Lipid nanoparticles for protein delivery

Abstract

The present invention relates to a lipid nanoparticle for protein delivery, and more particularly, to a lipid nanoparticle for delivering a plurality of medical proteins, such as a therapeutic protein or an enzyme replacement therapy (ERT) enzyme, into a cell or in a body. According to the present invention, a plurality of proteins such as a therapeutic protein or an enzyme replacement therapy (ERT) enzyme can be stably and efficiently delivered into a cell through the lipid nanoparticles, and thus the lipid nanoparticles can be effectively applied to the treatment and prevention of a plurality of diseases such as lysosomal storage diseases.

Description

Technical Field

[0001] The present invention relates to a lipid nanoparticle for protein delivery, and in particular to a lipid nanoparticle for delivering a variety of medical proteins such as therapeutic proteins or enzyme replacement therapy (ERT) enzymes into cells or in vivo. Background Art

[0002] In recent years, lipid nanoparticles (LNPs) have attracted much attention as a system for effectively delivering biological molecules such as messenger ribonucleic acid (mRNA) in vivo. The lipid nanoparticles mentioned above contain phospholipids, cholesterol, various ionic lipids, and phospholipids containing polyethylene glycol (PEG) in order to stably deliver substances into the body.

[0003] In addition, lysosomal storage disorders (LSDs) are relatively rare inherited metabolic diseases caused by defects in lysosomal function. Lysosomal storage disorders (LSDs) are usually caused by the deficiency of a single enzyme in the lysosome that is involved in the breakdown of metabolites. The storage of products due to insufficient enzyme activity can affect multiple organ systems and may also lead to severe symptoms and premature death. Most of the lysosomal storage diseases (LSDs) are also accompanied by important neurological-related symptoms ranging from progressive neurodegeneration, severe cognitive impairment to epilepsy, behavioral disorders and mental disorders.

[0004] Representative diseases of lysosomal storage diseases as mentioned above include Fabry disease, Gaucher disease and mucopolysaccharidosis, and enzyme replacement therapy (ERT) has become a standard treatment method. The enzyme replacement therapy mentioned above can reduce the concentration of harmful levels of substances stored in cells by regular intravenous injection of the enzyme protein that is lacking in the body (Concolino et al., Italian Journal of Pediatrics, 2018).

[0005] However, in order to improve the therapeutic efficiency of enzyme replacement therapy as described above, it is necessary to develop a delivery system that can reduce side effects such as undesirable immune responses and effectively and stably deliver enzyme proteins into cells, because this can reduce the dosage concentration of the enzyme or minimize the anti-drug antibody reaction. Summary of the invention

[0006] An exemplary object of the present invention is to provide a lipid nanoparticle for delivering a protein into a cell or into the body.

[0007] Another exemplary object of the present invention is to provide a lipid nanoparticle encapsulating a protein.

[0008] Another exemplary object of the present invention is to provide a composition for intracellular or in vivo delivery of a protein, comprising lipid nanoparticles encapsulating the protein.

[0009] Another exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating lysosomal storage diseases, comprising lipid nanoparticles encapsulating proteins.

[0010] Another exemplary object of the present invention is to provide a method for producing protein-encapsulated lipid nanoparticles.

[0011] Another exemplary object of the present invention is to provide a method for delivering enzyme replacement therapy (ERT) enzymes or therapeutic proteins into cells using lipid nanoparticles.

[0012] Another exemplary object of the present invention is to provide a method for treating diseases using lipid nanoparticles encapsulating enzyme replacement therapy (ERT) enzymes or therapeutic proteins.

[0013] The technical problems to be achieved by the technical ideas of the present invention disclosed in this specification are not limited to the problems for solving the problems mentioned in the above content, and a person skilled in the art will further clearly understand other problems not mentioned through the following description.

[0014] Next, it will be described in detail. In addition, each description and implementation disclosed in the present application may also be applicable to each other description and implementation. That is, all combinations of the various elements disclosed in the present application are included within the scope of the present application. In addition, it should not be considered that the scope of the present application is limited to the specific description recorded in the following content.

[0015] As one aspect for achieving the purpose of the present invention, the present invention provides a lipid nanoparticle for delivering a protein into the body. Specifically, the present invention provides a lipid nanoparticle for delivering a protein into a cell or into the body, comprising a lipid mixture consisting of hydrogenated soybean phosphatidylcholine (HSPC); cholesterol; dioleoyl-3-trimethylammonium propane (DOTAP); and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG).

[0016] The term "lipid nanoparticle (LNP)" in the present invention refers to a vesicle structure having adjacent lipid bilayers, such as a spherical vesicle, which can be used for delivery to a target location. As lipid nanoparticles as described above, liposomes, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and single-layer membrane structures (micelles) can be included.

[0017] The lipid nanoparticles of the present invention may contain more than one lipid. As an example, it may contain particulate lipids, polyethylene glycol (PEG)-lipids, phospholipids or cholesterol, etc. Among them, the particulate lipids may be cationic lipids. As an example of cationic lipids, it includes N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP); N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearoyl-N,N-dimethylammonium bromide (DDAB); N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); N-(1,2-dimyristoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N1,N3,N5-tri( 3-(Dididecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), liposome transfection reagent; 1,2-dilinoleoyl-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyl-N,N-dimethylaminopropane (DLenDMA), dioctadecyldimethylammonium (DODMA), distearyldimethylammonium (DSDMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and 1,2-dioleoyl-3-dimethylammonium propane (DODAP), etc.

[0018] In addition, other lipid components may also be included. As an example, phosphatidylcholine (PC) (e.g., 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC) and 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE)), sterol (e.g., cholesterol) and polyethylene glycol (PEG)-lipid conjugates (e.g., 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine-N-[folate (polyethylene glycol)-2000 (DOPE)) may be included). SPE-PEG2000)], 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-methoxy(polyethylene glycol) (DSPE-mPEG), L-α-hydrogenated phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), and other lipid molecules such as 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000 (C14-PEG2000)]).

[0019] Specifically, the lipid nanoparticles of the present invention may comprise a lipid mixture consisting of hydrogenated soybean phosphatidylcholine (HSPC); cholesterol; dioleoyl-3-trimethylammonium propane (DOTAP); and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG).

[0020] The ratio of each lipid as described above in the lipid nanoparticles is not particularly limited. For example, hydrogenated soybean phosphatidylcholine (HSPC) can occupy 8 to 15 mol% of the lipid mixture in the lipid nanoparticles, cholesterol can occupy 30 to 50 mol% of the lipid mixture in the lipid nanoparticles, dioleoyl-3-trimethylammonium propane (DOTAP) can occupy 40 to 60 mol% of the lipid mixture in the lipid nanoparticles, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG) can occupy 0.5 to 5 mol% of the lipid mixture in the lipid nanoparticles. More preferably, hydrogenated soybean phosphatidylcholine (HSPC) can occupy 9 mol% of the lipid mixture in the lipid nanoparticles, cholesterol can occupy 40 mol% of the lipid mixture in the lipid nanoparticles, dioleoyl-3-trimethylammonium propane (DOTAP) can occupy 50 mol% of the lipid mixture in the lipid nanoparticles, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG) can occupy 1 mol% of the lipid mixture in the lipid nanoparticles.

[0021] In addition, the molar ratio of hydrogenated soybean phosphatidylcholine (HSPC): cholesterol: dioleoyl-3-trimethylammonium propane (DOTAP): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG) in the lipid mixture can be 8-15:30-50:40-60:0.5-5. Preferably, the molar ratio of hydrogenated soybean phosphatidylcholine (HSPC): cholesterol: dioleoyl-3-trimethylammonium propane (DOTAP): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG) in the lipid mixture can be 8-15:30-50:40-60:0.5-5. The molar ratio of n-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG) can be 8-10:35-45:45-55:0.5-1.5. More preferably, the molar ratio of hydrogenated soybean phosphatidylcholine (HSPC):cholesterol:dioleoyl-3-trimethylammonium propane (DOTAP):1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG) in the lipid mixture can be 9:40:50:1, but is not limited to this.

[0022] The optimal molar ratio or molar % can be appropriately selected within the range according to the type of protein encapsulated in the lipid nanoparticles. As an example, when human iduronate-2-sulfatase (IDS) is encapsulated in lipid nanoparticles, preferably, the molar ratio of hydrogenated soybean phosphatidylcholine (HSPC): cholesterol: dioleoyl-3-trimethylammonium propane (DOTAP): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG) in the lipid mixture can be 9:40:50:1.

[0023] In the present invention, the ion size of lipid nanoparticles may affect drug release rate, biodistribution, mucosal adhesion, water absorption of cells, and buffer exchange and protein diffusion inside the nanoparticles. As an example, the diameter of lipid nanoparticles (LNPs) can be within the range of 10 to 500 nm, preferably, within the range of 70 to 120 nm, and more preferably, within the range of 75 to 95 nm.

[0024] In the present invention, the polydispersity index (PDI) of lipid nanoparticles is the result of measuring the heterogeneity of the molecular or particle size of the lipid mixture. As an example, the polydispersity index (PDI) of lipid nanoparticles can be in the range of 0.1 to 0.4, preferably, in the range of 0.1 to 0.2, and more preferably, in the range of 0.15 to 0.18.

[0025] In the present invention, the applicability of the protein is not particularly limited, and as an example, it may include an enzyme or a therapeutic protein. The therapeutic protein refers to a protein used to treat or prevent a disease or disorder, such as a recombinant protein, an antibody, a biosimilar, and a bioimproved drug. In the present invention, the applicability of the enzyme is not particularly limited, and it may be an enzyme that is lacking or absent in the body, and in particular, the enzyme may be an enzyme replacement therapy (ERT) enzyme.

[0026] In the present invention, the applicability of the enzyme is not particularly limited, and the enzyme may be an enzyme that is lacking or absent in the body, and in particular, the enzyme may be an enzyme replacement therapy (ERT) enzyme.

[0027] As another aspect for achieving the purpose of the present invention, the present invention provides a lipid nanoparticle encapsulated with a protein containing a lipid mixture consisting of hydrogenated soybean phosphatidylcholine (HSPC); cholesterol; dioleoyl-3-trimethylammonium propane (DOTAP); and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG).

[0028] The "lipid nanoparticles" and "proteins" are as described above.

[0029] The term "enzyme replacement therapy" in the present invention is a treatment method in which the enzyme lacking in the body is directly injected into the patient's vein. This is a representative treatment method for diseases such as lysosomal storage diseases (LSDs), such as Fabry disease, Gaucher disease, Pompe disease, and Hunter syndrome, and is also used to treat rare congenital syndromes such as severe combined immunodeficiency (ADA-SCID) caused by adenosine deaminase deficiency. In addition, enzyme replacement therapy can also be used when enzyme or protein deficiency occurs in substrate reduction therapy, gene therapy, and bone marrow-derived stem cell transplantation.

[0030] In the present invention, as an example, the enzyme is a lysosomal enzyme that can be used to treat lysosomal storage diseases, for example, idursulfase, laronidase, elosulfase alpha, galsulfase, vestronidase alpha, agalsidase beta, agalsidase alpha, pegunigalsidase alpha, imiglucerase, taliglucerase alpha, alglucerase, velaglucerase, alglucosidase alpha, avalglucosidase alpha, sebelipase alpha, cerliponase alpha. The enzyme may be, but is not limited to, adenosine deaminase or adenosine deaminase.

[0031] Specifically, the enzyme can be obtained by cloning the enzyme gene into a vector to produce an expression plasmid, transfecting the expression plasmid into a host cell and culturing it, then screening out a highly productive enzyme-expressing monoclonal cell strain and culturing it, and then isolating and purifying the protein.

[0032] In the present invention, the enzyme can be used to treat or prevent lysosomal storage diseases. Lysosomes are intracellular organelles that can distribute necessary components of substances produced in the body and excrete unnecessary components.

[0033] However, when there is a lack of enzymes in the body, the lysosomes will not be able to perform the above functions properly, and in the case described above, impurities will be stored in the body and cause damage to body tissues and organs.

[0034] There are more than 50 known lysosomal storage diseases. Due to genetic defects, the production of specific enzymes in the lysosomes is inhibited, resulting in the accumulation of products in the body and eventually causing serious effects on the bones, brain, skin, heart, and central nervous system.

[0035] Specifically, the lysosomal storage disease can be mucopolysaccharidosis type I (MPS I), mucopolysaccharidosis type II (MPS II), mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type VI (MPS VI), mucopolysaccharidosis type VII, Fabry disease, Gaucher disease, Gaucher disease type I, Pompe disease, lysosomal acid lipase deficiency (wolmandisease, CESD), neural ceroid lipofuscinosis (CLN2 disease) or severe combined immunodeficiency, but is not limited thereto.

[0036] As an example, Fabry disease is a progressive X-linked inborn error of glycosphingolipid metabolism caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) due to mutations in the α-galactosidase A (GLA) gene. Although an X-linked disease, females may also experience varying degrees of clinical symptoms. Fabry disease is a rare disease with an estimated incidence of only 1 in 40,000 males and 1 in 117,000 in the general population. For patients with Fabry disease, life expectancy is shortened without treatment, and death usually occurs in the 40s or 50s due to vascular disease affecting the kidneys, heart, and / or central nervous system. The enzyme deficiency results in intracellular accumulation of globulin trihexosylceramide (GL-3), a substrate in the vascular endothelium and visceral tissues throughout the body.

[0037] Cardiac disease due to Fabry disease occurs in most males and many females. Initial cardiac symptoms include left ventricular enlargement, valvular involvement, and conduction abnormalities. Aortic regurgitation is the most common valvular lesion that typically presents in childhood or adolescence. Cerebrovascular symptoms are primarily caused by multifocal small vessel involvement and may include, for example, thrombosis, transient ischemic attack, basilar artery ischemia and aneurysm, seizures, hemiparesis, unilateral sensory loss, aphasia, labyrinthine disorders, or cerebral hemorrhage. The average age of onset of cerebrovascular symptoms is 33.8 years. Personality changes and psychotic behaviors may occur with aging.

[0038] For Fabry disease, the current treatment method approved by the US Food and Drug Administration (FDA) is enzyme replacement therapy (ERT), including agalsidase alfa ( Shire Human Genetic Therapies), agalsidase beta ( Genzyme Corporation) and pegol alpha ( Protalix BioTherapeutics, Inc., etc. This type of enzyme replacement therapy (ERT) supplements the patient's insufficient α-galactosidase A (α-Gal A) activity by intravenously injecting a recombinant form of the enzyme. Although enzyme replacement therapy (ERT) is effective in many cases, there are still limitations in treatment. There is currently no evidence that enzyme replacement therapy (ERT) can reduce the risk of stroke, and the slow response of heart muscles and the subsequent removal of globulin trihexosylceramide (GL-3) in a certain cell type in the kidney have limitations. Some patients also show immune reactions to enzyme replacement therapy (ERT).

[0039] As another example, Gaucher Disease is also a representative lysosomal storage disease. It is a disease caused by a deficiency of the lysosomal hydrolase, β-glucocerebrosidase (β-GC), which leads to the storage of a sphingolipid, glucocerebroside, mainly in the reticuloendothelial system, such as the liver, spleen, central nervous system, bones, and lungs. It is also the most common lipid storage disease.

[0040] Gaucher disease is a disease caused by a genetic mutation in the glucocerebrosidase gene (GBA). It is inherited in an autosomal recessive manner and presents clinical symptoms of varying severity depending on the degree of deficiency in the enzyme activity of the glucocerebrosidase gene (GBA). Gaucher disease was once divided into three clinical types according to the classification method of Knudsen and Kaplan, based on whether the central nervous system is involved, the rate of disease progression, and the age of onset. Recently, it has been classified into three types: non-neuronopathic and acute or chronic neuropathic. At this time, the non-neuronopathic type is called type 1, the acute neuropathic type is called type 2, and the chronic neuropathic type is called type 3.

[0041] Enzyme replacement therapy is a typical treatment method for Gaucher disease that has been proven to be effective. In particular, it has been found to be very helpful in treating liver and spleen enlargement and hematological manifestations in Gaucher disease without neuroinvasive and chronic neuroinvasive forms. Gaucher disease is a disease that can show the effect of recombinant enzyme therapy. Since the acid β-glucosidase enzyme (imiglucerase, Since it was commercialized by Genzyme Co. in 1994 using recombinant DNA technology, it has been used as a standard treatment for type I Gaucher disease without neurological involvement.

[0042] In South Korea, a biosimilar to imiglucerase has also been developed. (Isu Abxis, Seongnam, South Korea) and has been commercialized and put into use. (Takeda Pharmaceutical Company, Tokyo, Japan) is used as a recombinant enzyme preparation. The recombinant enzyme as described above can improve the symptoms of the reticuloendothelial system, thereby further improving liver / splenomegaly. However, because the enzyme preparation as described above cannot cross the cerebrovascular barrier, it cannot prevent the progression of neurological symptoms in patients with neuroinvasive Gaucher disease. In addition, it has been known that there are limitations to the therapeutic effect for patients with more severe lymphadenopathy in the abdomen or mediastinum.

[0043] As another example, there are mucopolysaccharidosis (MPS) types I, II, IVA, VI, and VII.

[0044] Mucopolysaccharidosis type I (MPS I) is a rare recessive genetic disease with an estimated incidence of only 1 / 100,000 (Moore D et al., 2008, Orphanet Journal of Rare Diseases 3). Mucopolysaccharidosis type I (MPS I) is caused by the deficiency of α-1-iduronidase (IDUA), an enzyme required for the lysosomal metabolism of very common complex polysaccharides, namely heparan sulfate and dermatan sulfate. The polysaccharides described above, called glycosaminoglycans (GAGs), accumulate in the tissues of patients with MPS I, causing special storage lesions and a variety of disease sequelae. Patients may present with short stature, skeletal and joint deformities, coarse facial features, hepatosplenomegaly, cleft palate disease, obstructive sleep apnea, recurrent upper respiratory tract infections, hearing impairment, carpal tunnel syndrome, and visual impairment due to corneal opacity (Beck M, et al., 2014, The natural history of MPS I: global perspectives from the MPS I Registry. Genetics in medicine: official journal of the American College of Medical Genetics 16(10): 759-765). In addition, many patients present with symptoms related to the storage of glycosaminoglycans (GAGs) in the central nervous system, which may include hydrocephalus, spinal cord compression, and cognitive impairment in some patients.

[0045] Mucopolysaccharidosis type II (Hunter syndrome / MPS II) is a rare X-linked recessive genetic disease with an incidence of only 0.5 to 1.3 / 100,000 male newborns. The progressive and devastating disease as described above is caused by a genetic mutation in the human iduronate-2-sulfatase (IDS) gene, which further leads to a deficiency of the lysosomal storage enzyme human iduronate-2-sulfatase required for the lysosomal metabolism of heparan sulfate and dermatan sulfate.

[0046] Ubiquitous polysaccharides called glycosaminoglycans (GAGs) accumulate in tissues and organs of patients with MPS II, causing specific storage lesions and multiple disease sequelae. Morbidity and mortality rates are high in this patient group. Death (characterized by neurocognitive deterioration) has been reported at a mean age of 11.7 years in patients with severe phenotypes and 21.7 years in patients with mild or attenuated phenotypes.

[0047] Mucopolysaccharidosis type IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-6-sulfatase (GALNS) (Khan, et al., Mol Genet Metab., 2017; 120(1-2):78-95). The deficiency of the enzyme leads to the gradual accumulation of glycosaminoglycans (GAGs), chondroitin sulfate 6-sulfate (C6S) and keratan sulfate (KS) and causes a unique skeletal deformity throughout the body due to incomplete ossification and continuous growth imbalance, ultimately resulting in short neck and short trunk, cervical spinal cord compression, tracheal obstruction, pigeon chest, joint laxity, kyphoscoliosis, coxa valgum and genu valgum. Other clinical manifestations of the disease may also include hearing loss, heart valve disease and corneal opacity. More than 200 different mutations have been observed in patients, and the incidence in the United States is approximately 1 / 250,000.

[0048] By applying the lipid nano ion according to the present invention to enzyme replacement therapy, the lysosomal storage disease can be treated. Therefore, from the above viewpoint, the present invention provides a disease method utilizing enzyme replacement therapy (ERT) enzyme. In addition, in addition to the lysosomal storage disease, the protein lacking or at a lower level can also be effectively delivered to the body, thereby treating and preventing various diseases. Therefore, utilizing the lipid nanoparticle of the present invention equipped with therapeutic protein, a therapeutic method for multiple diseases can be provided. The therapeutic method as above can be achieved by delivering the enzyme replacement therapy (ERT) enzyme or therapeutic protein carried in the lipid nanoparticle to the intracellular mode.

[0049] As another aspect for achieving the object of the present invention, the present invention provides a composition for intracellular or in vivo delivery of a protein comprising the lipid nanoparticles encapsulating a protein or an enzyme.

[0050] The "protein" and "lipid nanoparticles" are as described above.

[0051] In the present invention, the composition may further contain a substance for improving the intracellular or in vivo delivery efficiency of lipid nanoparticles encapsulating proteins or enzymes, or the morphology of the composition may be modified by additional improvements.

[0052] As another aspect for achieving the object of the present invention, the present invention provides a composition for preventing or treating lysosomal storage diseases, comprising the protein-encapsulating lipid nanoparticles.

[0053] The “protein”, “lipid nanoparticles” and “lysosomal storage disease” are as described above.

[0054] The term "prevention" in the present invention refers to all actions that inhibit or delay the onset of lysosomal storage diseases by administering the pharmaceutical composition of the present invention, and the term "treatment" in the present invention refers to all actions that improve, alleviate or beneficially change the disease by administering the pharmaceutical composition of the present invention.

[0055] The term "pharmaceutical composition" in the present invention refers to a substance manufactured for the purpose of preventing or treating a disease, and can be formulated into various forms for use according to the usual methods. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions and syrups according to the route of administration, and can also be formulated into external preparations and sterile injection solutions for use. Specifically, the route of administration can include any appropriate route including a topical route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissues, and two or more routes can also be used in combination. An example of a combination of two or more routes refers to a situation where two or more dosage forms of drugs are combined according to the route of administration, for example, a certain drug can be first administered via an intravenous route, and then another drug can be administered via a topical route.

[0056] The pharmaceutical composition of the present invention can be manufactured into a form of a pharmaceutical composition for cancer treatment or prevention that contains an appropriate carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition, and the carrier may include a non-naturally occurring carrier. Specifically, the pharmaceutical composition can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and external preparations, suppositories, and sterile injection solutions according to the usual methods. The specific formulation method of the pharmaceutical composition belongs to the content known in the industry, for example, reference can be made to [Remington's Pharmaceutical Sciences (19th ed., 1995)] and the like. The document is regarded as a part of this specification.

[0057] The pharmaceutical composition of the present invention can be administered to animals such as rats, dogs, cats, cattle, horses, pigs and humans by various routes, preferably humans. All modes of administration are contemplated, such as oral administration, intravenous, intraarterial, intramuscular or subcutaneous injection, but are not limited thereto.

[0058] In the present invention, the treatment method also includes combination with currently known therapeutic therapies or co-administration with other currently known therapeutic agents.

[0059] As another form for achieving the purpose of the present invention, the present invention provides a method for producing protein-encapsulated lipid nanoparticles and nanoparticles obtained by the method as described above. Specifically, the method for producing protein-encapsulated lipid nanoparticles includes: (a) producing a lipid mixture containing hydrogenated soybean phosphatidylcholine (HSPC); cholesterol; dioleoyl-3-trimethylammonium propane (DOTAP); and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol) (DSPE-mPEG); (b) injecting a first solution formed by dissolving the lipid mixture in an alcohol solvent into an injection port of a (microfluidics) system; (c) injecting a second solution containing a protein into another injection port of a microfluidic system; and (d) purifying the protein-encapsulated lipid nanoparticles.

[0060] The "protein" and "lipid nanoparticles" are as described above.

[0061] In the present invention, the alcohol solvent may include methanol, ethanol, propanol or denatured alcohol, but is not limited thereto. Preferably, it may be ethanol.

[0062] In the present invention, the flow rate ratio of the ethanol phase (first solution) and the water phase (second solution) of the microfluidic system can be 1:3 to 1:5, but is not limited thereto. Preferably, it can be 1:3.

[0063] In the present invention, the flow rate of the microfluidic system may be 9 to 18 ml / min, but is not limited thereto. Preferably, the flow rate may be 15 ml / min.

[0064] In the present invention, the step of purifying the lipid nanoparticles in step (d) may include: removing the alcohol solvent and the unencapsulated protein in step (d). The purification of the lipid nanoparticles may be performed using a tangential flow filtration system equipped with a chromatographic column. Specifically, the lipid nanoparticles may be circulated through the chromatographic column and purified while adding a fresh buffer (20 mM sodium phosphate, 137 mM sodium chloride, pH 6±0.2) at the same rate as the permeate flowing out of the chromatographic column.

[0065] As another aspect for achieving the object of the present invention, the present invention provides a method for delivering an enzyme replacement therapy (ERT) enzyme or a therapeutic protein into cells using lipid nanoparticles.

[0066] The “enzyme replacement therapy (ERT)”, “enzyme”, “therapeutic protein” and “lipid nanoparticles” are as described above.

[0067] As another aspect for achieving the object of the present invention, the present invention provides a method for treating a disease using lipid nanoparticles encapsulating an enzyme replacement therapy (ERT) enzyme or a therapeutic protein.

[0068] The “enzyme replacement therapy (ERT)”, “enzyme”, “therapeutic protein” and “lipid nanoparticles” are as described above.

[0069] In the present invention, the "disease" may be a lysosomal storage disease. In addition, in addition to the lysosomal storage disease, the lacking or low-level protein can be effectively delivered to the body, thereby treating and preventing various diseases. Therefore, the lipid nanoparticles of the present invention encapsulated with therapeutic proteins or enzyme replacement therapy (ERT) enzymes can provide a variety of disease treatment methods.

[0070] The therapeutic method described above can be achieved by delivering enzyme replacement therapy (ERT) enzymes or therapeutic proteins encapsulated in lipid nanoparticles into cells.

[0071] The present invention can stably and effectively deliver various proteins such as therapeutic proteins or enzyme replacement therapy (ERT) enzymes into cells through lipid nanoparticles, thereby being effectively applicable to the treatment and prevention of various diseases such as lysosomal storage diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 An overall schematic diagram of lipid nanoparticle encapsulation based on a microfluidics system.

[0073] Figure 2The results are obtained by measuring the concentration of proteins taken into cells after normal fibroblasts and fibroblasts of Hunter syndrome patients were treated with human iduronate 2-sulfatase (IDS) and human iduronate 2-sulfatase / lipid nanoparticles (IDS / LNP) of the present invention.

[0074] Figure 3 The results are obtained by measuring the amount of heparan sulfate stored in fibroblasts of Hunter syndrome patients after the cells were treated with human iduronate-2-sulfatase (IDS) and human iduronate-2-sulfatase / lipid nanoparticles (IDS / LNP) of the present invention. DETAILED DESCRIPTION

[0075] Next, the present invention will be described in more detail by the following examples. However, these examples are only exemplary descriptions of the present invention, and the scope of the present invention is not limited by these examples.

[0076] Example 1. Preparation of protein-encapsulated lipid nanoparticles

[0077] 1.1. Production of enzyme protein

[0078] 1.1.1. Development of human iduronate-2-sulfatase (IDS) production cell line

[0079] After synthesizing the human iduronate-2-sulfatase (IDS) gene, it was cloned into the PanGen expression vector pPGXII (see Korean Patent No. 10-1385223). Figure 2 ) to produce an expression plasmid. The expression plasmid was transfected using the CHO DG44 host cell strain on a 24-well scale, and the transfection operation was performed using electroporation. After culturing in an incubator at 37°C and 5% CO2, the cells were cultured using a selective medium when they were fully grown so that only the transfected cells would grow. After about 2 weeks when the cells were fully grown, a portion of the culture solution was taken and the IDS-expressing cell population was screened by enzyme-linked immunosorbent assay (ELISA) analysis.

[0080] Based on the results of enzyme-linked immunosorbent assay (ELISA) analysis, cell populations were screened, and single clone selection was performed to obtain a stable cell line with high expression efficiency. The culture medium was dispensed into a 96-well plate at 0.5 cells / well, and the colonies formed were analyzed after about 4 weeks to screen out cell lines with high expression efficiency and culture them. When a sufficient number of cells was reached, the expression efficiency of the monoclonal cell lines was compared and analyzed by enzyme-linked immunosorbent assay (ELISA) analysis, and a monoclonal cell line with high productivity was selected as a candidate cell line. During the 90-day long-term subculture period, it was confirmed that the monoclonal cell line can stably express proteins, and the cell line with the highest productivity was selected as the final cell line. When the final cell line selected reached a sufficient number of cells, it was dispensed into a vial to produce a research cell bank, which was then stored in a liquid nitrogen storage tank.

[0081] 1.1.2. Cell culture for IDS production

[0082] After thawing, the IDS production cell line was subcultured at intervals of 2 to 3 days using a subculture medium (EX-CELL (+8mM L-glutamine) + 1% 2X Feed A+). As the subculture medium, 1% 2X Feed A+ additive was added to EX-CELL medium supplemented with 8mM L-glutamine.

[0083] After a total of 15 days of seed culture to reach a sufficient number of cells, IDS was produced in a 7.5 L bioreactor (New Brunswick scientific, Bioflo320) at a 5 L culture scale. 6 Cells were inoculated at a concentration of 10 cells / mL, and 12.5% ​​2X Feed A+ additive was added to EX-CELL medium supplemented with 8mM L-glutamine as a production medium. The culture conditions were set to a culture temperature of 37°C, 30% dissolved oxygen (DO), a pH of 7.0 to 7.2, and a stirring speed of 150 rpm, and the culture was carried out for a total of 11 days. During the culture period, Vi-CELL TMThe cell number and viability were analyzed by a counter, and the glucose content was maintained above 20 mM by daily measurement of pH and glucose and lactate content. The culture medium was harvested from the bioreactor after the culture was completed, and the cells were removed using a COHC depth filter and sterilized using a 0.22 μm polyethersulfone (PES) filter.

[0084] 1.1.3. IDS protein purification

[0085] IDS protein was separated and purified using cell culture medium. Chromatographic analysis was performed using a column filled with Capto MMC resin. After the column was installed in AKTA Pure (GE healthcare) and equilibrated with 20 mM sodium acetate / 150 mM sodium chloride (pH 4.3) equilibration buffer, the sample was loaded using culture medium adjusted to the same pH.

[0086] Next, the column was washed and the IDS protein was eluted using a 20 mM sodium acetate / 150 mM sodium chloride (pH 5.1) elution buffer. The chromatographic analysis using Blue Agarose 6FF was performed using the eluent. After the column was installed in AKTA Pure (GE healthcare) and equilibrated with a 20 mM sodium acetate / 50 mM sodium chloride (pH 4.0) equilibration buffer, a sample diluted to 1 / 3 with a dilution buffer (20 mM sodium acetate (pH 4.0)) was loaded.

[0087] Next, the chromatographic column was washed, and the IDS protein was eluted using a 20mM sodium phosphate (pH 6.6) elution buffer. The eluted IDS protein was used to perform a chromatographic analysis using Q agarose FF. After the chromatographic column was installed in AKTA Pure (GE healthcare) and equilibrated with a 20mM sodium phosphate (pH 6.6) equilibration buffer, the sample was loaded. After the chromatographic column was washed with a washing buffer (50mM acetic acid (pH 3.0)), the IDS protein was obtained by pouring a 50mM acetic acid / 150mM sodium chloride (pH 3.0) elution buffer. Before performing the next step of purification, the pH of the Q agarose FF eluate was lowered to 3.5 using 10% acetic acid, and then the virus extinguishing step was performed by reacting at room temperature for 180 minutes. After the virus inactivation was completed, the sample was immediately diluted to 1 / 3 using a dilution buffer (50 mM sodium acetate / 4,430 mM sodium chloride (pH 4.0)), and then a phenyl agarose chromatography analysis operation was performed. After the chromatographic column was installed in AKTA Pure (GE healthcare) and equilibrated with a 50 mM sodium acetate / 3,000 mM sodium chloride (pH 4.0) equilibration buffer, the sample was loaded. Next, the chromatographic column was washed, and the IDS protein was eluted using a 20 mM sodium phosphate (pH 6.0) elution buffer. The eluted IDS protein was made into a concentration of approximately 2 mg / mL in a 20 mM sodium phosphate / 137 mM sodium chloride (pH 6.0) buffer state through an ultrafiltration / diafiltration (UF / DF) process, and after the sample was recovered, polysorbate 20 was added to reach 0.022%.

[0088] 1.2. Fabrication of lipid nanoparticles

[0089] A cationic lipid mixture was dissolved in ethanol at a specific concentration, mainly in the range of 2.5 to 7.5 mg / mL of total lipid, and lipid nanoparticles (LNPs) were manufactured using a microfluidic system.

[0090] Specifically, the selected lipid mixture was injected into one of the two injection ports of a microfluidic system (micromixer system using a herringbone-shaped channel) manufactured by MOOGENE MEDI, and cationic lipid nanoparticles were produced according to a composition of less than 15% hydrogenated soybean phosphatidylcholine (HSPC), less than 50% cholesterol, less than 60% 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), and less than 5% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG).

[0091] 1.3. Preparation of protein-encapsulated lipid nanoparticles

[0092] Protein encapsulation by lipid nanoparticles was performed using a microfluidics system with a herringbone-shaped micromixer chip. The overall schematic diagram of lipid nanoparticle encapsulation according to the flow rate of the microfluidics system is shown in Figure 1 shown.

[0093] The selected lipid mixture was dissolved in ethanol at a specific concentration, mainly in the range of 2.5 to 7.5 mg / mL of total lipids, and injected into one of the two injection ports of the microfluidic system, and then the aqueous phase (20 mM sodium phosphate, 137 mM sodium chloride, pH 6±0.2) dissolved with protein was injected into the second injection port. Cationic lipid nanoparticles were manufactured according to the composition of hydrogenated soybean phosphatidylcholine (HSPC) of less than 15%, cholesterol of less than 50%, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) of less than 60%, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG) of less than 5%, and the size (size), polydispersity index (PDI) and encapsulation efficiency (encapsulation efficiency) of the lipid nanoparticles manufactured according to each lipid composition are shown in Table 1 below.

[0094]

Table 1

[0095]

[0096]

[0097] *“-” in encapsulation efficiency means not measured (NM, Not Measured).

[0098] The microfluidic system software controls multiple production parameters including the flow rate ratio (the ratio between the ethanol phase and the aqueous phase) and the total flow rate (the speed of injection through the two injection ports of the chip). Specifically, the flow rate ratio of ethanol phase (alcohol phase): aqueous phase (aqueous phase) = 1:3 to 1:5 and the flow rate (ml / min) of 9 to 18 ml / min were tested to select the optimal production parameters (Tables 2 and 3).

[0099]

Table 2

[0100]

[0101]

Table 3

[0102]

[0103] 1.4. Purification of lipid nanoparticles

[0104] A tangential flow filtration system (Repligen, Inc.) equipped with a modified polyethersulfone (mPES) hollow fiber column with a pore size of 750 kD was used. The lipid nanoparticles were purified using the KR2i TFF System. The lipid nanoparticle sample was circulated through the chromatographic column to remove ethanol and proteins not encapsulated in the lipid nanoparticles.

[0105] The sample was purified while adding fresh buffer (buffer, 20 mM sodium phosphate, 137 mM sodium chloride, pH 6 ± 0.2) at the same rate as the permeate flowing out of the column.

[0106] Example 2. Intracellular uptake of IDS-LNP

[0107] According to the method of Example 1, [iduronate-2-sulfatase protein-cationic lipid nanoparticles] (hereinafter referred to as "IDS / LNP") were prepared by encapsulating IDS, an enzyme protein lacking in patients with mucopolysaccharidosis II (Hunter syndrome / MPS II) type, into cationic lipid nanoparticles.

[0108] After normal human fibroblasts (CCD-986Sk, Korean Cell line Bank, KCLB21947) and Hunter syndrome patient fibroblasts (GM00615, Coriellnstitute, hereinafter referred to as "patient cells") were treated with various concentrations of IDS and IDS / LNP ranging from 1.25 to 80 nM for 6 hours, the amount of protein uptake into the cells was quantified by enzyme-linked immunosorbent assay (ELISA).

[0109] Specifically, the cells used in the experiment were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics at 5% CO2 and 37°C for normal human fibroblasts, and in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Minimum Essential Medium with Earle's salts, L-glutamine, sodium bicarbonate) for Hunter syndrome patients. The cells were seeded in 60 mm 24 hours before IDS / LNP treatment. 2 Cell culture plates (2.5×10 5 Cells were cultured on 5% fetal bovine serum-Iskoff's modified Dulbecco's medium (5% FBS-IMDM) or minimum essential medium (MEM) at 37°C for 24 hours after adding IDS control drug (ELAPRASE), IDS, and IDS / LNP on the day of drug treatment. The number of passages of patient cells used in the experiment was less than 10.

[0110] After 24 hours of reaction, the cells were washed 1 to 2 times with phosphate buffered saline (PBS), and then treated with 0.25% trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA), centrifuged, and washed with phosphate buffered saline (PBS) before only the cells were collected. The collected cells were dissolved in a radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors and phosphatase inhibitors (Sigma), and then centrifuged to collect only the supernatant for use as an analysis sample. For the analysis sample (whole cell lysate), the IDS taken into the cells was quantified using the Human Iduronate 2-Sulfatase (Human Iduronate 2-Sulfatase / IDS) enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Cat. DY2449-05) and the total amount of protein was quantified using the Bicinchoninic acid (BCA) protein analysis kit (Thermo Scientific), and the IDS protein taken into the cells per mg of protein was expressed in ng.

[0111] The results confirmed that the amount of IDS protein taken up into normal and patient cells was significantly higher in the case of IDS / LNP treatment than in the case of IDS treatment alone ( Figure 2 ).

[0112] Example 3. Confirmation of cellular activity of IDS / LNP

[0113] Mucopolysaccharidosis II (Hunter syndrome / MPS II) is a disease caused by the accumulation of polysaccharides called glycosaminoglycans (GAGs) in cells due to the deficiency of IDS in the patient's body. Heparan sulfate is a representative glycosaminoglycan (GAG).

[0114] To determine the cellular activity of IDS / LNPs, the reduction in intracellular sulfate accumulation was quantified by enzyme-linked immunosorbent assay (ELISA) after patient cells were treated with 5 to 40 nM concentrations of IDS protein and IDS / LNPs for 24 hours.

[0115] Specifically, the patient cells used in the experiment were cultured in a minimum essential medium (MEM) medium (containing Earle's salts, L-glutamine, and sodium bicarbonate) supplemented with 10% fetal bovine serum (FBS) at 5% CO2 and 37°C. The cells were seeded in 60 mm 24 hours before IDS / LNP treatment. 2 Cell culture plates (2.5×10 5 Cells were cultured on 100 cells / plate and IDS control drug (ELAPRASE), IDS and IDS / LNP were added to 5% fetal bovine serum-minimum essential medium (5% FBS-MEM) on the day of drug treatment and cultured at 37°C for 24 hours. The number of passages of patient cells used in the experiment was less than 10.

[0116] After 24 hours of reaction, the cells were washed once or twice with phosphate buffered saline (PBS), and then treated with 0.25% trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA), centrifuged, and washed with phosphate buffered saline (PBS) before being collected. The collected cells were dissolved in a radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors and phosphatase inhibitors (Sigma), and then centrifuged to collect only the supernatant for use as a sample for analysis. For the analytical samples (whole cell lysate), the intracellular heparan sulfate was quantified using the human heparan sulfate (HS) enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource, Cat. #MBS2515971) and the protein amount was quantified using the bicinchoninic acid (BCA) protein analysis kit (Thermo Scientific), and the heparan sulfate (HS) content (ng / mg) corrected by dividing the heparan sulfate (HS) concentration (ng / ml) by the protein concentration (mg / ml) is shown.

[0117] The results showed that the activity of the IDS / LNP treatment group was significantly higher than that of the IDS protein treatment group ( Figure 3 ).

[0118] In the above content, specific parts of the content of the present invention are described in detail, but people with general knowledge of the industry should understand that the specific descriptions described above are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the substantive scope of the present invention should be defined according to the attached claims and their equivalents.

Claims

1. A lipid nanoparticle for delivering a protein into a cell or into the body, The invention comprises a lipid mixture consisting of hydrogenated soybean phosphatidylcholine (HSPC); cholesterol; dioleoyl-3-trimethylammonium propane (DOTAP); and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-mPEG).

2. The lipid nanoparticle according to claim 1, The protein is an enzyme or a therapeutic protein.

3. The lipid nanoparticle according to claim 1, The content of hydrogenated soybean phosphatidylcholine (HSPC) is 8 to 15 mol % of the lipid mixture.

4. The lipid nanoparticle according to claim 1, The cholesterol content is 30 to 50 mol % of the lipid mixture.

5. The lipid nanoparticle according to claim 1, The molar ratio of hydrogenated soybean phosphatidylcholine (HSPC):cholesterol:dioleoyl-3-trimethylammonium propane (DOTAP):1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-mPEG) in the lipid mixture is 8-15:30-50:40-60:0.5-5.

6. A lipid nanoparticle encapsulated with a protein, The invention comprises a lipid mixture consisting of hydrogenated soybean phosphatidylcholine (HSPC); cholesterol; dioleoyl-3-trimethylammonium propane (DOTAP); and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-mPEG).

7. The lipid nanoparticle according to claim 6, The protein is an enzyme or a therapeutic protein.

8. The lipid nanoparticle according to claim 7, The enzyme is an enzyme replacement therapy (ERT) enzyme.

9. The lipid nanoparticle according to claim 8, The enzyme is a lysosomal enzyme.

10. The lipid nanoparticle according to claim 9, The enzyme is idursulfase, laronidase, elosulfase alpha, galsulfase, vestronidase alpha, agalsidase beta, agalsidase alpha, pegunigalsidase alpha, imiglucerase, taliglucerase alpha, alglucerase, velaglucerase, alglucosidase alpha, avalglucosidase alpha, sebelipase alpha, cerliponase alpha or adenosine deaminase.

11. The lipid nanoparticle according to claim 6, The molar ratio of hydrogenated soybean phosphatidylcholine (HSPC):cholesterol:dioleoyl-3-trimethylammonium propane (DOTAP):1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-mPEG) in the lipid mixture is 8-15:30-50:40-60:0.5-5.

12. The lipid nanoparticle according to claim 6, The lipid nanoparticles are used for treating lysosomal storage diseases.

13. The lipid nanoparticle according to claim 12, The lysosomal storage disease is mucopolysaccharidosis type I (MPSI), mucopolysaccharidosis type II (MPS II), mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type VI (MPS VI), mucopolysaccharidosis type VII, Fabry disease, Gaucher disease, Gaucher disease type I, Pompe disease, lysosomal acid lipase deficiency (Wolman disease, CESD), neural ceroid lipofuscinosis (CLN2 disease) or severe combined immunodeficiency.

14. A composition for intracellular or in vivo delivery of a protein, Comprising the lipid nanoparticles according to claim 6.

15. A pharmaceutical composition for preventing or treating lysosomal storage diseases, Comprising the lipid nanoparticles according to claim 6.

16. The pharmaceutical composition according to claim 15, The lysosomal storage disease is mucopolysaccharidosis type I (MPSI), mucopolysaccharidosis type II (MPS II), mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type VI (MPS VI), mucopolysaccharidosis type VII, Fabry disease, Gaucher disease, Gaucher disease type I, Pompe disease, lysosomal acid lipase deficiency (Wolman disease, CESD), neural ceroid lipofuscinosis (CLN2 disease) or severe combined immunodeficiency.

17. A method for producing protein-encapsulated lipid nanoparticles, comprising: (a) a step of preparing a lipid mixture comprising hydrogenated soybean phosphatidylcholine (HSPC); cholesterol; dioleoyl-3-trimethylammonium propane (DOTAP); and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-mPEG); (b) injecting a first solution formed by dissolving the lipid mixture in an alcohol solvent into an injection port of a (microfluidics) system; (c) a step of injecting a second solution containing a protein into another injection port of the microfluidic system; as well as, (d) A step of purifying the protein-encapsulated lipid nanoparticles.

18. The manufacturing method according to claim 17, The molar ratio of hydrogenated soybean phosphatidylcholine (HSPC):cholesterol:dioleoyl-3-trimethylammonium propane (DOTAP):1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-mPEG) in the lipid mixture of step (a) is 8-15:30-50:40-60:0.5-5.

19. The manufacturing method according to claim 17, The flow rate ratio of the ethanol phase and the water phase in the microfluidic system is 1:3 to 1:

5.

20. The manufacturing method according to claim 17, The flow rate of the microfluidic system was 9 to 18 ml / min.

21. The manufacturing method according to claim 17, The step of purifying the lipid nanoparticles in step (d) comprises: The step of removing the alcohol solvent and the unencapsulated protein in step (d).

22. A method for delivering enzyme replacement therapy (ERT) enzymes or therapeutic proteins into cells using lipid nanoparticles.

23. A method for treating a disease using lipid nanoparticles encapsulating an enzyme replacement therapy (ERT) enzyme or a therapeutic protein.

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