RU2799045C1 - Lipid nanoparticles for in vivo medicinal products delivery and their use - Google Patents

Abstract

FIELD: lipid nanoparticles.

SUBSTANCE: group of inventions relates to lipid nanoparticles for in vivo medicinal products delivery and their use. The lipid nanoparticle contains an ionizable lipid in which a 6-membered heterocyclic amine and an alkyl epoxide, a phospholipid, are linked; cholesterol, and a lipid-PEG (polyethylene glycol) conjugate in an amount of 0.5–5 mol.%.

EFFECT: lipid nanoparticles are specific for liver tissue, hepatocyte and/or LSEC (liver sinusoidal endothelial cell), have excellent biocompatibility, and can deliver a gene therapeutic agent with high efficiency.

14 cl, 10 dwg, 8 tbl, 10 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to lipid nanoparticles for in vivo drug delivery and their use.

PRIOR ART

In the pharmaceutical industry, a drug delivery system (DDS) designed to efficiently deliver the right amount of drug while reducing drug side effects and maximizing efficiency and action is a high value-added core technology that can bring economic benefits comparable to the development of new medicines and has great potential for success, and its goal is to improve the quality of patient care by increasing the effectiveness of medications.

Low solubility drug solubilization technology, which refers to drug absorption stimulation technology, which is one of the core technologies of the drug delivery system, is considered the most reasonable way to reduce the cost of developing new drugs and at the same time increase the additional efficacy of currently marketed drugs. In particular, the development of improved new drugs through the development of drug dissolution technology in a situation where the conditions for developing new drugs are poor, such as in Korea, is an area that can create huge value-added at low cost.

Gene therapy using a genetic drug delivery system is designed with great hope to change genetic binding and treat numerous diseases. With the successful and safe execution of this gene therapy, efficient gene delivery is one of the major challenges, and the viral delivery system has been proven to be effective for gene delivery. However, due to some defects such as immunogenicity, size limitation of the inserted DNA and difficulty in mass production, the use of viruses as a gene delivery system is limited. Non-viral gene carriers such as cationic liposomes and polymers have come to be noted as alternative means of the viral system.

The improved stability profile and ease of fabrication and operation of the polymer delivery system has initiated research to develop and synthesize a non-toxic and biodegradable polymer carrier for efficient and safe gene delivery. Poly(L-lysine), polyethyleneimine, star dendrimer, polyamidoamine dendrimer and cationic liposome, and the like. can self-assemble and compress plasmid DNA (pDNA) into a small structure sufficient to enter cells via endocytosis, and therefore they are widely studied as a non-viral gene delivery system.

Nucleic acids such as antisense RNA, siRNA (small interfering RNA) and the like are a material capable of inhibiting the expression of specific proteins in vivo and are regarded as an important tool for the treatment of cancer, genetic diseases, infectious diseases, autoimmune diseases and etc. (Novina and Sharp, Nature, 430, 161-164, 2004). However, nucleic acids such as siRNA are difficult to deliver directly to cells and are easily degraded by enzymes in the blood, so many studies are underway to overcome these difficulties. At present, the method for delivering nucleic acids to cells, which is a method of transfer by mixing with a positively charged lipid or polymer (called lipid-DNA conjugate (lipoplex) and polymer-DNA conjugate (polyplex), respectively) has been mainly used (Hirko et al. , Curr. Med. Chem., 10, 1185-93, 2003; Merdanetal., Adv. Drug. Deliv. Rev., 54, 715-58, 2002). The lipid-DNA conjugate is combined with a nucleic acid to efficiently deliver the nucleic acid to cells, and thus it is widely used at the cellular level, but in vivo, when administered topically, in many cases has the disadvantage of inducing inflammation in the body (Filonand and Phillips, Biochim, Biophys, Acta, 1329, 345-56, 1997).

In addition, such a non-viral delivery system has the problem of low transfection efficiency. Much effort has been put into improving the efficiency of transfection, but this is still far from a stable system. In addition, the carrier of a non-viral gene delivery system has a significantly high cytotoxicity due to poor biocompatibility and non-biodegradability.

In view of such prior art, as a result of the present inventors trying to develop a new particle which has excellent encapsulation efficiency and can efficiently deliver an anionic drug, a nucleic acid, and the like. into a target organ or cell, they have made the present invention by obtaining a lipid nanoparticle containing an ionizable lipid, a phospholipid, cholesterol, and a lipid-PEG (polyethylene glycol) conjugate, and confirmed that this lipid nanoparticle is specifically delivered to the liver tissue, and a drug such as an anionic compound or nucleic acid can be encapsulated with high efficiency.

DISCLOSURE OF THE INVENTION

TECHNICAL PROBLEM

The object of the present invention is to provide a lipid nanoparticle containing an ionizable lipid in which a 6-membered heterocyclic amine and an alkyl epoxide, a phospholipid, cholesterol and a lipid-PEG (polyethylene glycol) conjugate are linked.

Another object of the present invention is to provide a drug delivery composition (anionic drug, nucleic acid, or combination thereof) comprising (1) a lipid nanoparticle and (2) an anionic drug, nucleic acid, or combination thereof.

Yet another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a liver disease, comprising (1) a lipid nanoparticle and (2) an anionic drug, a nucleic acid, or a combination thereof.

TECHNICAL SOLUTION

This will be described in detail below. On the other hand, every description and embodiment disclosed in the present invention can be applied to every other description and embodiment. In other words, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, the scope of the present invention should not be considered to be limited by the specific description set forth below.

In one aspect, to solve the above problem, a lipid nanoparticle is provided that contains an ionizable lipid in which a 6-membered heterocyclic amine and an alkyl epoxide are combined; phospholipid; cholesterol; and a lipid-PEG (polyethylene glycol) conjugate.

The lipid nanoparticle according to one example is specific to liver tissue and has excellent biocompatibility, and can deliver a gene therapeutic agent and the like with high efficiency, and thus can be effectively used in related technical fields such as gene therapy, lipid nanoparticle mediated and imaging diagnostic technology.

Here, "ionizable lipid" or "lipidoid" means an amine-containing lipid that can be easily protonated, and for example, it can be a lipid whose state of charge changes depending on the surrounding pH. The ionizable lipid may be a lipid in which a 6-membered heterocyclic amine and an alkyl epoxide are combined. In particular, the ionizable lipid may be a compound having characteristics similar to a lipid obtained by the reaction of a 6-membered heterocyclic amine and an alkyl epoxide, and more specifically, it may be a compound obtained by a ring-opening reaction of an epoxide by reacting a 6-membered heterocyclic amine with alkyl epoxide.

In one example, the ionizable lipid may be a lipid in which a 6-membered heterocyclic amine and an alkyl epoxide are combined by reacting them in a 1:n molar ratio (n = number of primary amines contained in the 6-membered heterocyclic amine × 2 + number of secondary amines × 1). According to one specific example, it can be prepared by mixing 246 amine and 1,2-epoxydecane in a 1:4 molar ratio and reacting them at 700 to 800 rpm and 85 to 95 for 2 to 4 days.

The ionizable lipid may be protonated (positively charged) at a pH below the pKa of the cationic lipid, and it may be substantially neutral above the pKa. In one example, the lipid nanoparticle may contain a protonated ionizable lipid and/or an ionizable lipid that is neutral.

An ionizable lipid is an ionizable compound having lipid-like characteristics, and through electrostatic interaction with a drug (eg, an anionic drug and/or nucleic acid), can play the role of encapsulating the drug in a lipid nanoparticle with high efficiency.

The 6-membered heterocyclic amine may contain a piperazine or piperidine structure.

The 6-membered heterocyclic amine may be a chain or non-chain amine containing a tertiary amine, and in one example it may be one or more species selected from the group consisting of

и

And

In one example, the 6-membered heterocyclic amine may be one or more species selected from the group consisting of 1,4-bis(3-aminopropyl)piperazine, N-(3-aminopropyl)piperidine, (1-methyl-4 -piperidinyl)methanamine, 2-(4-methylpiperazin-1-yl)ethylamine, 1-(2-aminoethyl)piperazine and 1-(3-aminopropyl)piperazine.

Depending on the type of amine included in the ionizable lipid, (1) drug encapsulation efficiency, (2) PDI (polydispersity index), and/or (3) drug delivery efficiency to liver tissue and/or cells constituting the liver (e.g. , hepatocytes), and/or LSEC (liver sinusoidal endothelial cells), lipid nanoparticles can be different.

The lipid nanoparticle containing the amine-containing ionizable lipid may have one or more of the following characteristics:

(1) drug encapsulation with high efficiency;

(2) uniform particle size (or low PDI); and/or

(3) superior drug delivery efficiency to liver tissue and/or cells constituting the liver (eg, hepatocytes and/or LSEC).

According to one example, a lipid nanoparticle containing an ionizable lipid containing 1,4-bis(3-aminopropyl)piperazine (e.g., Cas Nos. 7209-38-3) may have one or more kinds of the following characteristics, compared to a lipid nanoparticle containing an ionizable lipid containing other types of amines:

(1) drug encapsulation with high efficiency;

(2) uniform particle size (or low PDI); and/or

(3) superior drug delivery efficiency to liver tissue and/or cells constituting the liver (eg, hepatocytes and/or LSEC).

The alkyl epoxide may have the structure of chemical formula 1 below:

[Chemical formula 1]

The alkyl epoxide may have a carbon number of C6 to C14, C6 to C12, C6 to C10, C8 to C14, C8 to C12, C8 to C10, C10 to C14, C10 to C12 or C10, and, for example, it may be 1,2-epoxydecane from C10. By setting the number of carbon atoms of the alkyl epoxide contained in the ionizable lipid in the above range, it is possible to ensure high encapsulation efficiency of the drug encapsulated in the lipid nanoparticle.

In one example, the ionizable lipid may have the general formula of chemical formula 2 below:

[Chemical Formula 2]

The structure of the chemical formula 2 is one example of the structure of the ionizable lipid according to one example, and the structure of the ionizable lipid may be different depending on the type of 6-membered heterocyclic amine and alkyl epoxide.

According to one example, a lipid nanoparticle containing an ionizable lipid having the structure of chemical formula 2 may have one or more kinds of the following characteristics compared to a lipid nanoparticle containing other types of ionizable lipids:

(1) drug encapsulation with high efficiency;

(2) uniform particle size (or low PDI); and/or

(3) superior drug delivery efficiency to liver tissue and/or cells constituting the liver (eg, hepatocytes and/or LSEC).

According to one example, a lipid nanoparticle may have a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. The pKa is the acid dissociation constant and refers to what is commonly used in as an index indicating the acid strength of the target substance. The pKa value of a lipid nanoparticle is important in terms of in vivo stability of the lipid nanoparticle and drug release from the lipid nanoparticle. In one example, a lipid nanoparticle having a pKa value in the above range can be safely delivered to a target organ (e.g. liver) and/or target cell (hepatocyte and/or LSEC) in vivo and reach the target organ and/or cell -target, and after endocytosis exhibits a positive charge to release the encapsulated drug through electrostatic interaction with the anionic protein of the endosome membrane.

The phospholipid of the elements of the lipid nanoparticle according to one example plays the role of coating and protecting the core formed by the interaction of the ionizable lipid and the drug in the lipid nanoparticle, and can facilitate the penetration through the cell membrane and exit from the endosome during intracellular drug delivery by binding to the phospholipid bilayer of the target cell .

As the phospholipid, a phospholipid which can promote the fusion of the lipid nanoparticle according to one example can be used without limitation, and for example, it can be one or more selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine ( POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn- glycero -3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn -glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] and the like. In one example, a DOPE-containing lipid nanoparticle can be effective for mRNA (small interfering RNA) delivery (superior mRNA drug delivery efficiency), and in another example, a DSPE-containing lipid nanoparticle can be effective for siRNA delivery (superior drug delivery efficiency). siRNA tools).

The cholesterol of the elements of the lipid nanoparticle according to one example can impart morphological rigidity to the lipid content of the lipid nanoparticle and be dispersed in the core and surface of the nanoparticle to improve the stability of the nanoparticle.

Here, "lipid-PEG (polyethylene glycol) conjugate", "lipid-PEG", "PEG-lipid", "PEG-lipid" or "lipid-PEG" refers to the form in which the lipid and PEG are conjugated, and means a lipid, in in which a polyethylene glycol (PEG) polymer, which is a hydrophilic polymer, is bonded to one end. The lipid-PEG conjugate contributes to the stability of the particle in the serum of the nanoparticle within the lipid nanoparticle and plays the role of preventing aggregation between the nanoparticles. In addition, the lipid-PEG conjugate can protect nucleic acids from a degrading enzyme during in vivo delivery of nucleic acids, and improve the stability of nucleic acids in vivo and increase the half-life of a drug encapsulated in a nanoparticle.

In a lipid-PEG conjugate, the PEG may be directly conjugated to a lipid or linked to a lipid via a linker moiety. Any linker moiety suitable for linking the PEG to the lipid can be used and, for example, it includes an ester-free linker moiety and an ester-containing linker moiety. The ester-free linker moiety includes not only amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea ( -NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH- ), ether, disulfide, but also combinations thereof (for example, a linker containing both a carbamate linker group and an amido linker group), but are not limited to them. The ester-containing linker moiety includes, for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)RON-O-), sulfonate ester, and combinations thereof.

In one example, the average molecular weight of the Lipid-Peg conjugate can be from 100 Dalton to 10,000 longtone, from 200 Dalton to 8000 Dalton, from 500 Dalton to 5000 Dalton, from 1000 Dalton to 3000 Dalton, from 1000 Dalton to 2600 Dalton, from 1,500 Dalton up to 2600 daltons, from 1500 daltons to 2500 daltons, from 2000 to 2600 daltons, from 2000 to 2500 daltons or 2000 daltons.

As the lipid in the lipid-PEG conjugate, any lipid capable of binding to polyethylene glycol can be used without limitation, and phospholipid and/or cholesterol, which are other elements of the lipid nanoparticle, can also be used. In particular, the lipid in the lipid-PEG conjugate may be ceramide, dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPE), distearoylphosphatidylethanolamine (DSPC), or cholesterol.

In one example, a lipid-PEG conjugate may be PEG linked to dialkyloxypropyl (PEG-DAA), PEG linked to diacylglycerol (PEG-DAG), PEG linked to a phospholipid such as phosphatidylethanolamine (PEG-PE), PEG conjugated with ceramide (PEG-CER, ceramide-PEG conjugate, ceramide-PEG, PEG-ceramide or PEG-ceramide conjugate), cholesterol or PEG conjugated to its derivative, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG -DMPE, PEG-DPPC, PEG-DSPE(DSPE-PEG) and mixtures thereof, and for example may be C16-PEG2000 ceramide (N-palmitoyl-sphingosine-1-{succinyl[methoxy-(polyethylene glycol)2000]} ), DMG-PEG 2000, 14:0 PEG2000 PE.

According to one example, a lipid nanoparticle containing a ceramide-PEG conjugate may have one or more of the following characteristics compared to a lipid nanoparticle containing other types of lipid-PEG conjugates:

(1) drug encapsulation with high efficiency;

(2) uniform particle size (or low PDI); and/or

(3) superior drug delivery efficiency to liver tissue and/or cells constituting the liver (eg, hepatocytes and/or LSEC).

The PEG in the lipid-PEG conjugate is a hydrophilic polymer and has the ability to inhibit the adsorption of serum proteins, increase the circulation time of lipid nanoparticles in the body, and can play the role of preventing aggregation between nanoparticles. In addition, the lipid-PEG conjugate may have an in vivo latent function to prevent degradation of the nanoparticles.

The PEG may be such that the functional group binds to the non-lipid side (functionalized PEG). In this case, the functional group that can be used may be one or more species selected from the group consisting of succinyl group, carboxylic acid, maleimide, amino group, biotin, cyanuric group and folate, and the like.

According to one example, the lipid-PEG conjugate may be contained in the lipid nanoparticle in an amount of from 0.1 to 15 mol% (mol.%), from 0.25 to 15 mol. %, from 0.5 to 15 mol. %, from 1 to 15 mol. %, from 1.5 to 15 mol. %, from 2 to 15 mol. %, from 2.5 to 15 mol. %, from 0.1 to 12.5 mol. %, from 0.25 to 12.5 mol. %, from 0.5 to 12.5 mol. %, from 1 to 12.5 mol. %, from 1.5 to 12.5 mol. %, from 2 to 12.5 mol. %, from 2.5 to 12.5 mol. %, from 0.1 to 10 mol. %, from 0.25 to 10 mol. %, from 0.5 to 10 mol. %, from 1 to 10 mol. %, from 1.5 to 10 mol. %, from 2 to 10 mol. %, from 2.5 to 10 mol. %, from 0.1 to 7.5 mol. %, from 0.25 to 7.5 mol. %, from 0.5 to 7.5 mol. %, from 1 to 7.5 mol. %, from 1.5 to 7.5 mol. %, from 2 to 7.5 mol. %, from 2.5 to 7.5 mol. %, from 0.1 to 5 mol. %, from 0.25 to 5 mol. %, from 0.5 to 5 mol. %, from 1 to 5 mol. %, from 1.5 to 5 mol. %, from 2 to 5 mol. %, from 2.5 to 5 mol. %, from 0.1 to 3 mol. %, from 0.25 to 3 mol. %, from 0.5 to 3 mol. %, from 1 to 3 mol. %, from 1.5 to 3 mol. %, from 2 to 3 mol. %, from 2.5 to 3 mol. %, from 0.1 to 2.5 mol. %, from 0.25 to 2.5 mol. %, from 0.5 to 2.5 mol. %, from 1 to 2.5 mol. %, from 1.5 to 2.5 mol. %, from 2 to 2.5 mol. %, from 0.1 to 4.5 mol. %, from 0.25 to 4.5 mol. %, from 0.5 to 4.5 mol. %, from 1 to 4.5 mol. %, from 1.5 to 4.5 mol. %, from 2 to 4.5 mol. %, from 2.5 to 4.5 mol. %, from 0.1 to 4 mol. %, from 0.25 to 4 mol. %, from 0.5 to 4 mol. %, from 1 to 4 mol. %, from 1.5 to 4 mol. %, from 2 to 4 mol. %, from 2.5 to 4 mol. %, from 0.1 to 3.5 mol. %, from 0.25 to 3.5 mol. %, from 0.5 to 3.5 mol. %, from 1 to 3.5 mol. %, from 1.5 to 3.5 mol. %, from 2 to 3.5 mol. %, from 2.5 to 3.5 mol. %, from 0.1 to 2.0 mol. %, from 0.1 to 1.5 mol. %, from 0.1 to 1.0 mol. %, from 0.5 to 2.0 mol. %, from 0.5 to 1.5 mol. %, from 0.5 to 1.0 mol. %, from 1.0 to 2.0 mol. %, from 1.0 to 1.5 mol. %, from 1.5 to 2.0 mol. %, 1.0 mol. % or 1.5 mol. %.

In one example, the effect of targeting liver tissue, hepatocytes and/or LSEC (drug delivery effect) of a lipid nanoparticle may depend on the content of the lipid-PEG conjugate contained in the lipid nanoparticle.

For example, a lipid nanoparticle containing a lipid-PEG conjugate in an amount of 0.1 to 15 mol. %, from 0.25 to 15 mol. %, from 0.5 to 15 mol. %, from 1 to 15 mol. %, from 1.5 to 15 mol. %, from 2 to 15 mol. %, from 2.5 to 15 mol. %, from 0.1 to 12.5 mol. %, from 0.25 to 12.5 mol. %, from 0.5 to 12.5 mol. %, from 1 to 12.5 mol. %, from 1.5 to 12.5 mol. %, from 2 to 12.5 mol. %, from 2.5 to 12.5 mol. %, from 0.1 to 10 mol. %, from 0.25 to 10 mol. %, from 0.5 to 10 mol. %, from 1 to 10 mol. %, from 1.5 to 10 mol. %, from 2 to 10 mol. %, from 2.5 to 10 mol. %, from 0.1 to 7.5 mol. %, from 0.25 to 7.5 mol. %, from 0.5 to 7.5 mol. %, from 1 to 7.5 mol. %, from 1.5 to 7.5 mol. %, from 2 to 7.5 mol. %, from 2.5 to 7.5 mol. %, from 0.1 to 5 mol. %, from 0.25 to 5 mol. %, from 0.5 to 5 mol. %, from 1 to 5 mol. %, from 1.5 to 5 mol. %, from 2 to 5 mol. %, from 2.5 to 5 mol. %, from 0.1 to 3 mol. %, from 0.25 to 3 mol. %, from 0.5 to 3 mol. %, from 1 to 3 mol. %, from 1.5 to 3 mol. %, from 2 to 3 mol. %, from 2.5 to 3 mol. %, from 0.1 to 2.5 mol. %, from 0.25 to 2.5 mol. %, from 0.5 to 2.5 mol. %, from 1 to 2.5 mol. %, from 1.5 to 2.5 mol. %, from 2 to 2.5 mol. %, from 0.1 to 4.5 mol. %, from 0.25 to 4.5 mol. %, from 0.5 to 4.5 mol. %, from 1 to 4.5 mol. %, from 1.5 to 4.5 mol. %, from 2 to 4.5 mol. %, from 2.5 to 4.5 mol. %, from 0.1 to 4 mol. %, from 0.25 to 4 mol. %, from 0.5 to 4 mol. %, from 1 to 4 mol. %, from 1.5 to 4 mol. %, from 2 to 4 mol. %, from 2.5 to 4 mol. %, from 0.1 to 3.5 mol. %, from 0.25 to 3.5 mol. %, from 0.5 to 3.5 mol. %, from 1 to 3.5 mol. %, from 1.5 to 3.5 mol. %, from 2 to 3.5 mol. %, from 2.5 to 3.5 mol. %, from 0.1 to 10 mol. %, from 0.1 to 5.0 mol. %, from 0.1 to 4.5 mol. %, from 0.1 to 4.0 mol. %, from 0.1 to 3.5 mol. %, from 0.1 to 3.0 mol. %, from 0.1 to 2.5 mol. %, from 0.1 to 2.0 mol. %, from 0.1 to 1.5 mol. %, from 0.1 to 1.0 mol. %, from 0.5 to 10 mol. %, from 0.5 to 5.0 mol. %, from 0.5 to 4.5 mol. %, from 0.5 to 4.0 mol. %, from 0.5 to 3.5 mol. %, from 0.5 to 3.0 mol. %, from 0.5 to 2.5 mol. %, from 0.5 to 2.0 mol. %, from 0.5 to 1.5 mol. %, from 0.5 to 1.0 mol. %, from 1.0 to 10 mol. %, from 1.0 to 5.0 mol. %, from 1.0 to 4.5 mol. %, from 1.0 to 4.0 mol. %, from 1.0 to 3.5 mol. %, from 1.0 to 3.0 mol. %, from 1.0 to 2.5 mol. %, from 1.0 to 2.0 mol. %, from 1.0 to 1.5 mol. %, from 1.5 to 10 mol. %, from 1.5 to 5.0 mol. %, from 1.5 to 4.5 mol. %, from 1.5 to 4.0 mol. %, from 1.5 to 3.5 mol. %, from 1.5 to 3.0 mol. %, from 1.5 to 2.5 mol. %, from 1.5 to 2.0 mol. %, 1.0 mol. % or 1.5 mol. % (compared to a lipid nanoparticle containing a lipid-PEG conjugate in an amount outside the above range) can have an excellent targeting effect on hepatocytes.

As another example, a lipid nanoparticle containing a lipid-PEG conjugate in an amount of from 0.1 to 15 mol. %, from 0.25 to 15 mol. %, from 0.5 to 15 mol. %, from 1 to 15 mol. %, from 1.5 to 15 mol. %, from 2 to 15 mol. %, from 2.5 to 15 mol. %, from 0.1 to 12.5 mol. %, from 0.25 to 12.5 mol. %, from 0.5 to 12.5 mol. %, from 1 to 12.5 mol. %, from 1.5 to 12.5 mol. %, from 2 to 12.5 mol. %, from 2.5 to 12.5 mol. %, from 0.1 to 10 mol. %, from 0.25 to 10 mol. %, from 0.5 to 10 mol. %, from 1 to 10 mol. %, from 1.5 to 10 mol. %, from 2 to 10 mol. %, from 2.5 to 10 mol. %, from 0.1 to 7.5 mol. %, from 0.25 to 7.5 mol. %, from 0.5 to 7.5 mol. %, from 1 to 7.5 mol. %, from 1.5 to 7.5 mol. %, from 2 to 7.5 mol. %, from 2.5 to 7.5 mol. %, from 0.1 to 5 mol. %, from 0.25 to 5 mol. %, from 0.5 to 5 mol. %, from 1 to 5 mol. %, from 1.5 to 5 mol. %, from 2 to 5 mol. %, from 2.5 to 5 mol. %, from 0.1 to 3 mol. %, from 0.25 to 3 mol. %, from 0.5 to 3 mol. %, from 1 to 3 mol. %, from 1.5 to 3 mol. %, from 2 to 3 mol. %, from 2.5 to 3 mol. %, from 0.1 to 2.5 mol. %, from 0.25 to 2.5 mol. %, from 0.5 to 2.5 mol. %, from 1 to 2.5 mol. %, from 1.5 to 2.5 mol. %, from 2 to 2.5 mol. %, from 0.1 to 4.5 mol. %, from 0.25 to 4.5 mol. %, from 0.5 to 4.5 mol. %, from 1 to 4.5 mol. %, from 1.5 to 4.5 mol. %, from 2 to 4.5 mol. %, from 2.5 to 4.5 mol. %, from 0.1 to 4 mol. %, from 0.25 to 4 mol. %, from 0.5 to 4 mol. %, from 1 to 4 mol. %, from 1.5 to 4 mol. %, from 2 to 4 mol. %, from 2.5 to 4 mol. %, from 0.1 to 3.5 mol. %, from 0.25 to 3.5 mol. %, from 0.5 to 3.5 mol. %, from 1 to 3.5 mol. %, from 1.5 to 3.5 mol. %, from 2 to 3.5 mol. %, from 2.5 to 3.5 mol. %, from 0.1 to 10 mol. %, from 0.1 to 5.0 mol. %, from 0.1 to 4.5 mol. %, from 0.1 to 4.0 mol. %, from 0.1 to 3.5 mol. %, from 0.1 to 3.0 mol. %, from 0.1 to 2.5 mol. %, from 0.1 to 2.0 mol. %, from 0.1 to 1.5 mol. %, from 0.1 to 1.0 mol. %, from 0.5 to 10 mol. %, from 0.5 to 5.0 mol. %, from 0.5 to 4.5 mol. %, from 0.5 to 4.0 mol. %, from 0.5 to 3.5 mol. %, from 0.5 to 3.0 mol. %, from 0.5 to 2.5 mol. %, from 0.5 to 2.0 mol. %, from 0.5 to 1.5 mol. %, from 0.5 to 1.0 mol. %, from 1.0 to 10 mol. %, from 1.0 to 5.0 mol. %, from 1.0 to 4.5 mol. %, from 1.0 to 4.0 mol. %, from 1.0 to 3.5 mol. %, from 1.0 to 3.0 mol. %, from 1.0 to 2.5 mol. %, from 1.0 to 2.0 mol. %, from 1.0 to 1.5 mol. %, from 1.5 to 10 mol. %, from 1.5 to 5.0 mol. %, from 1.5 to 4.5 mol. %, from 1.5 to 4.0 mol. %, from 1.5 to 3.5 mol. %, from 1.5 to 3.0 mol. %, from 1.5 to 2.5 mol. %, from 1.5 to 2.0 mol. %, 1.0 mol. %, or 1.5 mol. % (compared to a lipid nanoparticle containing a lipid-PEG conjugate in an amount outside the above range) can have an excellent targeting effect on LSEC.

According to one example, cholesterol can be contained in a lipid nanoparticle in an amount of from 10 to 60 mol. %, from 20 to 60 mol. %, from 30 to 60 mol. %, from 30 to 55 mol. %, from 30 to 52.5 mol. %, from 30 to 52 mol. %, from 30 to 51 mol. %, from 30 to 50 mol. %, from 30 to 47.5 mol. %, from 30 to 45 mol. %, from 30 to 44 mol. %, from 30 to 43.5 mol. %, from 30 to 43 mol. %, from 30 to 41.5 mol. %, from 30 to 40 mol. %, from 30 to 39.5 mol. %, from 35 to 60 mol. %, from 35 to 55 mol. %, from 35 to 52.5 mol. %, from 35 to 52 mol. %, from 35 to 51 mol. %, from 35 to 50 mol. %, from 35 to 47.5 mol. %, from 35 to 45 mol. %, from 35 to 44 mol. %, from 35 to 43.5 mol. %, from 35 to 43 mol. %, from 35 to 41.5 mol. %, from 35 to 40 mol. %, from 35 to 39.5 mol. %, from 37 to 60 mol. %, from 37 to 55 mol. %, from 37 to 52.5 mol. %, from 37 to 52 mol. %, from 37 to 51 mol. %, from 37.5 to 50 mol. %, from 37.5 to 47.5 mol. %, from 37.5 to 45 mol. %, from 37.5 to 44 mol. %, from 37.5 to 43.5 mol. %, from 37.5 to 43 mol. %, from 37.5 to 41.5 mol. %, from 37.5 to 40 mol. %, from 37.5 to 39.5 mol. %, from 39.5 to 60 mol. %, from 39.5 to 55 mol. %, from 39.5 to 52.5 mol. %, from 39.5 to 52 mol. %, from 39.5 to 51 mol. %, from 39.5 to 50 mol. %, from 39.5 to 47.5 mol. %, from 39.5 to 45 mol. %, from 39.5 to 44 mol. %, from 39.5 to 43.5 mol. %, from 39.5 to 43 mol. %, from 39.5 to 41.5 mol. %, from 39.5 to 40 mol. %, from 40 to 60 mol. %, from 40 to 55 mol. %, from 40 to 52.5 mol. %, from 40 to 52 mol. %, from 40 to 51 mol. %, from 40 to 50 mol. %, from 40 to 47.5 mol. %, from 40 to 45 mol. %, from 40 to 44 mol. %, from 40 to 43.5 mol. %, from 40 to 43 mol. %, from 40 to 41.5 mol. %, from 41.5 to 60 mol. %, from 41.5 to 55 mol. %, from 41.5 to 52.5 mol. %, from 41.5 to 52 mol. %, from 41.5 to 51 mol. %, from 41.5 to 50 mol. %, from 41.5 to 47.5 mol. %, from 41.5 to 45 mol. %, from 41.5 to 44 mol. %, from 41.5 to 43.5 mol. %, from 41.5 to 43 mol. %, from 43 to 60 mol. %, from 43 to 55 mol. %, from 43 to 52.5 mol. %, from 43 to 52 mol. %, from 43 to 51 mol. %, from 43 to 50 mol. %, from 43 to 47.5 mol. %, from 43 to 45 mol. %, from 43 to 44 mol. %, from 43 to 43.5 mol. %, from 43.5 to 60 mol. %, from 43.5 to 55 mol. %, from 43.5 to 52.5 mol. %, from 43.5 to 52 mol. %, from 43.5 to 51 mol. %, from 43.5 to 50 mol. %, from 43.5 to 47.5 mol. %, from 43.5 to 45 mol. %, from 43.5 to 44 mol. %, from 45 to 60 mol. %, from 45 to 55 mol. %, from 45 to 52.5 mol. %, from 45 to 52 mol. %, from 45 to 51 mol. %, from 45 to 50 mol. %, from 45 to 47.5 mol. %, from 47.5 to 60 mol. %, from 47.5 to 55 mol. %, from 47.5 to 52.5 mol. %, from 47.5 to 52 mol. %, from 47.5 to 51 mol. %, from 47.5 to 50 mol. %, from 50 to 60 mol. %, from 50 to 55 mol. %, from 50 to 52.5 mol. %, from 50 to 52 mol. %, from 50 to 52.5 mol. % from 50 to 51.5 mol. %, from 51 to 60 mol. %, from 51 to 55 mol. %, from 51 to 52.5 mol. %, or from 51 to 52 mol. %, from 51 to 60 mol. %, from 51 to 55 mol. %, from 51 to 52.5 mol. %, or from 51 to 52 mol. %.

According to one example, the amount of lipid-PEG-cholesterol conjugate may be contained in a lipid nanoparticle in an amount of 30 to 70 mol. %, from 40 to 70 mol. %, from 40 to 60 mol. %, from 40 to 55 mol. %, from 40 to 53.5 mol. %, from 40 to 50 mol. %, from 40 to 47.5 mol. %, from 40 to 45 mol. %, from 40 to 44.5 mol. %, from 42 to 60 mol. %, from 42 to 55 mol. %, from 42 to 53.5 mol. %, from 42 to 50 mol. %, from 42 to 47.5 mol. %, from 42 to 45 mol. %, from 42 to 44.5 mol. %, from 44 to 60 mol. %, from 44 to 55 mol. %, from 44 to 53.5 mol. %, from 44 to 50 mol. %, from 44 to 47.5 mol. %, from 44 to 45 mol. %, from 44 to 44.5 mol. %, from 44.5 to 60 mol. %, from 44.5 to 55 mol. %, from 44.5 to 53.5 mol. %, from 44.5 to 50 mol. %, from 44.5 to 47.5 mol. %, or from 44.5 to 45 mol. %.

According to one example, the ionizable lipid may be contained in the lipid nanoparticle in an amount of from 10 to 70 mol. %, from 10 to 60 mol. %, from 10 to 55 mol. %, from 10 to 50 mol. %, from 10 to 45 mol. %, from 10 to 42.5 mol. %, from 10 to 40 mol. %, from 10 to 35 mol. %, from 10 to 30 mol. %, from 10 to 26.5 mol. %, from 10 to 25 mol. %, from 10 to 20 mol. %, from 15 to 60 mol. %, from 15 to 55 mol. %, from 15 to 50 mol. %, from 15 to 45 mol. %, from 15 to 42.5 mol. %, from 15 to 40 mol. %, from 15 to 35 mol. %, from 15 to 30 mol. %, from 15 to 26.5 mol. %, from 15 to 25 mol. %, from 15 to 20 mol. %, from 20 to 60 mol. %, from 20 to 55 mol. %, from 20 to 50 mol. %, from 20 to 45 mol. %, from 20 to 42.5 mol. %, from 20 to 40 mol. %, from 20 to 35 mol. %, from 20 to 30 mol. %, from 20 to 26.5 mol. %, from 20 to 25 mol. %, from 25 to 60 mol. %, from 25 to 55 mol. %, from 25 to 50 mol. %, from 25 to 45 mol. %, from 25 to 42.5 mol. %, from 25 to 40 mol. %, from 25 to 35 mol. %, from 25 to 30 mol. %, from 25 to 26.5 mol. %, from 26.5 to 60 mol. %, from 26.5 to 55 mol. %, from 26.5 to 50 mol. %, from 26.5 to 45 mol. %, from 26.5 to 42.5 mol. %, 26.5 to 40 mol. %, from 26.5 to 35 mol. %, from 26.5 to 30 mol. %, from 30 to 60 mol. %, from 30 to 55 mol. %, from 30 to 50 mol. %, from 30 to 45 mol. %, from 30 to 42.5 mol. %, from 30 to 40 mol. %, from 30 to 35 mol. %, from 35 to 60 mol. %, from 35 to 55 mol. %, from 35 to 50 mol. %, from 35 to 45 mol. %, from 35 to 42.5 mol. %, from 35 to 40 mol. %, from 40 to 60 mol. %, from 40 to 55 mol. %, from 40 to 50 mol. %, from 40 to 45 mol. %, from 40 to 42.5 mol. %, from 42.5 to 60 mol. %, from 42.5 to 55 mol. %, from 42.5 to 50 mol. %, or from 42.5 to 45 mol. %.

According to one example, the phospholipid may be contained in the lipid nanoparticle in an amount of from 1 to 50 mol. %, from 5 to 50 mol. %, from 5 to 40 mol. %, from 5 to 30 mol. %, from 5 to 25 mol. %, from 5 to 20 mol. %, from 5 to 15 mol. %, from 5 to 13 mol. %, from 5 to 10 mol. %, from 10 to 30 mol. %, from 10 to 25 mol. %, from 10 to 20 mol. %, from 10 to 15 mol. %, from 10 to 13 mol. %, from 15 to 30 mol. %, from 15 to 25 mol. %, from 15 to 20 mol. %, from 20 to 30 mol. %, or from 20 to 25 mol. %.

Here "please. % (mole %, mole percent)" is expressed as a percentage by dividing the number of moles of a particular component by the sum of the moles of all components, then multiplying by 100 and expressed as a formula, for example, this can be Equation 1 below:

(Equation 1)

they say % = (moles of a specific component) / (sum of moles of all components) × 100

The lipid nanoparticle may contain an ionizable lipid : phospholipid : cholesterol : lipid-PEG conjugate in a molar ratio of 20 to 50 : 10 to 30 : 20 to 60 : 0.1 to 10, in a molar ratio of 20 to 50 : 10 up to 30: from 20 to 60: from 0.25 to 10, in a molar ratio from 20 to 50: from 10 to 30: from 30 to 60: from 0.25 to 10, in a molar ratio from 20 to 50: from 10 up to 30: from 30 to 60: from 0.1 to 5, in a molar ratio from 20 to 50: from 10 to 30: from 30 to 60: from 0.5 to 5, in a molar ratio from 25 to 45: from 10 up to 25 : 40 to 50 : 0.5 to 3, in molar ratio 25 to 45 : 10 to 20 : 40 to 55 : 0.5 to 3, in molar ratio 25 to 45 : 10 up to 20 : 40 to 55 : 1.0 to 1.5, in a molar ratio of 40 to 45 : 10 to 15 : 40 to 45 : 0.5 to 3.0, in a molar ratio of 40 to 45 : 10 to 15 : 40 to 45 : 0.5 to 3, in a molar ratio of 40 to 45 : 10 to 15 : 40 to 45 : 1 to 1.5, in a molar ratio of 25 to 30: 17 to 22; 50 to 55: 0.5 to 3.0, in a molar ratio of 25 to 30: 17 to 22; 50 to 55: 1.0 to 2.5, or in a molar ratio of 25 to 30: 17 to 22; 50 to 55: 1.5 to 2.5. According to one example, by keeping the sum of moles of lipid-PEG conjugate and cholesterol among the components contained in the lipid nanoparticle constant, the number of moles of cholesterol decreases as the number of moles of lipid-PEG conjugate increases, and thus the molar ratio of the components can be maintained.

Here, the molar ratio means the ratio of moles, and "parts by mass" means the mass ratio in which each component is contained.

In one example, the lipid nanoparticle may contain an ionizable lipid in an amount of 20 to 50 parts by weight, a phospholipid in an amount of 10 to 30 parts by weight, cholesterol in an amount of 20 to 60 parts by weight (or 20 to 60 parts by weight) , and a lipid-PEG conjugate 0.1 to 10 parts by weight (or 0.25 to 10 parts by weight, 0.5 to 5 parts by weight). The lipid nanoparticle may contain an ionizable lipid in an amount of 20 to 50% by mass, a phospholipid in an amount of 10 to 30% by mass, a cholesterol in an amount of 20 to 60% by mass (or 30 to 60% by mass) and a lipid conjugate. -PEG 0.1 to 10% by weight (or 0.25 to 10% by weight, 0.5 to 5% by weight) based on the total weight of the nanoparticles. In another example, the lipid nanoparticle may contain an ionizable lipid in an amount of 25 to 50% by weight, a phospholipid in an amount of 10 to 20% by weight, a cholesterol in an amount of 35 to 55% by weight, and a lipid-PEG conjugate in an amount of 0, 1 to 10% by weight (or 0.25 to 10% by weight, 0.5 to 5% by weight) based on the total weight of the nanoparticles.

A lipid nanoparticle containing an ionizable lipid, cholesterol, a phospholipid and/or a lipid-PEG conjugate in the above range (molar ratio, parts by weight and/or % by weight) can have excellent (1) lipid nanoparticle stability, (2) efficiency drug encapsulation and/or (3) drug delivery efficiency to liver tissue and/or cells (e.g., hepatocytes and/or LSEC) compared to a lipid nanoparticle containing an ionizable lipid, cholesterol, phospholipid, and/or or a lipid- PEG outside of the above range.

The lipid nanoparticle according to one example may have an average diameter of 20 nm to 200 nm, 20 to 180 nm, 20 nm to 170 nm, 20 nm to 150 nm, 20 nm to 120 nm, 20 nm to 100 nm, 20 nm to 90 nm, 30 nm to 200 nm, 30 to 180 nm, 30 nm to 170 nm, 30 nm to 150 nm, 30 nm to 120 nm, 30 nm to 100 nm, 30 nm nm to 90 nm, 40 nm to 200 nm, 40 to 180 nm, 40 nm to 170 nm, 40 nm to 150 nm, 40 nm to 120 nm, 40 nm to 100 nm, 40 nm to 90 nm, 50 nm to 200 nm, 50 to 180 nm, 50 nm to 170 nm, 50 nm to 150 nm, 50 nm to 120 nm, 50 nm to 100 nm, 50 nm to 90 nm , 60 nm to 200 nm, 60 to 180 nm, 60 nm to 170 nm, 60 nm to 150 nm, 60 nm to 120 nm, 60 nm to 100 nm, 60 nm to 90 nm, 70 nm to 200 nm, 70 to 180 nm, 70 nm to 170 nm, 70 nm to 150 nm, 70 nm to 120 nm, 70 nm to 100 nm, 70 nm to 90 nm, 80 nm to 200 nm, 80 to 180 nm, 80 nm to 170 nm, 80 nm to 150 nm, 80 nm to 120 nm, 80 nm to 100 nm, 80 nm to 90 nm, 90 nm to 200 nm , 90 to 180 nm, 90 nm to 170 nm, 90 nm to 150 nm, 90 nm to 120 nm, or 90 nm to 100 nm, for easy injection into liver tissue, hepatocytes and/or LSEC (sinusoidal liver endothelial cells). When the size of the lipid nanoparticle is smaller than the above range, it is difficult to maintain stability because the surface area of the lipid nanoparticle is excessively increased, and thus the delivery to the target tissue and/or the effect of the drug may be reduced.

The targeting effect on liver tissue, hepatocytes and/or LSEC (drug delivery effect) of a lipid nanoparticle according to one example may depend on the size of the lipid nanoparticle. For example, in the case of a lipid nanoparticle with a diameter of 40 to 120 nm, 30 to 100 nm, 35 to 95 nm, 40 to 90 nm, 45 to 90 nm, 50 to 90 nm, 55 to 85 nm, from 60 to 80 nm, 70 to 90 nm, 70 to 80 nm, 50 to 70 nm, or 60 to 70 nm, the targeting effect on hepatocytes can be excellent (compared to nanoparticles having a diameter outside the above range). As another example, in the case of a lipid nanoparticle having a diameter of 20 to 200 nm, 20 to 180 nm, 40 to 180 nm, 40 to 170 nm, 50 to 160 nm, 70 to 180 nm, 70 up to 170 nm, 75 to 170 nm, 75 to 165 nm, 70 to 150 nm, 70 to 130 nm, 75 to 130 nm, 80 to 120 nm, 85 to 120 nm, approximately 90 to 120 nm, 90 to 10 nm, 90 to 100 nm, 80 to 110 nm, 80 to 100 nm, 85 to 95 nm, about 90 nm or 90 nm, LSEC targeting effect can be excellent (compared to with a nanoparticle having a diameter outside the above range).

The lipid nanoparticle can specifically target liver tissue. A lipid nanoparticle according to one example can very closely mimic the metabolic behavior of natural lipoproteins and thus can be successfully applied to the process of lipid metabolism in the liver and the therapeutic mechanism thereby.

The lipid nanoparticle can target hepatocytes. When the content of the lipid-PEG contained in the lipid nanoparticle is from 0.1 to 15 mol. %, from 0.25 to 15 mol. %, from 0.5 to 15 mol. %, from 1 to 15 mol. %, from 1.5 to 15 mol. %, from 2 to 15 mol. %, from 2.5 to 15 mol. %, from 0.1 to 12.5 mol. %, from 0.25 to 12.5 mol. %, from 0.5 to 12.5 mol. %, from 1 to 12.5 mol. %, from 1.5 to 12.5 mol. %, from 2 to 12.5 mol. %, from 2.5 to 12.5 mol. %, from 0.1 to 10 mol. %, from 0.25 to 10 mol. %, from 0.5 to 10 mol. %, from 1 to 10 mol. %, from 1.5 to 10 mol. %, from 2 to 10 mol. %, from 2.5 to 10 mol. %, from 0.1 to 7.5 mol. %, from 0.25 to 7.5 mol. %, from 0.5 to 7.5 mol. %, from 1 to 7.5 mol. %, from 1.5 to 7.5 mol. %, from 2 to 7.5 mol. %, from 2.5 to 7.5 mol. %, from 0.1 to 5 mol. %, from 0.25 to 5 mol. %, from 0.5 to 5 mol. %, from 1 to 5 mol. %, from 1.5 to 5 mol. %, from 2 to 5 mol. %, from 2.5 to 5 mol. %, from 0.1 to 3 mol. %, from 0.25 to 3 mol. %, from 0.5 to 3 mol. %, from 1 to 3 mol. %, from 1.5 to 3 mol. %, from 2 to 3 mol. %, from 2.5 to 3 mol. %, from 0.1 to 2.5 mol. %, from 0.25 to 2.5 mol. %, from 0.5 to 2.5 mol. %, from 1 to 2.5 mol. %, from 1.5 to 2.5 mol. %, from 2 to 2.5 mol. %, from 0.1 to 4.5 mol. %, from 0.25 to 4.5 mol. %, from 0.5 to 4.5 mol. %, from 1 to 4.5 mol. %, from 1.5 to 4.5 mol. %, from 2 to 4.5 mol. %, from 2.5 to 4.5 mol. %, from 0.1 to 4 mol. %, from 0.25 to 4 mol. %, from 0.5 to 4 mol. %, from 1 to 4 mol. %, from 1.5 to 4 mol. %, from 2 to 4 mol. %, from 2.5 to 4 mol. %, from 0.1 to 3.5 mol. %, from 0.25 to 3.5 mol. %, from 0.5 to 3.5 mol. %, from 1 to 3.5 mol. %, from 1.5 to 3.5 mol. %, from 2 to 3.5 mol. %, from 2.5 to 3.5 mol. %, from 0.1 to 2.0 mol. %, from 0.1 to 1.5 mol. %, from 0.1 to 1.0 mol. %, from 0.5 to 2.0 mol. %, from 0.5 to 1.5 mol. %, from 0.5 to 1.0 mol. %, from 1.0 to 2.0 mol. %, from 1.0 to 1.5 mol. %, from 1.5 to 2.0 mol. %, 1.0 mol. % or 1.5 mol. %, the drug delivery efficiency (hepatocyte targeting efficiency) to hepatocytes by the lipid nanoparticle can be excellent.

The lipid nanoparticle can target LSECs (liver sinusoidal endothelial cells). When the content of the lipid-PEG contained in the lipid nanoparticle is from 0.1 to 15 mol. %, from 0.25 to 15 mol. %, from 0.5 to 15 mol. %, from 1 to 15 mol. %, from 1.5 to 15 mol. %, from 2 to 15 mol. %, from 2.5 to 15 mol. %, from 0.1 to 12.5 mol. %, from 0.25 to 12.5 mol. %, from 0.5 to 12.5 mol. %, from 1 to 12.5 mol. %, from 1.5 to 12.5 mol. %, from 2 to 12.5 mol. %, from 2.5 to 12.5 mol. %, from 0.1 to 10 mol. %, from 0.25 to 10 mol. %, from 0.5 to 10 mol. %, from 1 to 10 mol. %, from 1.5 to 10 mol. %, from 2 to 10 mol. %, from 2.5 to 10 mol. %, from 0.1 to 7.5 mol. %, from 0.25 to 7.5 mol. %, from 0.5 to 7.5 mol. %, from 1 to 7.5 mol. %, from 1.5 to 7.5 mol. %, from 2 to 7.5 mol. %, from 2.5 to 7.5 mol. %, from 0.1 to 5 mol. %, from 0.25 to 5 mol. %, from 0.5 to 5 mol. %, from 1 to 5 mol. %, from 1.5 to 5 mol. %, from 2 to 5 mol. %, from 2.5 to 5 mol. %, from 0.1 to 3 mol. %, from 0.25 to 3 mol. %, from 0.5 to 3 mol. %, from 1 to 3 mol. %, from 1.5 to 3 mol. %, from 2 to 3 mol. %, from 2.5 to 3 mol. %, from 0.1 to 2.5 mol. %, from 0.25 to 2.5 mol. %, from 0.5 to 2.5 mol. %, from 1 to 2.5 mol. %, from 1.5 to 2.5 mol. %, from 2 to 2.5 mol. %, from 0.1 to 4.5 mol. %, from 0.25 to 4.5 mol. %, from 0.5 to 4.5 mol. %, from 1 to 4.5 mol. %, from 1.5 to 4.5 mol. %, from 2 to 4.5 mol. %, from 2.5 to 4.5 mol. %, from 0.1 to 4 mol. %, from 0.25 to 4 mol. %, from 0.5 to 4 mol. %, from 1 to 4 mol. %, from 1.5 to 4 mol. %, from 2 to 4 mol. %, from 2.5 to 4 mol. %, from 0.1 to 3.5 mol. %, from 0.25 to 3.5 mol. %, from 0.5 to 3.5 mol. %, from 1 to 3.5 mol. %, from 1.5 to 3.5 mol. %, from 2 to 3.5 mol. %, from 2.5 to 3.5 mol. %, from 0.1 to 2.0 mol. %, from 0.1 to 1.5 mol. %, from 0.1 to 1.0 mol. %, from 0.5 to 2.0 mol. %, from 0.5 to 1.5 mol. %, from 0.5 to 1.0 mol. %, from 1.0 to 2.0 mol. %, from 1.0 to 1.5 mol. %, from 1.5 to 2.0 mol. %, 1.0 mol. %, or 1.5 mol. %, the drug delivery efficiency (LSEC targeting efficiency) in the LSEC by the lipid nanoparticle can be excellent.

Here, "targeting" and "localization" in liver tissue, hepatocytes, and/or LSEC of a lipid nanoparticle may mean internalization into tissue or cells, and may mean internalization within the nucleus, since it can penetrate the nuclear membrane.

As another aspect, there is provided a drug delivery composition comprising (1) a lipid nanoparticle and (2) an anionic drug, a nucleic acid, or a combination thereof (combination of anionic drug and nucleic acid). The drug may be an anionic drug, a nucleic acid, or a combination thereof (anionic drug and nucleic acid).

The drug delivery composition may be a bioactive agent such as an anionic drug and/or nucleic acid or the like that can be encapsulated within a lipid nanoparticle and a bioactive agent such as an anionic drug and/or nucleic acid , can be encapsulated within a lipid nanoparticle with stable and high efficiency, and thus the delivery composition can exhibit an excellent therapeutic effect. In addition, there is the advantage of different control of the types of drugs encapsulated in lipid nanoparticles depending on the purpose of the treatment.

The lipid nanoparticle may have an anionic drug and/or nucleic acid encapsulated within (the lipid nanoparticle). With respect to the lipid nanoparticle in which the anionic drug and/or nucleic acid is encapsulated (within the lipid nanoparticle), it is the same as the lipid nanoparticle described above.

In one example, the weight ratio of ionizable lipid and drug (anionic drug, nucleic acid, or combination thereof) contained in a lipid nanoparticle may be 1 to 20:1, 1 to 15:1, 1 to 10:1, 5 to 20:1, 5 to 15:1, 5 to 10:1, 7.5 to 20:1, 7.5 to 15:1, or 7.5 to 10:1.

In one example, when (1) an ionizable lipid; and (2) the drug (anionic drug, nucleic acid, or combination thereof) is contained in a weight ratio within the above range, the encapsulation efficiency of the drug (anionic drug, nucleic acid, or combination thereof) within the lipid nanoparticle, and/or the drug delivery efficiency means can be higher than lipid nanoparticles containing (1) ionizable lipid; and (2) an anionic drug, nucleic acid, or combination thereof in a weight ratio outside the above range.

The lipid nanoparticle in which the anionic drug and/or nucleic acid is encapsulated may have an average diameter of 20 nm to 200 nm, 20 to 180 nm, 20 nm to 170 nm, 20 nm to 150 nm, 20 nm to 120 nm, 20 nm to 100 nm, 20 nm to 90 nm, 30 nm to 200 nm, 30 nm to 180 nm, 30 nm to 170 nm, 30 nm to 150 nm, 30 nm to 120 nm , 30 nm to 100 nm, 30 nm to 90 nm, 40 nm to 200 nm, 40 to 180 nm, 40 nm to 170 nm, 40 nm to 150 nm, 40 nm to 120 nm, from 40 nm to 100 nm, 40 nm to 90 nm, 50 nm to 200 nm, 50 to 180 nm, 50 nm to 170 nm, 50 nm to 150 nm, 50 nm to 120 nm, 50 nm up to 100 nm, 50 nm to 90 nm, 60 nm to 200 nm, 60 to 180 nm, 60 nm to 170 nm, 60 nm to 150 nm, 60 nm to 120 nm, 60 nm to 100 nm , 60 nm to 90 nm, 70 nm to 200 nm, 70 to 180 nm, 70 nm to 170 nm, 70 nm to 150 nm, 70 nm to 120 nm, 70 nm to 100 nm, 70 nm to 90 nm, 80 nm to 200 nm, 80 to 180 nm, 80 nm to 170 nm, 80 nm to 150 nm, 80 nm to 120 nm, 80 nm to 100 nm, 80 nm to 90 nm, 90 nm to 200 nm, 90 to 180 nm, 90 nm to 170 nm, 90 nm to 150 nm, 90 nm to 120 nm, or 90 nm to 100 nm, so that insertion into tissue liver hepatocytes and/or LSEC (liver sinusoidal endothelial cells) is mild.

When the size of the lipid nanoparticle is less than the lower limit of the above range, (1) during the systemic circulation of the lipid nanoparticle, the binding of apolipoproteins (e.g., ApoE (e.g., ApoE3)) present in the blood is reduced, and therefore the amount of lipid nanoparticles entering the cells , can be reduced and/or (2) the surface area of the lipid nanoparticle is excessively increased, and therefore it is difficult to maintain the stability and therefore the efficiency of drug delivery to the target tissue (or target cell) and/or the therapeutic effect of the drug that carries lipid nanoparticle, can be reduced.

A lipid nanoparticle having a diameter in the above range has superior drug delivery efficiency to a target organ and/or cell compared to a lipid nanoparticle having a diameter greater than the upper limit of the above range.

In one example, a drug delivery composition comprising (1) a lipid nanoparticle and (2) an anionic drug, a nucleic acid, or a combination thereof may be a hepatocyte drug delivery composition.

According to one example, the diameter of a lipid nanoparticle contained in a composition for delivering a drug (anionic drug, nucleic acid, or combination thereof) to hepatocytes can be 40 to 120 nm, 30 to 100 nm, 35 to 95 nm, 40 up to 90 nm, 45 to 90 nm, 50 to 90 nm, 55 to 85 nm, 60 to 80 nm, 70 to 90 nm, 70 to 80 nm, 50 to 70 nm or 60 to 70 nm . In drug delivery to hepatocytes, the diameter of the pores leading from the lumen of the sinusoids to the hepatocytes is about 140 nm in mammals and about 100 nm in humans, so a drug delivery composition having a diameter in this range can have excellent drug delivery efficiency in hepatocytes compared to a lipid nanoparticle having a diameter outside the above range.

According to one example, the lipid nanoparticle, which is part of the composition for drug delivery to hepatocytes, may contain a lipid-PEG conjugate in an amount of from 0.1 to 15 mol. %, from 0.25 to 15 mol. %, from 0.5 to 15 mol. %, from 1 to 15 mol. %, from 1.5 to 15 mol. %, from 2 to 15 mol. %, from 2.5 to 15 mol. %, from 0.1 to 12.5 mol. %, from 0.25 to 12.5 mol. %, from 0.5 to 12.5 mol. %, from 1 to 12.5 mol. %, from 1.5 to 12.5 mol. %, from 2 to 12.5 mol. %, from 2.5 to 12.5 mol. %, from 0.1 to 10 mol. %, from 0.25 to 10 mol. %, from 0.5 to 10 mol. %, from 1 to 10 mol. %, from 1.5 to 10 mol. %, from 2 to 10 mol. %, from 2.5 to 10 mol. %, from 0.1 to 7.5 mol. %, from 0.25 to 7.5 mol. %, from 0.5 to 7.5 mol. %, from 1 to 7.5 mol. %, from 1.5 to 7.5 mol. %, from 2 to 7.5 mol. %, from 2.5 to 7.5 mol. %, from 0.1 to 5 mol. %, from 0.25 to 5 mol. %, from 0.5 to 5 mol. %, from 1 to 5 mol. %, from 1.5 to 5 mol. %, from 2 to 5 mol. %, from 2.5 to 5 mol. %, from 0.1 to 3 mol. %, from 0.25 to 3 mol. %, from 0.5 to 3 mol. %, from 1 to 3 mol. %, from 1.5 to 3 mol. %, from 2 to 3 mol. %, from 2.5 to 3 mol. %, from 0.1 to 2.5 mol. %, from 0.25 to 2.5 mol. %, from 0.5 to 2.5 mol. %, from 1 to 2.5 mol. %, from 1.5 to 2.5 mol. %, from 2 to 2.5 mol. %, from 0.1 to 4.5 mol. %, from 0.25 to 4.5 mol. %, from 0.5 to 4.5 mol. %, from 1 to 4.5 mol. %, from 1.5 to 4.5 mol. %, from 2 to 4.5 mol. %, from 2.5 to 4.5 mol. %, from 0.1 to 4 mol. %, from 0.25 to 4 mol. %, from 0.5 to 4 mol. %, from 1 to 4 mol. %, from 1.5 to 4 mol. %, from 2 to 4 mol. %, from 2.5 to 4 mol. %, from 0.1 to 3.5 mol. %, from 0.25 to 3.5 mol. %, from 0.5 to 3.5 mol. %, from 1 to 3.5 mol. %, from 1.5 to 3.5 mol. %, from 2 to 3.5 mol. %, from 2.5 to 3.5 mol. %, from 0.1 to 2.0 mol. %, from 0.1 to 1.5 mol. %, from 0.1 to 1.0 mol. %, from 0.5 to 2.0 mol. %, from 0.5 to 1.5 mol. %, from 0.5 to 1.0 mol. %, from 1.0 to 2.0 mol. %, from 1.0 to 1.5 mol. %, from 1.5 to 2.0 mol. %, 1.0 mol. % or 1.5 mol. %, and a lipid nanoparticle containing a lipid-PEG conjugate in the above range can have excellent drug delivery efficiency specifically to hepatocytes (or targeting to hepatocytes).

In one example, a lipid nanoparticle in a hepatocyte drug delivery composition may contain an ionizable lipid:phospholipid:cholesterol:lipid-PEG conjugate in the range described above, or in a molar ratio of 20 to 50:10 to 30 : 30 to 60 : 0.5 to 5, in a molar ratio of 25 to 45 : 10 to 25 : 40 to 50 : 0.5 to 3, in a molar ratio of 25 to 45 : 10 to 20 : 40 to 55 : 0.5 to 3 or in a molar ratio of 25 to 45 : 10 to 20 : 40 to 55 : 1.0 to 1.5. A lipid nanoparticle containing components in a molar ratio in the above range may have excellent efficiency in delivering a drug specifically to hepatocytes (or targeting hepatocytes).

In one example, a drug delivery composition comprising (1) a lipid nanoparticle and (2) an anionic drug, nucleic acid, or combination thereof may be a drug delivery composition in an LSEC.

When the diameter of the lipid nanoparticle contained in the LSEC drug delivery composition is similar to or slightly smaller than the pore diameter, the LSEC drug delivery effect can be excellent. In one example, the diameter of a lipid nanoparticle in an LSEC drug delivery composition may be 20 to 200 nm, 20 to 180 nm, 40 to 180 nm, 40 to 170 nm, 50 to 160 nm, 70 to 180 nm, 70 to 170 nm, 75 to 170 nm, 75 to 165 nm, 70 to 150 nm, 70 to 130 nm, 75 to 130 nm, 80 to 120 nm, 85 up to 120 nm, about 90 to 120 nm, 90 to 110 nm, 80 to 110 nm, 80 to 100 nm, 85 to 95 nm, about 90 nm or 90 nm.

According to one example, the lipid nanoparticle, which is part of the composition for drug delivery in LSEC, may contain a lipid-PEG conjugate in an amount of from 0.1 to 15 mol. %, from 0.25 to 15 mol. %, from 0.5 to 15 mol. %, from 1 to 15 mol. %, from 1.5 to 15 mol. %, from 2 to 15 mol. %, from 2.5 to 15 mol. %, from 0.1 to 12.5 mol. %, from 0.25 to 12.5 mol. %, from 0.5 to 12.5 mol. %, from 1 to 12.5 mol. %, from 1.5 to 12.5 mol. %, from 2 to 12.5 mol. %, from 2.5 to 12.5 mol. %, from 0.1 to 10 mol. %, from 0.25 to 10 mol. %, from 0.5 to 10 mol. %, from 1 to 10 mol. %, from 1.5 to 10 mol. %, from 2 to 10 mol. %, from 2.5 to 10 mol. %, from 0.1 to 7.5 mol. %, from 0.25 to 7.5 mol. %, from 0.5 to 7.5 mol. %, from 1 to 7.5 mol. %, from 1.5 to 7.5 mol. %, from 2 to 7.5 mol. %, from 2.5 to 7.5 mol. %, from 0.1 to 5 mol. %, from 0.25 to 5 mol. %, from 0.5 to 5 mol. %, from 1 to 5 mol. %, from 1.5 to 5 mol. %, from 2 to 5 mol. %, from 2.5 to 5 mol. %, from 0.1 to 3 mol. %, from 0.25 to 3 mol. %, from 0.5 to 3 mol. %, from 1 to 3 mol. %, from 1.5 to 3 mol. %, from 2 to 3 mol. %, from 2.5 to 3 mol. %, from 0.1 to 2.5 mol. %, from 0.25 to 2.5 mol. %, from 0.5 to 2.5 mol. %, from 1 to 2.5 mol. %, from 1.5 to 2.5 mol. %, from 2 to 2.5 mol. %, from 0.1 to 4.5 mol. %, from 0.25 to 4.5 mol. %, from 0.5 to 4.5 mol. %, from 1 to 4.5 mol. %, from 1.5 to 4.5 mol. %, from 2 to 4.5 mol. %, from 2.5 to 4.5 mol. %, from 0.1 to 4 mol. %, from 0.25 to 4 mol. %, from 0.5 to 4 mol. %, from 1 to 4 mol. %, from 1.5 to 4 mol. %, from 2 to 4 mol. %, from 2.5 to 4 mol. %, from 0.1 to 3.5 mol. %, from 0.25 to 3.5 mol. %, from 0.5 to 3.5 mol. %, from 1 to 3.5 mol. %, from 1.5 to 3.5 mol. %, from 2 to 3.5 mol. %, from 2.5 to 3.5 mol. %, from 0.1 to 2.0 mol. %, from 0.1 to 1.5 mol. %, from 0.1 to 1.0 mol. %, from 0.5 to 2.0 mol. %, from 0.5 to 1.5 mol. %, from 0.5 to 1.0 mol. %, from 1.0 to 2.0 mol. %, from 1.0 to 1.5 mol. %, from 1.5 to 2.0 mol. %, 1.0 mol. % or 1.5 mol. %, and a lipid nanoparticle containing a lipid-PEG conjugate in the above range can have excellent drug delivery efficiency specifically in LSEC (or targeting LSEC).

In one example, a lipid nanoparticle in a composition for drug delivery to an LSEC may contain an ionizable lipid:phospholipid:cholesterol:lipid-PEG conjugate in the range described above, or in a molar ratio of 20 to 50:10 to 30 : 30 to 60 : 0.5 to 5, in a molar ratio of 25 to 45 : 10 to 25 : 40 to 50 : 0.5 to 3, in a molar ratio of 25 to 45 : 10 to 20 : 40 to 55 : 0.5 to 3 or in a molar ratio of 25 to 45 : 10 to 20 : 40 to 55 : 1.0 to 1.5. A lipid nanoparticle containing components in a molar ratio in the above range may have excellent drug delivery efficiency specifically to the LSEC (or targeting to the LSEC).

A lipid nanoparticle according to one example can deliver a therapeutic agent to liver tissue, hepatocytes and/or LSECs with high efficiency due to properties specific to liver tissue, and can be effectively used for a drug for the treatment of liver diseases, in a gene delivery method for treatment and the like, and a therapeutic agent mediated by the liver, which mediate this highly effective liver tissue, hepatocytes and/or LSEC-specific lipid nanoparticles. In addition, the lipid nanoparticle according to one example forms a stable complex with a gene drug such as a nucleic acid and the like, and exhibits low cytotoxicity and efficient uptake by cells, and thus it effectively delivers the gene drug, such as nucleic acid.

The lipid nanoparticle is as described above.

A lipid nanoparticle according to one example exhibits a positive charge under acidic pH conditions, showing a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7, and can encapsulate a drug with high efficiency through easy complexation with a drug through electrostatic interaction with a therapeutic agent such as a nucleic acid and an anionic drug (e.g. protein) having a negative charge, and can be effectively used as a composition for intracellular or in vivo drug delivery means (eg nucleic acid).

Here, "encapsulation" refers to the encapsulation of a substance for delivery for its effective environment and in vivo implementation, and the drug encapsulation efficiency (encapsulation efficiency) means the content of the drug encapsulated in the lipid nanoparticle in relation to the total content of the drug used for preparation.

The anionic or nucleic acid drug encapsulation efficiency of the delivery composition can be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 94% or more. more, more than 80% to 99% or less, more than 80% to 97% or less, more than 80% to 95% or less, 85% or more to 95% or less, 87% or more to 95 % or less, 90% or more to 95% or less, 91% or more to 95% or less, 91% or more to 94% or less, more than 91% to 95% or less, 92% or more or more to 99% or less, 92% or more to 97% or less, or 92% or more to 95% or less.

According to one example, the encapsulation efficiency can be calculated using commonly used methods, and for example, the drug encapsulation efficiency can be calculated according to the following Equation 2, with the treatment of a lipid nanoparticle with Triton-X according to one example, and measuring the fluorescence intensity of the treated with Triton-X and untreated with Triton-X lipid nanoparticles in a specific wavelength band (eg, excitation: about 480-490 nm, emission: about 520-530 nm).

(Equation 2)

Drug encapsulation efficiency (%) = (Fluorescence intensity (fluorescence) of Triton-X treated lipid nanoparticle - Fluorescence intensity (fluorescence) of non-Triton-X treated lipid nanoparticle) / (Fluorescence intensity (fluorescence) of Triton-X treated lipid nanoparticle) × 100

The drug delivery composition of one example may contain Cas9 mRNA with high encapsulation efficiency. The previously known Cas9 mRNA delivery composition has limitations when used as a Cas9 mRNA delivery composition because it contains Cas9 mRNA in a low ratio. On the other hand, the lipid nanoparticle according to one example may contain Cas9 mRNA with high encapsulation efficiency, in particular, an encapsulation efficiency of 70% or more, and thus can be effectively used for gene editing therapy.

The anionic drug may be an anionic biopolymer-drug conjugate such as various kinds of peptides, protein drugs, protein-nucleic acid structures or hyaluronic acid-peptide conjugates, hyaluronic acid-protein conjugates that have an anion, and the like. Non-limiting examples of protein drugs can be apoptosis inducing factors (e.g., cytochrome c, caspase 3/7/8/9, etc.) and include gene editing proteins such as Cas 9, cpf1, which are "scissors" for gene editing, and various intracellular proteins (eg, transcription factors), and the like.

The nucleic acid may be one or more species selected from the group consisting of small interfering RNA (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer , messenger ribonucleic acid (mRNA), transport ribonucleic acid (tRNA), antisense oligonucleotide, short hairpin RNA (shRNA), miRNA, ribozyme, nucleic acid peptide (PNA), DNAzyme and single guide RNA (sgRNA) for gene editing, etc. .p., but not limited to.

Here, the term "siRNA" refers to a double-stranded RNA (duplex RNA) that can induce RNAi (RNA interference) by cleaving a specific mRNA, or a single-stranded RNA that has a double-stranded form within a single-stranded RNA. It consists of an RNA sense strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having its complementary sequence. Since siRNA can inhibit target gene expression, it is provided by an efficient gene knockdown technique or gene therapy technique. Bonding between double strands is accomplished by hydrogen bonds between nucleotides, and not all nucleotides within a double strand need to be complementary and fully linked.

The length of the siRNA can be from about 15 to 60, in particular from about 15 to 50, from about 15 to 40, from about 15 to 30, from about 15 to 25, from about 16 to 25, from about 19 to 25, from about 20 to 25 or about 20 to 23 nucleotides. The siRNA length means the number of nucleotides on one side of the double-stranded RNA, i.e. the number of base pairs, and in the case of single-stranded RNA, means the length of the double strand within the single-stranded RNA. In addition, siRNA may be composed of nucleotides introduced with various functional groups in order to increase blood stability or weaken the immune response, and the like.

Here, the term "antisense oligonucleotide" may be modified at the position of one or more bases, sugars, or scaffolds to improve efficiency (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). The oligonucleotide backbone can be modified with phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl, cycloalkyl, short chain heteroatom heterocyclic intersaccharide coupling, and the like. In addition, the antisense oligonucleotide may contain one or more protected sugar moieties. The antisense oligonucleotide may contain a modified base. The modified base includes hypoxanthine, 6-methyladenine, 5-me-pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl-HMC, gentiobiosyl-HMC, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 2,6-diaminopurine, and the like.

Here, "single-stranded deoxyribonucleic acid (ssDNA)" means a single-stranded oligonucleotide that selectively binds to a specific target DNA and induces an antigenic effect.

Here, "aptamer" means an oligonucleotide (typically about 20-80 nucleotides of DNA or RNA) that binds to a specific target. Preferably here "aptamer" means an oligonucleotide aptamer (eg DNA or RNA aptamer).

Here, "mRNA" means a synthetic mRNA (mRNA transcribed in vitro) capable of expressing a gene.

Here, "shRNA" means a single-stranded RNA of 50 to 70 nucleotides in length and forms a stem-loop (stem/loop) structure in vivo. On either side of the 5 to 10 nucleotide loop, a complementary long 19 to 29 nucleotide long RNA pairs to form a double-stranded stem.

Here "miRNA (miRNA)" means a single-stranded RNA molecule that controls gene expression and consists of 21-23 nucleotides in full length. miRNA is an oligonucleotide that is not expressed in the cell and has a short stem-loop structure. miRNA has full or partial homology with one, two or more mRNA (messenger RNA) and suppresses the expression of the target gene due to complementary binding to mRNA.

Here, "ribozyme" is a type of RNA, and is an RNA that recognizes the nucleotide sequence of a particular RNA and performs the same function as an enzyme that cuts it by itself. A ribozyme is the complementary nucleotide sequence of a target messenger RNA strand and consists of a region that specifically binds and a region that cuts the target RNA.

Here, "DNAzyme" is a single-stranded DNA molecule with enzymatic activity, and DNAzyme, consisting of 10-23 nucleotides (DNAzyme 10-23), cuts the RNA chain at a certain position under physiologically similar conditions. DNAzyme 10-23 cuts any reactive purines and pyrimidines without base pairing. DNAzyme 10-23 consists of an active site (catalytic domain) of the enzyme, consisting of 15 conserved nucleotide sequences (for example, 5'-GGCTAGCTACAACGA-3'), and an RNA substrate binding domain of approximately 7-8 nucleotide sequences of DNA, which recognize RNA substrates to the left and right of the active domain of the enzyme described above.

Here, "PNA (peptide-nucleic acid)" is a molecule having all the properties of nucleic acids and proteins, and means a molecule capable of complementary binding to DNA or RNA. PNA was first reported in 1999 as analogous to DNA in which the nitrogenous bases are linked by peptide bonds (document [Nielsen PE, Egholm M, Berg RH, Buchardt O, "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide", Science 1991, Vol. 254: pp 1497-1500]). PNA does not occur in nature and is artificially synthesized by a chemical method. PNA induces a hybridization reaction with the natural nucleic acid of a complementary nucleotide sequence to form a double strand. PNA/DNA double strands are more stable than DNA/DNA double strands of the same length. N-(2-aminoethyl)glycine, which is repeatedly linked by amide bonds, is most often used as a peptide backbone, and in this case, the peptide-nucleic acid backbone is electrically neutral, in contrast to the natural nucleic acid backbone. The 4 nucleotides present in PNA have almost the same spatial size and distance between nucleotides as in the case of natural nucleic acid. PNA is not only chemically more stable than natural nucleic acid, but also biologically stable because it is not cleaved by nuclease or protease.

Here, "sgRNA" is an oligonucleotide (usually an RNA molecule) that binds to a specific DNA target, and means a complex single RNA molecule consisting of RNA with regularly alternating short palindromic repeats (crispr RNA) and trans-encoded crRNA (tracrRNA). It is an RNA molecule that is used to recognize a specific DNA sequence using the Cas9 nuclease in the CRISPR system and provides selective gene cleavage, and contains approximately 20 nucleotide (nt) sequence capable of complementary binding to DNA, and has a total length of 100 nt.

Here, "gene editing protein" refers to Cas9, spCas9, cjCas9, casX, CasY, and Cpf1, and the like. and refers to a protein that recognizes the nucleotide sequence of a target DNA using sgRNA to cause DNA cleavage.

The target cell into which the drug and/or nucleic acid is delivered by the lipid nanoparticle according to one example may be a hepatocyte and/or LSEC in vivo or isolated in vivo. A drug delivery composition and/or complex of drug (anionic drug, nucleic acid, or combinations thereof) and lipid nanoparticles according to one example can target or specifically target a hepatocyte and/or LSEC. Accordingly, a lipid nanoparticle or drug or nucleic acid delivery composition comprising a lipid nanoparticle according to one example may be for the treatment of acute or chronic liver diseases such as liver fibrosis, cirrhosis, hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.), and, in addition, it can be used as a composition for the delivery of a therapeutic agent (drug) absorbed through the liver.

In another aspect, a pharmaceutical composition for the prevention or treatment of a liver disease is provided, comprising (1) a lipid nanoparticle and (2) an anionic drug, a nucleic acid, or a combination thereof.

The lipid nanoparticle included in a pharmaceutical composition for preventing or treating a liver disease is the same as the lipid nanoparticle included in the aforementioned drug delivery composition.

The anionic drug and nucleic acid included in the pharmaceutical composition for preventing or treating liver disease are the same as the anionic drug and nucleic acid included in the aforementioned drug delivery composition.

A pharmaceutical composition for the prevention or treatment of a liver disease, according to one example, may contain a lipid nanoparticle encapsulated with an anionic drug and/or a nucleic acid.

The liver disease may be one or more selected from the group consisting of ATTR amyloidosis, hypercholesterolemia, hepatitis B virus infection, acute liver failure, cirrhosis, and liver fibrosis.

The anionic drug may have the effect of preventing or treating a liver disease.

The nucleic acid may have the effect of preventing or treating a liver disease and, for example, it may be an siRNA and/or miRNA that can inhibit expression such as (1) TTR (transthyretin) ((e.g. human TTR (protein: accession numbers in GenBank NP_000362.1; gene: GenBank accession number HM_000371.4 etc.), mouse TTR (protein: GenBank accession number; NP_038725.1; gene: GenBank accession number HM_013697.5 etc.) ), (2) PCSK9 (proprotein convertase subtilisin/kexin type 9) ((e.g., human PCSK9 (protein: GenBank accession number NP_777596.2; gene: GenBank accession number HM_174936.4, etc.), mouse PCSK9 ( protein: GenBank accession number NP_705793.1, gene: GenBank accession number HM_153565.2, etc.), (3) HBV (hepatitis B virus) (for example, HBV genotype A (for example, GenBank accession number: X02763 , X51970, or AF090842); HBV genotype B (eg, GenBank accession number: D00329, AF100309, or AB033554); HBV genotype C (eg, GenBank accession number: X04615, M12906, AB014381, AB042285, AB042284, AB0 42283, АВ042282, AB026815, AB026814, AB026813, AB026812 or AB026811); HBV genotype D (eg, GenBank accession number: X65259, M32138, or X85254); HBV genotype E (for example, GenBank accession number: X75657 or AB032431); HBV genotype F (for example, GenBank accession number: X69798, AB036910, or AF223965); HBV genotype G (eg, GenBank accession number: AF160501, AB064310, or AF405706), HBV genotype H (eg, AY090454, AY090457, or AY090460) (4) Bax (BCL2 associated X)) ((eg, human Bax (protein: numbers GenBank access numbers NP_001278357.1, NP_001278358.1, NP_001278359.1, NP_001278360.1, NP_004315.1; gene: GenBank access numbers HM_001291428.2, HM_001291429.2, HM_00129 1430.1, HM_001291431.2, HM_004324.4, and etc.), mouse Bax (protein: GenBank accession number NP_031553.1; gene: GenBank accession number HM_007527.3, etc.), (5) VEGF (vascular endothelial growth factor) (e.g., VEGFA ((e.g. human VEGFA (protein: GenBank access numbers NP_001020537.2, NP_001020538.2, NP_001020539.2, NP_001020540.2, NP_001020541.2; gene: GenBank access numbers HM_003376.6, HM_0010253 66.3, HM_001025367.3 , HM_001025368.3, HM_001025369.3, etc.), mouse VEGFA (protein: GenBank accession number NP_001020421.2, NP_001020428.2, NP_001103736.1, NP_001103737.1, NP_0011037 38.1; gene: access number in GenBank HM_001025250.3, HM_001025257.3, HM_001110266.1, HM_001110267.1, HM_001110268.1, etc.); VEGFB ((e.g. human VEGFB (protein: GenBank accession numbers NP_001230662.1, NP_003368.1; gene: GenBank accession numbers HM_003377.5, HM_001243733.2, etc.), mouse VEGFB (protein: GenBank accession number GenBank NP_001172093.1, NP_035827.1; gene: GenBank access number HM_001185164.1, HM_011697.3, etc.) VEGFC (e.g. human VEGFC (protein: GenBank access number NP_005420.1; gene: access number in GenBank HM_005429.5, etc.), mouse VEGFC (protein: GenBank accession number; NP_033532.1; gene: GenBank accession number HM_009506.2, etc.)), and/or (6) PDGF (platelet growth factor) (e.g., PDGFA ((e.g., human PDGFA (protein: GenBank accession numbers NP_002598.4, NP_148983.1; gene: GenBank accession numbers HM_002607.5, HM_033023.4, etc.) , mouse PDGFA (protein: GenBank accession number NP_032834.1, NP_001350200.1; gene: GenBank accession number HM_008808.4, HM_001363271.1, etc.)); access to GenBank NP_002599.1, NP_148937.1; gene: GenBank accession number HM_033016.3, HM_002608.4, etc.), mouse PDGFB (protein: GenBank accession number NP_035187.2; gene: GenBank accession number HM_011057.4, etc.)); PDGFC ((e.g., human PDGFC (protein: GenBank access numbers NP_057289.1; gene: GenBank access numbers HM_016205.3, etc.), mouse PDGFC (protein: GenBank access numbers NP_064355.1, NP_001344675.1 ; gene: GenBank accession number HM_019971.3, HM_001357746.1, etc.)); .4, HM_025208.5, etc.), mouse PDGFD (protein: GenBank accession number NP_082200.1, NP_001344326.1, NP_001344327.1; gene: GenBank accession number HM_027924.3, HM_001357397.1, HM_001357 398. 1, etc.)).

The pharmaceutical composition can be administered in various ways, including parenteral administration to mammals, including humans, and parenteral administration can be administered intravenously, subcutaneously, intraperitoneally, or topically, and the dosage varies depending on the condition and body weight of the patient, the degree of disease, the form of the drug, the method administration and timing, but may be appropriately selected by those skilled in the art.

When preparing a pharmaceutical composition according to one example, it is prepared using a diluent or excipient such as filler, filler, binder, wetting agent, disintegrant, surfactant, and the like, which are commonly used.

The composition for parenteral administration includes a sterilized aqueous solution, a non-aqueous solvent, a suspended solvent, an emulsion, a lyophilized composition, a suppository, and the like.

As the non-aqueous solvent and the suspended solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, an injectable ester such as ethyl oleate, and the like can be used. Witepsol, macrogol, tween 61, cocoa butter, lauric oil, glycerin, gelatin and the like are used as the basis of the suppository.

The pharmaceutical composition according to one example is administered at a pharmaceutically effective dose. Here, "pharmaceutically effective dose" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the level of effective dose may be determined depending on factors including the type of disease of the patient, severity, drug activity, sensitivity to drug, time of administration, route of administration and rate of elimination, period of treatment and concomitant drugs, as well as other factors well known in the medical field. The pharmaceutical composition according to one example may be administered as a single therapeutic agent, or may be administered in combination with other therapeutic agents, and may also be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. It is important to administer the amount that can provide the maximum effect with the minimum amount without side effects, taking into account all of the above factors, and this can be easily determined by those skilled in the art.

In particular, an effective dose of the compound of the present invention may vary depending on the age, sex and body weight of the patient, and may be administered daily or every other day, or administered by division from 1 to 3 times a day. However, it may be increased or decreased depending on the route of administration, severity of obesity, sex, body weight, age, and the like, and therefore, the above dose does not limit the scope of the present invention in any way.

In one specific example, the pharmaceutical composition may be administered at a dose of 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 1 to 10 mg/kg, or 1 to 5 mg/kg, based on drug concentration. agent (anionic drug, nucleic acid or combination thereof) contained in the pharmaceutical composition.

Another aspect relates to a method for preventing or treating liver disease, comprising administering a composition (eg, a pharmaceutical composition for preventing or treating liver disease) containing (1) a lipid nanoparticle and (2) an anionic drug, a nucleic acid, or a combination thereof. The lipid nanoparticle, the anionic drug, the nucleic acid, the pharmaceutical composition, and the liver disease are as described above.

According to one example, the method for preventing or treating a liver disease may further comprise confirming (selecting) a patient in need of preventing and/or treating a liver disease prior to administering the composition.

The subject to which the method of treatment is applied means a mammal, including mice, livestock, and the like, including humans, which has or may have a liver disease, but is not limited to this. A pharmaceutical composition containing a lipid nanoparticle according to one example can effectively deliver an anionic drug and/or nucleic acid to the liver and thus can effectively treat a subject.

In one example, a method for preventing or treating liver disease can be provided, comprising administering to a patient a pharmaceutically effective dose of a composition (e.g., a pharmaceutical composition for preventing or treating liver disease) comprising (1) a lipid nanoparticle and (2) an anionic drug, a nucleic acid, or their combination. A pharmaceutically effective dose is as described above and a suitable total daily dose may be determined by treatment within sound medical judgment and may be administered in a single dose or divided into multiple doses. However, a particular therapeutically effective dose for a particular patient will be administered differently depending on various factors, including the particular composition, the patient's age, body weight, general health, sex and diet, time of administration, route of administration and rate of elimination of the composition, period of treatment. and drugs used with or simultaneously with a particular composition, in addition to the type and extent of response to be achieved and whether another agent is used, if necessary, and similar factors well known in the pharmaceutical field.

BENEFICIAL EFFECTS

According to one example, the lipid nanoparticle is specific to liver tissue, has excellent biocompatibility, and can deliver a gene therapy agent with high efficiency, and thus can be effectively used in related technical fields such as lipid nanoparticle mediated gene therapy.

BRIEF DESCRIPTION OF GRAPHICS

On FIG. 1a shows an example of the structure of a lipid nanoparticle according to one example, and FIG. 16 shows an image of an observation of a nanoparticle according to one example using Cryo-TEM.

On FIG. 2 shows the result of ¹ H NMR (room temperature, 400 MHz) of 246-C10 in CDCl ₃ .

On FIG. 3a (241-C10 LNP to 243-C10 LNP) and FIG. 3b (from 244-C10 LNP to 246-C10 LNP) shows the result of measuring the fluorescence intensity of each lipid nanoparticle in a solution having a pH range of pH 4.1 to pH 9.6.

On FIG. 4a and Fig. 4b presents the results showing the efficiency of intracellular gene delivery of each nanoparticle. In particular, in FIG. 4a shows the luminescence intensity measured upon transformation of LNP-encapsulated luciferase-encoding mRNA (luc mRNA) into a HeLa cell followed by cell dissolution, and FIG. 4b shows the luminescence intensity measured upon transformation of mRNA encapsulated in LNP luc into a hepatocyte followed by cell dissolution. On FIG. 4b, +ApoE refers to the ApoE3-treated group, and -ApoE refers to the ApoE3-untreated group.

On FIG. 5a shows drug distribution upon in vivo delivery in a mouse treated with 244-C10 LNP to 246-C10 LNP with encapsulated Luc mRNA, and FIG. 5b shows the distribution of the drug upon delivery to each organ of a mouse removed from a mouse injected with 246-C10 LNP with encapsulated Luc mRNA.

On FIG. 6 shows the drug delivery efficiency of the lipid nanoparticle and the size of the nanoparticle as a function of the lipid-PEG content of the lipid nanoparticle in a mouse injected with 246-C10 LNP containing 1.0 to 2.5 mol of lipid-PEG. . %.

On FIG. 7 shows the result of confirming the ability to target hepatocytes depending on the concentration of siFVII administered encapsulated in a lipid nanoparticle by FVII expression.

On FIG. 8 shows the size of the lipid nanoparticle and the PDI value of the lipid nanoparticle as a function of the content of the lipid-PEG constituting the lipid nanoparticle (left table), and shows the result of confirming the efficiency of in vivo drug delivery to the hepatocyte by FVII expression (right graph).

On FIG. 9 shows the size of the lipid nanoparticle and the PDI value of the lipid nanoparticle as a function of the content of the lipid-PEG constituting the lipid nanoparticle (left table), and shows the result of confirming in vivo drug delivery efficiency in LSEC by FVIII expression (right graph).

On FIG. 10 shows the result of measuring the efficiency of intracellular siRNA delivery of a lipid nanoparticle containing ceramide-PEG or DSPE-PEG.

METHOD FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail using the following examples, but its scope is not limited to the following examples.

Hereinafter, the present invention will be described in more detail by examples. These examples are only intended to describe the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples in accordance with the gist of the present invention.

Example 1 Preparation of Ionizable Lipids

Example 1-1. Obtaining ionizable lipids

Ionizable lipids were synthesized by reacting amine-based compounds of Table 1 below, comprising a 6-membered heterocyclic tertiary amine, and 1,2-epoxydecane (hereinafter C10) (Sigma-Aldrich, USA) in a 1:n molar ratio (n=primary amine × 2 + secondary amine × 1).

Specifically, each of the amines 241 to 246 of Table 1 and the epoxide (C10) were added in a 1:n molar ratio (n = primary amine × 2 + secondary amine × 1) in a 5 ml vial with a magnetic bar and subjected to interaction in a stirrer at 750 rpm and 90°C for 3 days. Then, after purification on a WELUX fine silica column (Intertec, Korea), the molecular weight of each ionizable lipid resulting from this interaction was calculated and stored at a concentration of 100 mg/ml using ethanol. The ionizable lipid made using amine 241 and C10 was named "241-C10" and other ionizable lipids made using other kinds of amines were named in the same way, i.e. "name of the amine used (241-246)-C10".

Example 1-2. Confirmation of the obtained ionizable lipids

1H NMR was performed to confirm the ionizable lipids obtained in Example 1-1. In particular, 5 μg of the ionizable lipid (246-C10) synthesized in Example 1-1 was prepared by dilution in CDCl ₃ (sigma, USA) at a concentration of 0.5 ml to 100 mmol. Then, 0.5 ml was placed in a 400 MHz NMR tube and the top was sealed and then sealed with parafilm to obtain NMR spectra using an Agilent 400MHZ FT-HMR (Agilent, USA) and the result is shown in FIG. 2. As shown in FIG. 2, it can be seen that the signal representing each 246-C10 functional group was saturated.

In addition, MS analysis was performed to confirm the ionizable lipids (241-C10 to 246-C10) obtained in Example 1-1. Specifically, ionizable lipids were diluted with ethanol to a concentration of 0.5 ppm or less, and MS analysis was performed. For analysis, Agilent Technologies 6230 LC/MS equipment (Palo Alto, USA) and Zorbax SB-C18 (100 mm × 2.1 mm, inner diameter, 3.5 µm) from Agilent Technologies were used, which were used for the separation tube, and carried out a gradient elution with two solvents: distilled water (A) containing 0.1% formic acid, and acetonitrile (B). The mobile phase solvent gradient was maintained for 4 minutes until the organic solvent ratio of acetonitrile (B) increased initially from 30% to 80% over 2 minutes, followed by reduction of the organic solvent ratio back to 30%, and stabilized. The mobile phase flow rate was 300 μl/min and then the analyzer injection volume was 2 μl. The result of the MS analysis is shown in Table 2 below. As shown in Table 2, it was possible to confirm that the measured m/z ratio and the calculated m/z ratio of ionizable lipids were almost identical.

Based on this result, it was possible to confirm that ionizable lipids were indeed obtained in Example 1-1.

Example 2 Preparation of lipid nanoparticles

Example 2-1. Preparation of lipid nanoparticles

Ionizable lipids (241-C10 to 246-C10) obtained in Example 1-1, cholesterol (cholesterol powder, BioReagent suitable for cell culture, ≥99%, sigma, Korea), phospholipid (DSPC) (Avanti, USA) and a lipid-PEG conjugate (ceramide-PEG conjugate; C16 PEG2000 Ceramide, Avanti, USA) was dissolved in ethanol in a molar ratio of 42.5:13:43:1.5.

Ethanol, in which ionizable lipids, cholesterol, phospholipid, and lipid-PEG were dissolved, and acetate buffer were mixed using a microfluidic mixer (Benchtop Nanoassemblr; PNI, Canada) at a flow rate of 12 ml/min in a volume ratio of 1:3, thus thereby obtaining lipid nanoparticles (LNP).

Example 2-2. Production of lipid nanoparticles with encapsulated nucleic acids

Ionizable lipids (241-C10 to 246-C10) obtained in Example 1-1, cholesterol (cholesterol powder, BioReagent suitable for cell culture, ≥99%, sigma, Korea), phospholipid (DSPC or DOPE) (18: 0 PC (DSPC), 18:1 (Δ9-Cis) PE (DOPE), Avanti, USA, and lipid-PEG conjugate (ceramide-PEG conjugate; C16 PEG2000 Ceramid, Avanti, USA) were dissolved in ethanol. , mRNA (luciferase mRNA; SEQ ID NO: 1) in an amount of 30 μg was diluted in 0.75 ml of sodium citrate, or siRNA (siFVII; SEQ ID NO: 2 and 3 were mixed in the same molar ratio, or siFVIII; SEQ ID NO: 4 to 11 were mixed in the same molar ratio, or siLuc: SEQ ID NOs: 12 and 13 were mixed in the same molar ratio) in an amount of 30 μg was diluted in 0.75 ml of sodium acetate (50 mm) to prepare an aqueous phase.

The siRNA sequences used were as follows: SEQ ID NO: 2 (FVII target siRNA_sense; 5'-GGAUCAUCUCAAGUCUUACdtdt-3'), SEQ ID NO: 3 (FVII target siRNA_antisense; 5'-GUAAGACUUGAGAUGAUCCdtdt-3'), SEQ ID NO: 4 ( FVIII target miRNA_sense_1; 5'CUUAUAUCGUGGAGAAUUAdtdt-3') SEQ ID NO: 5 (FVIII target miRNA_antisense_1; 5'-UAAUUCUCCACGAUAUAAGdtdt-3'), SEQ ID NO: 6 (FVIII target miRNA_sense_2; 5'-UCAAAGGAUUCGAUGGU AUdtdt-3'), SEQ ID NO: 7 (FVIII targeted minerink_antismed_2; 5'-Auaccauchaucuucuugadtttt-3 '), Seq ID No: 8 (FVIII targeted minerals_3; 5'-cAAAGAAGUAUUUDTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'), seQ ID NO: 9 (FVIII I targeted minerink_antismed_3; 5'- AUAAUCACUAGUGCUCUUGdtdt-3'), SEQ ID NO: 10 (FVIII target miRNA_sense_4; 5'-GGGCACCACUCCUGAAAUAdtdt-3'), SEQ ID NO: 11 (FVIII target miRNA_antisense_4; 5'-UAUUUCAGGAGUGGUGCCCdtdt-3'), SEQ ID NO: 12 ( siLuc sense; 5'-AACGCUGGGCGUUAAUCAAdtdt-3'), SEQ ID NO: 13 (siLuc_antisense; 5'-UUGAUUAACGCCCAGCGUUdtdt-3').

An aqueous phase (sodium acetate or sodium citrate) in which an organic phase (ethanol) has been dissolved, in which ionizable lipids, cholesterol, a phospholipid and a lipid-PEG conjugate (hereinafter referred to as lipid-PEG) have been dissolved, and an RNA therapeutic agent (nucleic acid) has been dissolved acid) was mixed using a microfluidic mixer (Benchtop Nanoassemblr; PNI, Canada) at a flow rate of 12 ml/min to obtain lipid nanoparticles (LNP) in which the nucleic acid was encapsulated. (1) To obtain a lipid nanoparticle in which mRNA is encapsulated, ionizable lipid : phospholipid (DOPE) : cholesterol : lipid-PEG (ceramide C16-PEG2000) was dissolved in ethanol in a molar ratio of 26.5 : 20: from 52.5 to 51 : 1.0 to 2.5 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100), and the organic phase and the aqueous phase are mixed so that the mass ratio of mRNA (luciferase mRNA; SEQ ID NO: 1 ): ionizable lipid was 1:10, and thus a lipid nanoparticle was obtained. (2) To obtain a lipid nanoparticle encapsulating siRNA, ionizable lipid : phospholipid (DSPC) : cholesterol : lipid-PEG (ceramide C16-PEG2000) was dissolved in ethanol in a molar ratio of 42.5 : 13 : 44 to 39.5 : 0.5 to 5.0 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100), and the organic phase and the aqueous phase are mixed so that the mass ratio of siRNA (siFVII; SEQ ID NO: 2 and 3 were mixed in the same molar ratio, or siFVIII; SEQ ID NOs: 4-11 were mixed in the same molar ratio, or siLuc: SEQ ID NOs: 12 and 13 were mixed in the same ratio): ionizable lipid was 1:7.5 , and thus a lipid nanoparticle (LNP) was obtained.

The resulting LNPs were dialyzed against PBS for 16 hours using a 3500 MWCO dialysis cassette to remove ethanol and adjust body pH and nanoparticle pH.

The lipid nanoparticles containing the ionizable lipid "241-C10" were named "241-C10 LNP", and the lipid nanoparticles prepared using the amine-containing ionizable lipid (including the lipid nanoparticles in which the nucleic acid was encapsulated) were named "containing amine name (from 214 to 246)-С10 LNP".

Example 2-3. Results of Observations of Lipid Nanoparticles with Encapsulated Nucleic Acids

Lipid nanoparticles encapsulated with siLuc (SEQ ID NOs: 12 and 13) were prepared using a ceramide-PEG conjugate (Ceramide C16-PEG2000) as in Example 2-2. The resulting lipid nanoparticles (containing 1.5 mole % ceramide-PEG conjugate) were coated onto a Cu-grid 200 mesh carbon film at 60 µg based on siRNA concentration and immersed in vitrobot liquefied ethane (about -170 degrees or less ) and deep-frozen until prepared and then observed with Cryo-TEM (Tecnai F20, FEI) and the result is shown in FIG. 16. As shown in FIG. 16, solid-shaped spherical particles were observed.

Example 3 pKa of lipid nanoparticles

In the present example, the pKa of each lipid nanoparticle (LNP) obtained in Example 2-1 was calculated using in vitro TNS analysis. Anionic TNS becomes lipophilic when interacting with a positively charged ionizable lipid, and as the pH value approaches the pKa value of each LNP, the lipophilicity of TNS decreases and more water molecules quench TNS fluorescence, and hence lipid nanoparticles having a pKa of 6.0 to 7.0 have excellent in vivo drug delivery efficiency, and the lipid nanoparticles exhibiting an "s-curve" in the fluorescence versus pH plot means that they easily interact with the endosome membrane and can easily leave the endosome during acidification. .

More specifically, the pH of a solution containing 20 mM sodium phosphate, 25 mM citrate, 20 mM ammonium acetate and 150 mM NaCl with 0.1 N. NaOH and/or 0.1 N. HCl with an interval of 0.5 from pH 4.1 to pH 9.6 for the preparation of solutions of various pH units. 100 μl of each solution having each pH (pH in the range of 0.5 from pH 4.1 to pH 9.6) was added to a black 96-well plate, and each was added to the solution having a pH in the range to obtain the final concentration 6 µmol using stock solution TNS 300 µmol. Lipid nanoparticles from 241-C10 LNP to 246-C10 LNP were added to the mixed solution so that the final concentration was 20 micromoles. Fluorescence intensity was measured by excitation at 325 nm and emission at 435 nm with Tecan equipment, and the fluorescence intensity for each lipid nanoparticle was displayed in FIG. 3a and Fig. 3b, and the pKa for each lipid nanoparticle was calculated as the pH value reaching half of the maximum fluorescence, and the result is shown in Table 3 below. As shown in FIG. 3b, it can be seen that lipid nanoparticles from 244-C10 LNP to 246-C10 LNP exhibit an s-shaped fluorescent titration curve by non-linear regression.

As shown in Table 3, it was confirmed that the lipid nanoparticles according to one example showed a pKa range of 6.0 to 7.0, in which in vivo safety and drug release are excellent.

The LNPs in which the nucleic acid was encapsulated, obtained by the method of Example 2-2, also showed the same pattern depending on the type of ionizable lipids contained (the type of amine contained in the ionizable lipids).

Example 4 Characterization of Lipid Nanoparticles

Example 4-1. Particle size measurement

In the present example, the size of the lipid nanoparticles (LNP; containing 1.5 mole % lipid-PEG) into which the mRNA obtained in Example 2-2 was encapsulated was measured. It was diluted with PBS so that the concentration of RNA (luciferase mRNA; SEQ ID NO: 1) contained in each lipid nanoparticle obtained in Example 2-2 was 1 μg/ml, and the diameter and polydispersity index (PDI) of the LNP were measured using dynamic light scattering (DLS) in a Malvern Zetasizer Nano (Malvern Instruments, UK) and the result is described in Table 4 below.

As confirmed in Table 4, the lipid nanoparticles according to one example showed a particle size that is easily introduced into hepatocytes and provides excellent drug release, and it could be found that the PDI values were small and the particles were uniform in the order of 241-C10 LNP > 243-С10 LNP > 242-С10 LNP = 245-С10 LNP > 244-С10 LNP > 246-С10 LNP.

Example 4-2. Measuring Encapsulation Efficiency

The encapsulation efficiency (drug encapsulation efficiency, %) of each LNP (containing 1.5 mole % lipid-PEG) in which the siRNA (siFVII siRNA) was encapsulated as a nucleic acid drug was measured using the Ribogreen assay (Quant-iT™ RiboGreen® RNA, Invitrogen). The LNPs encapsulated with the nucleic acid drug obtained in Example 2-2 were diluted with 1×TE buffer 50 µl in a 96-well plate so that the final siRNA concentration was about 4-7 µg/ml for the non-Triton treated group. -X (Triton-x LNP(-)), 1×TE buffer 50 μl was added, and for the Triton-X (Triton-x LNP(+)) treated group, 50 μl of 2% Triton-X buffer was added. Upon incubation at 37° C. for 10 minutes, the encapsulated nucleic acid is released by LNP degradation with Triton-X. Then 100 µl Ribogreen reagent was added to each well. The fluorescence intensity (FL) of Triton LNP(-) and Triton LNP(+) was measured by the wavelength bandwidth (excitation: 485 nm, emission: 528 nm) in an Infinite® 200 PRO NanoQuant (Tecan), and drug encapsulation efficiency (efficiency encapsulation %) was calculated according to the following Equation 3. The drug encapsulation efficiency (%) for each LNP is shown in Table 5 below as the average of the results measured in duplicate.

(Equation 3)

Drug Encapsulation Efficiency (%) = (Triton LNP(+) Fluorescence Intensity Triton LNP(-) Fluorescence Intensity) / (Triton LNP(+) Fluorescence Intensity) × 100

As shown in Table 5, it was confirmed that the lipid nanoparticles according to one example can encapsulate a drug with high efficiency.

Example 5 Confirmation of Intracellular Nucleic Acid Delivery Using Lipid Nanoparticles

Example 5-1. The effect of nucleic acid delivery depending on the types of ionizable lipids that make up the LNP

The day before transfection, LNP according to one example into HeLa cells (Korea Cell Line Bank) was aliquoted at 0.01×10 ⁶ cells/well in a white plate (96 wells) and cultured at 37°C, about 0.5- 3% CO ₂ in DMEM (SH30022, Hyclone, USA). After mixing LNPs (from 241-C10 LNP to 246-C10 LNP containing 1.5 mol.% lipid-PEG), in which mRNA (luc mRNA; SEQ ID NO: 1) encoding the luciferase gene with ApoE3, 0.1 µg/ml, by pipetting and then incubating at room temperature for 10 minutes, they were processed (1 oong/well per mRNA contained in the lipid nanoparticles) in HeLa cells. ApoE3 binds to the LNP surface and plays a role in allowing LNP to enter the cell via endocytosis via the LDL receptor expressed on the cell surface.

After 24 hours, after treatment with 100 µl/well of Bright-Glo™ Luciferase Assay solution (promega, USA) and keeping them at room temperature for 10 minutes, the luminescence intensity of dissolved cells was measured using an Infinite M200 luminescence measuring device (Tecan, USA), and the result is shown in Fig. 4a. As shown in FIG. 4a, 244-C10 LNP, 245-C10 LNP, and 246-C10 LNP having a pKa range of 6.0 to 7.0 showed strong luminescence intensity, and among them, 246-C10 LNP had the highest luminescence intensity, and hence it can be seen that 246-C10 LNP has the highest intracellular drug delivery efficiency.

Example 5-2. Confirmation of nucleic acid delivery to hepatocytes

Luminescence intensity was measured by delivering luc mRNA to hepatocytes using the 246-C10 lipid nanoparticles obtained in Example 2-2, thereby confirming gene expression.

More specifically, after combining 246-C10 LNP (containing 1.5 mol% lipid-PEG) in which luc mRNA (SEQ ID NO: 1) was encapsulated with ApoE3 5 μg/ml, LNP was introduced into a hepatocyte cell line (Nexel , Korea), aliquots of 1×105 cells/well at 0.2 μg/well, 0.5 μg/well, or 1 μg/well, depending on the concentration of mRNA contained in the nanoparticle. After 6 hours, treated with Bright-Glo™ Luciferase Assay (promega, USA) 100 μg/well and left at room temperature for 10 minutes, and then measured the luminescence intensity of dissolved cells using an Infinite M200 luminescence meter. (Tecan, USA) and the result is shown in FIG. 4b.

As shown in FIG. 4b, it was confirmed that the lipid nanoparticle according to one example was easily introduced into cells by binding to ApoE3, increased the amount of delivered drug (nucleic acid) depending on the concentration, and could deliver the drug to hepatocytes with high efficiency.

Example 6 Confirmation of In Vivo Expression Using Lipid Nanoparticles

As confirmed in Example 5-1, in vivo drug delivery efficiency and biodistribution from 244-C10 LNP to 246-C10 LNP showing excellent gene expression effect (gene delivery effect) in vitro was confirmed in the present example.

244-C10 to 246-C10 LNP lipid nanoparticles (containing 1.5 mol % lipid-PEG) were prepared into which luc mRNA (SEQ ID NO: 1) was encapsulated according to the method of Example 2-2, and each nanoparticle was subjected to dialysis in PBS for 16 hours to remove ethanol. 3 hours after intravenous (i.v.) administration of the lipid nanoparticle encapsulated with mRNA to 7-week-old female C57BL/6 mice (Orient Bio) in the amount of 0.1 mg/kg per mRNA contained in the lipid nanoparticle , luciferin 0.25 mg/kg was injected intraperitoneally, bioluminescence was confirmed using IVIS equipment (PerkinElmer, USA), and the result was displayed in FIG. 5a.

Mice injected with LNP 246-C10 encapsulated luc mRNA were sacrificed and organs were harvested, and the biodistribution of lipid nanoparticles was confirmed in each organ by IVIS equipment, and the result is shown in FIG. 5 B.

As shown in FIG. 5a, mice injected with lipid nanoparticles from 244-C10 LNP to 246-C10 LNP with encapsulated luc mRNA showed high luminescence intensity, which is consistent with the result of Example 5-1. In particular, as shown in FIG. 5a and Fig. 5b, it was confirmed by system imaging and ex vivo organ imaging that LNP 246-C10 with encapsulated luc mRNA showed a high luminescence intensity, especially in the liver, and thus it could be confirmed that the lipid nanoparticle according to one example showed high biodistribution in the liver.

Example 7. Confirmation of the composition ratio of lipid nanoparticles optimal for delivery of nucleic acids

In the present example, it was necessary to confirm the ratio of the composition of lipid nanoparticles with the highest efficiency of drug delivery, especially to the liver, in vivo.

In the production of a lipid nanoparticle, a lipid nanoparticle (246-C10 LNP) into which luc mRNA (SEQ ID NO: 1) was encapsulated was obtained according to the method of Example 2-2 by mixing lipid-PEG (C16-PEG2000 ceramide) from 1.0 to 2.5 mol. %. The mass ratio of ionizable lipid to mRNA contained in the lipid nanoparticle was 10:1, and the molar ratio of ionizable lipid (246-C10) : phospholipid (DOPE) : cholesterol : lipid-PEG (C16-PEG2000 ceramide) was 26.5 in LNP : 20 : 52.5 to 51: 1.0 to 2.5 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100).

For 246-C10 LNP, in which the lipid-PEG was contained in an amount of 1.0 mol. %, 1.5 mol. % or 2.5 mol. % and luc mRNA was encapsulated, similarly to the method of Example 6, 3 hours after intravenous (IV) administration of a lipid nanoparticle with encapsulated C57BL/6 mRNA to female 7-week-old mice (Orient Bio) at a dose of 0.1 mg/kg calculated per luc mRNA contained in the lipid nanoparticle, luciferin 0.25mg/kg was injected intraperitoneally through IVIS equipment (PerkinElmer, USA) to confirm bioluminescence, and the result was displayed in FIG. 6, and the size of lipid nanoparticles according to lipid-PEG content was measured in the same manner as in the method of Example 4-1, and is set forth in Table 6 below and shown in FIG. 6.

As shown in FIG. 6, it could be confirmed that the group administered with the lipid nanoparticle according to one example had excellent drug delivery efficiency to the liver, and the size of LNPs containing 1.5 mol. % lipid-PEG, ranged from about 70 nm.

Example 8 Confirmation of the effect of hepatocyte-specific drug delivery

Example 8-1. Confirmation of FVII knockout effect using lipid nanoparticles

FVII is expressed specifically in hepatocytes, and therefore, in the present example, the ability to target lipid nanoparticles to hepatocytes according to one example was confirmed by the knockout effect of FVII (factor VII) using siFVII.

Thus, the concentration based on the concentration of siRNA contained in the lipid nanoparticle was 0.03 mg/kg, 0.1 mg/kg, or 0.3 mg/kg 3 days after administration of the 246-C10 lipid nanoparticle (containing lipid-PEG 1.5 mol %), in which the target FVII siRNA (SEQ ID NOs: 2 and 3) obtained in Example 2-2 was encapsulated, was administered intravenously to 7-week-old female C57BL/6 mice, 20 g, blood was collected through the tail vein and blood analysis were carried out according to the coaset FVII assay kit protocol, and a blood standard curve was generated from PBS-infused mice, and FVII expression was measured, and the result is shown in FIG. 7. As shown in FIG. 7, since FVII expression was inhibited in vivo depending on the concentration of siRNA encapsulated in the 246-C10 lipid nanoparticle, it was confirmed that the lipid nanoparticle according to one example can deliver the nucleic acid to hepatocytes as a target.

Example 8-2. The effect of drug delivery to hepatocytes depending on the content of lipid-PEG

Received lipid nanoparticle (246-C10 LNP), which was encapsulated siFVII (SEQ ID No: 2 and 3) according to the method of Example 2-2, by varying the content of lipid-PEG, which is part of the lipid nanoparticle, from 0.5 to 5 .0 mol. %. The mass ratio of ionizable lipid : miRNA contained in the lipid nanoparticle was 7.5:1, and the molar ratio of ionizable lipid (246-C10) : phospholipid (DSPC) : cholesterol : lipid-PEG (C16-PEG2000 ceramide), included in the composition LNP was 42.5 : 13 : 44 to 39.5 : 0.5 to 5.0 (adjusting cholesterol and lipid-PEG so that the total sum of the molar ratio is 100).

The diameter and polydispersity index of the lipid nanoparticles obtained above were measured in the same manner as in the method of Example 4-1, and are shown in Table 7 below and in FIG. 8 (table on the left).

Thus, the concentration based on the concentration of siRNA contained in the lipid nanoparticle was 0.2 mg/kg, 3 days after the lipid nanoparticle was encapsulated (containing lipid-PEG from 0.5 to 5 mol.%), in which was encapsulated with siFVII was intravenously administered to 7-week-old female C57BL/6 mice weighing 20 g, blood was collected through the tail veins, and similarly to the method of Example 8-1, using the coaset FVII assay kit, FVII expression was measured, and the result was displayed in FIG. 8 (graph on the right). As shown in FIG. 8, it was confirmed that with the introduction of the lipid nanoparticle according to one example, the expression of FVII in vivo decreased, and with the introduction of the lipid nanoparticle with a lipid-PEG content of 0.5 to 5.0 mol. % FVII expression was excellently suppressed.

Example 9 Effect of LSEC-Specific Drug Delivery

Since FVIII is specifically expressed in LSEC (liver sinusoidal endothelial cells), in this example, the ability of lipid nanoparticles to LSEC to target according to one of the examples was confirmed by the knockout effect of FVIII (factor VIII) using siFVIII and the drug, and the effect of delivery depending from the content of lipid-PEG.

By varying the content of lipid-PEG in the lipid nanoparticle from 0.5 to 5.0 mol. % received lipid nanoparticles (246-C10 LNP), in which siFVIII was encapsulated (SEQ ID NOs: 4-11) by the method of Example 2. the ratio of ionizable lipid (246-C10) : phospholipid (DSPC) : cholesterol : lipid-PEG (C16-PEG2000 ceramide) in LNP was 42.5 : 13 : 44 to 39.5 : 0.5 to 5.0 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100).

The diameter and PDI of the lipid nanoparticles obtained above were measured in the same manner as in Example 4-1 and are shown in Table 8 below and in FIG. 9 (table on the left).

Thus, the concentration based on the concentration of siRNA contained in the lipid nanoparticle was 0.5 mg/kg, 2 days after the administration of the lipid nanoparticle (containing lipid-PEG from 0.5 to 5 mol.%), in which siFVIII was encapsulated , was intravenously administered to 7-week-old female C57BL/6 mice weighing 20 g, blood was collected through the tail veins, and similarly to the method of Example 8-1, using the coaset FVII assay kit, FVIII expression was measured, and the result was displayed in FIG. 9 (graph on the right). As shown in FIG. 9, it was confirmed that when the lipid nanoparticle according to one example was administered, the expression of FVIII in vivo decreased, and the lipid nanoparticle according to one example could target LSEC, and when the lipid nanoparticle with a lipid-PEG content was from 0.5 up to 5.0 mol. %, FVIII expression was excellently suppressed.

Example 10 Effect of Drug Delivery Depending on Types of Lipid-PEG Conjugate

Lipid nanoparticles (containing a lipid-PEG conjugate from 0.25 to 10.0 mol.%), including a ceramide-PEG conjugate (C16-PEG 2000 ceramide; Avanti, USA) or PEG-DSPE (Avanti, USA) as a lipid conjugate -PEG, was obtained similarly to the method of Example 2-2. The mass ratio of ionizable lipid : siRNA (siLuc) contained in the lipid nanoparticle was 7.5:1, and the molar ratio of ionizable lipid (2464-C10) : phospholipid (DSPC) : cholesterol : lipid-PEG (ceramide-PEG or PEG- DSPE) contained in LNP was 42.5 : 13 : 44.25 to 34.5 : 0.25 to 10 (cholesterol and lipid-PEG are adjusted so that the total sum of the molar ratio is 100). The sequence of the siLuc used (siRNA targeting the luciferase gene; SEQ ID NOs: 12 and 13) was described in Example 2-2.

One day prior to transfection of LNP into cells according to one example, HeLa cells (Korea Cell Line Bank) were aliquoted at 0.01×10 ⁶ cells/well in a white plate (96 wells) and cultured at 37°C, about 0. 5-3% CO ₂ in DMEM (SH30022, Hyclone, USA). 24 hours after treatment of the lipid nanoparticle encapsulated with siLuc in the HeLa-Luc cell line at a wavelength of 10 nm, depending on the concentration of siRNA, Bright-Glo™ Luciferase Assay solution (promega, USA) was treated at 100 µg/well each and left at room temperature for 10 min, and then the luminescence intensity was measured for the dissolved cells using an Infinite M200 luminescence measuring device (Tecan, USA), and the result was displayed in FIG. 10. The measured result was represented by the mean ± standard deviation (SD). The value of the result was statistically tested by Student's method, and the case of p<0.05 or more was determined as statistically significant.

As shown in FIG. 10, the lipid nanoparticle according to one example had an excellent effect of delivering a nucleic acid to cells, and in particular, in the case of including a ceramide-PEG conjugate as a lipid-PEG conjugate, the effect of delivering a nucleic acid was excellent.

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SEQUENCE LIST

<110> Ewha University - Industry Collaboration Foundation

<120> Lipid nanoparticles for in vivo drug delivery

and their application

<130>OPP20204870KR

<150> KR 10-2020-0005642

<151> 2020-01-15

<150> KR 10-2020-0040586

<151> 2020-04-02

<160> 13

<170> KoPatentIn 3.0

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guuccauucc aucacgguuu uggaauguuu acuacacucg gauauuugau auguggauuu 780

cgagucgucu uaauguauag auuugaagaa gagcuguuuc ugaggagccu ucaggauuac 840

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uauguaaaca auccggaagc gaccaacgcc

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cugauuaagu acaaaggcua ucagguggcu cccgcugaau uggaauccau cuugcuccaa 1380

1440

cccgccgccg uuguuguuuu ggagcacgga aagacgauga cggaaaaaga gaucguggau 1500

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aaggccaaga aggcgggaaa gaucgccgug uaa 1653

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<---

Claims (18)

1. Lipid nanoparticle specifically targeting liver tissue, hepatocyte and/or LSEC (liver sinusoidal endothelial cell) containing an ionizable lipid in which a 6-membered heterocyclic amine and an alkyl epoxide are bound; phospholipid; cholesterol; and a lipid-PEG (polyethylene glycol) conjugate, wherein the 6-membered heterocyclic amine is selected from the group consisting of

where the alkyl epoxide has a number of carbon atoms from C6 to C14, where the phospholipid is one or more species selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG) , dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1, 2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], where the specified lipid nanoparticle contains a lipid-PEG conjugate in an amount of from 0.5 to 5 mol.%. 2. The lipid nanoparticle according to claim 1, wherein the alkyl epoxide is 1,2-epoxydecane. 3. The lipid nanoparticle according to claim 1, wherein the phospholipid is DOPE or DSPC. 4. The lipid nanoparticle of claim 1, wherein the lipid in the lipid-PEG conjugate is one or more species selected from the group consisting of ceramide, dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE) and cholesterol. 5. A lipid nanoparticle according to claim 1 containing an ionizable lipid: phospholipid: cholesterol: lipid-PEG conjugate in a molar ratio of 20 to 50 : 10 to 30 : 30 to 60 : 0.5 to 5. 6. Lipid nanoparticle according to claim 1, having pKa from 6.0 to 7.0. 7. Lipid nanoparticle according to claim 1, specifically targeting liver tissue. 8. Lipid nanoparticle according to claim 1, targeting a hepatocyte. 9. Lipid nanoparticle according to claim 1 targeting LSEC. 10. Composition for drug delivery, specifically targeting liver tissue, containing (1) a lipid nanoparticle according to any one of paragraphs. 1-9 and (2) an anionic drug, a nucleic acid, or a combination thereof. 11. The drug delivery composition of claim 10, wherein the anionic drug, nucleic acid, or combination thereof is encapsulated within a lipid nanoparticle. 12. The drug delivery composition of claim 10, wherein the lipid nanoparticle has an average diameter of 30 nm to 150 nm. 13. The drug delivery composition of claim 10, wherein the anionic drug is one or more species selected from the group consisting of a peptide, a drug protein, a protein-nucleic acid structure, and an anionic biopolymer-drug conjugate. 14. The drug delivery composition of claim 10, wherein the nucleic acid is one or more species selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transport ribonucleic acid (tRNA), antisense oligonucleotide, short hairpin RNA (shRNA), microRNA, ribozyme, peptide nucleic acid (PNA), and DNA winter.

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