500 nm, 5 to 450 nm, 5 to 400 nm, 5 to 350 nm, 5 to 300 nm, 5 to 500 nm. 250 nm, 5 to 240 nm, 5 to 230 nm, 5 to 220 nm, 5 to 210 nm, 5 to 200 nm, 10 to 200 nm.
본 발명 지질 나노입자는 양이온성 지질, 보조지질, 세포 사멸 유도 물질 등을 유기 용매에 각 몰백분율로 용해후 혼합시켜 제조할 수 있다. 유기 용매는 특별히 제한되지 않으나, 클로로포흠, 메탄올, 에탄올, 펜탄, 헥산, 벤젠 등을 사용할 수 있다. 또한, 지질 나노입자의 형성을 위하여, 가열, 증발, 나노입자 형성 용액 등을 사용하여 제조할 수 있으나, 이에 제한되는 것은 아니다.The lipid nanoparticles of the present invention can be prepared by dissolving cationic lipids, auxiliary lipids, cell death-inducing substances, etc. in an organic solvent at each molar percentage and then mixing them. The organic solvent is not particularly limited, but chlorophyll, methanol, ethanol, pentane, hexane, benzene, etc. can be used. Additionally, to form lipid nanoparticles, they can be prepared using heating, evaporation, nanoparticle forming solution, etc., but are not limited thereto.
본 발명에서, 지질 나노입자는 A) a) 양이온성 지질 DMKD(O,O′-dimyristyl-N-lysyl aspartate), b) 인지질; c) 스테롤계 지질; 및 d) 세포사멸 유도 물질을 유기용매에 용해시키는 단계; B) 유기용매를 제거하는 단계; 및 C) 초음파 처리 및 압출하는 단계를 포함하여 제조할 수 있다.In the present invention, lipid nanoparticles include A) a) cationic lipid DMKD (O,O′-dimyristyl-N-lysyl aspartate), b) phospholipid; c) sterol lipids; and d) dissolving the apoptosis-inducing substance in an organic solvent; B) removing the organic solvent; and C) ultrasonic treatment and extrusion.
본 발명의 일 양태에서, 상기 단계 A)의 양이온성 지질, 인지질, 스테롤계 지질, 세포사멸 유도 물질은 각 몰 백분율에 따를 수 있다.In one aspect of the present invention, the cationic lipids, phospholipids, sterol lipids, and apoptosis-inducing substances in step A) may be used in accordance with their respective molar percentages.
또한, 본 발명은 a) DMKD(O,O′-dimyristyl-N-lysyl aspartate); b) 인지질; c) 스테롤계 지질; d) 세포사멸 유도 물질; 및 e) 핵산을 포함하는 지질 나노입자 복합체에 관한 것이다.In addition, the present invention provides a) DMKD (O,O'-dimyristyl-N-lysyl aspartate); b) phospholipids; c) sterol lipids; d) Apoptosis-inducing substances; and e) lipid nanoparticle complexes containing nucleic acids.
본 발명에서 지질 나노입자 복합체는 핵산이 지질 내부에 캡슐화된 상태로, 핵산이 뉴클레아제 등으로부터의 분해, 세포막 투과 등이 가능하다.In the present invention, the lipid nanoparticle complex is a state in which nucleic acids are encapsulated inside lipids, and the nucleic acids can be decomposed by nucleases, etc., and penetrate cell membranes.
본 발명 지질 나노입자 복합체는 면역세포와 비면역세포의 구분없이 핵산을 전달 할 수 있다. 또한, 본 발명 지질 나노입자 복합체는 비면역세포와 비교하여 면역세포에서 효과적으로 핵산을 전달할 수 있어, 면역세포가 직접 항원을 만들어낼 수 있도록 하여 정상세포에서의 항원 과발현에 따른 부작용을 현저히 감소시킬 수 있다.The lipid nanoparticle complex of the present invention can deliver nucleic acids without distinguishing between immune cells and non-immune cells. In addition, the lipid nanoparticle complex of the present invention can effectively deliver nucleic acids to immune cells compared to non-immune cells, allowing immune cells to directly produce antigens, significantly reducing side effects caused by antigen overexpression in normal cells. there is.
본 발명에서, a) 내지 d)를 포함하는 지질 나노입자 및 e) 핵산을 교반하여 지질 나노 입자 복합체를 제조할 수 있다. 교반 시간, 교반 속도, 온도 등의 조건은 제조된 나노입자의 특성, mRNA의 특성 등에 따라 달리할 수 있다.In the present invention, a lipid nanoparticle complex can be prepared by stirring lipid nanoparticles containing a) to d) and e) nucleic acid. Conditions such as stirring time, stirring speed, and temperature may vary depending on the characteristics of the manufactured nanoparticles, the characteristics of the mRNA, etc.
본 발명에서, 지질 나노입자 또는 지질 나노입자 복합체를 통해 제조하는 경우, a) 양이온성 지질, b) 인지질, c) 스테롤계 지질, d) 세포사멸 유도 물질, 및 e) 핵산의 농도는 각각 0.1 내지 100 mg/mL의 임의의 농도를 사용할 수 있다. In the present invention, when manufacturing lipid nanoparticles or lipid nanoparticle complexes, the concentrations of a) cationic lipid, b) phospholipid, c) sterol-based lipid, d) apoptosis-inducing substance, and e) nucleic acid are each 0.1. Any concentration from 100 mg/mL can be used.
본 발명에서 "핵산"은 단일가닥 또는 이중가닥 형태의 데옥시리보뉴클레오티드 또는 리보뉴클레오티드 및 이들의 중합체를 의미한다. 핵산의 예로 데옥시리보 핵산(DNA), 리보핵산(RNA), 포스포르아미데이트, 메틸 포스포네이트 등을 포함한다.In the present invention, “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Examples of nucleic acids include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), phosphoramidate, methyl phosphonate, etc.
본 발명의 일 양태에서, 상기 핵산은 DNA, siRNA, mRNA, 안티센스 RNA, 단일가닥 RNA, 또는 마이크로 RNA이다.In one aspect of the invention, the nucleic acid is DNA, siRNA, mRNA, antisense RNA, single-stranded RNA, or micro RNA.
또한, 본 발명의 일 양태에서, 상기 핵산은 DNA, siRNA, 또는 mRNA이다.Additionally, in one aspect of the invention, the nucleic acid is DNA, siRNA, or mRNA.
또한, 구체적인 본 발명의 일 양태에서, 상기 핵산은 mRNA이다.Additionally, in one specific aspect of the present invention, the nucleic acid is mRNA.
본 발명의 일 양태에서, 상기 a) 내지 d)의 지질 나노입자 및 e) 핵산은 N:P 비율을 1 : 2 내지 4, 구체적으로 1 : 2.2 내지 3.8, 1 : 2.3 내지 3.7, 1 : 2.4 내지 3.6, 1 : 2.5 내지 3.5일 수 있다.In one aspect of the present invention, the lipid nanoparticles of a) to d) and e) nucleic acids have an N:P ratio of 1:2 to 4, specifically 1:2.2 to 3.8, 1:2.3 to 3.7, 1:2.4. to 3.6, 1:2.5 to 3.5.
본 발명의 일 양태에서, 상기 지질 나노입자 또는 지질 나노입자 복합체는 DMKD를 사용하지 않는 경우와 비교하여, 더 높은 핵산 발현을 나타낸다.In one aspect of the invention, the lipid nanoparticle or lipid nanoparticle complex exhibits higher nucleic acid expression compared to the case without DMKD.
또한, 본 발명의 일 양태에서, 상기 지질 나노입자 또는 지질 나노입자 복합체는 DMKD를 사용하지 않는 경우와 비교하여, 핵산 발현이 5% 이상, 구체적으로 10% 이상, 15% 이상, 20% 이상, 25% 이상, 30% 이상, 35% 이상, 40% 이상, 45% 이상, 50% 이상, 55% 이상, 60% 이상, 65% 이상, 70% 이상, 75% 이상, 80% 이상, 85% 이상, 90% 이상, 95% 이상, 100% 이상 더 높은 핵산 발현을 나타낸다.In addition, in one aspect of the present invention, the lipid nanoparticle or lipid nanoparticle complex has nucleic acid expression of 5% or more, specifically 10% or more, 15% or more, 20% or more, compared to the case where DMKD is not used. More than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more, 90% or more, 95% or more, or 100% or more higher nucleic acid expression.
또한, 본 발명의 일 양태에서, 상기 지질 나노입자 또는 지질 나노입자 복합체는 포스파티딜세린(phosphatidylserine)을 사용하지 않는 경우와 비교하여, 핵산 발현이 5% 이상, 구체적으로 10% 이상, 15% 이상, 20% 이상, 25% 이상, 30% 이상, 35% 이상, 40% 이상, 45% 이상, 50% 이상 더 높은 핵산 발현을 나타낸다.In addition, in one aspect of the present invention, the lipid nanoparticle or lipid nanoparticle complex has nucleic acid expression of 5% or more, specifically 10% or more, 15% or more, compared to the case where phosphatidylserine is not used. It shows higher nucleic acid expression by more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, and more than 50%.
본 발명의 일 양태에서, 상기 지질 나노입자 복합체는 5일 이상 입자의 크기를 유지할 수 있다. 구체적인 본 발명의 일 양태에서, 상기 지질 나노입자 복합체는 10일 이상, 더 구체적으로, 20일 이상, 30일 이상, 40일 이상, 50일 이상, 60일 이상, 70일 이상, 80일 이상, 90일 이상, 100일 이상, 110일 이상, 120일 이상 크기를 유지할 수 있다. In one aspect of the present invention, the lipid nanoparticle complex can maintain the particle size for more than 5 days. In one specific aspect of the present invention, the lipid nanoparticle complex is maintained for at least 10 days, more specifically, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, The size can be maintained for more than 90 days, more than 100 days, more than 110 days, and more than 120 days.
또한, 본 발명은 상기 지질 나노입자 복합체를 포함하는 약학적 조성물에 관한 것이다.Additionally, the present invention relates to a pharmaceutical composition containing the lipid nanoparticle complex.
또한, 본 발명은 지질 나노입자 복합체를 포함하는 백신에 관한 것이다.Additionally, the present invention relates to a vaccine comprising a lipid nanoparticle complex.
본 발명의 일 양태에서, 상기 지질 나노입자 복합체는 약학적으로 허용가능한 염, 또는 보조제(adjuvant)를 더 포함할 수 있다. In one aspect of the present invention, the lipid nanoparticle complex may further include a pharmaceutically acceptable salt or adjuvant.
또한, 본 발명은 상기 지질 나노입자 복합체를 투여하는 방법을 제공한다.Additionally, the present invention provides a method of administering the lipid nanoparticle complex.
또한, 상기 약학적 조성물은 약학적으로 허용 가능한 성분을 추가로 포함할 수 있다. Additionally, the pharmaceutical composition may further include pharmaceutically acceptable ingredients.
본 발명의 화합물 투여 경로는 예를 들면, 경구 또는 비경구 경로로 투여될 수 있다. 여기서, 비경구는 광의의 투여 경로를 의미하며, 예를 들면, 정맥내, 동맥내, 복강내, 근육내, 피하, 비내, 설하, 척추강내(초내, intrathecal), 흡입, 눈, 직장, 질, 뇌실 투여 등을 포함한다.The route of administration for the compound of the present invention may be, for example, oral or parenteral route. Here, parenteral refers to a broad route of administration, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intranasal, sublingual, intrathecal, inhalation, ocular, rectal, vaginal, Including intraventricular administration, etc.
상기 조성물을 제제화할 경우, 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조된다.When formulating the composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
경구투여를 위한 고형 제제에는 정제, 환자, 산제, 과립제, 캡슐제, 트로키제 등이 포함되며, 이러한 고형 제제는 하나 이상의 본 발명에 따른 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로스(sucrose) 또는 락토오스(lactose) 또는 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc. These solid preparations include one or more compounds according to the present invention and at least one or more excipients, such as starch, calcium carbonate, It is prepared by mixing sucrose, lactose, or gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제, 동결건조제제, 좌제 등이 포함된다.Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc.
비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.
본 발명에 따른 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
구체적으로, 본 발명에 따른 화합물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.01 mg 내지 100 mg, 보다 구체적으로 체중 1 kg 당 0.1 mg 내지 15 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the compound according to the present invention may vary depending on the patient's age, gender, and weight, and is generally 0.01 mg to 100 mg per kg of body weight, more specifically 0.1 mg to 15 mg per kg of body weight. It can be administered daily or every other day, or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.
이하, 본 발명을 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail through examples and experimental examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 한정되는 것은 아니다.However, the following examples and experimental examples only illustrate the present invention, and the content of the present invention is not limited to the following examples and experimental examples.
<실시예 1> 양이온성 지질 나노입자(cationic lipid nanoparticle, LNP) 제조<Example 1> Preparation of cationic lipid nanoparticle (LNP)
<실시예 1-1> 시료의 준비<Example 1-1> Preparation of samples
DSPE-PEG2000-amine(1,2-distearoyl-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] 및 콜레스테롤(cholesterol) 및 포스파티딜세린(phosphatidylserine)은 Avanti Polar Lipid, Inc., (Alabaster, USC)로부터 구입하였다. 양이온성 지질 DMKD(O,O-dimyristyl-N-lysyl glutamate)는 대한민국공개특허 10-2002-0062479호에 따라 합성하였다.DSPE-PEG2000-amine (1,2-distearoyl-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] and cholesterol and phosphatidylserine were purchased from Avanti Polar Lipid, Inc., (Alabaster) , USC) The cationic lipid DMKD (O,O-dimyristyl-N-lysyl glutamate) was synthesized according to Korean Patent Publication No. 10-2002-0062479.
<실시예 1-2> 양이온성 지질 나노입자 제조<Example 1-2> Preparation of cationic lipid nanoparticles
도 1의 모식도와 같이 양이온성 지질 나노입자(cationic lipid nanoparticle, LNP)를 초음파 처리 및 압출에 의해 제조하였다. 구체적으로, 양이온성 지질인 DMKD, 보조지질인 포스파티딜세린, DSPE-PEG2000-amine, 콜레스테롤(Cholesterol)을 클로로포름 및 메탄올 혼합 용액(2:1, v/v)에 용해시켰다. 이 때, DMKD-PS는 DMKD, 포스파티딜세린, DSPE-PEG200-amine, 콜레스테롤을 각각 48 : 2.5 : 18 : 24.5 몰비로 하여 제조하였다. 이후, 유기용매를 N2 가스 스트림 하에 증발시켜 양이온성 지질 박막을 형성시키고, 이후 1시간 동안 진공 건조하여 잔류 유기용매를 제거하고 1 mL 식염수로 수화하였다. 이후, 실온에서 20분 간격으로 10분씩 3회 초음파 처리 및 압출을 통해 나노입자의 크기를 균일화하였다. As shown in the schematic diagram of Figure 1, cationic lipid nanoparticles (LNPs) were manufactured by sonication and extrusion. Specifically, the cationic lipid DMKD, the auxiliary lipids phosphatidylserine, DSPE-PEG2000-amine, and cholesterol were dissolved in a mixed solution of chloroform and methanol (2:1, v/v). At this time, DMKD-PS was prepared using DMKD, phosphatidylserine, DSPE-PEG200-amine, and cholesterol at a molar ratio of 48:2.5:18:24.5, respectively. Afterwards, the organic solvent was evaporated under a N 2 gas stream to form a cationic lipid thin film, which was then vacuum dried for 1 hour to remove the remaining organic solvent and hydrated with 1 mL saline solution. Afterwards, the size of the nanoparticles was homogenized through ultrasonic treatment and extrusion three times for 10 minutes at 20-minute intervals at room temperature.
<실시예 2> mRNA-양이온성 지질 나노입자 제조<Example 2> Preparation of mRNA-cationic lipid nanoparticles
양이온성 지질 나노입자로 mRNA를 캡슐화하기 위하여, mRNA와 양이온성 지질을 1:3 N:P ratio로 첨가한 후, 상온에서 30분 간 교반하여 mRNA와 복합체를 형성한 지질 나노입자를 제조하였다. To encapsulate mRNA with cationic lipid nanoparticles, mRNA and cationic lipid were added at a 1:3 N:P ratio and stirred at room temperature for 30 minutes to prepare lipid nanoparticles that formed a complex with mRNA.
상기 실시예 1 및 2에서 제조된 나노입자의 크기 및 표면전하는 Zetasizer Nano-ZS90(Malvern Instrument Ltd., Malvern, UK)을 사용하여 DLS(dynamic light scattering)으로 3회 측정하였고 GraphPad Prism 소프트웨어(GraphPad Software, Inc., Ca, USA)로 통계적으로 분석하였으며, 그 결과는 하기 표 1에 나타난 바와 같다.The size and surface charge of the nanoparticles prepared in Examples 1 and 2 were measured three times by dynamic light scattering (DLS) using Zetasizer Nano-ZS90 (Malvern Instrument Ltd., Malvern, UK) and measured using GraphPad Prism software (GraphPad Software). , Inc., Ca, USA), and the results are shown in Table 1 below.
[a: 크기(지름), 다분산지수, 제타전위는 입자 분석기로 3회 측정][a: Size (diameter), polydispersity index, and zeta potential were measured three times using a particle analyzer]
[b: 크기(지름), 다분산지수, 제타전위 평균 ± 표준편차(S.D)][b: Size (diameter), polydispersity index, zeta potential mean ± standard deviation (S.D)]
<실험예 1> GFP mRNA-양이온성 지질 나노입자의 특성 확인<Experimental Example 1> Confirmation of characteristics of GFP mRNA-cationic lipid nanoparticles
상시 실시예 2에서 GFP mRNA를 사용하여 GFP mRNA-양이온성 지질 나노입자를 제조하였으며, 생리식염수 용액 100 μL에 넣고 37 ℃에서 배양하여 이의 안정성을 확인하였다. 실험결과는 도 2에 나타난 바와 같으며, GFP mRNA-양이온성 지질 나노입자는 4일까지는 입자의 크기가 커지나 5일째에는 감소하였다. 또한, GFP mRNA-양이온성 지질 나노입자가 4개월 후에도 크기 변화가 거의 없는 것을 확인하여, 안정성이 우수한 것을 확인하였다.GFP mRNA-cationic lipid nanoparticles were prepared using GFP mRNA in Example 2, and their stability was confirmed by placing them in 100 μL of physiological saline solution and culturing them at 37°C. The experimental results are as shown in Figure 2, and the size of the GFP mRNA-cationic lipid nanoparticles increased until the 4th day, but decreased on the 5th day. In addition, it was confirmed that the GFP mRNA-cationic lipid nanoparticles showed little change in size even after 4 months, confirming that they had excellent stability.
<실험예 2> mRNA <Experimental Example 2> mRNA in vitroin vitro 형질감염 transfection
<실험예 2-1> GFP mRNA로 형질감염된 비면역세포 및 면역세포의 공초점 현미경(Confocal Microscopy) 분석<Experimental Example 2-1> Confocal Microscopy Analysis of Non-Immune Cells and Immune Cells Transfected with GFP mRNA
비면역세포 CT 26 및 면역세포 RAW 264.7의 녹색형광단백질 발현여부를 공초점 레이저 스캐닝 현미경(LSM 710, Zeiss)로 분석하였다.The expression of green fluorescent protein in non-immune cells CT 26 and immune cells RAW 264.7 was analyzed using a confocal laser scanning microscope (LSM 710, Zeiss).
구체적으로, 상기 실시예에서 제조된 mRNA-양이온성 지질 나노입자의 세포 내 흡수를 확인하기 위하여, 상기 실시예에서 제조된 양이온성 지질 나노입자 30 μg을 세포에 처리한 후, 37 ℃에서, 24 시간 동안 배양하였다. 처리 후, 모든 세포를 PBS(pH 7.4)로 2회 세척하고 2% 파라포름알데히드(paraformaldehyde)로 4 ℃, 어두운 곳에서 10분간 고정시켰다. 이후, 비면역세포 CT 26 및 면역세포 RAW 264.7를 DAPI(4’,6-diamidino-2-phenylindole) 용액(Vector lab, Burlingame, USA)으로 어두운 곳에서 10분간 염색하고, 슬라이드에 고정시킨 후, 공초점 레이저 스캐닝 현미경을 사용하여 관찰하였다. 또한, 대조군, 리포펙타민(Lipofectamine) 사용군, DMKD-chol(포스파티딜 미포함) 군, DMKD-PS(실시예에 따라 제조)으로 나누어 실험을 진행하였다.Specifically, to confirm the cellular uptake of the mRNA-cationic lipid nanoparticles prepared in the above example, 30 μg of the cationic lipid nanoparticles prepared in the above example were treated with cells, and then incubated at 37° C. for 24 hours. It was cultured for some time. After treatment, all cells were washed twice with PBS (pH 7.4) and fixed with 2% paraformaldehyde at 4°C in the dark for 10 minutes. Afterwards, non-immune cell CT 26 and immune cell RAW 264.7 were stained with DAPI (4',6-diamidino-2-phenylindole) solution (Vector lab, Burlingame, USA) for 10 minutes in the dark and fixed on a slide. Observation was made using a confocal laser scanning microscope. In addition, the experiment was conducted by dividing the group into a control group, a Lipofectamine group, a DMKD-chol (without phosphatidyl) group, and DMKD-PS (prepared according to the example).
실험결과는 도 3에 나타난 바와 같으며, 비면역세포 및 면역세포 모두에서 DMKD-PS 군이 다른 군에 비해 신호가 높게 나타나 단백질 발현이 가장 많은 것을 확인하였다.The experimental results are as shown in Figure 3, and it was confirmed that the DMKD-PS group showed a higher signal than the other groups in both non-immune cells and immune cells, showing the highest protein expression.
<실험예 2-2> GFP mRNA로 형질감염된 비면역세포 및 면역세포의 유세포 분석(flow cytometry analysis)<Experimental Example 2-2> Flow cytometry analysis of non-immune cells and immune cells transfected with GFP mRNA
비면역세포 CT 26 및 면역세포 RAW 264.7의 녹색형광단백질 발현 여부를 유세포 분석기(BD biosciences, San Jose, CA, USA)로 분석하였다. The expression of green fluorescent protein in non-immune cells CT 26 and immune cells RAW 264.7 was analyzed by flow cytometry (BD biosciences, San Jose, CA, USA).
구체적으로, 비면역세포 CT 26 세포 및 면역세포 Raw 264.7을 6-웰 플레이트에 웰당 2 × 105 세포 밀도로 접종하고 상기 실시예에서 제조된 양이온성 지질 나노입자를 30 μg을 처리한 후 24, 48시간 동안 배양하였다. 이후, 모든 세포를 PBS(pH 7.4)로 2회 세척하고 2% 파라포름알데히드(paraformaldehyde)로 4 ℃ 어두운 곳에서 10분간 고정시킨 후 FACS Calibur 유세포 분석기(Becton Dickinson)로 분석하였다. 또한, 대조군, 리포펙타민(Lipofectamine) 사용군, DMKD-chol(포스파티딜세린 미포함) 군, DMKD-PS(실시예에 따라 제조)으로 나누어 실험을 진행하였다.Specifically, non-immune CT 26 cells and immune cells Raw 264.7 were inoculated into a 6-well plate at a density of 2 × 10 5 cells per well and treated with 30 μg of the cationic lipid nanoparticles prepared in the above example 24, Cultured for 48 hours. Afterwards, all cells were washed twice with PBS (pH 7.4), fixed with 2% paraformaldehyde at 4°C in the dark for 10 minutes, and then analyzed using a FACS Calibur flow cytometer (Becton Dickinson). In addition, the experiment was conducted by dividing the group into a control group, a group using Lipofectamine, a DMKD-chol (without phosphatidylserine) group, and DMKD-PS (prepared according to the example).
실험결과는 도 4 내지 7에 나타난 바와 같으며, 비면역세포 및 면역세포 모두에서 DMKS-PS 군이 다른 군에 비해 현저하게 많은 양의 단백질 발현이 일어나는 것을 확인하였다. 또한, 도 7에 나타난 바와 같이, 면역세포에서 DMKS-PS군이 다른 군에 비해 48시간 후 압도적으로 많은 양의 단백질 발현이 일어나는 것을 확인하였다.The experimental results are as shown in Figures 4 to 7, and it was confirmed that a significantly higher amount of protein expression occurred in the DMKS-PS group compared to the other groups in both non-immune cells and immune cells. Additionally, as shown in Figure 7, it was confirmed that an overwhelming amount of protein expression occurred in the DMKS-PS group in immune cells after 48 hours compared to the other groups.
Claims (16)
a) DMKD (O,O′-dimyristyl-N-lysyl aspartate); b) phospholipids; c) sterol lipids; and d) lipid nanoparticles containing an apoptosis-inducing substance.
상기 인지질은
1,2-디팔미토일-sn-글리세로-3-포스포에탄올아민(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPE),
디스테아로일포스파티딜에탄올아민-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine),
디스테아로일포스파티딜에탄올말레이미드-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide),
1,2-디스테아로일-sn-글리세로-3-포스포콜린(1,2-Distearoyl-sn-glycero-3-phosphocholine, DSPC),
1,2-디팔미토일-sn-글리세로-3-포스포콜린(1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC),
1,2-디라우로일-sn-글리세로-3-포스포콜린(1,2-dilauroylsn-glycero-3-phosphocholine, DLPC),
1,2-디미리스토일-sn-글리세로-3-포스포콜린(1,2-dimyristoyl-snglycero-3-phosphocholine, DMPC),
1,2-디올레오일-sn-글리세로-3-포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC),
1,2-디미리스토일-sn-글리세로-3-포스포에탄올아민(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPE), 및
1,2-디올레오일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE)로 구성된 군으로부터 선택된 1종 이상인, 지질 나노입자.
According to paragraph 1,
The phospholipids are
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
Distearoylphosphatidylethanolamine-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine),
Distearoylphosphatidylethanol maleimide-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide),
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and
At least one lipid nanoparticle selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE).
상기 인지질은
1,2-디팔미토일-sn-글리세로-3-포스포에탄올아민(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPE),
디스테아로일포스파티딜에탄올아민-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine) 및
디스테아로일포스파티딜에탄올말레이미드-폴리에틸렌글리콜(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide)
로 구성된 군으로부터 선택된 1종 이상인, 지질 나노입자.
According to paragraph 1,
The phospholipids are
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
Distearoylphosphatidylethanolamine-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000, DSPE-PEG2000-amine) and
Distearoylphosphatidylethanol maleimide-polyethylene glycol (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000, DSPE-PEG2000-maleimide)
At least one lipid nanoparticle selected from the group consisting of.
상기 스테롤계 지질은 콜레스테롤(cholesterol), 시토스테롤(sitosterol), 스티그마스테롤(stigmasterol), 캄페스테롤(campesterol), 및 에르고스테롤(ergosterol)로 구성된 군으로부터 선택된 1종 이상인, 지질 나노입자.
According to paragraph 1,
The sterol-based lipid is one or more selected from the group consisting of cholesterol, sitosterol, stigmasterol, campesterol, and ergosterol, lipid nanoparticles.
상기 스테롤계 지질은 콜레스테롤(cholesterol)인, 지질 나노입자.
According to paragraph 1,
Lipid nanoparticles wherein the sterol-based lipid is cholesterol.
상기 세포사멸 유도 물질은 포스파티딜 세린(phosphatidylserine)인, 지질 나노입자.
According to paragraph 1,
The apoptosis-inducing substance is phosphatidylserine, a lipid nanoparticle.
상기 DMKD(O,O′-dimyristyl-N-lysyl aspartate)는 나노입자 대비 몰백분율 45 내지 65 mol% 인, 지질 나노입자.
According to paragraph 1,
The DMKD (O,O'-dimyristyl-N-lysyl aspartate) is a lipid nanoparticle with a molar percentage of 45 to 65 mol% relative to the nanoparticle.
상기 인지질은 나노입자 대비 몰백분율 14 내지 24 mol%인, 지질 나노입자.
According to paragraph 1,
Lipid nanoparticles wherein the phospholipids have a molar percentage of 14 to 24 mol% relative to the nanoparticles.
상기 스테롤계 지질은 나노입자 대비 몰백분율 18 내지 30 mol%인, 지질 나노입자.
According to paragraph 1,
The sterol-based lipid is a lipid nanoparticle with a mole percentage of 18 to 30 mol% relative to the nanoparticle.
상기 세포사멸 유도 물질은 나노입자 대비 몰백분율 1 내지 4 mol%인, 지질 나노입자.
According to paragraph 1,
The apoptosis-inducing substance is a lipid nanoparticle with a molar percentage of 1 to 4 mol% compared to the nanoparticle.
상기 지질 나노입자는 크기가 1 내지 500 nm인, 지질 나노입자.
According to paragraph 1,
The lipid nanoparticles are lipid nanoparticles having a size of 1 to 500 nm.
a) DMKD (O,O′-dimyristyl-N-lysyl aspartate); b) phospholipids; c) sterol lipids; d) Apoptosis-inducing substances; and e) lipid nanoparticle complexes containing nucleic acids.
상기 핵산은 DNA, siRNA, mRNA, 안티센스 RNA, 단일가닥 RNA, 또는 마이크로 RNA인, 지질 나노입자 복합체.
According to clause 12,
The lipid nanoparticle complex, wherein the nucleic acid is DNA, siRNA, mRNA, antisense RNA, single-stranded RNA, or micro RNA.
상기 핵산은 mRNA인, 지질 나노입자 복합체.
According to clause 12,
A lipid nanoparticle complex, wherein the nucleic acid is mRNA.
A pharmaceutical composition comprising the lipid nanoparticle complex according to any one of claims 12 to 14.
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|---|---|---|---|---|
| KR20210091120A (en) | 2018-08-29 | 2021-07-21 | 트랜슬레이트 바이오 인코포레이티드 | Improved Process for Making mRNA-Loaded Lipid Nanoparticles |
| KR20210105889A (en) | 2018-11-09 | 2021-08-27 | 아뷰터스 바이오파마 코포레이션 | Lipid Nanoparticle Formulation |
| KR20210135494A (en) | 2019-01-31 | 2021-11-15 | 모더나티엑스, 인크. | Method for preparing lipid nanoparticles |
-
2022
- 2022-04-11 KR KR1020220044633A patent/KR20230145791A/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210091120A (en) | 2018-08-29 | 2021-07-21 | 트랜슬레이트 바이오 인코포레이티드 | Improved Process for Making mRNA-Loaded Lipid Nanoparticles |
| KR20210105889A (en) | 2018-11-09 | 2021-08-27 | 아뷰터스 바이오파마 코포레이션 | Lipid Nanoparticle Formulation |
| KR20210135494A (en) | 2019-01-31 | 2021-11-15 | 모더나티엑스, 인크. | Method for preparing lipid nanoparticles |
Non-Patent Citations (1)
| Title |
|---|
| 음이온성 지질을 포함한 지질나노입자의 제조 및 물리적 특성, 이정은 외, Journal of the Korean Chemical Society, 2008, Vol. 52, No.3 |
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