Lipid nanoparticles and method for producing same

Abstract

The present invention relates to lipid nanoparticles and a method for producing the same. More specifically, the present invention relates to particles comprising an ionizable lipid and a polyethylene glycol derivative moiety (PEG moiety)-degradable linking functional group-lipid conjugate, which are characterized by minimizing side effects in the body, effectively delivering the nanoparticles to target cells, and efficiently transporting pharmacologically active substances to the cytoplasm.

Description

[Technical Field]

[0001] The present invention relates to lipid nanoparticles and a method for producing the same, and more particularly to lipid nanoparticles and a method for producing the same, which contain an ionizable lipid and a polyethylene glycol derivative moiety (PEG moiety)-degradable linking functional group-lipid conjugate, thereby minimizing in vivo side effects and effectively delivering the nanoparticles to target cells and efficiently transporting pharmacologically active substances into the cytoplasm.

[0002] [Background technology]

[0003] Even the same drug can have a wide range of effects depending on the delivery method. The route of administration and drug form that efficiently delivers the required amount of drug, maximizing efficacy and effectiveness while minimizing side effects, is called a drug delivery system (DDS). In the pharmaceutical formulation industry, drug delivery systems can generate economic profits comparable to new drug development and can be said to be a high-value-added core technology with a high probability of success.

[0004] Solubilization technology for poorly soluble drugs, which belongs to drug absorption enhancement technology and is one of the core technologies of drug delivery systems, is considered the most rational method to increase the added value of pharmaceuticals while reducing the development cost of new drug substances. In particular, in a poor environment for new drug development like Korea, the development of improved new drugs through the development of drug solubilization technology can create enormous added value at low cost.

[0005] Among various drug delivery systems, the development of gene therapy using gene drug delivery systems has been expanding in recent years. For gene therapy to be successful and safe, it is important to deliver genes or gene regulatory factors to the desired tissue. Representative examples of such genes or gene regulatory factors include mRNA and siRNA. Nucleic acids such as mRNA have the function of expressing specific proteins, making them capable of complementing proteins lacking due to genetic factors. Furthermore, the use of mRNA that expresses cancer markers or viral surface proteins can enable the development of anticancer drugs and vaccines that activate immune responses in the body. Furthermore, nucleic acids such as siRNA, which can suppress the expression of specific proteins in the body, have been attracting attention as important tools for the treatment of cancer, genetic diseases, infectious diseases, autoimmune diseases, and the like. However, nucleic acids such as mRNA and siRNA are difficult to deliver directly into cells and are easily degraded by enzymes in the blood, and much research is being conducted to overcome these issues.

[0006]

[0007] Properties and limitations of cationic liposomes or cationic polymer-based nanoparticles

[0008] To effectively deliver such substances, non-viral gene carriers such as cationic liposomes and polymers have been developed. Their improved stability profiles and ease of manufacture and manipulation have accelerated research into the design and synthesis of non-toxic, biodegradable polymeric carriers for effective and safe gene delivery. Poly(L-lysine), polyethyleneimine, starburst, polyamidoamine dendrimers, and cationic liposomes have been widely investigated as non-viral gene carriers because they can self-assemble independently by combining with anionic oligos and compact plasmid DNA (pDNA) into structures small enough for cellular uptake by endocytosis.

[0009] However, nanoparticles prepared using cationic liposomes or cationic polymers have limitations, such as their inadequacy for use as therapeutic agents. This method involves mixing cationic liposomes or polymers with anionic oligos to produce nanoparticles through electrostatic attraction. Therefore, to produce stable particles, an excess of cationic material relative to the anionic charge must be used. Nanoparticles cannot be formed using the same molar ratio of cations and anions; instead, an excess of cations, at least several to even several tens of times, must be used. Therefore, nanoparticles prepared using this method consistently maintain a cationic charge because the remaining cations, neutralized by interaction with anions, remain in excess. However, positively charged nanoparticles are known to induce side effects due to unwanted interactions with various cells, including immune cells, in the body, making it difficult to develop therapeutic agents using such nanoparticles.

[0010]

[0011] Characteristics of lipid nanoparticles using ionized lipids and their manufacturing method

[0012] In recent years, effective therapeutic agents have been developed using a new type of lipid nanoparticle as a non-viral gene delivery vehicle that overcomes the drawbacks of cationic nanoparticles (Non-Patent Documents 1-9). Lipid nanoparticles are particulate drug delivery vehicles that utilize phospholipids and cholesterol, which are substances present in the body, and therefore have high bioavailability and affinity, enable controlled drug release, and are highly stable against degradation by enzymes. In particular, the essential difference that distinguishes these lipid nanoparticles from existing cationic nanoparticles is the inclusion of ionized lipids as a component. Ionized lipids are characterized by being cationic at acidic pH but maintaining an electrically neutral state at neutral pH. Therefore, nanoparticles manufactured using ionized lipids are electrically neutral in the bloodstream at neutral pH, thereby dramatically eliminating in vivo side effects caused by the cationic nature of cationic polymers or cationic liposomes.

[0013] Another difference lies in the method of producing lipid nanoparticles. The specific method of producing lipid nanoparticles is as follows: Lipid nanoparticles are produced by mixing lipid components dissolved in an organic phase with oligos dissolved in an aqueous buffer solution. As the organic and aqueous phases are mixed, the polarity of the solvent changes, causing the lipid components contained in the organic phase to independently form particles in the mixed solution. When the ionized lipids contained in the organic phase are mixed with an acidic buffer solution containing oligos, the ionized lipids transition to a cationic state. The cationic ionized lipids and anionic oligos bond through electrostatic attraction to form nanoparticles containing the oligos.

[0014] Another feature of lipid nanoparticles using ionized lipids is that they are prepared by incorporating a lipid for preventing nanoparticle aggregation into the organic phase. The anti-aggregation lipid has a molecular structure in which a lipid component is bound to a hydrophilic substance. The hydrophilic substance can be a biological substance such as carbohydrates or proteins, or a synthetic substance such as polyol or PEG. A typical example is a substance in the form of a PEG-lipid conjugate. In PEG-lipid conjugates, the lipid component is hydrophobic and therefore moves toward the interior of the particles during lipid nanoparticle formation, while the PEG moiety is hydrophilic and moves toward the exterior of the particles (Non-Patent Documents 11 and 12). If a lipid for preventing particle aggregation, such as a PEG-lipid conjugate, is not included as a component of the nanoparticle surface during this process, nanoparticles will continue to aggregate together. This makes it extremely difficult to produce nanoparticles of a relatively uniform size, and non-uniform nanoparticles have poor efficacy.

[0015] Another reason PEG is typically used as a hydrophilic substance in lipids for anti-aggregation is that PEG, which forms the surface of lipid nanoparticles, also affects the biological properties of the lipid nanoparticles. Generally, when nanoparticles are injected into the body, they suffer from the drawback of being lost through phagocytosis by macrophages due to adsorption with proteins, lipids, and immune components in the bloodstream. To overcome this drawback, it is known to modify the surface with PEG, a hydrophilic polymer with excellent biocompatibility (U.S. Patent No. US9,999,673 B2; Korean Patent Publication No. 10-2018-0032652; WO 2020 / 061284). PEG is most commonly used because it has no side effects and can be easily applied to lipid nanoparticles and other drug delivery systems (Non-Patent Document 10).

[0016]

[0017] Disadvantages of using PEG-lipid conjugates and limitations of existing solutions

[0018] PEG is an essential component in the production process of lipid nanoparticles. It has the distinct advantage of reducing many side effects by suppressing nonspecific interactions between nanoparticles and other substances in vivo. However, it also has fundamental limitations. PEG's ability to reduce nonspecific interactions between nanoparticles and other biological components in vivo ultimately results in the inhibition of interactions between nanoparticles and target cells. As a result, PEG makes it difficult to deliver nanoparticles into target cells, significantly reducing their pharmacological efficacy (Non-Patent Documents 13-15).

[0019] PEG inhibits the interaction between nanoparticles and target cells. To address this issue, two approaches have been devised. The first approach involves separating PEG from lipid nanoparticles after infusion into the body. Generally, the lipid component of PEG-lipid conjugates is composed of two fatty acids. Reducing the number of carbon atoms in the fatty acid reduces the hydrophobic nature of the fatty acid, allowing the PEG-lipid conjugate to separate from the nanoparticles and be easily removed during in vivo circulation. This eliminates the interfering effect of PEG, thereby maintaining nanoparticle efficacy. It is known that increasing the number of carbon atoms in the fatty acid can prevent the PEG-lipid conjugate from being removed from the nanoparticles, resulting in reduced efficacy (Non-Patent Documents 14-16). However, removing the PEG-lipid conjugate, a nanoparticle component, in this manner can destabilize the particles themselves and potentially result in the loss of other components (Non-Patent Document 17).

[0020] A second approach to addressing the issue of reduced efficacy of lipid nanoparticles delivered to target cells by PEG is to attach a ligand capable of binding to a cellular receptor to one end of PEG (Non-Patent Document 16). Because the ligand at the PEG end binds to specific cellular receptors, nanoparticles containing the ligand can be delivered to intracellular endosomes by the receptor. However, even if nanoparticles can be effectively delivered to intracellular endosomes using the ligand, the pharmacologically active substance contained in the nanoparticles, such as nucleic acids, must be delivered from the endosome to the cytoplasm in order to exert its pharmacological effect. This requires a fusion process between the lipid nanoparticles and the endosomal lipid membrane, but the PEG surrounding the nanoparticles hinders this process. As a result, although attaching a ligand to the end of PEG can deliver nanoparticles into the endosomes of target cells, it is difficult to deliver the active substance from the endosome to the cytoplasm, and therefore, significant improvement in efficacy cannot be expected (Non-Patent Document 16).

[0021]

[0022] Application of PEG-degradable functional group-lipid conjugates to ionized lipid nanoparticles

[0023] To solve the problem of reduced efficacy due to PEG in endosomes, a method for removing PEG that covers the surface of lipid particles within endosomes is typically used. Because the pH inside endosomes is acidic, a PEG-degradable functional group-lipid conjugate is included as a component, so that the site linking PEG to the lipid component contains a functional group that can be hydrolyzed under acidic conditions. This is known to effectively remove PEG from the surface of nanoparticles within endosomes (Non-Patent Documents 18 and 19).

[0024] However, the conditions for producing nanoparticles using PEG-degradable functional group-lipid conjugates containing degradable functional groups that degrade under acidic conditions as components are limited. This is because the degradable functional groups of PEG-degradable functional group-lipid conjugates are unstable under acidic conditions, and therefore the nanoparticle production conditions can only be applied to nanoparticles produced under neutral or alkaline conditions. However, lipid nanoparticles, which have recently been widely used in the development of various oligo-specific therapeutic agents, contain ionized lipids as components. As mentioned above, lipid nanoparticles containing oligo components must be produced under acidic pH conditions. For this reason, when producing lipid nanoparticles containing ionized lipid components, it is considered essentially impossible to use PEG-degradable functional group-lipid conjugates, which contain functional groups that are hydrolyzed under acidic conditions, as components, and no such method has been attempted to date.

[0025] The present inventors have made extensive efforts to solve the above problems and have found that lipid nanoparticles containing an ionized lipid component can be stably prepared using a polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate. In particular, the PEG moiety-degradable functional group-lipid conjugate remains stable during the acidic conditions used in the lipid nanoparticle preparation process, but is effectively hydrolyzed under the acidic conditions of the endosome, thereby promoting fusion of the lipid nanoparticle with the endosomal lipid membrane, thereby dramatically improving oligonucleotide delivery efficiency. Furthermore, when such lipid nanoparticles are prepared and used as therapeutic agents, they minimize in vivo side effects due to the nanoparticle components, effectively deliver the nanoparticles to target cells, and efficiently shed endosomes within the target cells to deliver pharmacologically active substances such as nucleic acids to the cytoplasm. Based on this finding, the present invention was completed.

[0026]

[0027]

[0028] [Preliminary Technology Documents] [License]

[0029] [License 1] U.S. Register of Registrations Publication No. 9,999,673 B2

[0030] [License 2] Korean Publication Patent No. 10-2018-0032652

[0031] [License 3] International Publication No. WO2020 / 061284

[0032]

[0033] [Non-licensed literature]

[0034] [Non-licensed Document 1] Moss, KH, Popova, P., Hadrup, SR, Astakhova, K. & Taskova, M. Lipid Nanoparticles for Delivery of Therapeutic RNA Oligonucleotides. Mol Pharm 16, 2265-2277 (2019).

[0035] [Non-licensed Document 2] Kulkarni, JA, Witzigmann, D., Chen, S., Cullis, PR & van der Meel, R. Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics. Acc Chem Res 52, 2435-2444 (2019).

[0036] [Non-patent document 3] Buck, J., Grossen, P., Cullis, PR, Huwyler, J. & Witzigmann, D. Lipid-Based DNA Therapeutics: Hallmarks of Non-Viral Gene Delivery. ACS Nano 13, 3754-3782 (2019).

[0037] [Non-patent document 4] Akinc, A. et al.The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat Nanotechnol14, 1084-1087 (2019).

[0038] [Non-patent document 5] Springer, AD & Dowdy, SF GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics. Nucleic Acid Ther 28, 109-118 (2018).

[0039] [Non-patent document 6] Kulkarni, JA, Cullis, PR & van der Meel, R. Lipid Nanoparticles Enabling Gene Therapies: From Concepts to Clinical Utility. Nucleic Acid Ther 28, 146-157 (2018).

[0040] [Non-Patent Document 7] Rietwyk, S. & Peer, D. Next-Generation Lipids in RNA Interference Therapeutics. ACS Nano 11, 7572-7586 (2017).

[0041] [Non-patent document 8] Fang, Y. et al.Cleavable PEGylation: a strategy for overcoming the "PEG dilemma" in efficient drug delivery. Drug Deliv 24, 22-32 (2017).

[0042] [Non-Patent Document 9] Cullis, PR & Hope, MJ Lipid Nanoparticle Systems for Enabling Gene Therapies. Mol Ther 25, 1467-1475 (2017).

[0043] [Non-Patent Document 10] Kumar, V. et al.Shielding of Lipid Nanoparticles for siRNA Delivery: Impact on Physicochemical Properties, Cytokine Induction, and Efficacy. Mol Ther Nucleic Acids 3, e210 (2014).

[0044] [Non-Patent Document 11] Belliveau, NM et al.Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA. Mol Ther Nucleic Acids 1, e37 (2012).

[0045] [Non-Patent Document 12] Sato, Y. et al. Elucidation of the physicochemical properties and potency of siRNA-loaded small-sized lipid nanoparticles for siRNA delivery. J Control Release 229, 48 - 57 (2016).

[0046] [Non-Patent Document 13] Bao, Y. et al. Effect of PEGylation on biodistribution and gene silencing of siRNA / lipid nanoparticle complexes. Pharm Res 30, 342 - 51 (2013).

[0047] [Non-Patent Document 14] Mui, B.L. et al. Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles. Mol Ther Nucleic Acids 2, e139 (2013).

[0048] [Non-Patent Document 15] Chen, S. et al. Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration. J Control Release 196, 106 - 12 (2014).

[0049] [Non-Patent Document 16] Akinc, A. et al.Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther 18, 1357-64 (2010).

[0050] [Non-Patent Document 17] Chen, S. et al.Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA. J Control Release235, 236-244 (2016).

[0051] [Non-Patent Document 18] Bargh, JD, Isidro-Llobet, A., Parker, JS & Spring, DR Cleavable linkers in antibody-drug conjugates. Chem Soc Rev 48, 4361-4374 (2019).

[0052] [Non-Patent Document 19] Zhao, G. et al. Smart pH-sensitive nanoassemblies with cleavable PEGylation for tumor targeted drug delivery. Sci Rep 7, 3383 (2017).

[0053]

[0054] Summary of the Invention

[0055] The object of the present invention is to provide lipid nanoparticles that minimize in vivo side effects due to the components of the nanoparticles, effectively deliver the nanoparticles to target cells, and efficiently deliver pharmacologically effective substances such as nucleic acids to the cytoplasm by shedding endosomes in the target cells.

[0056] To achieve the above-mentioned objectives, the present invention provides lipid nanoparticles comprising: (a) a lipid formulation comprising an ionizable lipid and a polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate; and (b) a drug, a nucleic acid, or a combination thereof encapsulated within the lipid formulation.

[0057] The present invention also provides a method for producing the lipid nanoparticles, comprising the steps of: (a) mixing an organic solution containing a lipid formulation including an ionizable lipid and a polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate with a buffer solution containing a drug, a nucleic acid, or a combination thereof, and adjusting the pH; and (b) removing the solvent from the mixed solution.

[0058] [Brief explanation of the drawings]

[0059] [Figure 1] According to one embodiment of the present invention, lipid nanoparticles containing PEG2K-Hz-DSG or PEG2K-PE-DSG at 1, 2, and 5 mol% of the total lipids constituting the nanoparticles were prepared, and graph (A) shows the size measured by DLS, and graph (B) shows the numerical value of the size.

[0060] [Figure 2]1 shows graphs of the luciferase expression inhibitory efficacy measured by treating cells constitutively expressing luciferase with lipid nanoparticles containing luciferase-targeting siRNA according to one embodiment of the present invention, where (A) is lipid nanoparticles containing 1, 2, and 5 mol% of PEG2K-Hz-DSG or PEG2K-PE-DSG, (B) is lipid nanoparticles containing 1, 2, and 5 mol% of PEG1K-Hz-DSG or PEG1K-PE-DSG, and (C) is lipid nanoparticles containing 1, 2, 5, 10, 20, 30, and 40 mol% of PEG600-Hz-DSG.

[0061] [Figure 3] Figures show the effect of PEG content in lipid nanoparticles on cellular uptake efficiency, according to one embodiment of the present invention. Figure A shows the cell uptake efficiency of lipid nanoparticles containing 2, 5, and 10 mol% PEG-DMG relative to total lipids, analyzed by flow cytometry (FACS). Figure B shows the luciferase expression inhibitory efficacy of nanoparticles containing 0, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mol% PEG-DMG, which were prepared with luciferase-targeting siRNA and treated with cells expressing luciferase. Figures C and D show the luciferase expression inhibitory efficacy of lipid nanoparticles containing 1, 2, 5, and 10 mol% PEG-DMG or PEG600-Hz-DSG, respectively, in different cell lines. Figures E and F show EC50 values.

[0062] [Figure 4] 1 shows data on the size of lipid nanoparticles containing 1.5 mol% PEG-DMG (A) or 10 mol% PEG600-Hz-DSG (B) measured over time under acidic conditions according to one embodiment of the present invention.

[0063] [Figure 5]This is a graph showing the results of measuring the luciferase expression inhibitory efficacy of lipid nanoparticles containing 5 or 10 mol% of PEG600-Hz-DSG prepared according to one embodiment of the present invention, pretreated at pH 4 or 7, and then applied to a cell line.

[0064] [Figure 6] According to one embodiment of the present invention, lipid nanoparticles containing 1, 2, 5, and 10 mol% of PEG600-Hz-DSG, PEG600-Ester-DSG, or PEG600-PhHz-DSG were prepared and treated with a cell line to measure the luciferase expression inhibitory efficacy. Graph (A) shows the results, and data (B) shows the size of the lipid nanoparticles measured under neutral or acidic conditions.

[0065] [Figure 7] According to one embodiment of the present invention, lipid nanoparticles containing 10 mol% PEG600-Hz-DSG or 1.5 mol% PEG-DMG and a fluorescent dye were prepared, and the fusion strength with the liposome lipid membrane was measured under the corresponding pH conditions. (A) shows the results. (B) shows the results of measuring the hemolysis reaction under pH 4.5 and pH 7.4 conditions using lipid nanoparticles containing 5 or 10 mol% GalNAc-PEG600-Hz-DSG or PEG-DMG.

[0066] [Figure 8] In one embodiment of the present invention, lipid nanoparticles containing PEG-DMG at 1.5 mol% and 5 mol% of the total lipids and lipid nanoparticles containing GalNAc-PEG2K-Hz-DSG at 5 mol% of the total lipids were prepared by adding a FRET fluorescent dye. These were then loaded into rat plasma, and the change in the fluorescence intensity contained in the nanoparticles was measured over time.

[0067] [Figure 9] Lipid nanoparticles containing 10 mol% GalNAc-PEG600-Hz-DSG were mixed with peripheral blood mononuclear cells (PBMCs) isolated from rats, and the relative amount of cytokines produced was analyzed by q-PCR.

[0068] [Figure 10] 1 shows data obtained by administering lipid nanoparticles containing PEG2K-PE-DSG or PEG2K-Hz-DSG to mice according to one embodiment of the present invention, and measuring the content of Factor 7 in the serum.

[0069] [Figure 11] According to one embodiment of the present invention, lipid nanoparticles containing 10 mol% PEG600-Hz-DSG or a mixed lipid of 9.5 mol% PEG600-Hz-DSG and 0.5 mol% GalNAc-PEG2K-Hz-DSG were administered to mice, and the serum Factor 7 content was measured.

[0070] [Figure 12] 1 shows data on the relative viability of cell lines measured after treating each cell line with lipid nanoparticles containing PEG-DMG or PEG-Hz-DSG according to one embodiment of the present invention.

[0071]

[0072] DETAILED DESCRIPTION OF THE INVENTION

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein and the experimental procedures described below are those well known and commonly employed in the art.

[0074]

[0075] The present invention has confirmed that when lipid nanoparticles containing a polyethylene glycol derivative moiety of Chemical Formula 1-degradable functional group-lipid and an ionized lipid as components are prepared and used as a therapeutic agent, the nanoparticles can minimize side effects in vivo due to the nanoparticle components, effectively deliver the nanoparticles to target cells, and efficiently shed endosomes in the target cells to deliver pharmacologically effective substances such as nucleic acids to the cytoplasm.

[0076] Thus, in one aspect, the present invention relates to lipid nanoparticles comprising: (a) a lipid formulation comprising an ionizable lipid and a polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate; and (b) a drug, a nucleic acid, or a combination thereof encapsulated within the lipid formulation.

[0077]

[0078] In another aspect, the present invention relates to a method for producing the lipid nanoparticles, which comprises the steps of: (a) mixing an organic solution containing a lipid formulation including an ionizable lipid and a polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate with a buffer solution containing a drug, a nucleic acid, or a combination thereof, and adjusting the pH; and (b) removing the solvent from the mixed solution.

[0079]

[0080] The present invention will be described in detail below.

[0081] As used herein, the terms "polyethylene glycol," "PEG," "PEG moiety," and "PEG derivative" include functional groups corresponding to polyethylene glycol and may be used interchangeably. Unless otherwise indicated herein or clearly contradicted by context, combinations of the foregoing elements in all possible variations are included.

[0082] The lipid nanoparticles of the present invention comprise: (a) a lipid formulation comprising an ionizable lipid and a PEG moiety-degradable functional group-lipid conjugate; and (b) a drug, a nucleic acid, or a combination thereof encapsulated within the lipid formulation.

[0083] In the present invention, the PEG moiety-degradable functional group-lipid conjugate may be represented by Chemical Formula 1.

[0084] [Chemical formula 1]

[0085] TIFF0007792441000001.tif21128

[0086] In chemical formula 1,

[0087] a is 0 or 1,

[0088] L is a targeting ligand;

[0089] M is H, OH, a single bond, O, S, C(O), NHC(O), C(O)NH, OC(O), or C(O)O;

[0090] P is CH2O(CH2CH2O)qCH2 or CH2CH2O(CH2CH2O)qCH2, where q is an integer from 2 to 120;

[0091] L6 is C(O)NH-N=CR4, R4C=N-NHC(O), NH-N=CR4, R4C=N-NH, C(O)O, OC(O), OC(O)O, ON=CR4, R4C=NO, SS, S or trans-cyclooctene;

[0092] Or, TIFF0007792441000002.tif23128, where R4 is H, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C3-C10 Cycloalkyl, C6-C 20 aryl or heterocycle, which is a radical containing a heteroatom selected from fluorine, oxygen, sulfur, and nitrogen; Z is NH, O, or S; d is an integer from 1 to 10; and e is an integer from 1 to 10;

[0093] T is a single bond or 1,4-C6H4O-;

[0094] R1 and R3 are each independently -YR, and R2 is -CH2-YR, where Y is a single bond, O, S, C(O), C(O)O, OC(O), C(O)NH, or NHC(O), and R is H, C 10 ~C 20 It is an alkyl, alkenyl or sterol.

[0095]

[0096] The present invention may also include a ligand at one end of the PEG moiety-degradable functional group-lipid conjugate.

[0097] The ligand (L) of the PEG moiety-degradable functional group-lipid conjugate may be represented by the following chemical formula 2:

[0098] [Chemical formula 2]

[0099] TIFF0007792441000003.tif33128

[0100] In chemical formula 2,

[0101] a, b, and c are 0 or 1, but at least one of a, b, and c must be 1;

[0102] X1, X2 and X3 are targeting ligands;

[0103] L1, L'1 and L"1 are a single bond, O, S, C(O), NHC(O), C(O)NH, OC(O) or C(O)O;

[0104] L2, L'2 and L"2 are (CH2) n or (OCH2CH2) m where n is an integer from 1 to 20, and m is an integer from 1 to 10;

[0105] L3, L ’ 3 and L ” 3 is a single bond, O, S, C(O), NHC(O), C(O)NH, OC(O) or C(O)O;

[0106] L4, L'4 and L"4 are (CH2) n where n is an integer from 1 to 20;

[0107] L5, L'5 and L"5 are a single bond, O, S, C(O), NHC(O), C(O)NH, OC(O) or C(O)O.

[0108] In the present invention, X1, X2, and X3 may be selected from the group consisting of N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-galactose, D-galactose, N-acetyl-D-glucosamine, N-acetyl-D-glucosamine, D-glucose, D-mannose, L-fucose, carbohydrate derivatives, folate, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CADY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single-chain variable fragment (scFv), and aptamer. Here, the antigen-binding fragment of an antibody or antibody fragment means a fragment that retains the antigen-binding function, and includes Fab, F(ab'), F(ab')2, Fv, and the like.

[0109] In the present invention, examples of antibodies that can be used as ligands include, but are not limited to, anti-CD3, anti-CD19, anti-CD20, anti-CD22, anti-CD33, anti-CD38, anti-CD54, anti-CD74, anti-CD138, anti-CD166, anti-CD209, anti-cMET, anti-EGFR, anti-HER2, anti-HIV-gp120, anti-HLA-DR, and anti-transferrin receptor (TfRscFv).

[0110] In the present invention, the polyethylene glycol (PEG) moiety-degradable functional group-lipid conjugate may be preferably represented by the following chemical formula 3 (PEG-Hz-lipid).

[0111] [Chemical formula 3]

[0112] TIFF0007792441000004.tif21142

[0113] In Chemical Formula 3, n is an integer of 2 to 120.

[0114] In the present invention, the PEG moiety-degradable functional group-lipid conjugate is preferably a ligand-PEG moiety-degradable functional group-lipid conjugate (chemical formula 1, where a is 1), and more preferably may be represented by chemical formula 4 (GalNAc-PEG-Hz-Lipid).

[0115] [Chemical formula 4]

[0116] TIFF0007792441000005.tif42149

[0117] In Chemical Formula 4, n is an integer of 2 to 120.

[0118]

[0119] In the present invention, the polyethylene glycol moiety composition may be a mixture of a PEG moiety-degradable functional group-lipid conjugate (in Chemical Formula 1, a is 0) and a ligand-PEG moiety-degradable functional group-lipid conjugate (in Chemical Formula 1, a is 1).

[0120] In the present invention, the molar ratio of the compound in which a is 0 in Chemical Formula 1 to the compound in which a is 1 may be (0.01 to 99.9):(0.01 to 99.9), preferably (50 to 99.9):(0.01 to 50), more preferably (70 to 99):(1 to 30), and most preferably (90 to 95):(5 to 10). That is, the ligand-PEG moiety-degradable functional group-lipid conjugate may constitute 0.01 to 100 mol %, preferably 0.01 to 20 mol %, and more preferably 5 to 10 mol % of the mixture of the ligand-PEG moiety-degradable functional group-lipid conjugate and the PEG moiety-degradable functional group-lipid conjugate. In this case, the ligand-PEG moiety-degradable functional group-lipid conjugate may vary depending on the ligand to be bound. The N-acetyl-D-galactosamine (GalNAc) used in the examples of the present invention can exhibit the efficacy of lipid nanoparticles even when used in small amounts (5 to 10 mol%).

[0121] In the present invention, the size of the lipid nanoparticles may be 20 to 200 nm, preferably 30 to 200 nm.

[0122] In the PEG moiety-degradable functional group-lipid conjugate of the present invention, the structural features of the PEG moiety are as follows:

[0123] 1. The PEG moiety has a molecular weight of 100 to 5000 and a single or branched chemical structure. The PEG moiety content may be 0.5 to 50 mol %, preferably 0.5 to 40 mol %, and more preferably 0.5 to 30 mol % of the total lipid components constituting the lipid nanoparticles. If the PEG moiety content is less than 0.5 mol %, it is difficult to obtain nanoparticles of uniform size during lipid nanoparticle production, and other lipid components constituting the nanoparticles are exposed to the surface, which can easily induce nonspecific interactions. If the PEG moiety content is more than 50 mol %, the PEG moiety-degradable functional group-lipid conjugate component is not incorporated into the lipid nanoparticles, resulting in the formation of independent micelles. Furthermore, the PEG molecular weight and the lipid nanoparticle content are inversely correlated. When PEG with a high molecular weight around 5000 is used, the content in lipid nanoparticles is 20 mol % or less. However, when the PEG molecular weight is 100, the content in lipid nanoparticles can be increased to 50 mol %. As the molecular weight increases, it becomes more difficult to form uniform lipid nanoparticles due to the steric effect of PEG. In addition, the hydrophilic PEG portion increases more than the hydrophobic lipid portion, which means that there is a strong tendency for the PEG portion to form independent micelles without binding to the lipid nanoparticles.

[0124] 2. A part or all of a polyethylene glycol (PEG) moiety-degradable functional group-lipid conjugate can be used by binding a ligand capable of binding to a cellular receptor to one end of the PEG moiety. The ligand can be in the form of a sugar or carbohydrate composed of N-acetylgalactosamine, glucose, mannose, fucose, etc., a peptide containing RGD, etc., a small molecule such as folate, a nucleic acid such as an aptamer, or a protein such as an antibody or an antigen-recognizing portion of an antibody, which can selectively bind to a receptor expressed on a specific cell. More specifically, these include N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-galactose, D-galactose, N-acetyl-D-glucosamine, N-acetyl-D-glucosamine, D-glucose, D-mannose, L-fucose, carbohydrate derivatives, folate, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CADY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single-chain variable fragment (scFv), or aptamer.

[0125] 3. The other end of the PEG moiety is connected with a linker, which contains a chemical functional group that decomposes under specific conditions or a substrate for a biological hydrolase, such as -C(O)-NH-N=CR-, -RC=N-NH-C(O)-, -NH-N=CR-, -RC=N-NH-, -C(O)-O-, -OC(O)-, -OC(O)-O-, -ON=CR-, -RC=NO-, -SS-, -S-, or -trans-cyclooctene-, where R is H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, or the like.

[0126] 4. The linker is linked to a hydrophobic substance such as a lipid in the form of a fatty acid or a sterol.

[0127]

[0128] Among the components of the lipid nanoparticles according to the present invention, the ionizable lipid is an ionizable compound bound to a lipid component, and can play a role in enabling drugs (e.g., anionic drugs and / or nucleic acids) to be encapsulated in the lipid nanoparticles with high efficiency through electrostatic interactions with the drug. The ionizable lipid carries a neutral charge at neutral pH and a positive charge at acidic pH.

[0129] In the present invention, the ionizable lipids include (6Z,9Z,28Z,31Z)-heptatriaconta 6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid (SM -102), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), dioctadecylamidoglycylcarboxyspermine (DOGS), spermine cholesteryl carbamate (GL-67); bis-guanidinium-spermine amidine-cholesterol (BGTC), 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), Nt-butyl-N'-tetradecylamino-propionamidine (diC14-amidine);Dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxyl)propyl)-N-2-(sperm The alkyl ester may be selected from the group consisting of N-(1-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE);

[0130] Preferably, it may be a compound represented by Chemical Formula 5 (DLin-MC3-DMA), Chemical Formula 6 (ALC-0315), or Chemical Formula 7 (Lipid H(SM-102)).

[0131] [Chemical formula 5]

[0132] TIFF0007792441000006.tif16128

[0133] [Chemical formula 6]

[0134] TIFF0007792441000007.tif35128

[0135] [Chemical formula 7]

[0136] TIFF0007792441000008.tif27128

[0137]

[0138] The lipid nanoparticles according to the present invention may further comprise a sterol lipid and a neutral lipid.

[0139] Among the components of the lipid nanoparticles according to the present invention, the neutral lipids play a role in enveloping and protecting the core formed by the interaction of the ionized lipid and the drug within the lipid nanoparticles, and bind to the lipid bilayer of the target cells, facilitating passage through the cell membrane and escape from the endosome during delivery of the drug into the cells. The neutral lipids among the components of the lipid nanoparticles according to the present invention can be any phospholipid or sphingolipid that can promote fusion of lipid particles, and are preferably DOPE (dioleoylphosphatidylethanolamine), DSPC (distearoylphosphatidylcholine), POPC (palmitoyloleoylphosphatidylcholine), EPC (egg phosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DOPG (dioleoylphosphatidylglycerol), DPPG (dip almitoylphosphatidylglycerol), DSPE(distearoylphosphatidylethanolamine), PE(Phosphatidylethanolamine), DPPE(dipalmitoylphosphatidylethanola mine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]), ceramide, or sphingomyelin.

[0140] Among the components of the lipid nanoparticles according to the present invention, the sterol lipid imparts morphological rigidity to the lipid packing within the lipid nanoparticles and plays a role in improving the stability of the nanoparticles by being dispersed in the core and surface of the nanoparticles. Cholesterol and cholesterol derivatives including cholesteryl esters can be used.

[0141]

[0142] In the present invention, the drug may be one or more selected from the group consisting of peptides, protein drugs, protein-nucleic acid constructs, and anionic biopolymer-drug conjugates.

[0143] In the present invention, the nucleic acid may be one or more selected from the group consisting of single-stranded siRNA, double-stranded siRNA, rRNA, DNA, cDNA, plasmid, aptamer, mRNA, tRNA, lncRNA, piRNA, circRNA, saRNA, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme.

[0144] In the present invention, siRNA may target genes such as ACACA, ANGPTL3, AOC3, MAP3K5, CCR2, CCR5, DGAT2, DPP4, FASN, HSD17B13, KHK, LGALS3, LOXL2, METAP2, MPC, NOX1, NOX4, PNPLA3, SCD, SGLT1, and SGLT2 of metabolic diseases including non-alcoholic steatohepatitis, genes such as HBV-S, HBV-P, and HBV-X of hepatitis B virus (HBV) as examples of viral diseases, the TTR gene of TTR-mediated amyloisosis, the ALAS1 gene of acute hepatic porphyria, and primary hyperhydroxylacturia (PHA). HAO1 gene in hyperoxaluria, PCSK9 gene in hypercholesterolemia, AT gene in hemophilia, C5 gene in complement-mediated disease, AAT gene in Alpha-1 liver disease, AGT gene in hypertension, AAT gene in Alpha-1 antitrypsin deficiency, APOC3 gene in hypertriglyceridemia, ANGPTL3 gene in hypertriglyceridemia, LA gene in cardiovascular disease, HSP47 gene in idiopathic pulmonary fibrosis, LDHA gene in primary hyperoxaluria, CTGF in climatic and keloid scar diseases, CTNNB1, HSP90, KRAS, EPHA2, BCL2L12, PKN3, HIF-2alpha gene in cancer, cystic fibrosis The target genes include, but are not limited to, the alphaENaC gene in idiopathic fibrosis (IDF) disease.

[0145] In the present invention, the mRNA may be a vaccine using an mRNA that expresses an antigen, an immunotherapeutic agent using an mRNA that expresses an antibody, an mRNA that expresses a protein missing due to gene mutation, or an mRNA and sgRNA that express a Cas protein and guide RNA using CRISPR technology for specific DNA gene editing. Examples of antigen-expressing mRNA for vaccine development include rabies virus glycoprotein mRNA for a rabies vaccine, glycoprotein mRNA and stabilized prefusion F protein mRNA for respiratory syncytial virus vaccines, Zika virus structural protein mRNA for Zika virus vaccines, viral antigen protein mRNA for chikungunya virus vaccines, petameric viral antigen and gB protein mRNA for cytomegalovirus vaccines, HA protein mRNA for influenza vaccines, and HIV immunogen protein mRNA for generating T cell activity in HIV vaccines. Examples of mRNA for the development of anticancer drugs or vaccines include mRNA expressing specific overexpressed protein antigens derived from patients, antigen-expressing mRNAs including PAP, PSA, PSCA, PSMA, and STEAP1 for prostate cancer, MUC1, CEA, Her-2neu, telomerase, survivin, and MAGE-A1 mRNA for renal cancer, Melan-A, tyrosinase, gp100, MAGE-A1, MAGE-A3, and survivin mRNA for melanoma, and cytokine mRNAs including IL-23, IL-36, IL-12, IL-15, GM-CSF, INF-alpha, IL-7, and IL-2 as vaccines or therapeutic agents for various solid cancers or blood cancers.Furthermore, examples of mRNA that directly expresses an antibody in vivo using an mRNA that expresses an antibody include, but are not limited to, mRNA that expresses a monoclonal antibody that neutralizes chikungunya virus.

[0146] The lipid nanoparticles of the present invention may be prepared by (a) mixing an organic solution containing a lipid formulation including an ionizable lipid and a polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate with a buffer solution containing a drug, a nucleic acid, or a combination thereof, and adjusting the pH; and (b) removing the solvent from the mixed solution.

[0147] The lipid nanoparticles according to the present invention can be prepared by mixing lipid components dissolved in an organic phase with oligos dissolved in an aqueous buffer solution. As the organic and aqueous solutions are mixed, the polarity of the solvent changes, allowing the lipid components contained in the organic phase to independently form particles in the mixed solution. When the ionized lipids contained in the organic phase are mixed with an acidic buffer solution containing oligos, the ionized lipids transition to a cationic state. The cationic ionized lipids and the anionic oligos bind to each other through electrostatic attraction, forming nanoparticles in which the oligos are encapsulated within the lipid nanoparticles.

[0148] In the method for producing lipid nanoparticles according to the present invention, the mixing ratio (volume ratio) of the organic solution to the buffer solution may be 1:1 to 1:100.

[0149]

[0150] Efficiency depending on the PEG content in lipid nanoparticles

[0151] Lipid nanoparticles are typically prepared with 1-2 mol% PEG-lipid conjugates. If lipid nanoparticles are prepared without PEG, the resulting particles tend to aggregate. To prevent this, a certain amount of PEG-lipid conjugate must be included. The lipid portion of the PEG-lipid conjugate is located in the hydrophobic environment that constitutes the interior of the resulting nanoparticles, while the hydrophilic PEG portion is located on the surface of the particles, preventing aggregation between nanoparticles. However, nanoparticles with too much PEG also inhibit the interaction between lipid nanoparticles and cells due to the steric effect of the PEG exposed on the particle surface. As a result, lipid nanoparticles are not effectively delivered to cells, resulting in side effects.

[0152] When lipid nanoparticles are prepared using PEG-DMG (1,2-dimyristoyl-3-PEG-glycerol), a PEG-lipid conjugate commonly used in lipid nanoparticle preparation, increasing the PEG content reduces the efficiency of cellular uptake and mRNA degradation by siRNA. However, because some or all of the PEG moiety-degradable functional group-lipid conjugates of the present invention contain a ligand at one end of the PEG that specifically binds to a receptor expressed in a specific cell, lipid nanoparticles prepared using the PEG moiety-degradable functional group-lipid conjugates of the present invention have been shown to have high cellular uptake efficiency regardless of the content of the PEG moiety-degradable functional group-lipid conjugate. This indicates that the steric effect of PEG can be used to overcome the inhibition of cellular uptake.

[0153]

[0154] Confirmation of PEG removal from lipid nanoparticles

[0155] For lipid nanoparticles to deliver active substances such as nucleic acids into cells and exert their effects, a two-step delivery process is required. First, lipid nanoparticles must enter the cell's endosomes through cellular uptake. Second, the active substance must be able to escape the endosome and be delivered to the cytoplasm. It is widely known that lipid nanoparticles are taken up into the cell's endosomes through interaction with cells, and the lipid components of the lipid nanoparticles fuse with the endosomal lipid membrane, simultaneously delivering the active substance to the cytoplasm. However, when the surface of lipid nanoparticles is coated with PEG, interaction with the endosomal lipid membrane is inhibited, making delivery to the cytoplasm difficult. Therefore, PEG on the surface of lipid nanoparticles not only inhibits cell uptake efficiency by inhibiting cell interaction, but also interferes with the interaction between nanoparticles successfully taken up into endosomes and the endosomal lipid membrane, resulting in a two-fold inhibitory effect on nanoparticle efficiency.

[0156] Therefore, for effective interaction between the endosomal lipid membrane and lipid nanoparticles, it is advantageous to remove PEG within the endosome. However, nanoparticles using commonly used PEG-lipid conjugates, such as PEG-DMG, lack a functional group that can remove PEG, resulting in PEG remaining on the particle surface even within endosomes. In contrast, the PEG moiety-degradable functional group-lipid conjugates described in this invention contain a chemical functional group that is hydrolyzed under acidic conditions between the PEG and lipid. For example, the PEG moiety-degradable functional group-lipid conjugate shown in Chemical Formula 3 has two lipids and PEG linked via a hydrazone functional group (-C=NNH-). The hydrazone functional group is stable at neutral pH but is hydrolyzed under acidic conditions. When the size of lipid nanoparticles prepared using the PEG moiety-degradable functional group-lipid conjugate according to the present invention is observed over time under acidic conditions, it is found that the size gradually increases due to the hydrolysis of the hydrazone functional group, removal of PEG from the particle surface, and fusion of the lipid components that make up the particles. Furthermore, it is found that the fusion between particles continues over time, resulting in a gradual increase in size. This is a phenomenon in which the successful hydrolysis of the hydrazone functional group under acidic conditions triggers fusion between particles, which also acts as a factor that smoothly induces fusion between the endosomal lipid membrane and the nanoparticles.

[0157]

[0158] Lipid nanoparticles induce hemolysis

[0159] A well-known method for assessing the interaction between lipid nanoparticles and endosomal lipid membranes is to contact nanoparticles with red blood cells and measure the degree of red blood cell destruction, i.e., hemolysis. Here, the red blood cell membrane is considered as a surrogate for the endosomal lipid membrane. Increased hemolysis can be interpreted as more efficient fusion of lipid nanoparticles with the endosomal lipid membrane.

[0160] Ionizable lipids, one of the components of lipid nanoparticles, are known to play a central role in the interaction with the endosomal lipid membrane. Ionizable lipids often contain amine groups, which are uncharged at neutral pH but positively charged under the acidic conditions of the endosomal environment. The optimal pKa of the amine group is known to be approximately 5 to less than 7. Therefore, to examine the degree to which lipid nanoparticles fuse with the endosomal lipid membrane, red blood cells and nanoparticles were mixed at a pH lower than the pKa of the amine group, and the hemolysis reaction was measured and evaluated. The ionizable lipid used in this example was DLin-MC3-DMA. The pKa of this amine group is approximately 6.5, which means that the amine group is positively charged at pH 4. The positive charge facilitates fusion through electrostatic attraction with the negative charge of the endosomal lipid membrane. However, when PEG is distributed on the surface of nanoparticles, it not only creates a blocking effect by weakening the electrostatic attraction, but also hinders direct contact between the surface of the nanoparticles and the lipid membrane of the endosome.

[0161] Therefore, lipid nanoparticles containing general PEG-DMG as a component, which does not contain degradable functional groups, show reduced hemolysis as the PEG content increases, whereas lipid nanoparticles containing PEG moiety-degradable functional group-lipid conjugates as a component effectively induce hemolysis regardless of the content of PEG moiety-degradable functional group-lipid conjugates, because PEG is removed by hydrolysis under acidic conditions.

[0162]

[0163] Relationship between the length of lipids constituting PEG moiety-degradable functional group-lipid conjugates and the structural stability of lipid nanoparticles

[0164] Although PEG surrounding the surface of lipid nanoparticles is necessary for the lipid nanoparticle manufacturing process, it has a detrimental effect on pharmacological efficacy. Therefore, to facilitate spontaneous removal of PEG from lipid nanoparticles during circulation in the bloodstream, lipids linked to PEG are typically myristic acid (14 carbons). The fewer carbons in the lipid moiety, the less interaction there is with other lipid components that make up the lipid nanoparticle, making it easier for the PEG-lipid conjugate to be separated and removed from the lipid nanoparticle in vivo. Lipid nanoparticles manufactured using substances with relatively long fatty acids, such as palmitic acid (16 carbons) or stearic acid (18 carbons), have difficulty removing PEG during circulation through the bloodstream, making it difficult for them to enter cellular endosomes, resulting in reduced efficacy.

[0165] However, this method of removing PEG during circulation can be a significant destabilizing factor in terms of the structural stability of lipid nanoparticles. As PEG-lipids begin to separate and be removed from lipid nanoparticles, the stability of the particles themselves decreases, leading to the loss of other lipid components that make up the lipid nanoparticles. While PEG removal is essential for lipid nanoparticles to have adequate efficacy, lipid nanoparticles prepared using such short PEGs have weakened particle stability.

[0166] On the other hand, the PEG moiety-degradable functional group-lipid conjugate described in the present invention contains a degradable functional group that removes PEG under acidic conditions, allowing the use of a lipid composed of a relatively long hydrocarbon, for example, 18 carbon atoms. The PEG moiety-degradable functional group-lipid conjugate remains a component of the nanoparticle until it reaches the acidic conditions of the endosome where the degradable functional group can be degraded, thereby maintaining the stability of the particle during circulation through the bloodstream.

[0167]

[0168] Lipid nanoparticles induce immune responses

[0169] Ionizable lipids, which make up lipid nanoparticles, are known to stimulate immune cells and induce immune responses. Increasing the PEG content of lipid nanoparticles can suppress direct interactions between immune cells and lipid nanoparticles, minimizing immunological side effects caused by the lipid components. However, increasing the PEG content inevitably leads to severe inhibition of cellular uptake of nanoparticles and endosomal escape within the cells, making it difficult to increase the PEG content beyond the minimum required amount.

[0170] However, lipid nanoparticles containing the PEG moiety-degradable functional group-lipid conjugates of the present invention contain degradable functional groups that can increase cellular uptake efficiency using ligands and remove PEG within endosomes. Therefore, when the PEG moiety-degradable functional group-lipid conjugates presented in the present invention are used, the efficacy of the prepared lipid nanoparticles does not decrease even when the PEG content is increased, and at the same time, immune response induction can be suppressed. Therefore, when lipid nanoparticles using the PEG moiety-degradable functional group-lipid conjugates are mixed with peripheral blood mononuclear cells (PBMCs) and the level of cytokine induction by the lipid nanoparticles is measured, it is found that the level is similar to that of the PBS buffer solution-treated group used as a negative control group.

[0171]

[0172] The present invention will be described in more detail below using examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0173]

[0174]

[0175] [Example]

[0176] Example 1: Synthesis of polyethylene glycol (PEG) moiety-degradable functional group-lipid conjugate

[0177] Example 1-1: 2-(2-(2-azidoethoxy)ethoxy)ethanol

[0178] TIFF0007792441000009.tif14165

[0179] Sodium azide (1.54 g, 23.72 mmol) was added to a solution of 2-(2-(2-azidoethoxy)ethoxy)ethanol (2.00 g, 11.86 mmol) in water (40 mL) and stirred at 90° C. for 18 hours. The reaction mixture was extracted three times with dichloromethane, dried over magnesium sulfate, and then dried under reduced pressure to give the compound (2.22 g) in 98.5% yield.

[0180] 1 H NMR (400 MHz, CDCl3) δ 3.75-3.70 (m, 2H), 3.69-3.65 (m, 6H), 3.63-3.59 (m, 2H), 3.42-3.36 (m, 2H), 2.31 (s, 1H).

[0181]

[0182] Example 1-2: 2-methyl-3,4, 6 -Tri-O-acetyl-1,2-deoxy-alpha-D-galactopyrano[2,1,d]-2-oxazoline

[0183] TIFF0007792441000010.tif34130

[0184] Trimethylsilyl trifluoromethanesulfonate (0.2 mL, 1.13 mmol) was added to a solution of peracetylated galactosamine (400 mg, 1.03 mmol) dissolved in DCE (22 mL) at room temperature and stirred at 50 °C for 4 hours. The reaction temperature was lowered to room temperature, and triethylamine was slowly added and stirred for 15 minutes. After the reaction was complete, the organic layer was extracted with water and CHCl, dried over magnesium sulfate, filtered, and distilled under reduced pressure. The mixture obtained after vacuum distillation was separated by column chromatography (CHCl:MeOH = 40:1) to obtain the desired compound (260 mg, 0.77 mmol) in 78% yield.

[0185] 1 H NMR (400 MHz, CDCl3) δ 5.99 (d, J = 6.8 Hz, 1H), 5.46 (t, J = 3.0 Hz, 1H), 4.90 (dd, J = 7.5, 3.3 Hz, 1H), 4.26-4.08 (m, 3H), 3.99 (m, 1H), 2.12 (s, 3H), 2.07 (s, 3H), 2.07 (s, 3H), 2.05 (d, J = 1.2 Hz, 3H).

[0186]

[0187] Example 1-3: 5-Acetamino-2-(acetoxymethyl)-6-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate

[0188] TIFF0007792441000011.tif29159

[0189] 2-Methyl-3,4, dissolved in DCE (7.2 mL) 6 -tri-O-acetyl-1,2-deoxy-alpha-D-galactopyrano[2,1,d]-2-oxazoline (390 mg, 1.18 mmol) and 2-(2-(2- AzideThe 4Å molecule (190 mg) was added to a mixed solution of ethoxyethoxyethanol (228 mg, 1.30 mmol) and stirred for 5 minutes. Trimethylsilyl trifluoromethanesulfonate (104 μL, 0.59 mmol) was added at room temperature and stirred for 16 hours. After the reaction was completed, the 4Å molecule was removed by filtration, and water and CHCl were added. 。 The organic solvent layer Separate, After drying over magnesium sulfate and filtration, the mixture was distilled under reduced pressure and separated by column chromatography (EA:MeOH=9:1) to give the desired compound (460 mg, 0.91 mmol) in 77% yield.

[0190] 1 H NMR (400 MHz, CDCl3): δ6.14 (d, J = 9.3 Hz, 1H), 5.32 (dd, J = 3.4, 1.1 Hz, 1H), 5.05 (dd, J = 11.2, 3.4 Hz, 1H), 4.78 (d, J = 8.6 Hz, 1H), 4.22 (dt, J= 11.2, 8.9 Hz, 1H), 4.19-4.10 (m, 2H), 3.93-3.83 (m, 3H), 3.76-3.65 (m, 4H), 3.63 (dd, J = 7.1, 3.2 Hz, 4H), 3.51-3.39 (m, 2H), 2.15 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.98 (s, 3H).

[0191]

[0192] Examples 1-4: 2-(2-(2-aminoethoxy)ethoxy)ethyl 2-acetamino-3,4,6-tri-O-acetyl-2-dioxy-α-D-galactopyranoside

[0193] TIFF0007792441000012.tif19165

[0194] To a solution of 5-acetamino-2-(acetoxymethyl)-6-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (240 mg, 0.476 mmol) dissolved in THF (10 mL), PPh3 (150 mg, 0.571 mmol) was added at room temperature and stirred for 48 hours. HO (26 μL, 1.427 mmol) was added and stirred for 24 hours. Toluene (10 mL) and trifluoroacetic acid (73 μL, 0.951 mmol) were then added to generate the salt. Water and CHCl2 were added. After that, The extracted aqueous layer was distilled under reduced pressure to give the desired compound (270 mg, 0.91 mmol) in 98% yield.

[0195] 1 H NMR (400 MHz, CD3OD): δ5.35 (d, J = 3.2 Hz, 1H), 5.05 (dd, J = 11.2, 3.2 Hz, 1H), 4.58 (d, J = 8.4 Hz, 1H), 4.16-3.96 (m, 4H), 3.64-3.74 (m, 10H), 3.24 (m, 2H), 2.14 (s, 3H), 2.03 (s, 3H), 1.95 (s, 3H), 1.94 (s, 3H).

[0196]

[0197] Examples 1-5: Tris{[2-( tert- (butoxycarbonyl)ethoxy]methyl}methylamine

[0198] TIFF0007792441000013.tif36149

[0199] To a solution of Tris (1.21 g, 10.00 mmol) dissolved in DMSO (2.0 mL) was added 5.0 M sodium hydroxide (0.2 mL) at 15° C., followed by stirring for 5 minutes. tTo the reaction mixture, 2.5 g (5.00 mmol) of butyl acrylate was slowly added dropwise. Water (0.2 mL) was then added, followed by stirring at room temperature under argon for 24 hours. After the reaction was completed, the mixture was distilled under reduced pressure and separated by column chromatography (EA:Hex=2:1) ​​to obtain the desired compound (2.5 g, 5.00 mmol) in a 50% yield.

[0200] 1 H NMR (CDCl3, 400 MHz): δ3.65 (t, J = 6.0 Hz, 6H), 3.32 (s, 6H), 2.46 (t, J = 6.0 Hz, 6H), 1.45 (s, 27H).

[0201]

[0202] Example 1-6: (R)-((2,3-bis(octadecyloxy)propoxy)methyl)benzene

[0203] TIFF0007792441000014.tif34150

[0204] 1-Bromooctadecane (732 mg, 2.20 mmol) and potassium hydroxide (123 mg, 2.20 mmol) were added to a solution of (R)-3-benzyloxy-1,2-propanediol (106 mg, 0.55 mmol) dissolved in toluene (2.5 mL) at room temperature, and the mixture was refluxed at 110°C for 24 hours. After the reaction was completed, water and CHCl were added. Ta. The extracted organic solvent layer was dried over magnesium sulfate and filtered. 、 The mixture obtained by vacuum distillation was separated by column chromatography (Hex:EA=10:1) to give the desired compound (300 mg, 0.44 mmol) in 80% yield.

[0205] 1H NMR (400 MHz, CDCl3) δ 7.32-7.21 (m, 5H), 4.55 (s, 2H), 3.64-3.48 (m, 7H), 3.43 (t, J = 6.7 Hz, 2H), 1.61-1.51 (m, 4H), 1.39-1.21 (m, 60H), 0.88 (t, J = 6.8 Hz, 6H).

[0206]

[0207] Example 1-7 (S)-2,3-bis(octadecyloxy)propan-1-ol

[0208] TIFF0007792441000015.tif26128

[0209] (R)-((2,3-bis(octadecyloxy)propoxy)methyl)benzene (50 mg, 0.07 mmol) was dissolved in CHCl (4 mL), and Pd / C (10 mg) was added dropwise. The mixture was stirred under hydrogen at room temperature for 20 hours. After the reaction was completed, the mixture was filtered using Celite 545 with ethyl acetate solvent and then distilled under reduced pressure to obtain the desired compound (40 mg, 0.07 mmol) in a 92% yield.

[0210] 1 H NMR (CDCl3) δ 3.75-3.37 (m, 9H), 2.17 (m, 1H), 1.55 (m, 4H), 1.38-1.21 (m, 60H), 0.88 (t, J = 6.8 Hz, 6H).

[0211]

[0212] Examples 1-8: (R)-2,3-bis(octadecyloxy)propanal

[0213] TIFF0007792441000016.tif35130

[0214] A solution of Dess-Martin periodinane (88 mg, 0.206 mmol) dissolved in CHCl (3.7 mL) was slowly added dropwise to a solution of (S)-2,3-bis(octadecyloxy)propan-1-ol (110 mg, 0.184 mmol) dissolved in CHCl (1.3 mL) at 0°C, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, water, CHCl, and aqueous sodium bicarbonate solution were added. Extracted organic solvents Layer, Chi Sodium bisulfate Wash with the solution Dry over magnesium sulfate and filter 、 The mixture obtained by vacuum distillation was separated by column chromatography (Hex:EA=20:1) to give the desired compound (83 mg, 0.15 mmol) in 80% yield.

[0215] 1 H NMR (CDCl3) δ 3.81-3.37 (m, 9H), 1.65-1.51 (m, 4H), 1.38-1.21 (m, 60H), 0.88 (t, J = 6.8 Hz, 6H).

[0216]

[0217] Examples 1-9: Carboxymethyl-PEG600-Cbz-hydrazide (M n of PEG=600)

[0218] TIFF0007792441000017.tif13164

[0219] Poly(ethylene glycol) bis(carboxymethyl)ether in DMF (5.8 mL) (M n =600) After dissolving (1 g, 1.72 mmol), CbzNHNH2 (300 mg, 1.81 mmol), hydroxybenzotriazole (244 mg, 1.81 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (320 μL, 1.81 mmol) were added dropwise at 0 °C. The mixture was stirred at room temperature under argon for 18 hours. After the reaction was complete, water, CHCl, and an aqueous solution of sodium bicarbonate were added. The separated aqueous phase was 3 PO 4 Acidify with CH 2 Cl 2 Extracted with. The organic layer was distilled under reduced pressure to give the product in 40% yield.

[0220] 1 H NMR (400 MHz, CD3OD) δ 7.39-7.30 (m, 5H), 5.15 (s, 2H), 4.13 (s, 2H), 4.10 (s, 2H), 3.73-3.60 (m, 70H).

[0221]

[0222] Examples 1-10: Tris{[2-(t-butoxycarbonyl)ethoxy]methyl}methylamide-PEG600-Cbz-hydrazide (M n of PEG=600)

[0223] TIFF0007792441000018.tif28164

[0224] DMF (4 mL) Carboxymethyl-PEG600-Cbz-hydrazide (M n =600) (870 mg, 1.16 mmol), Tris{[2-( t- After dissolving 526 mg (1.04 mmol) of butoxycarbonyl)ethoxy]methyl}methylamine, hydroxybenzotriazole (157 mg, 1.16 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (248 μL, 1.40 mmol), and diisopropylethylamine (243 μL, 1.40 mmol) were added dropwise at 0°C. The mixture was stirred under argon at room temperature for 18 hours. After the reaction was completed, water, CHCl, H 3 PO Added 4. The extracted organic solvent layer was diluted with NaHCO 3 Washed with liquid. The organic layer was dried over magnesium sulfate, filtered, and then distilled under reduced pressure. The resulting mixture was separated by column chromatography (CH2Cl2:MeOH = 15:1) to give the desired compound in 40% yield.

[0225] 1H NMR (400 MHz; CD3OD) δ 7.38-7.31 (m, 5H), 5.16 (s, 2H), 4.30-4.13 (m, 3H), 4.13 (s, 2H), 3.90 (s, 2H), 3.77-3.60 (m, 70H), 2.46 (t,J = 6.0 Hz, 6H), 1.46 (s, 27H).

[0226]

[0227] Examples 1-11: Tris{[2-(carboxymethyl)ethoxy]methyl}methylamide-PEG600-Cbz-hydrazide (M n of PEG=600)

[0228] TIFF0007792441000019.tif25164

[0229] Tris{[2-(t-butoxycarbonyl)ethoxy]methyl}methylamide-PEG600-Cbz-hydrazide (570 mg, 0.46 mmol) was dissolved in 85% formic acid (4.0 mL) and stirred at room temperature for 18 hours. After the reaction was completed, the desired compound was obtained in 99% yield by distillation under reduced pressure at 50°C.

[0230] 1 H NMR (CD3OD) δ 7.38-7.30 (m, 5H), 5.15 (s, 2H), 4.30-4.17 (m, 3H), 4.13 (s, 2H), 3.90 (s, 2H), 3.78-3.63 (m, 70H), 2.53 (t, J = 6.0Hz, 6H).

[0231]

[0232] Examples 1-12: Tri-(Paracetyl-GalNAc) -PEG600-Cbz hydrazide

[0233] TIFF0007792441000020.tif31164

[0234] DMF (4.4 mL) Tris{[2-(carboxymethyl)ethoxy]methyl}methylamide-PEG600-Cbz-hydrazide ( 0.5mmole ) , 2-(2-(2-aminoethoxy)ethoxy)ethyl 2-acetamino-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosideAfter dissolving (1.58 mmol), hydroxybenzotriazole (214 mg, 1.58 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (280 μL, 1.58 mmol), and diisopropylethylamine (550 μL, 3.16 mmol) were added dropwise at 0°C. The mixture was stirred under argon at room temperature for 18 hours. Diisopropylethylamine (550 μL, 3.16 mmol) was then added dropwise under argon at room temperature, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, water, CHCl, H 3 PO Add 4 Et Extracted organic solvent layer Separate and prepare NaHCO 3 Wash with liquid, It was dried over magnesium sulfate, filtered and then distilled under reduced pressure. obtained The mixture was separated by column chromatography (CH2Cl2:MeOH=10:1) to give the desired compound in 23% yield.

[0235] 1 H NMR (400 MHz, CD3OD) δ 7.40-7.31 (m, 5H), 5.34 (d, J = 3.0 Hz, 3H), 5.07 (dd, J = 11.2, 3.3 Hz, 3H), 5.16 (s, 2H), 4.64 (t, J = 8.5 Hz, 3H), 4.16-4.02 (m, 16H), 3.95-3.91 (m, 6H), 3.77-3.61 (m, 106H), 3.55 (t, J = 5.6 Hz, 6H), 3.38 (t, J = 5.6 Hz, 6H), 2.45 (t, J= 6.0 Hz, 6H), 2.14 (s, 9H), 2.03 (s, 9H), 1.95 (s, 9H), 1.93 (s, 9H).

[0236]

[0237] Examples 1-13: Tri-(Paracetyl-GalNAc) -PEG600-hydrazide

[0238] TIFF0007792441000021.tif31166

[0239] MeOH (2 mL) Tri-(Paracetyl-GalNAc) After dissolving PEG600-Cbz hydrazide (373 mg, 0.16 mmol), Pd / C (13 mg) and acetic acid (28 μL, 0.48 mmol) were added dropwise. The mixture was stirred under hydrogen at room temperature for 20 hours. After the reaction was completed, the mixture was filtered using Celite 545 with methanol solvent and then distilled under reduced pressure to obtain the desired compound (120 mg, 0.05 mmol) in 99% yield.

[0240] 1 H NMR (400 MHz, CD3OD) δ 1.94 (s, 9H), 5.34 (d, J = 3.0 Hz, 3H), 5.07 (dd, J = 11.2, 3.4 Hz, 3H), 4.64 (d, J= 8.5 Hz, 3H), 4.17-3.91 (m, 16H), 3.77-3.61 (m, 106H), 3.56 (t, J = 5.5 Hz, 6H), 3.39 (t, J = 5.5 Hz, 6H), 2.46 (t, J = 5.9 Hz, 6H), 2.15 (s, 9H), 2.03 (s, 9H), 1.95 (s, 9H), 1.94 (s, 9H).

[0241]

[0242] Examples 1-14: Tri-(paraacetyl-GalNAc)-PEG600-(dioctadecyloxy-propylidene)-acylhydrazone

[0243] TIFF0007792441000022.tif24166

[0244] In a mixed solution of EtOH (1 mL) and CH2Cl2 (0.1 mL) Tri-(Paracetyl-GalNAc)-PEG600-hydrazide (0.05 mmol) and (R)-2,3-bis(octadecyloxy)propanal (39 mg, 0.06 mmol) were dissolved in the solution, and the mixture was stirred under argon at room temperature for 3 hours. After the reaction was completed, the mixture was distilled under reduced pressure. . profit The resulting mixture was separated by column chromatography (CH2Cl2:MeOH=10:1) to give the desired compound in 26% yield.

[0245] 1 H NMR (CDCl3) δ 9.92 (s, 1H), 7.43 (d, J= 6.9 Hz, 1H), 7.19 (m, 2H), 7.03 (s, 1H), 6.6 (s, 1H), 5.32 (d, J = 3.0 Hz, 3H), 5.16 (dd, J = 11.3, 3.0 Hz, 3H), 4.78 (d, J = 8.5 Hz, 3H), 4.15-3.40 (m, 134H), 2.45 (t, J = 5.9 Hz, 6H), 2.13 (s, 9H), 2.03 (s, 9H), 1.98 (s, 9H), 1.94 (s, 9H), 1.54-1.48 (m, 4H), 1.24 (m, 60H), 0.86 (t, J = 6.8 Hz, 6H).

[0246]

[0247] Examples 1-15: Tri-(GalNAc)-PEG600-(dioctadecyloxy-propylidene)-acylhydrazone (3GalNAc-PEG600-Hz-DSG)

[0248] TIFF0007792441000023.tif88146

[0249]

[0250] In a mixed solution of MeOH (1 mL) and CH2Cl2 (0.1 mL), Tri-(paraacetyl-GalNAc)-PEG600-(dioctadecyloxy-propylidene)-acylhydrazone After dissolving (0.013 mmol), sodium methoxide (0.7 mg, 0.013 mmol) was added dropwise. The mixture was stirred at room temperature under argon for 2 hours. After the reaction was completed, the mixture was treated with Amberlite IR-120 ion exchange resin and then distilled under reduced pressure to obtain the desired compound in 26% yield.

[0251] 1H NMR (CDCl3) δ 9.97(s, 1H), 8.54 (s, 1H), 7.90(m, 2H), 7.72 (m, 1H), 4.47 (m, 3H), 4.15-3.44 (m, 144H), 2.47 (t, J= 5.9 Hz, 6H), 2.00 (s, 9H), 1.55 (m, 4H), 1.24 (m, 60H), 0.87 (t, J = 6.8 Hz, 6H).

[0252]

[0253] Examples 1-16: Carboxymethyl-PEG2000-Cbz-hydrazide (M n of PEG=2000)

[0254] TIFF0007792441000024.tif12166

[0255] The desired compound was obtained in a yield of 40% by the same method as in Examples 1-9.

[0256] 1 H NMR (400 MHz, CD3OD) δ 7.39-7.30 (m, 5H), 5.15 (s, 2H), 4.13 (s, 2H), 4.10 (s, 2H), 3.73-3.68 (m, 190H).

[0257]

[0258] Examples 1-17: Tris{[2-(t-butoxycarbonyl)ethoxy]methyl}methylamide-PEG2000-Cbz-hydrazide (M n of PEG=2000)

[0259] TIFF0007792441000025.tif28166

[0260] The desired compound was obtained in a yield of 30% by the same method as in Examples 1-10.

[0261] 1H NMR (400 MHz; CD3OD) δ 7.38-7.31 (m, 5H), 5.16 (s, 2H), 4.30-4.13 (m, 3H), 4.13 (s, 2H), 3.90 (s, 2H), 3.77-3.60 (m, 190H), 2.46 (t, J= 6.0 Hz, 6H), 1.46 (s, 27H).

[0262]

[0263] Examples 1-18: Tris{[2-(carboxymethyl)ethoxy]methyl}methylamide-PEG2000-Cbz-hydrazide (M n of PEG=2000)

[0264] TIFF0007792441000026.tif23166

[0265] The desired compound was obtained in 99% yield by the same method as in Examples 1-11.

[0266] 1 H NMR (CD3OD) δ 7.38-7.30 (m, 5H), 5.15 (s, 2H), 4.12 (s, 4H), 3.78-3.96 (m, 190H), 2.53 (t, J = 6.0 Hz, 6H).

[0267]

[0268] Examples 1-19: Tri-(Paracetyl-GalNAc)-PEG2K-Cbz Hydrazide

[0269] TIFF0007792441000027.tif33168

[0270] The desired compound was obtained in a yield of 30% by the same method as in Examples 1-12.

[0271] 1H NMR (400 MHz, CD3OD) δ 7.40-7.31 (m, 5H), 5.34 (d, J = 3.0 Hz, 3H), 5.16 (s, 2H), 5.07 (dd, J = 11.2, 3.3 Hz, 3H), 4.64 (t, J = 8.5 Hz, 3H), 4.16-4.02 (m, 16H), 3.95-3.91 (m, 6H), 3.77-3.61 (m, 226H), 3.55 (t, J = 5.6 Hz, 6H), 3.38 (t, J= 5.6 Hz, 6H), 2.45 (t, J = 6.0 Hz, 6H), 2.14 (s, 9H), 2.03 (s, 9H), 1.95 (s, 9H), 1.93 (s, 9H).

[0272]

[0273] Example 1-20: Tri-(Paracetyl-GalNAc)-PEG2K-hydrazide

[0274] TIFF0007792441000028.tif30168

[0275] In Examples 1-13, the desired compound was obtained using the same method, with a yield of 99%.

[0276] 1 H NMR (400 MHz, CD3OD) δ 5.34 (d, J = 3.0 Hz, 3H), 5.07 (dd, J = 11.2, 3.4 Hz 3H), 4.64 (d, J = 8.5 Hz, 3H), 4.17-3.91 (m, 16H), 3.77-3.60 (m, 226H), 3.56 (t, J = 5.5 Hz, 6H), 3.39 (t, J = 5.5 Hz, 6H), 2.46 (t, J = 5.9 Hz, 6H), 2.15 (s, 9H), 2.03 (s, 9H), 1.95 (s, 9H), 1.94 (s, 9H).

[0277]

[0278] Example 1-21: Tri-(paraacetyl-GalNAc)-PEG2K-(dioctadecyloxypropylidene)-acylhydrazone

[0279] TIFF0007792441000029.tif23168

[0280] The desired compound was obtained in a yield of 37% by the same method as in Examples 1-14.

[0281] 1 H NMR (CDCl3) δ 9.95 (s, 1H), 7.43 (d, J= 6.9 Hz, 1H), 7.19 (m, 2H), 7.03 (s, 1H), 6.6 (s, 1H), 5.32 (d, J = 3.0 Hz, 3H), 5.16 (dd, J = 11.3, 3.0 Hz, 3H), 4.78 (d, J = 8.5 Hz, 3H), 4.15-3.40 (m, 226H), 2.45 (t, J = 5.9 Hz, 6H), 2.13 (s, 9H), 2.03 (s, 9H), 1.98 (s, 9H), 1.94 (s, 9H), 1.54-1.48 (m, 4H), 1.24 (m, 60H), 0.86 (t, J = 6.8 Hz, 6H).

[0282]

[0283] Examples 1-22: Tri-(GalNAc)-PEG2K-(dioctadecyloxy-propylidene)-acylhydrazone (3GalNAc-PEG2K-Hz-DSG)

[0284] TIFF0007792441000030.tif88146

[0285] The desired compound was obtained in a yield of 26% by the same method as in Examples 1-15.

[0286] 1H NMR (CDCl3) δ 9.93 (s, 1H), 8.54 (s, 1H), 7.90 (m, 2H), 7.72 (m, 1H), 4.47 (m, 3H), 4.15-3.44 (m, 236H), 2.47 (t, J= 5.9 Hz, 6H), 2.00 (s, 9H), 1.55 (m, 4H), 1.24 (m, 60H), 0.87 (t, J = 6.8 Hz, 6H).

[0287]

[0288] Example 1-23. Methyl-PEG600-Cbz hydrazide (M n of PEG=600)

[0289] JPEG0007792441000031.jpg14168

[0290] in 2.7mL DMF α-Methyl-ω-carboxymethyl-poly(ethylene glycol) ether (M n of PEG=600) After dissolving the HCl (150 mg, 0.27 mmol), CbzNHNH (50 mg, 0.3 mmol), EDCI (58 μL, 0.33 mmol), and HOBt (44 mg, 0.33 mmol) were added at 0 °C and stirred at room temperature for one day. After the reaction was completed, the reaction mixture was transferred to DCM (40 mL), washed with diluted HPO (1 M, 40 mL), NaHCO (1 M, 40 mL) aqueous solution (40 mL), and then dried over magnesium sulfate. The reaction mixture was separated on a silica gel column using DCM / methanol (10 / 1, v / v) to obtain 175 mg of compound in 91% yield.

[0291] 1 H NMR (400 MHz, MeOH-d4) δ 7.39-7.30 (m, 5H), 5.16 (s, 2H), 4.20 (s, 2H), 3.68-3.52 (m, 50H). 3.37 (s, 3H)

[0292]

[0293] Examples 1-24: Methyl-PEG600-hydrazide (M n of PEG=600)

[0294] TIFF0007792441000032.tif14168

[0295] in methanol (2.8 mL) Methyl-PEG600- Cbz hydrazide (175 mg, 0.25 mmol) was dissolved, Pd / C (18 mg, 10 w / w%) was added, and the mixture was stirred under hydrogen for 20 hours. After the reaction was completed, the mixture was filtered through Celite 545 to give 147 mg of the compound in 95% yield.

[0296] 1 H NMR (400 MHz, CDCl3) δ 4.14-4.01 (m, 2H), 3.61 (m, 50 H), 3.34 (s, 3H)

[0297]

[0298] Examples 1-25: Methyl-PEG600-(dioctadecyloxy-propylidene) acylhydrazone (mPEG600-Hz-DSG)

[0299] JPEG0007792441000033.jpg22170

[0300] The product of Example 1-24 (79 mg, 0.127 mmol) was dissolved in 1.3 mL of ethanol, and the aldehyde of Example 1-8 (113 mg, 0.19 mmol) was added. The mixture was stirred at 50°C for 18 hours to complete the reaction. The reaction mixture was then column-eluted. Chromatography The compound was purified by HPLC to give 123 mg in a yield of 85%.

[0301] 1 H NMR (400 MHz, CDCl3) δ 9.97(s, 1H), 7.38 (d, J = 7.0 Hz, 1H), 4.15 (m, 3 H), 3.65-3.40 (m, 56 H), 3.36(s, 3H), 1.52(m, 4H), 1.23 (m, 60 H), 0.86 (t, J = 6.4 Hz, 3H).

[0302]

[0303] Examples 1-26: Methyl-PEG2K-Cbz-hydrazide (M n of PEG=2000)

[0304] TIFF0007792441000034.tif23170

[0305] The same method as in Example 1-23 was used to obtain 160 mg of the compound in 95% yield.

[0306] 1 H NMR (400 MHz, MeOH-d4) δ 7.39-7.30 (m, 5H), 5.16 (s, 2H), 4.20 (s, 2H), 3.68-3.52 (m, 190H). 3.36 (s, 3H)

[0307]

[0308] Examples 1-27: Methyl-PEG2K-hydrazide (M n of PEG=2000)

[0309] TIFF0007792441000035.tif27170

[0310] The same method as in Example 1-24 was used to obtain 110 mg of the compound in 75% yield.

[0311] 1 H NMR (400 MHz, CDCl3) δ 4.14-4.01 (m, 2H), 3.61 (m, 190 H), 3.34 (s, 3H)

[0312]

[0313] Examples 1-28: Methyl-PEG2K-(dioctadecyloxy-propylidene)-acylhydrazone (mPEG2K-Hz-DSG)

[0314] JPEG0007792441000036.jpg25170

[0315] The same method as in Example 1-25 was used to obtain 57 mg of the compound in 53% yield.

[0316] 1 H NMR (400 MHz, CDCl3) δ 9.95(s, 1H), 7.36 (d, J = 7.0 Hz, 1H), 4.14 (m, 3 H), 3.60-3.40 (m, 200 H), 3.34(s, 3H), 1.51(m, 4H), 1.21 (m, 60 H), 0.84 (t, J = 6.4 Hz, 3H).

[0317]

[0318] Example 1-29: (R)-1-(4-(2,3-bis(octadecyloxy)propoxy)phenyl)ethan-1-one

[0319] TIFF0007792441000037.tif22170

[0320] The product of Example 1-7 (59.8 mg, 100 μmol) was dissolved in 2 mL of THF, and 4-hydroxyacetophenone (15.0 mg, 110 μmol), PPh3 (39.4 mg, 150 μmol), and DIAD (30 μL, 150 μmol) were added at 0°C. The mixture was stirred at room temperature for 12 hours to complete the reaction. The reaction mixture was extracted three times with dichloromethane and washed with brine. The organic layer was dried over anhydrous magnesium sulfate and evaporated under reduced pressure. Silica gel The resulting product was purified by chromatography to give 55.0 mg of the product in 77% yield.

[0321] 1H-NMR (400 MHz, CDCl3) δ 7.92 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 4.19-4.05 (m, 2H), 3.79 (quin, J = 5.1 Hz, 1H), 3.64-3.57 (m, 4H), 3.46 (t, J = 6.6 Hz, 2H), 2.55 (s, 3H), 1.61-1.50 (m, 4H), 1.32-1.21(m, 60H), 0.88 (t, J = 6.8 Hz).

[0322]

[0323] Examples 1-30: Methyl-PEG600-(2,3-dioctadecyloxypropoxy)phenyl-acetohydrazone (mPEG600-PhHz-DSG)

[0324] TIFF0007792441000038.tif60128

[0325] m-PEG-hydrazide M in ethanol (4 mL) n PEG550 (CreativePEGworks) (200 mg, 364 μmol) was dissolved, and the resultant product from Example 1-29 (260 mg, 364 μmol) and acetic acid (100 μL, 3.64 mmol) were added. The mixture was stirred at 85°C for 16 hours. The reaction product was purified by column purification to obtain 265 mg of the product in 66% yield.

[0326]

[0327] Examples 1-31: Methyl-PEG600-(2,3-dioctadecyloxypropoxy)carbonyl-methyl ether (mPEG600-Ester-DSG)

[0328] TIFF0007792441000039.tif51170

[0329] in DCM (30.0 mL) ω-(methoxypolyethylene glycol) carboxylic acid (M n =600)After dissolving (100 mg, 0.156 mmol), (S)-2,3-bis(octadecyloxy)propan-1-ol (187 mg, 0.312 mmol), 4-dimethylaminopyridine (23 mg, 0.187 mmol), and dicyclohexylcarbodiimide (37 mg, 0.180 mmol) were added dropwise. The mixture was stirred under nitrogen at room temperature for 24 hours. After the reaction was completed, the reaction mixture was CH 2 Cl 2 and Filtered through Celite 545 . Yes The organic solvent layer Wash with water, After drying over magnesium sulfate and filtration, the mixture was distilled under reduced pressure and separated by column chromatography (DCM:MeOH=20:1) to give the desired compound (68 mg, 0.64 mmol) in 41% yield.

[0330] 1 H NMR (500 MHz, CDCl3) δ 4.24 (t, 2H), 3.60-3.70 (m, 33H), 3.42 (s, 2H), 1.96-1.98 (d, 3H), 1.75-1.76 (d, 3H), 1.29 (s, 60H), 0.92 (t, 6H)

[0331]

[0332] Example 2: Preparation of lipid nanoparticles

[0333] To confirm whether the PEG moiety-degradable functional group-lipid conjugate described in this invention, i.e., a PEG derivative containing a functional group that degrades under acidic conditions, can stably form nanoparticles under acidic conditions, two types of nanoparticles were prepared. One nanoparticle contained PEG2K-Hz-DSG (Chemical Formula 3), which contains a hydrazone functional group that is hydrolyzed under acidic conditions. The other nanoparticles were prepared using PEG2K-PE-DSG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000, Avanti), which is not hydrolyzed under acidic conditions. Two solutions were prepared using a conventional method for preparing nanoparticles containing ionized lipids. One was an organic solution containing lipid components, prepared by dissolving the lipid components that make up the lipid nanoparticles in ethanol. To prepare lipid nanoparticles containing 1 mol% PEG2K-Hz-DSG or PEG2K-PE-DSG, DLin-MC3-DMA, DSPC (distearoylphosphatidylcholine), cholesterol, PEG2K-Hz-DSG, or PEG2K-PE-DSG were prepared at a total concentration of 4.5 mg / mL, maintaining a molar ratio of 50:10:39:1. To prepare lipid nanoparticles containing 2 mol% and 5 mol% PEG2K-Hz-DSG or PEG2K-PE-DSG, respectively, solutions containing 2 and 5 times the PEG content used to prepare the 1 mol% PEG lipid nanoparticles were prepared.

[0334] Separately, siRNA was prepared at a concentration of 0.046 mg / mL in a citrate buffer solution at pH 4. Lipid nanoparticles were prepared by mixing the ethanol solution and the aqueous buffer solution at a volume ratio of 3:1 using a micromixer chip or T-line chip. The flow rate of the mixed solution was 4 mL / min. The solution that passed through the chip was further mixed with buffer at a volume ratio of 1:1 to adjust the pH to 7, and then dialyzed against a pH 7.5 PBS buffer solution for 24 hours. The particle size of the prepared lipid nanoparticles was measured using a dynamic light scattering (DLS) device. If the hydrazone of PEG2K-Hz-DSG were hydrolyzed by PEG and lipid (DSG) under the acidic conditions used to prepare lipid nanoparticles, the lipid components should precipitate as heterogeneously sized precipitates, similar to the preparation of nanoparticles without a PEG moiety-degradable functional group-lipid conjugate. However, as can be seen from Figure 2, nanoparticles containing PEG2K-Hz-DSG as a component form nanoparticles of uniform size, similar to nanoparticles containing PEG2K-PE-DSG, and the larger the PEG content, the smaller the nanoparticles formed. This example confirmed that a PEG moiety-degradable functional group-lipid conjugate containing a functional group that is degraded under acidic conditions can stably form nanoparticles without being degraded under the acidic conditions used to produce lipid nanoparticles.

[0335]

[0336] Example 3: Comparison of efficacy of lipid nanoparticles containing PEG-Hz-DSG or PEG-PE-DSG

[0337] To confirm the efficacy of lipid nanoparticles containing PEG-Hz-DSG or PEG-PE-DSG, lipid nanoparticles containing siRNA targeting luciferase were prepared using the method described in Example 2. A cell line that constitutively expresses luciferase was treated with lipid nanoparticles at various concentrations relative to the siRNA concentration, and the degree of inhibition of luciferase activity was evaluated.

[0338] To confirm the effect of the molecular weight and content of PEG in lipid nanoparticles on efficacy, PEG2000 (PEG2K-Hz-DSG or PEG2K-PE-DSG) or PEG1000 (PEG1K-Hz-DSG or PEG1K-PE-DSG) were prepared at 1, 2, and 5 mol%, respectively. In the case of PEG600-Hz-DSG, lipid nanoparticles containing 1, 2, 5, 10, 20, 30, and 40 mol% were also prepared and evaluated to provide a wider range of PEG content.

[0339] Lipid nanoparticles containing PEG-PE-DSG either had no efficacy regardless of the PEG content (Figure 2A) or their efficacy decreased as the PEG content increased (Figure 2B). However, PEG-Hz-DSG was confirmed to be able to effectively inhibit luciferase expression regardless of the PEG content. In particular, lipid nanoparticles containing PEG600-Hz-DSG were confirmed to have improved efficacy by more effectively inhibiting luciferase expression up to a content of 20 mol%.

[0340]

[0341] Example 4: Comparison of cell uptake and target inhibition efficiency depending on the PEG content in lipid nanoparticles

[0342] PEG-DMG, a commonly used lipid nanoparticle manufacturing method, is known to decrease cellular uptake efficiency as the PEG content increases. To confirm this, lipid nanoparticles containing 2, 5, and 10 mol% PEG-DMG were prepared with the addition of 0.2 mol% of the fluorescent dye DiI (1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and then treated with HepG2 cells for 24 hours. Analysis of the cellular uptake efficiency of the nanoparticles using a flow cytometer (FACS) confirmed that increasing the PEG content decreased cellular uptake (Figure 3A).

[0343] Decreased uptake results in reduced efficacy of lipid nanoparticles. When preparing lipid nanoparticles containing PEG-DMG at 0, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mol%, siRNA targeting luciferase was also included. These were then applied to cells expressing luciferase, and luciferase activity was measured. As can be seen from graph B in Figure 3, the highest efficacy was observed in the 0.5-2 mol% PEG content range. No efficacy was observed in lipid nanoparticles containing 0-0.2 mol% PEG. Above 2 mol%, the efficacy of luciferase inhibition decreased with increasing PEG content. This is because very low PEG content leads to nonuniform particle formation, and PEG content above a certain level makes cellular uptake and endosomal escape difficult.

[0344] To directly compare the efficacy of lipid nanoparticles containing different amounts of PEG-DMG and PEG600-Hz-DSG, we prepared lipid nanoparticles containing luciferase-targeting siRNA at 1, 2, 5, and 10 mol% of the corresponding PEG moiety-degradable functional group-lipid conjugate. The lipid nanoparticles were then applied to HEPG2 and HEK293 cell lines, which constitutively express luciferase, and luciferase expression levels were measured after 24 hours. The efficacy of lipid nanoparticles containing PEG-DMG decreased with increasing PEG content, whereas the efficacy of lipid nanoparticles containing PEG600-Hz-DSG remained stable or even increased with increasing PEG content (Figures 3C and 3D). The EC50 values ​​calculated based on this data are shown in Figures 3E and 3F. In the HEPG2 cell line, the EC50 values ​​of lipid nanoparticles containing 10% each of PEG-DMG and PEG600-Hz-DSG were 8.9 nM and 0.2 nM, respectively, a difference of approximately 45-fold. In the HEK293 cell line, the values ​​were 26 nM and 0.72 nM, respectively, a difference of approximately 36-fold.

[0345]

[0346] Example 5: Confirmation of PEG removal from lipid nanoparticles

[0347] After lipid nanoparticles enter the cell's endosomes, removal of PEG within the endosome is advantageous for efficient interaction between the endosomal lipid membrane and the lipid nanoparticles. Lipid nanoparticles using conventional PEG-lipid conjugates lacking degradable functional groups maintain relatively stable particle structures under acidic conditions that mimic the internal environment of the endosome. In contrast, lipid nanoparticles containing functional groups that degrade under acidic conditions, such as PEG-Hz-DSG, become unstable due to the removal of PEG under acidic conditions. To confirm this, size changes were measured in buffer solutions of pH 7.4, 5.5, 5.0, and 4.5 for nanoparticles containing 1.5 mol% PEG-DMG (a typical lipid nanoparticle production ratio) and lipid nanoparticles containing 10 mol% PEG600-Hz-DSG (used in the present invention).

[0348] When observing the change in particle size over time under different pH conditions for nanoparticles produced using PEG-DMG, it was found that there was no significant change (Figure 4A). On the other hand, the size of lipid nanoparticles containing PEG600-Hz-DSG remained stable under neutral conditions (pH 7.4), but interparticle fusion was activated in acidic pH environments (Figure 4B). Furthermore, under each acidic condition, interparticle fusion continued and progressed over time, resulting in a steady increase in size. This indicates that the PEG that constitutes the nanoparticle surface is effectively removed under acidic conditions.

[0349] Therefore, PEG-Hz-DSG, which contains functional groups that are hydrolyzed under acidic conditions, such as hydrazone, maintains structural stability under the acidic conditions (pH 4, 20-40% ethanol buffer solution) used to prepare lipid nanoparticles (Figure 1). However, after removing the ethanol, it was confirmed that the hydrazone was rapidly hydrolyzed at acidic pH, resulting in the separation of the PEG and lipid moieties.

[0350]

[0351] Example 6: Confirmation of the hydrolysis time point of degradable functional groups

[0352] To determine whether the timing of PEG removal from the lipid nanoparticle surface affects efficacy, we prepared lipid nanoparticles containing 5 or 10 mol% PEG600-Hz-DSG, including luciferase-targeting siRNA. The lipid nanoparticles were treated with pH 7 or 4 buffer for 24 hours, and then treated with a cell line that constitutively expresses luciferase for 72 hours to measure luciferase expression levels.

[0353] Lipid nanoparticles pretreated with a pH 7 buffer solution were able to effectively suppress luciferase expression, but no efficacy was observed for lipid nanoparticles from which PEG had been removed in a pH 4 buffer solution (Figure 5). This indicates that efficacy is only achieved when PEG is removed after the lipid nanoparticles enter the cell's endosomes, but nanoparticles from which PEG was removed before they were uptaken by the cells lose their efficacy.

[0354]

[0355] Example 7: Evaluation of the effect of the chemical structure of the degradable functional group on efficacy

[0356] The hydrolysis half-life under acidic conditions varies depending on the chemical structure of the degradable functional group in the PEG moiety-degradable functional group-lipid conjugate component. To confirm the effect of hydrolysis sensitivity under acidic conditions on the efficacy of lipid nanoparticles, lipid nanoparticles containing 1, 2, 5, and 10 mol% of PEG derivatives with degradable functional groups as esters (PEG600-Ester-DSG, Chemical Formula 8) or arylhydrazones (PEG600-PhHz-DSG, Chemical Formula 9) were prepared.

[0357] [Chemical formula 8]

[0358] JPEG0007792441000040.jpg20147

[0359] [Chemical formula 9]

[0360] TIFF0007792441000041.tif26168

[0361] The lipid nanoparticles were prepared by incorporating siRNA targeting luciferase, and after 24 hours of treatment with a cell line that constitutively expresses luciferase, luciferase activity was measured (Figure 6A).

[0362] It was found that the efficiency of lipid nanoparticles improved as the content of PEG600-Hz-DSG and PEG600-Ester-DSG increased, and it was confirmed that the hydrazone functional group showed superior efficiency compared to the ester functional group.

[0363] To confirm the correlation between these results and the sensitivity of each functional group to hydrolysis under acidic conditions, lipid nanoparticles containing 10 mol% of the corresponding PEG moiety-degradable functional group-lipid conjugate were transferred to PBS pH 7.4 or citric acid pH 4.5 buffer solution, and the change in particle size was measured using dynamic light scattering (Figure 6B). Particle size increased in the order of functional groups: hydrazone, ester, and arylhydrazone, indicating that each functional group is more susceptible to hydrolysis under acidic conditions in that order. In other words, Figures 6A and 6B confirm that the more active the hydrolysis and the more effectively PEG is removed, the better the efficiency.

[0364]

[0365] Example 8: Evaluation of lipid membrane fusion and hemolysis induction of lipid nanoparticles

[0366] To investigate the degree to which lipid nanoparticles fuse with endosomal lipid membranes, liposome lipid membranes or red blood cells were mixed with the nanoparticles at a pH lower than the pKa of the ionized lipid, and the lipid membrane fusion reaction or hemolysis reaction was measured. The ionized lipid used in this study was DLin-MC3-DMA, whose amine group pKa is approximately 6.5, and at pH 4, the amine group is positively charged. The positive charge facilitates fusion through electrostatic attraction with the negative charge of the endosomal lipid membrane. However, when PEG is distributed on the nanoparticle surface, it not only creates a shielding effect by weakening the electrostatic attraction, but also prevents direct contact between the nanoparticle surface and the endosomal lipid membrane.

[0367] To evaluate the fusion efficiency of lipid nanoparticles with endosomal lipid membranes, lipid nanoparticles containing 10 mol% PEG600-Hz-DSG or 1.5 mol% PEG-DMG, a compound commonly used for lipid nanoparticle production, were prepared, but each nanoparticle contained 0.5 mol% of 18:1 NBD PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(ammonium salt)) and 18:1 Liss Rhod PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(ammonium salt)).

[0368] Liposomes were prepared to simulate the endosomal lipid membrane. Soy PS:DOPC:DOPE were dissolved in chloroform at a 25:25:50 mol% ratio to a total concentration of 1 mM. The chloroform was removed under reduced pressure in a round-bottom flask. 1 mL of PBS was added and sonication was performed to generate liposomes.

[0369] Lipid nanoparticles were mixed with liposomes in buffer solutions at pH 7.4, 6.5, 6.0, 5.5, 5.0, and 4.5. When the NBD and Liss Rhod contained in the lipid nanoparticles were in close proximity, no fluorescent signal was detected. However, as the nanoparticles fused with the lipid membrane of the liposome, the relative distance between the fluorescent lipids increased, resulting in an increase in the NBD fluorescent signal, which could be used to measure the lipid membrane fusion efficiency. The lipid membrane fusion efficiency of the nanoparticles was determined by measuring the NBD fluorescent signal at wavelengths of ex 475 / em 540.

[0370] Fluorescence signal generation measurements confirmed that lipid nanoparticles containing PEG-DMG or PEG600-Hz-DSG fused with the lipid membrane of liposomes under acidic conditions (Figure 7A). In particular, lipid nanoparticles containing 10% PEG600-Hz-DSG, which has a higher PEG content, began to fuse with the lipid membrane at a relatively higher pH than PEG-DMG lipid nanoparticles containing 1.5% PEG, generating a stronger fluorescence signal. Because both nanoparticles used the same ionized lipid, the effect of the positive charge of the ionized lipid was the same. However, the PEG in PEG-DMG lipid nanoparticles remained intact on the particle surface, whereas the PEG in PEG600-Hz-DSG was removed by hydrolysis of the hydrazone group, demonstrating a relatively superior lipid membrane fusion efficiency.

[0371] The fusion efficiency of lipid nanoparticles with lipid membranes was also confirmed by hemolysis experiments using red blood cells. Figure 7B shows the results of hemolysis induction assays using lipid nanoparticles containing 5 and 10 mol% PEG-DMG, a commonly used compound for lipid nanoparticle production, at pH 4.5 and 7.4. It can be seen that hemolysis is suppressed when the PEG content increases from 5% to 10%. This indicates that increasing the PEG content inhibits the lipid membrane fusion ability due to the blocking effect of PEG. In contrast, lipid nanoparticles prepared using ligand-containing PEG600-Hz-DSG exhibited hydrolysis under acidic conditions, and therefore lipid nanoparticles with an increased PEG derivative content of 10% also effectively induced hemolysis.

[0372] For reference, at a neutral pH of 7.4, the amine groups of the ionized lipids are electrically neutral, so neither of the two types of lipid nanoparticles induces hemolysis. In other words, at a neutral pH, the lipid nanoparticles do not interact with other cells around them, but when they reach endosomes, the lipid nanoparticles containing PEG derivatives fuse with the endosomal lipid membrane relatively easily.

[0373]

[0374] Example 9: Relationship between the length of lipids constituting PEG moiety-degradable functional group-lipid conjugates and the structural stability of lipid nanoparticles

[0375] As confirmed by the above examples, PEG surrounding the surface of lipid nanoparticles is necessary for the lipid nanoparticle manufacturing process but has a detrimental effect on pharmacological efficacy. Therefore, PEG-DMG, in which PEG is linked to a hydrocarbon fatty acid such as myristic acid (14 carbons), is commonly used to facilitate spontaneous removal of PEG from lipid nanoparticles during circulation in the bloodstream. Lipid nanoparticles manufactured using substances with relatively long fatty acids, such as palmitic acid (16 carbons) or stearic acid (18 carbons), have difficulty in entering cellular endosomes due to inefficient removal of PEG during circulation through the bloodstream, resulting in reduced efficacy.

[0376] However, the use of PEG-lipid conjugates with short fatty acids and subsequent PEG removal during circulation can significantly destabilize the structural stability of lipid nanoparticles. As the PEG-lipid conjugates begin to separate and be removed from the lipid nanoparticles, the stability of the particles themselves decreases, and other lipid components that make up the lipid nanoparticles also begin to be washed away. To confirm this, we compared the structural stability of lipid nanoparticles containing 1.5% or 5% PEG-DMG (lipid carbon length: 14) with that of lipid nanoparticles containing 5% PEG moiety-degradable functional group-lipid conjugates (chemical formula 4) with a fatty acid length of 18.

[0377] Fluorescent 18:1 NBD-PE and 18:1 Liss Rhod PE were added at 1 mol% each to prepare lipid nanoparticles. The lipid nanoparticles were placed in rat plasma at 37°C, and samples were collected at the designated times to measure the NBD fluorescence intensity (Figure 8). If the particles were stable, the two fluorescent dyes would be relatively close together within the nanoparticles, resulting in a small fluorescent signal from the NBD fluorescent dye. If the nanoparticle structure were to become unstable, this would induce the release of nanoparticle components, which would also release the fatty acids labeled with each fluorescent dye, resulting in an increase in fluorescent signal. As can be seen from the data, lipid nanoparticles prepared using PEG-DMG with short fatty acid lengths exhibited a short increase in fluorescent signal due to their unstable particle stability, whereas the PEG moiety-degradable functional group-lipid conjugate with long fatty acid lengths exhibited a relatively slow increase in fluorescent signal. In other words, lipid nanoparticles that intentionally use PEG-lipid conjugates composed of short fatty acids to remove PEG from lipid nanoparticles are subject to rapid elimination of PEG and other lipid components during circulation through the bloodstream, whereas lipid nanoparticles produced using the PEG moiety-degradable functional group-lipid conjugates described in the present invention are able to maintain a relatively stable structure.

[0378]

[0379] Example 10: Measurement of immune response induction by lipid nanoparticles

[0380] Ionized lipids that make up lipid nanoparticles can induce unwanted immune responses through interactions with immune cells. PEG can be used to minimize these side effects. However, as seen in the above examples, increasing the PEG content can effectively suppress unwanted interactions and reduce immune responses. However, increasing the PEG content beyond the minimum required amount inevitably leads to severe inhibition of cellular uptake of nanoparticles and endosomal escape within the cells.

[0381] In contrast, lipid nanoparticles containing PEG moiety-degradable functional group-lipid conjugates described in the present invention contain chemical functional groups that can increase cellular uptake efficiency using ligands and remove PEG within endosomes. Therefore, when using PEG moiety-degradable functional group-lipid conjugates, the efficacy of the resulting lipid nanoparticles does not decrease even when the PEG content is increased, while at the same time suppressing the induction of immune responses. To confirm this, lipid nanoparticles containing 10 mol% of the ligand-containing PEG moiety-degradable functional group-lipid conjugate were mixed with peripheral blood mononuclear cells (PBMCs) extracted from rats, and the extent to which the lipid nanoparticles induced cytokines was measured. As shown in Figure 9, the level of immune response induction by lipid nanoparticles containing PEG moiety-degradable functional group-lipid conjugates was found to be similar to that of the PBS buffer solution-treated negative control group.

[0382]

[0383] Example 11: Efficacy evaluation of lipid nanoparticles in animals

[0384] Using lipid nanoparticles that demonstrated efficacy in cell experiments, animal experiments were conducted to confirm their efficacy in vivo. Lipid nanoparticles containing 5 mol% PEG2K-PE-DSG or PEG2K-Hz-DSG were prepared, and each lipid nanoparticle contained siRNA targeting Factor 7. Eight-week-old C57BL / 6 female mice (Raon Bio) were intravenously injected with PBS or each lipid nanoparticle at a dose of 0.3 or 1 mg / kg siRNA. The injection volume was maintained at 10 mL / kg. Forty-eight hours after injection, serum Factor 7 levels were quantified using a Factor 7 assay kit (Abcam), and the levels were compared with those in the negative control group (PBS) (Figure 10).

[0385] Similar to the results of the cell experiments (Figure 2A), lipid nanoparticles containing hydrazone, a degradable functional group, dose-dependently suppressed the expression of Factor 7, whereas no efficacy was observed in lipid nanoparticles without degradable functional groups.

[0386]

[0387] Example 12: Efficacy evaluation of lipid nanoparticles containing ligands

[0388] To selectively deliver nanoparticles to tissues expressing specific receptors, lipid nanoparticles can be prepared by incorporating a ligand that specifically binds to the receptor. To confirm the effect of ligand-containing lipid nanoparticles, lipid nanoparticles containing 10 mol% PEG600-Hz-DSG were prepared, as were lipid nanoparticles containing a mixture of 0.5 mol% GalNac-PEG2K-Hz-DSG and 9.5 mol% PEG600-Hz-DSG for specific delivery to liver tissue. Animal experiments were conducted using the same method as described in Example 11, administering 0.1 mg / kg of siRNA to mice.

[0389] As a result, it was found that lipid nanoparticles containing liver-specific ligands exhibited superior efficacy compared to lipid nanoparticles without ligands (Figure 11).

[0390]

[0391] Example 13: Cytotoxicity evaluation of lipid nanoparticles

[0392] The relative toxicity of lipid nanoparticles containing commonly used PEG-DMG and lipid nanoparticles containing the PEG moiety-degradable functional group-lipid conjugate used in the present invention was evaluated. To this end, lipid nanoparticles containing 1.5 mol% PEG-DMG and lipid nanoparticles containing a mixture of GalNAc-PEG2K-Hz-DSG and PEG600-Hz-DSG at 1 mol% and 9 mol%, respectively, were prepared. Cell lines, including HepG2, HEK293, H460, and A549, were treated with siRNA at different concentrations in the lipid nanoparticles. After 72 hours, the activity of each cell line was measured using a CellTiter-Glo kit (Promega) (Figure 12).

[0393] When cells are treated with lipid nanoparticles containing PEG-DMG at high concentrations, many of the cells die, whereas the lipid nanoparticles containing 10 mol% of the PEG moiety-degradable functional group-lipid conjugate used in the present invention were found to induce relatively little cell death and to have very low toxicity.

[0394]

[0395] The therapeutic agent using lipid nanoparticles according to the present invention can minimize in vivo side effects caused by the components of the nanoparticles, effectively deliver the nanoparticles to target cells, and shed endosomes in the target cells to efficiently deliver pharmacologically active substances such as nucleic acids to the cytoplasm.

[0396] The lipid nanoparticles according to the present invention are coated on the outside with PEG, thereby suppressing nonspecific interactions. Furthermore, by attaching a ligand to the PEG terminus, they can be delivered only to target cells. Lipid nanoparticles taken up into endosomes by the ligand and cellular receptors undergo degradation of the degradable functional groups within the endosome, removing the PEG from the outside of the nanoparticles, thereby increasing the interaction between the surface of the lipid nanoparticles and the endosomal lipid membrane. Furthermore, the lipid component linking the PEG can contain a relatively long-chain fatty acid, which provides structural stability to the lipid nanoparticles and helps maintain the stability of the particles during processing into cells.

[0397]

[0398] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the claims and their equivalents.

Claims

1. Lipid nanoparticles comprising: (a) a lipid formulation comprising an ionizable lipid and a polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate; and (b) a drug, a nucleic acid, or a combination thereof, encapsulated within said lipid formulation; wherein the ionizable lipid is cationic at acidic pH and maintains an electrically neutral state at neutral pH; The polyethylene glycol moiety-degradable functional group-lipid conjugate is represented by Chemical Formula 1: 【Chemistry 1】 In chemical formula 1, a is 0 or 1; L is a targeting ligand or H; M is H, OH, a single bond, O, S, C(O), NHC(O), C(O)NH, OC(O) or C(O)O, provided that when a is 0, M is H or OH; P is CH ２ O (CH ２ CH ２ O) qCH ２ or CH ２ CH ２ O (CH ２ CH ２ O) qCH ２ where q is an integer from 2 to 120; L ６ is C(O)NH-N=CR ４ , R ４ C=N-NHC(O), NH-N=CR ４ , R ４ C═N—NH, ​​C(O)O, or OC(O), where R ４ is H, C １ -C ２０ Alkyl, C ２ -C ２０ Alkenyl, C ２ -C ２０ Alkynyl, C ３ -C １０ Cycloalkyl, C ６ -C ２０ C, which is an aryl or a radical containing a heteroatom selected from fluorine, oxygen, sulfur and nitrogen ６ -C ２０ It is a heterocycle, T is a single bond or 1,4-C ６ H ４ O-, R １ and R ３ are each independently -Y-R, and R ２ is -CH ２ -Y-R, where Y is a single bond, O, S, C(O), C(O)O, OC(O), C(O)NH, or NHC(O), and R is H, C １０ ~C ２０ Alkyl, or C １０ ~C ２０ is alkenyl, The targeting ligand L is represented by Chemical Formula 2: 【Transformation 3】 In chemical formula 2, a, b, and c are 0 or 1, but at least one of a, b, and c must be 1; X １ , X ２ and X ３ is a targeting ligand and is selected from the group consisting of N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-galactose, D-galactose, N-acetyl-D-glucosamine, D-glucose, D-mannose, L-fucose, carbohydrate derivatives, folic acid, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CADY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single chain variable fragment (scFv), and aptamer; L １ , L' １ and L” １ is a single bond, O, S, C(O), NHC(O), C(O)NH, OC(O) or C(O)O, L ２ , L' ２ and L” ２ is (CH ２ ) ｎ or (OCH ２ CH ２ ) m, where n is an integer from 1 to 20 and m is an integer from 1 to 10; L ３ , L' ３ and L” ３ is a single bond, O, S, C(O), NHC(O), C(O)NH, OC(O) or C(O)O, L ４ , L' ４ and L” ４ is (CH ２ ) ｎ where n is an integer from 1 to 20; L ５ , L' ５ and L” ５ is a single bond, O, S, C(O), NHC(O), C(O)NH, OC(O) or C(O)O.

2. The lipid nanoparticle of claim 1, wherein the polyethylene glycol moiety-degradable functional group-lipid conjugate is represented by chemical formula 3: 【Chemistry 4】 In Chemical Formula 3, n is an integer from 2 to 120.

3. The lipid nanoparticle of claim 1, wherein the polyethylene glycol moiety-degradable functional group-lipid conjugate is represented by chemical formula 4. 【Transformation 5】 In Chemical Formula 4, n is an integer from 2 to 120.

4. The lipid nanoparticle of claim 1, wherein the polyethylene glycol moiety-degradable functional group-lipid conjugate is a mixture of a compound in which a is 0 in Chemical Formula 1 and a compound in which a is 1.

5. The lipid nanoparticles according to claim 4, characterized in that the molar ratio of the compound in which a is 0 to the compound in which a is 1 is 0.01 to 99.9:0.01 to 99.

9.

6. The lipid nanoparticle according to claim 1, wherein the content of the polyethylene glycol moiety-degradable functional group-lipid conjugate in the lipid nanoparticle is 0.5 to 50 mol% of the total lipid components constituting the lipid nanoparticle.

7. The lipid nanoparticles according to claim 1, characterized in that the size of the lipid nanoparticles is 20 to 200 nm.

8. The ionizable lipids were (6Z,9Z,28Z,31Z)-heptatriaconta 6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid (SM-102), 1 -linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin -K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), dioctadecylamidoglycylcarboxyspermine (DOGS), spermine cholesteryl carbamate (GL-67); bis-guanidinium-spermidine -cholesterol (BGTC), 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), N-t-butyl-N'-tetradecylamino-propionamidine (diC14-amidine);Dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxyl)propyl)-N-2-(sperminecarboxamido)ethyl The lipid nanoparticles according to claim 1, characterized in that the lipid nanoparticles are at least one selected from the group consisting of N-(1-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE).

9. The lipid nanoparticle of claim 1, further comprising a sterol lipid and a neutral lipid.

10. The lipid nanoparticle of claim 9, wherein the sterol lipid is cholesterol or a cholesteryl ester.

11. The lipid nanoparticle of claim 9, wherein the neutral lipid is a phospholipid or a sphingolipid.

12. The phospholipids include DOPE (dioleoylphosphosphatidylethanolamine), DSPC (distearoylphosphosphatidylcholine), POPC (palmitoyloleoylphosphosphatidylcholine), EPC (egg phosphotidylcholine), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylp phosphatidylcholine), DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylp phosphatidylglycerol), DSPE (distearoylphosphotidylthanolamine), PE (Phospha tidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (1,2-di oleoyl-sn-glycero-3-phosphoethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]), ceramide, sphingomyelin, and mixtures thereof. The lipid nanoparticles according to claim 11, characterized in that they are selected from the group consisting of ceramide, sphingomyelin, and mixtures thereof.

13. The lipid nanoparticle according to claim 1, wherein the drug is at least one selected from the group consisting of a peptide, a protein drug, a protein-nucleic acid structure, and an anionic biopolymer-drug conjugate.

14. The lipid nanoparticle according to claim 13, wherein the nucleic acid is one or more selected from the group consisting of single-stranded siRNA, double-stranded siRNA, rRNA, DNA, cDNA, plasmid, aptamer, mRNA, tRNA, lncRNA, piRNA, circRNA, saRNA, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme.

15. 10. A method for producing lipid nanoparticles according to claim 1, comprising the steps of: (a) mixing an organic solution containing a lipid formulation including an ionizable lipid and a polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate with a buffer solution containing a drug, a nucleic acid, or a combination thereof, and adjusting the pH to acidic; and (b) removing the solvent from the mixed solution;

16. The method for producing lipid nanoparticles according to claim 15, wherein the mixing ratio of the organic solution to the buffer solution is 1:1 to 1:100 by volume.

17. The method for producing lipid nanoparticles according to claim 15, wherein the polyethylene glycol moiety-degradable functional group-lipid conjugate is represented by Chemical Formula 3: 【Chemistry 9】 In Chemical Formula 3, n is an integer from 2 to 120.

18. The method for producing lipid nanoparticles according to claim 15, wherein the polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate is represented by Chemical Formula 4: 【Chemistry 10】 In Chemical Formula 4, n is an integer from 2 to 120.

19. The method for producing lipid nanoparticles according to claim 15, wherein the polyethylene glycol moiety (PEG moiety)-degradable functional group-lipid conjugate is a mixture of a compound in which a is 0 in Chemical Formula 1 and a compound in which a is 1.

20. The method for producing lipid nanoparticles according to claim 19, characterized in that the molar ratio of the compound in which a is 0 to the compound in which a is 1 is 0.01 to 99.9:0.01 to 99.

9.

21. The method for producing lipid nanoparticles according to claim 15, characterized in that the content of the polyethylene glycol moiety in the lipid nanoparticles is 0.5 to 50 mol% of the total lipid components constituting the lipid nanoparticles.

22. The method for producing lipid nanoparticles according to claim 15, wherein the size of the lipid nanoparticles is 20 to 200 nm.

23. The ionizable lipids were (6Z,9Z,28Z,31Z)-heptatriaconta 6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid (SM-102), 1 -linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin -K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), dioctadecylamidoglycylcarboxyspermine (DOGS), spermine cholesteryl carbamate (GL-67); bis-guanidinium-spermidine -cholesterol (BGTC), 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), N-t-butyl-N'-tetradecylamino-propionamidine (diC14-amidine);Dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxyl)propyl)-N-2-(sperminecarboxamido)ethyl)- The method for producing lipid nanoparticles according to claim 15, characterized in that the lipid nanoparticle is at least one selected from the group consisting of N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE).

24. The method for producing lipid nanoparticles according to claim 15, further comprising a sterol lipid and a neutral lipid.

25. The method for producing lipid nanoparticles according to claim 24, wherein the sterol lipid is cholesterol or a cholesteryl ester.

26. The method for producing lipid nanoparticles according to claim 24, wherein the neutral lipid is a phospholipid.

27. The phospholipids include DOPE (dioleoylphosphosphatidylethanolamine), DSPC (distearoylphosphosphatidylcholine), POPC (palmitoyloleoylphosphosphatidylcholine), EPC (egg phosphotidylcholine), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylph osphatidylcholine), DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylpho sphatidylglycerol), DSPE (distearoylphosphatidylethanolamine), PE (phosphatid ylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (1,2-dioleo 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]), ceramide, or sphingomyelin, and mixtures thereof. The method for producing lipid nanoparticles according to claim 26, characterized in that the lipid nanoparticles are selected from the group consisting of ceramide, sphingomyelin, and mixtures thereof.

28. The method for producing lipid nanoparticles according to claim 15, wherein the drug is at least one selected from the group consisting of peptides, protein drugs, protein-nucleic acid structures, and anionic biopolymer-drug conjugates.

29. The method for producing lipid nanoparticles according to claim 28, wherein the nucleic acid is at least one selected from the group consisting of single-stranded siRNA, double-stranded siRNA, rRNA, DNA, cDNA, plasmid, aptamer, mRNA, tRNA, lncRNA, piRNA, circRNA, saRNA, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme.

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