CN117615753A

CN117615753A - Lipid nanoparticle and method for preparing the same

Info

Publication number: CN117615753A
Application number: CN202280047870.7A
Authority: CN
Inventors: 李泰雨; 白有美; 柳智原; 张多慧
Original assignee: Taina Treatment Co
Current assignee: Taina Treatment Co
Priority date: 2021-06-24
Filing date: 2022-06-23
Publication date: 2024-02-27

Abstract

The present invention relates to lipid nanoparticles and methods of making the same, and more particularly, to particles comprising ionizable lipids and polyethylene glycol moieties (PEG moieties) -degradable linking functional groups-lipid conjugates characterized by minimized in vivo side effects and efficient transfer of the nanoparticles into target cells, thereby delivering pharmaceutically effective substances into the cytoplasm.

Description

Lipid nanoparticle and method for preparing the same

Technical Field

The present invention relates to lipid nanoparticles and a method of preparing the same, and more particularly, to lipid nanoparticles comprising ionizable lipids and polyethylene glycol derivative moieties (PEG moieties) -degradable linking functional group-lipid conjugates, thereby minimizing in vivo side effects, effectively delivering the nanoparticles to target cells, and effectively transporting pharmacological substances into the cytoplasm, and a method of preparing the same.

Background

The efficacy of a drug varies greatly depending on the method of administration. A drug system having a drug administration route effective in delivering the required amount of drug by minimizing side effects of the drug while maximizing efficacy and effect thereof is called a "Drug Delivery System (DDS)". In the pharmaceutical formulation industry, drug delivery systems are considered as high value core technologies that have a high potential for success and offer economic benefits comparable to new drug development.

Solubilization of poorly soluble drugs, which is a key technology in drug delivery systems, is a drug absorption enhancement, and is considered to be the most rational approach to reduce the cost of new drug development and increase the added value of drugs. In particular, in the case where the conditions for developing the korean novel drug are deficient, the development of the improved novel drug based on the development of the drug solubilization technology can create a great additional value at low cost.

Recently, development of gene therapy using a gene drug delivery system among various drug delivery systems is gradually expanding. For successful and safe gene therapy, it is important to deliver the gene or gene regulatory factor to the desired tissue. Representative examples of such genes or gene regulatory factors include mRNA and siRNA. Since nucleic acids such as mRNA function to express specific proteins, they can compensate for protein deficiency due to genetic factors. In addition, anticancer drugs and vaccines can be developed that use mRNA expressing cancer markers and viral surface proteins to activate immune responses in vivo. In addition, nucleic acids such as siRNA are substances capable of inhibiting the expression of specific proteins in vivo, and are attracting attention as a key tool for the treatment of cancer, genetic diseases, infectious diseases, and autoimmune diseases. However, it is difficult to deliver nucleic acids such as mRNA and siRNA directly into cells, and nucleic acids are easily degraded by enzymes in the blood. Accordingly, research is actively being conducted to overcome this problem.

Characteristics and limitations of nanoparticles based on cationic liposomes or cationic polymers.

In order to deliver these substances efficiently, non-viral gene vectors such as cationic liposomes and polymers have been developed, and the improved stability characteristics of the vectors, as well as ease of manufacture and handling, have accelerated research into the design and synthesis of non-toxic and biodegradable polymeric vectors for efficient and safe gene delivery. Poly (L-lysine), polyethylenimine, starburst, polyamide-amine dendrimers, cationic liposomes, and the like are capable of spontaneously self-assembling with anionic oligomers and compressing plasmid DNA (pDNA) into a structure small enough to introduce the plasmid DNA (pDNA) into cells by endocytosis, and thus are being widely studied as non-viral gene vectors.

However, nanoparticles prepared using cationic liposome or cationic polymer based methods have limitations that are not suitable for development into therapeutic agents. The method is characterized in that cationic liposomes or polymers are mixed with anionic oligonucleotides (oligos) and nanoparticles are prepared in a dense manner by electrostatic attraction. Thus, in order to produce stable particles, an excess of cationic species compared to the amount of anionic charge must be used. Since nanoparticles cannot be formed when the same molar ratio of cations and anions is used, an excess of cations is used, more particularly at least several times to several tens times. Thus, the nanoparticles prepared in this way are neutralized by the interaction with anions, and the remaining cations are still present in excess in the nanoparticles. Thus, the prepared nanoparticles always maintain cationic charge. However, positively charged nanoparticles are known to have the disadvantage of causing side effects due to unwanted interactions with various cells including immune cells in vivo. Therefore, it is difficult to develop therapeutic agents using these nanoparticles.

Characteristics of lipid nanoparticles using ionizable lipids and methods of making the same.

Recently, effective therapeutic agents have been developed using novel lipid nanoparticles, which overcome the disadvantages of cationic nanoparticles, as non-viral gene vectors (non-patent documents 1 to 9). Lipid nanoparticles are particulate drug carriers with high bioavailability and affinity because they use substances present in vivo such as phospholipids and cholesterol, are capable of releasing and controlling drugs, are highly stable against enzymatic degradation, and the like. In particular, the essential difference between such lipid nanoparticles and conventional cationic nanoparticles is that the lipid nanoparticles comprise ionizable lipids as components. The ionizable lipids are characterized in that they are cationic at acidic pH, but remain electrically neutral at neutral pH. Thus, nanoparticles prepared using ionizable lipids are electrically neutral in the blood stream at neutral pH, and thus in vivo side effects caused by the cationic nature of the cationic polymer or cationic liposome can be significantly eliminated.

Another distinction between them relates to a method of preparing lipid nanoparticles. More particularly, the method of preparing lipid nanoparticles is described below. Lipid nanoparticles are prepared by mixing lipid components dissolved in an organic phase with oligonucleotides dissolved in an aqueous buffer solution. Since the polarity of the solvent changes with the mixing of the organic phase with the aqueous solution, the lipid component contained in the organic phase spontaneously forms particles in the mixed solution. At this time, when the ionizable lipid contained in the organic phase is mixed with an aqueous buffer solution containing the oligonucleotide under acidic conditions, the ionizable lipid becomes cationic, and the cationic ionizable lipid is linked to the anionic oligonucleotide by electrostatic attraction, thereby preparing the nanoparticle containing the oligonucleotide.

Another feature of the lipid nanoparticle based on ionizable lipids is that the lipid nanoparticle is prepared by incorporating a lipid in order to prevent aggregation of the nanoparticle in the organic phase. The lipid for preventing aggregation of nanoparticles has a molecular structure in which the lipid is linked to a hydrophilic substance. The hydrophilic material may be a biological material such as a carbohydrate or protein, or may be a synthetic material such as a polyol and PEG. A representative example thereof is PEG-lipid conjugate. In the PEG-lipid conjugate, the lipid is hydrophobic and thus is guided to the inside of the particle at the time of formation of the lipid nanoparticle, whereas PEG is hydrophilic and thus is located on the surface of the lipid nanoparticle and is guided to the outside of the particle (non-patent documents 11 to 12). In this process, nanoparticles can be continuously aggregated with each other unless a lipid for preventing aggregation of particles, such as PEG-lipid conjugate, is included as a component to constitute the nanoparticle surface. Thus, it is difficult to prepare nanoparticles having a relatively uniform size, and it is also difficult to achieve the desired efficacy from non-uniform nanoparticles.

Another reason that PEG is commonly used as a hydrophilic substance of lipids to prevent aggregation is that PEG constituting the surface of the lipid nanoparticle also affects the biological properties of the lipid nanoparticle. In general, injection of nanoparticles into the body disadvantageously causes loss due to phagocytosis by macrophages through adsorption of proteins, lipids and immune components in the blood. To solve this problem, it is known that this disadvantage can be overcome by modifying the surface of nanoparticles with PEG, a hydrophilic polymer having excellent biocompatibility (U.S. Pat. No. 9,999,673B2; korean patent publication No. 10-2018-0032652, WO 2020/061284). PEG is most commonly used because it has no side effects on living bodies and is easily applied to lipid nanoparticles and other drug delivery systems (non-patent document 10).

Disadvantages of using PEG-lipid conjugates and limitations of conventional solutions.

Although PEG is an essential component in the preparation of lipid nanoparticles and has the significant advantage of reducing many side effects by inhibiting non-specific interactions of the nanoparticles with other substances in the body, it still has fundamental limitations. The fact that PEG reduces non-specific in vivo interactions between nanoparticles and other biological components ultimately results in inhibition of interactions between nanoparticles and target cells. Thus, PEG makes it difficult to deliver nanoparticles into target cells, so that pharmacological efficiency is significantly reduced (non-patent documents 13 to 15).

PEG inhibits interactions between nanoparticles and target cells. To solve this problem, two methods are devised. One is a method of separating PEG from lipid nanoparticles after the nanoparticles are incorporated into the body. Typically, the lipid component of PEG-lipid conjugates consists of two fatty acid chains. When the hydrophobicity of the fatty acid is reduced by reducing the number of carbons constituting the fatty acid, the PEG-lipid conjugate is separated from the nanoparticle and easily removed while the nanoparticle circulates in the body. Thus, the efficiency of the nanoparticle is maintained because the interference effect caused by PEG is eliminated. It is known that PEG-lipid conjugates are not removed from nanoparticles when the number of carbons constituting fatty acids increases, so that the efficacy thereof is reduced (non-patent documents 14 to 16). However, when the PEG-lipid conjugate as the nanoparticle component is removed in this way, disadvantageously, the particles become unstable and other components constituting the particles are lost (non-patent document 17).

Another method for solving the problem of reduced delivery efficiency of lipid nanoparticles to target cells due to PEG is to attach a ligand capable of binding to a cell receptor to one end of PEG (non-patent document 16). Since the ligand at the end of PEG binds to the receptor of a particular cell, the nanoparticle containing the ligand may be delivered to the endosome within the cell through the receptor. However, even though the nanoparticle can be efficiently delivered to an endosome within a cell by using a ligand, in order to obtain pharmacological efficacy, it is necessary to deliver a nucleic acid, which is a pharmacological substance contained in the nanoparticle, from the endosome into the cytoplasm. For this purpose, a fusion process is required through interactions between the lipid nanoparticle and the endosomal lipid membrane, but PEG around the outside of the nanoparticle would interfere with this function. Thus, attaching a ligand to the end of PEG, while enabling the nanoparticle to be delivered into the endosome of the target cell, does not enable the delivery of the active substance from the endosome into the cytoplasm. Therefore, this method cannot significantly improve efficiency (non-patent document 16).

Use of PEG-degradable functional group-lipid conjugates in ionizable lipid nanoparticles.

To address the reduced efficacy caused by PEG in endosomes, methods of removing PEG in endosomes around the surface of lipid particles are typically used. Since the pH inside the endosome is acidic, a substance in the form of a PEG-degradable functional group-lipid conjugate is included as a component, so that the site connecting PEG and lipid has a functional group that is hydrolyzable under acidic conditions. Thus, PEG constituting the nanoparticle surface in the interior can be effectively removed (non-patent documents 18 to 19).

However, the conditions under which nanoparticles of PEG-degradable functional group-lipid conjugate type substances containing a degradable functional group that degrades under acidic conditions can be prepared are inevitably limited. This is because the degradable functional group of the PEG-degradable functional group-lipid conjugate is unstable under acidic conditions, and thus the preparation conditions of the nanoparticle can only be applied to the nanoparticle prepared under neutral pH or alkaline conditions. However, the lipid nanoparticles widely used in various oligonucleotide-based therapies recently developed contain an ionizable lipid as a component, and as described above, the lipid nanoparticles containing an oligonucleotide component using the ionizable lipid must be prepared at an acidic pH. For this reason, when preparing lipid nanoparticles comprising ionizable lipids, the use of a component having a functional group that hydrolyzes under acidic conditions (i.e., PEG-degradable functional group-lipid conjugate) as a component is considered to be substantially impossible and has not been tried so far.

Thus, as a result of long-term efforts to solve the problem, the present inventors found that lipid nanoparticles comprising an ionizable lipid as a 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 under the acidic conditions of the process of preparing the lipid nanoparticle, but is effectively hydrolyzed under the acidic conditions of the endosome, which facilitates fusion of the endosome lipid membrane and the lipid nanoparticle, and significantly improves the oligonucleotide delivery efficiency. The present inventors have found that when these lipid nanoparticles are prepared and used as therapeutic agents, they can minimize in vivo side effects due to the composition of the nanoparticles, effectively transfer the nanoparticles to target cells, and are suitable for effectively delivering pharmacological substances such as nucleic acids from endosomes within the target cells to cytoplasm. Based on this, the present invention has been completed.

[ Prior literature ]

[ patent literature ]

U.S. Pat. No. 9,999,673B2

Korean patent laid-open publication No. 10-2018-0032652

International publication WO 2020/061284

[ non-patent literature ]

1.Moss,K.H.,Popova,P.,Hadrup,S.R.,Astakhova,K.&Taskova,M.Lipid Nanoparticles for Delivery of Therapeutic RNAOligonucleotides.Mol Pharm 16,2265-2277(2019).

2.Kulkarni,J.A.,Witzigmann,D.,Chen,S.,Cullis,P.R.&van der Meel,R.Lipid Nanoparticle Technology for Clinical Translation of siRNATherapeutics.Acc Chem Res 52,2435-2444(2019).

3.Buck,J.,Grossen,P.,Cullis,P.R.,Huwyler,J.&Witzigmann,D.Lipid-Based DNATherapeutics:Hallmarks of Non-Viral Gene Delivery.ACS Nano 13,3754-3782(2019).

4.Akinc,A.et al.The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs.Nat Nanotechnol 14,1084-1087(2019).

5.Springer,A.D.&Dowdy,S.F.GalNAc-siRNA Conjugates:Leading the Way for Delivery of RNAi Therapeutics.Nucleic Acid Ther 28,109-118(2018).

6.Kulkarni,J.A.,Cullis,P.R.&van der Meel,R.Lipid Nanoparticles Enabling Gene Therapies:From Concepts to Clinical Utility.Nucleic Acid Ther28,146-157(2018).

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

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

9.Cullis,P.R.&Hope,M.J.Lipid Nanoparticle Systems for Enabling Gene Therapies.Mol Ther 25,1467-1475(2017).

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).

11.Belliveau,N.M.et al.Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA.Mol Ther Nucleic Acids 1,e37(2012).

12.Sato,Y.et al.Elucidation of the physicochemical properties and potency of siRNA-loaded small-sized lipid nanoparticles for siRNAdelivery.J Control Release 229,48-57(2016).

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

14.Mui,B.L.et al.Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNALipid Nanoparticles.Mol Ther Nucleic Acids 2,e139(2013).

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).

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

17.Chen,S.et al.Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA.J Control Release 235,236-244(2016).

18.Bargh,J.D.,Isidro-Llobet,A.,Parker,J.S.&Spring,D.R.Cleavable linkers in antibody-drug conjugates.Chem Soc Rev 48,4361-4374(2019).

19.Zhao,G.et al.Smart pH-sensitive nanoassemblies with cleavable PEGylation for tumor targeted drug delivery.Sci Rep 7,3383(2017)。

Disclosure of Invention

Technical problem

Accordingly, the present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide lipid nanoparticles that minimize in vivo side effects due to the composition of the nanoparticles, effectively deliver the nanoparticles to target cells, and effectively transport pharmacological substances such as nucleic acids from endosomes in the target cells into the cytoplasm.

Technical proposal

In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of a lipid nanoparticle comprising: (a) Lipid formulations comprising an ionizable lipid and a polyethylene glycol moiety (PEG moiety) -degradable functional group-lipid conjugate; and (b) a drug, nucleic acid, or combination thereof encapsulated in a lipid formulation.

According to another aspect of the present invention, there is provided a method of preparing lipid nanoparticles comprising (a) mixing an organic solution comprising a lipid formulation comprising an ionizable lipid and a polyethylene glycol moiety (PEG moiety) -degradable functional group-lipid conjugate with a buffer solution comprising a drug, a nucleic acid, or a combination thereof to adjust pH, and (b) removing solvent from the solution.

Drawings

Fig. 1 shows a graph (a) showing the size of lipid nanoparticles containing 1, 2 and 5mol% PEG2K-Hz-DSG or PEG2K-PE-DSG based on the total lipid weight of the nanoparticles according to an embodiment of the present invention, measured by DLS, and a graph (B) showing the quantitative result thereof.

Fig. 2 is a graph showing efficacy of inhibiting luciferase expression when a constitutive luciferase-expressing cell is treated with lipid nanoparticles prepared by incorporating sirnas targeting luciferase according to embodiments of the invention, wherein a is a graph showing lipid nanoparticles containing 1, 2 and 5mol% PEG2K-Hz-DSG or PEG2K-PE-DSG, B is a graph showing lipid nanoparticles containing 1, 2 and 5mol% PEG1K-Hz-DSG or PEG1K-PE-DSG, and C is a graph showing lipid nanoparticles containing 1, 2, 5, 10, 20, 30, or 40mol% PEG 600-Hz-DSG.

FIG. 3 illustrates the effect of PEG content in lipid nanoparticles according to an embodiment of the present invention on cellular uptake efficiency, wherein A shows a graph showing the efficacy of inhibiting luciferase expression when a cell constitutively expressing luciferase is treated with lipid nanoparticles prepared by incorporating siRNA targeting luciferase into nanoparticles containing 0, 0.1, 0.2, 0.5, 1, 2, 5 or 10mol% of PEG-DMG according to an embodiment of the present invention, and C and D are graphs showing the efficacy of inhibiting luciferase expression in different cell lines of lipid nanoparticles containing 1, 2, 5 and 10mol% of PEG-DMG or PEG600-Hz-DSG, respectively, analyzed by flow cytometry (FACS) based on total lipid weight ₅₀ Is a data of (a) a data of (b).

FIG. 4 shows data of the change in size of lipid nanoparticles containing 1.5mol% PEG-DMG (A) or 10mol% PEG600-Hz-DSG (B) under acidic conditions according to an embodiment of the present invention.

FIG. 5 is a graph showing the efficacy of lipid nanoparticles containing 5 or 10mol% PEG600-Hz-DSG in inhibiting luciferase expression after pretreatment of a cell line with lipid nanoparticles at pH 4 or 7 and then treatment of the cell line with lipid nanoparticles according to embodiments of the invention.

FIG. 6 is a graph (A) showing the efficacy of lipid nanoparticles comprising 1, 2, 5 or 10mol% PEG600-Hz-DSG, PEG 600-ester-DSG or PEG600-PhHz-DSG to inhibit luciferase expression after treatment of a cell line with lipid nanoparticles and data (B) of the size of the lipid nanoparticles under neutral or acidic conditions, according to embodiments of the invention.

FIG. 7 shows data (A) of the intensity of fusion of lipid nanoparticles prepared according to an embodiment of the present invention comprising 10mol% PEG600-Hz-DSG or 1.5mol% PEG-DMG and fluorescent dye with liposome lipid membranes at the corresponding pH conditions and data (B) of hemolysis of lipid nanoparticles comprising 5 or 10mol% GalNAc-PEG600-Hz-DSG or PEG-DMG at pH 4.5 and pH 7.4.

Fig. 8 shows data of fluorescence intensity over time contained in lipid nanoparticles when lipid nanoparticles containing 1.5mol% and 5mol% PEG-DMG and lipid nanoparticles containing 5mol% GalNAc-PEG2K-Hz-DSG based on the total weight of lipids prepared using FRET fluorescent dye were immersed in rat plasma according to an embodiment of the present invention.

FIG. 9 shows data of relative amounts of prepared cytokines measured by q-PCR when lipid nanoparticles containing 10mol% GalNAc-PEG600-Hz-DSG were mixed with Peripheral Blood Mononuclear Cells (PBMC) isolated from rats.

Figure 10 shows data for the amount of factor 7 in serum of mice administered with lipid nanoparticles containing PEG2K-PE-DSG or PEG2K-Hz-DSG, according to an embodiment of the invention.

FIG. 11 shows data of the content of factor 7 in serum of mice administered with lipid nanoparticles of mixed lipids containing 10mol% PEG600-Hz-DSG or 9.5mol% PEG600-Hz-DSG, and 0.5mol% GalNAc-PEG2K-Hz-DSG, according to an embodiment of the present invention.

Figure 12 shows data of relative viability of each cell line treated with lipid nanoparticles containing PEG-DMG or PEG-Hz-DSG according to an embodiment of the invention.

Best mode for carrying out the invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the terms used herein are well known in the art and are commonly used.

The invention is based on the following findings: when lipid nanoparticles comprising an ionizable lipid and a polyethylene glycol derivative moiety-a degradable functional group-lipid as components are prepared and used as therapeutic agents, they can minimize in vivo side effects due to the components of the nanoparticles, effectively transfer the nanoparticles to target cells, and are suitable for effectively delivering pharmacological substances such as nucleic acids from endosomes within the target cells to the cytoplasm. Based on this, the present invention has been completed.

In one aspect, the present invention relates to a lipid nanoparticle comprising (a) an ionizable lipid and a lipid formulation of a polyethylene glycol moiety (PEG moiety) -degradable functional group-lipid conjugate, and (b) a drug, a nucleic acid, or a combination thereof encapsulated in the lipid formulation.

In another aspect, the invention relates to a method of preparing the lipid nanoparticle comprising (a) mixing an organic solution comprising a lipid formulation comprising an ionizable lipid and a polyethylene glycol moiety (PEG moiety) -degradable functional group-lipid conjugate with a buffer solution comprising a drug, a nucleic acid, or a combination thereof to adjust pH, and (b) removing the solvent from the solution.

Hereinafter, the present invention will be described in detail.

As used herein, the terms "polyethylene glycol", "PEG moiety" and "PEG derivative" include functional groups corresponding to polyethylene glycol, and are used interchangeably. Combinations of these elements are included in all possible variations unless otherwise indicated herein or clearly contradicted by context.

Lipid nanoparticles according to the present invention include (a) a lipid formulation comprising an ionizable lipid and a polyethylene glycol moiety (PEG moiety) -degradable functional group-lipid conjugate, and (b) a drug, nucleic acid, or combination thereof encapsulated in the lipid formulation.

In the present invention, the PEG moiety-degradable functional group-lipid conjugate may be represented by the following formula 1.

[ 1]

Wherein the method comprises the steps of

a is 0 or 1 and the number of the groups,

l is a targeting ligand which is a ligand,

m is H, OH, a single bond, O, S, C (O), NHC (O), C (O) NH, OC (O) or C (O) O,

p is H ₂ O(CH ₂ CH ₂ O) _q CH ₂ Or CH (CH) ₂ CH ₂ O(CH ₂ CH ₂ O) _q CH ₂ Wherein q is an integer of 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、OC(O)、OC(O)O、O-N＝CR ₄ 、R ₄ c=n-O, S-S, S or trans-cyclooctene,

or (b)Wherein R is 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 Aryl or heterocycle which is a radical containing heteroatoms 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,

t is a single bond or 1,4-C ₆ H ₄ O-，

R ₁ And R is ₃ Each independently is-Y-R, R ₂ is-CH ₂ Y-R, wherein 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, alkenyl or sterol.

Furthermore, the ligand may be present at one end of the PEG moiety-degradable functional group-lipid conjugate.

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

[ 2]

Wherein the method comprises the steps of

a. b and c are 0 or 1, with the proviso that at least one of a, b or c is 1,

X ₁ 、X ₂ and X ₃ Is a targeting ligand, which is a target ligand,

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 Wherein n is an integer of 1 to 20, and m is an integer of 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 Wherein n is an integer of 1 to 20, and

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.

In the present invention, X ₁ 、X ₂ And X ₃ May be selected from the group consisting of: N-acetyl-D-galactosamine (GalNAc), N-acetyl-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 antibodies, antigen binding fragments or antibody fragments, single chain variable fragments (scFv) and aptamers. Here, an antigen-binding fragment of an antibody or antibody fragment refers to a fragment having an antigen-binding abilityFragments include Fab, F (ab') 2, fv, and the like.

Examples of antibodies that may be used as ligands in the present invention include, but are not limited to, anti-CD 3, anti-CD 19, anti-CD 20, anti-CD 22, anti-CD 33, anti-CD 38, anti-CD 54, anti-CD 74, anti-CD 138, anti-CD 166, anti-CD 209, anti-cMET, anti-EGFR, anti-HER 2, anti-HIV-gp 120, anti-HLA-DR, anti-transferrin receptor (TfRscFv), and the like.

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

[ 3]

Wherein n is an integer from 2 to 120.

In the present invention, the PEG moiety-degradable functional group-lipid conjugate is preferably a ligand-PEG moiety-degradable functional group-lipid conjugate (a is 1 in formula 1), more preferably represented by the following formula 4 (GalNAc-PEG-Hz-lipid).

[ 4]

Wherein n is an integer from 2 to 120.

In the present invention, the ligand-PEG moiety-degradable functional group-lipid conjugate may be a mixture of a PEG moiety-degradable functional group-lipid conjugate (a in formula 1 is 0) and a ligand-PEG moiety-degradable functional group-lipid conjugate (a in formula 1 is 1).

In the present invention, the molar ratio of the compound of formula 1 in which a is 0 to the compound of formula 1 in which a is 1 is (0.01 to 99.9): (0.01 to 99.9), preferably (50 to 99.9): (0.01 to 50), 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 be present in an amount of 0.01 to 100mol%, preferably 0.01 to 20mol%, more preferably 5 to 10mol% 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 which it is bound. Although the amount of N-acetyl-D-galactosamine (GalNAc) used in the embodiments of the present invention is small (5 to 10 mol%), it also shows the efficacy of lipid nanoparticles.

In the present invention, the size of the lipid nanoparticle may be 20 to 200nm, preferably 30 to 200nm.

The structural features of the PEG moiety in the PEG moiety-degradable functional group-lipid conjugate according to the present invention are as follows.

The peg moiety has a molecular weight of 100 to 5,000 and has a single or branched chemical structure. The content of the PEG moiety may be 0.5 to 50mol%, preferably 0.5 to 40mol%, more preferably 0.5 to 30mol% of the total lipid constituting the lipid nanoparticle. When the content of the PEG moiety is less than 0.5mol%, it is difficult to obtain nanoparticles having a uniform size when preparing lipid nanoparticles, and other lipid components constituting the nanoparticles may be exposed to the surface, easily causing non-specific interactions. When the content exceeds 50mol%, there is a problem in that the PEG moiety-degradable functional group-lipid conjugate component is not contained in the lipid nanoparticle and independent micelles are formed. Furthermore, the molecular weight of PEG and the content of lipid nanoparticles are inversely related to each other. When PEG having a large molecular weight of about 5,000 is used, the content of PEG in the lipid nanoparticle is 20mol% or less, but when PEG having a molecular weight of 100 is used, the content of PEG in the lipid nanoparticle may be increased to 50mol%. With increasing molecular weight, it is difficult to form uniform lipid nanoparticles due to the steric effect caused by PEG, and hydrophilic PEG moieties are increased as compared to hydrophobic lipid moieties, thus having a strong tendency to form independent micelles instead of binding to lipid nanoparticles.

2. Some or all of the polyethylene glycol (PEG) moiety-degradable functional group-lipid conjugates may be used by attaching a ligand capable of binding to a cell receptor at one end of the PEG moiety. The ligand includes a ligand that can be selectively linked to a receptor expressed in a specific cell in the form of a sugar or carbohydrate including N-acetylgalactosamine, glucose, mannose, fucose, etc., a peptide containing RGD, etc., a small molecule such as folic acid, a nucleic acid such as an aptamer, and a protein such as an antibody or an antigen recognition portion antibody. More particularly, the ligand comprises: N-acetyl-D-galactosamine (GalNAc), N-acetyl-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 antibodies, antigen binding fragments or antibody fragments, single chain variable fragments (scFv), aptamers, and the like.

The other end of the peg moiety is attached via a linker comprising a chemical functional group or a substrate for a biohydrolase that degrades under specific conditions. For example, linkers include-C (O) -NH-n=cr-, -rc=n-NH-C (O) -, -NH-n=cr-, -rc=n-NH-, -C (O) -O-, -O-C (O) -O-, -O-n=cr-, -rc=n-O-, -S-, -S-or-trans-cyclooctene-, wherein R is H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, and the like.

4. The linker is attached to a hydrophobic substance, such as a fatty acid based lipid or sterol.

Among the many components of lipid nanoparticles according to the present invention, ionizable lipids are ionizable compounds that are linked to the lipid component and encapsulate drugs (e.g., anionic drugs and/or nucleic acids) in the nanoparticle with high efficiency via electrostatic interactions. The ionizable lipid is neutral at neutral pH and positively charged at acidic pH.

In the present invention, the ionizable lipid may include at least one selected from the group consisting of: 4- (dimethylamino) butanoic acid (6Z, 9Z,28Z, 31Z) -heptadecane-6,9,28,31-tetralin-19-yl ester (DLin-MC 3-DMA), [ (4-hydroxybutyl) azetidino ] bis (2-hexyldecanoate) (ALC-0315), 8- [ (2-hydroxyethyl) [ 6-oxomethylene-6- (undecyloxy) hexyl ] amino ] -octanoic acid (SM-102), 1-oleoyl-2-oleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-dioleoyl-3-dimethylaminopropane (DLin-DAP), 1, 2-dioleoyloxy-N, N-dimethylaminopropane (DLin-DAP), 1, 2-dioleoyl-4-dimethylaminomethyl-1, 3-dioleoyl-1-dioleyl ] -1, 2-dioleoyl-1-dioleyl-3-dioleyl-1-dioleyl-2-DA (DLN), 1, 2-dioleoyl-3-dioleyl-3-dA (DLN-DAP), n-dimethyl- (2, 3-dioleoyloxy) propylamine (DODMA), dioctadecyl amidoglycyl-spermine (DOGS), spermine cholesterol carboamide (GL-67), biguanidino-spermidine-cholesterol (BGTC), 3 beta- (N- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol), 1'- (2- (4- (2- ((2- (bis (2-hydroxydecyl) amino) ethyl) piperazin-1-yl) ethylazadiyl) didodecyl-2-ol (C12-200), N-t-butyl-N' -tetradecylaminopropionamidine (bic 14 amidine (diC-amide)), dimethyl Dioctadecyl Ammonium Bromide (DDAB), N- (1, 2-dimyristoyloxy propan-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N-dioleoyl-N, N-dimethyl ammonium chloride (DOUK-2-yl) ethylamidopropyl ammonium bromide (DORIE), N- (1- (2, 3-dioleoyloxy) propyl) -N-2- (spermine carboxamido) ethyl) -N, N-dimethyl ammonium trifluoroacetate (DOSPA), 1, 2-dioleoyltrimethylpropane ammonium chloride (DOTAP), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTMA), and aminopropyl-dimethyl-bis (dodecyloxy) -propane ammonium bromide (GAP-DLRIE).

Preferably, the ionizable lipid may be a compound represented by formula 5 (DLin-MC 3-DMA), formula 6 (ALC-0315), or formula 7 (lipid H (SM-102)).

[ 5]

[ 6]

[ 7]

Lipid nanoparticles according to the present invention may also include sterol lipids and neutral lipids.

Among the many components of lipid nanoparticles according to the present invention, neutral lipids function to surround and protect the core within the lipid nanoparticle formed by the interaction of the ionizable lipid and the drug, and bind to the lipid bilayer of the target cell to facilitate the drug to pass through the cell membrane and escape from the endosome during delivery of the drug to the cell. Among the components of the lipid nanoparticle according to the present invention, neutral lipid may be used without limitation as long as it is a phospholipid or sphingolipid capable of promoting the fusion of lipid particles, and is preferably DOPE (dioleoyl phosphatidylethanolamine), DSPC (distearoyl phosphatidylcholine), POPC (palmitoyl phosphatidylcholine), EPC (egg yolk phosphatidylcholine), DOPC (dioleoyl phosphatidylcholine), DPPC (dipalmitoyl phosphatidylcholine), DOPG (dioleoyl phosphatidylglycerol), DPPG (dipalmitoyl phosphatidylglycerol), DSPE (distearoyl phosphatidylethanolamine), PE (phosphatidylethanolamine), DPPE (dipalmitoyl phosphatidylethanolamine), DOPE (1, 2-dioleoyl-sn-glycerol-3-phosphorylethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate), DOPS (1, 2-dioleoyl-sn-glycerol-3-phosphate choline), or sphingomyelin (1, 2-dioleoyl-sn-3-phosphate).

Among the numerous components of the lipid nanoparticle according to the present invention, sterol lipids impart morphological rigidity to the lipid filled with the lipid nanoparticle, which is dispersed on the core and surface of the nanoparticle to increase the stability of the nanoparticle, and may be cholesterol and cholesterol derivatives including cholesterol esters.

In the present invention, the medicine may include at least one selected from the group consisting of: peptides, protein drugs, protein-nucleic acid structures, and anionic biopolymer-drug conjugates.

In the present invention, the nucleic acid may be 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.

In the present invention, the siRNA may target genes of metabolic diseases such as ACACA, ANGPTL3, AOC3, MAP3K5, CCR2, CCR5, DGAT2, DPP4, FASN, HSD17B13, KHK, LGALS3, LOXL2, METAP2, MPC, NOX1, NOX4, PNPLA3, SCD, SGLT1 and SGLT2, genes of Hepatitis B Virus (HBV), such as the TTR gene of HBV-S, HBV-P and HBV-X, TTR mediated amyloidosis, the ALAS1 gene of acute hepatoporphyrin, the HAO1 gene of primary hyperoxaluria, the PCSK9 gene of hypercholesterolemia, the C5 gene of complement mediated diseases, the AAT gene of alpha-1 liver disease, the AGT gene of hypertension, the AAT gene of alpha-1 anti-deficiency diseases, the apl gene of hypertriglyceridemia, the apl 3 gene of hypertriglyceridemia, the gptl gene of alpha-1 and SGLT2, the tsche 3, the angustic fibrous diseases of the vascular type, the capillary of the capillary, and the capillary of the lung, and the capillary of the lung, the capillary, the vascular diseases, and the vascular diseases.

In the present invention, mRNA is a vaccine using mRNA expressing an antigen, an immunotherapeutic agent using mRNA expressing an antibody, mRNA expressing a defective protein due to gene mutation or the like, or mRNA expressing Cas protein and guide RNA, sgRNA, or the like, which is subjected to specific DNA gene editing using CRISPR. Examples of antigen expressed mRNAs for vaccine development include glycoprotein mRNAs for rabies virus of rabies vaccine, glycoprotein mRNAs and stable pre-fusion F protein mRNAs for respiratory syncytial virus vaccine, zika virus structural protein mRNAs from Zika virus vaccine, virus antigen protein mRNAs from chikungunya virus vaccine, petameric virus antigen from cytomegalovirus vaccine, gB protein mRNAs, influenza vaccine HA protein mRNAs, T cell activated HIV immunogen protein mRNAs for HIV vaccine, and the like. Examples of mRNAs useful in the development of anticancer drugs or anticancer vaccines include mRNAs expressing specific over-expressed protein antigens from patients, mRNAs expressing antigens including PAP, PSA, PSCA, PSMA and STEAP1 in prostate cancer, MUC1, CEA, her-2neu, telomerase, survivin, and MAGE-A1 mRNAs, melan-A, tyrosinase, gp100, MAGE-A1, MAGE-A3, survivin mRNAs, and cytokine mRNAs including IL-23, IL-36, IL-12, IL-15, GM-CSF, INF-alpha, IL-7, IL-2, and the like as vaccines or treatment methods for various solid or blood cancers. In addition, examples of the mRNA directly expressing the antibody in vivo using the mRNA expressing the antibody include mRNA expressing a chikungunya virus neutralizing monoclonal antibody, but are not limited thereto.

The lipid nanoparticle according to the present invention may be prepared by the following method: (a) Mixing an organic solution comprising a lipid formulation containing an ionizable lipid and a polyethylene glycol moiety (PEG moiety) -degradable functional group-lipid conjugate with a buffer solution comprising a drug, a nucleic acid, or a combination thereof to adjust pH, and (b) removing the solvent from the solution.

The lipid nanoparticle according to the present invention may be prepared by mixing a lipid component dissolved in an organic phase with an oligonucleotide dissolved in an aqueous buffer solution. Since the polarity of the solvent changes with the mixing of the organic phase with the aqueous solution, the lipid component contained in the organic phase spontaneously forms particles in the mixed solution. At this time, when the ionizable lipid containing organic phase is mixed with the oligonucleotide-containing acidic aqueous buffer solution, the ionizable lipid becomes cationic, and the cationic ionizable lipid is linked to the anionic oligonucleotide by electrostatic attraction, thereby preparing the oligonucleotide-containing nanoparticle.

In the method of preparing 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.

Depending on the efficiency of the PEG content in the lipid nanoparticle.

PEG-lipid conjugates are typically used in amounts of 1 to 2mol% to prepare lipid nanoparticles. When lipid nanoparticles are prepared without PEG, the resulting particles aggregate with each other. To prevent this, a predetermined amount of PEG-lipid conjugate should be incorporated to prepare lipid nanoparticles. The lipid portion of the incorporated PEG-lipid conjugate is located in a hydrophobic environment that constitutes the interior of the resulting nanoparticle, and the hydrophilic PEG portion is located on the surface of the particle to inhibit aggregation between the nanoparticles. On the other hand, nanoparticles with too high a PEG content can inhibit interactions between lipid nanoparticles due to the steric effect of PEG exposed on the particle surface. Thus, lipid nanoparticles have the side effect of not being efficiently delivered into cells.

When PEG-DMG (1, 2-dimyristoyl-3-PEG-glycerol, a PEG-lipid conjugate commonly used to prepare lipid nanoparticles) is used to prepare lipid nanoparticles, as the PEG content increases, the cellular uptake efficiency decreases and the ability of siRNA to degrade mRNA also decreases. However, since some or all of the PEG moiety-degradable functional group-lipid conjugates according to the present invention contain a ligand that specifically binds to a receptor expressed in a specific cell at one end of PEG, lipid nanoparticles prepared using the PEG moiety-degradable functional group-lipid conjugates have high cell uptake efficiency regardless of the content of the PEG moiety-degradable functional group-lipid conjugates. In other words, it can be seen that the use of ligands can overcome the inhibition of cellular uptake due to the steric effect of PEG.

Demonstration of removal of PEG from lipid nanoparticles.

In order for lipid nanoparticles to deliver an effective substance, such as a nucleic acid, into a cell to provide efficacy, a two-step delivery process is required. First, lipid nanoparticles must enter the endosome through a cellular uptake process. In the second step, it is critical that the active ingredient effectively escapes from the endosome and is delivered into the cytoplasm. It is well known that lipid nanoparticles are encapsulated in endosomes of cells due to interactions with the cells, and that the lipid component of the lipid nanoparticles can fuse with the lipid membrane constituting the endosome while delivering the active ingredient into the cytoplasm. However, when the surface of the lipid nanoparticle is surrounded by PEG, its interaction with the endosomal lipid membrane is inhibited, and thus it is difficult to deliver into the cytoplasm. Thus, PEG on the surface of the lipid nanoparticle inhibits interactions with cells, thereby reducing the cellular uptake efficiency of the lipid nanoparticle and interfering with interactions between the nanoparticle and the endosomal lipid membrane that are successfully encapsulated in the endosome. Thus, PEG double inhibits the efficiency of the particles.

Therefore, for efficient interaction between endosomal lipid membranes and lipid nanoparticles, it is advantageous to remove PEG from the endosomes. However, nanoparticles comprising common PEG-lipid conjugates such as PEG-DMG do not have functional groups capable of removing PEG. Thus, PEG remains on the particle surface, even in the endosome. In another aspect, the PEG moiety-degradable functional group-lipid conjugates described in the present invention comprise a chemical functional group between PEG and lipid that hydrolyzes under acidic conditions. For example, the PEG moiety-degradable functional group-lipid conjugate shown in formula 3 has a configuration in which two lipid chains are linked to PEG through a hydrazone functional group (-c=nnh-). The hydrazone functional group is stable at neutral pH but hydrolyzes under acidic conditions. When the change in size of the lipid nanoparticle prepared using the PEG moiety-degradable functional group-lipid conjugate according to the present invention with time is observed under acidic conditions, PEG is removed from the particle surface because the hydrazone functional group is hydrolyzed, and the size gradually increases due to continuous fusion of lipid components constituting the particle. Furthermore, it can be seen that over time, the particles fuse together continuously and increase in size. This is a phenomenon in which the hydrazone function is successfully hydrolyzed under acidic conditions, which in turn initiates fusion between particles, and this also promotes fusion between endosomal lipid membranes and nanoparticles.

Induction of hemolysis of lipid nanoparticles.

One well known method for assessing the interaction between lipid nanoparticles and endosomal lipid membranes is to measure the extent of destruction of erythrocytes, i.e. hemolysis, when the nanoparticles are brought into contact with erythrocytes. Here, erythrocyte membranes are considered as substitutes for lipid membranes of endosomes. Frequent occurrence of hemolysis suggests that the lipid nanoparticle fuses more effectively with the endosomal lipid membrane.

Ionizable lipids are one component of lipid nanoparticles that are known to play a critical role in interactions with endosomal lipid membranes. Ionizable lipids are uncharged at neutral pH, but generally contain amine groups, so they are positively charged in the acidic environment of the endosome. The amine groups have an optimum pKa of less than 5 to 7. Thus, to determine the extent to which lipid nanoparticles fuse with endosomal lipid membranes, erythrocytes were mixed with the nanoparticles at a pH below the pKa of the amine groups, and hemolysis was measured. The ionizable lipid used in this example is DLin-MC3-DMA. The amine group of this material has a pKa of about 6.5 and is positively charged at pH 4. The positive charge readily fuses with the negative charge of the endosomal lipid membrane by electrostatic attraction. However, when PEG is located on the surface of the nanoparticle, it exhibits a shielding effect of reducing electrostatic attraction and an effect of preventing direct contact between the nanoparticle surface and the lipid membrane of the endosome.

Thus, as the PEG content increases, the hemolysis reaction of lipid nanoparticles comprising conventional PEG-DMG (without degradable functional groups) is inhibited. On the other hand, since PEG of the PEG moiety-degradable functional group-lipid conjugate is removed by hydrolysis under acidic conditions, the lipid nanoparticle comprising the PEG moiety-degradable functional group-lipid conjugate as a component effectively induces a hemolysis reaction regardless of the content of the functional group-lipid conjugate.

The length of the lipid constituting the PEG moiety-degradable functional group-lipid conjugate is related to the structural stability of the lipid nanoparticle.

PEG around the surface of the lipid nanoparticle is necessary during the lipid nanoparticle preparation process, but has drawbacks in terms of pharmacological efficacy. Thus, the lipid attached to the PEG is typically myristate with 14 carbon atoms to facilitate the spontaneous removal of PEG from the lipid nanoparticle while the lipid nanoparticle is circulating in the blood stream in vivo. This is because as the number of carbon atoms in the lipid fraction decreases, the interactions with other lipid components constituting the lipid nanoparticle decrease, thus allowing the PEG-lipid conjugate to be relatively easily separated and removed from the lipid nanoparticle in vivo. As a result, lipid nanoparticles prepared using substances having relatively long fatty acids such as palmitate (16 carbon atoms) and stearate (18 carbon atoms) cannot be easily incorporated into intracellular bodies because they cannot smoothly remove PEG in circulation through the blood stream, and thus the lipid nanoparticle efficacy is reduced.

However, this method of removing PEG during circulation is very unstable in view of the structural stability of the lipid nanoparticle. This is because when PEG-lipids begin to be separated and removed from the lipid nanoparticle, other lipid components constituting the lipid nanoparticle also begin to be lost due to the decrease in particle stability. In order for the lipid nanoparticle to have proper efficiency, PEG removal is necessary. However, lipid nanoparticles prepared using PEG having a small lipid length have poor particle stability.

In another aspect, the PEG moiety-degradable functional group-lipid conjugates disclosed herein comprise a degradable functional group that is capable of removing PEG under acidic conditions and thus can be used to form relatively longer hydrocarbons, such as lipids having 18 carbon atoms. Since the PEG moiety-degradable functional group-lipid conjugate remains as a component of the nanoparticle throughout until the acidic condition of the endosome is established in which the degradable functional group can be degraded, it maintains the stability of the particle during circulation through the blood stream.

Induction of immune responses by lipid nanoparticles.

Ionizable lipids constituting lipid nanoparticles are known to stimulate immune cells and trigger immune responses. Increasing the PEG content of the lipid nanoparticles can inhibit direct interactions between immune cells and the lipid nanoparticles, thereby minimizing immune side effects caused by the lipid component. However, it is difficult to increase the PEG content beyond the minimum amount required, because increasing the PEG content inevitably may severely inhibit the uptake efficiency of nanoparticles by cells and escape from endosomes in cells.

However, the lipid nanoparticle comprising the PEG moiety-degradable functional group-lipid conjugate of the present invention may use a ligand to increase cellular uptake efficiency and have a degradable functional group that can remove PEG in endosomes. Thus, when the PEG moiety-degradable functional group-lipid conjugate present in the present invention is used, even if the PEG content is increased, induction of immune response can be suppressed without decreasing the efficacy of the prepared lipid nanoparticle. Thus, when lipid nanoparticles using PEG moiety-degradable functional group-lipid conjugates were mixed with Peripheral Blood Mononuclear Cells (PBMC) and the degree of cytokine induction by the lipid nanoparticles was measured, the cytokine induction level was similar to that of PBS buffer-treated group used as a negative control.

Hereinafter, the present invention will be described in more detail with reference to examples. It will be apparent to those skilled in the art, however, that these examples are for illustration of the present invention only and should not be construed as limiting the scope of the invention.

Examples (example)

Example 1: synthesis of polyethylene glycol (PEG) moiety-degradable functional group-lipid conjugate

Example 1-1:2- (2- (2-azidoethoxy) ethoxy) ethanol

Sodium azide (1.54 g,23.72 mmol) was added to an aqueous solution (40 mL) of 2- (2- (2-azidoethoxy) ethoxy) ethanol (2.00 g,11.86 mmol) and stirred at 90℃for 18 hours. The reaction product was extracted three times with methylene chloride and dried over magnesium sulfate under reduced pressure to obtain the compound (2.22 g) in 98.5% yield.

¹ H NMR(400MHz,CDCl ₃ )δ3.75-3.70(m,2H),3.69-3.65(m,6H),3.63-3.59(m,2H),3.42-3.36(m,2H),2.31(s,1H)。

Examples 1-2: 2-methyl-3, 4, 5-tri-O-acetyl-1, 2-deoxy-alpha-D-galactopyranoso [2,1, D]- 2-oxazolines

Trimethylsilicone triflate (0.2 mL,1.13 mmol) was added to a solution of peracetylated galactosamine (peracetylated galactosamine) (400 mg,1.03 mmol) in DCE (22 mL) at room temperature followed by stirring at 50℃for 4 hours. The reaction temperature was lowered to room temperature, and triethylamine was slowly added thereto, followed by stirring for 15 minutes. After the reaction is completed, the mixture is prepared by mixing water and CH ₂ Cl ₂ The organic solvent layer extracted in the above was dried over magnesium sulfate, filtered and distilled under reduced pressure. The mixture obtained by distillation under reduced pressure was purified by column chromatography (CH ₂ Cl ₂ Meoh=40:1) to give the desired compound (260 mg,0.77 mmol) in 78% yield.

¹ H NMR(400MHz,CDCl ₃ )δ5.99(d,J＝6.8Hz,1H),5.46(t,J＝3.0Hz,1H),4.90(dd,J＝7.5,3.3Hz,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＝1.2Hz,3H)。

Examples 1-3: diacetic acid 5-acetamido-2- (acetoxymethyl) -6- (2- (2- (2-azidoethoxy) group) Ethoxy) tetrahydro-2H-pyran-3, 4-diyl ester

Will beMolecular sieves (190 mg) added to 2-methyl-3,4,5-tri-O-acetyl-1, 2-deoxy-alpha-D-galactopyranoso [2,1, D ]]-2-oxazoline (2-methyl-3, 4,5-tri-O-acetyl-1, 2-deoxy-alpha-D-galactose [2,1, D)]A solution of 2-oxoline in DCE (7.2 mL) and a mixed solution of 2- (2- (2-aminoethoxy) ethoxy) ethanol (228 mg,1.30 mmol) (390 mg,1.18 mmol) were stirred for 5 minutes. Trimethylsilicone triflate (104. Mu.L, 0.59 mmol) was added to the resulting mixture at room temperature, followed by stirring for 16 hours. After the completion of the reaction, the reaction mixture,filtering the reaction product to remove->Molecular sieves, and will be in water and CH ₂ Cl ₂ The organic solvent layer extracted in the above was dried over magnesium sulfate, filtered and distilled under reduced pressure. The mixture obtained by distillation under reduced pressure was separated by column chromatography (EA: meoh=9:1) to obtain the desired compound (460 mg,0.91 mmol) in 77% yield.

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

Examples 1 to 4:2- (2- (2-Aminoethoxy) ethoxy) ethyl 2-acetamido-3, 4, 6-tri-O-acetyl- 2-deoxy-alpha-D-galactopyranoside

PPh at room temperature ₃ (150 mg,0.571 mmol) was added to a solution of 5-acetamido-2- (acetoxymethyl) -6- (2- (2- (2-azidoethoxy) ethoxy) tetrahydro-2H-pyran-3,4-diyl diacetate (5-acetamido-2- (2- (2-azidoethoxy) method) tetra hydro-2H-pyran-3,4-diyl diacetate) (240 mg,0.476 mmol) in THF (10 mL) and stirred for 48 hours. Will H ₂ O (26. Mu.L, 1.427 mmol) was added to the reaction product, which was then stirred for 24 hours. Then, toluene (10 mL) and trifluoroacetic acid (73. Mu.L, 0.951 mmol) were sequentially added to prepare a salt. Will be under water and CH ₂ Cl ₂ The aqueous layer extracted in (b) was distilled under reduced pressure to obtain the desired compound (270 mg,0.91 mmol) in 98% yield.

¹ H NMR(400MHz,CD ₃ OD):δ5.35(d,J＝3.2Hz,1H),5.05(dd,J＝11.2,3.2Hz,1H),4.58(d,J＝8.4Hz,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)。

Examples 1 to 5: tris { [2- (tert-butoxycarbonyl) ethoxy ]]Methyl } methylamine

5.0M sodium hydroxide (0.2 mL) was added to a solution of Tris (1.21 g,10.00 mmol) in DMSO (2.0 mL) at 15℃and then stirred for 5 minutes. To this was slowly added dropwise tert-butyl acrylate (5.0 mL,34 mmol). Then, water (0.2 mL) was added to the reaction product, followed by stirring at room temperature under argon for 24 hours. After completion of the reaction, the mixture obtained by distillation under reduced pressure was separated by column chromatography (EA: hex=2:1) to obtain the desired compound (2.5 g,5.00 mmol) in 50% yield.

¹ H NMR(CDCl ₃ ,400MHz):δ3.65(t,J＝6.0Hz,6H),3.32(s,6H),2.46(t,J＝6.0Hz,6H),1.45(s,27H)。

Examples 1 to 6: (R) - ((2, 3-bis (octadecyloxy) propoxy) methyl) benzene

1-bromooctadecane (732 mg,2.20 mmol) and potassium hydroxide (123 mg) were added to a solution of (R) -3-benzyloxy-1, 2-propanediol (106 mg,0.55 mmol) in toluene (2.5 mL) at room temperature, followed by reflux at 110℃for 24 hours. After the reaction is completed, the mixture is prepared by mixing water and CH ₂ Cl ₂ The organic solvent layer extracted in the above was dried over magnesium sulfate, filtered and distilled under reduced pressure. The mixture obtained by distillation under reduced pressure was separated by column chromatography (Hex: ea=10:1) to obtain the desired compound (300 mg,0.44 mmol) in 80% yield.

¹ H NMR(400MHz,CDCl ₃ )δ7.32-7.21(m,5H),4.55(s,2H),3.64-3.48(m,7H),3.43(t,J＝6.7Hz,2H),1.61-1.51(m,4H),1.39-1.21(m,60H),0.88(t,J＝6.8Hz,6H)。

Examples 1 to 7: (S) -2, 3-bis (octadecyloxy) propan-1-ol

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

¹ H NMR(CDCl ₃ )δ3.75-3.37(m,9H),2.17(m,1H),1.55(m,4H),1.38-1.21(m,60H),0.88(t,J＝6.8Hz,6H)。

Examples 1 to 8: (R) -2, 3-bis (octadecyloxy) propanal

Dess-Martin periodate (Dess-Martin periodinane) (88 mg,0.206 mmol) CH at 0deg.C ₂ Cl ₂ (3.7 mL) solution was slowly added dropwise to (S) -2, 3-bis (octadecyloxy) propan-1-ol (110 mg,0.184 mmol) in CH ₂ Cl ₂ (1.3 mL) and then stirred at room temperature for 2 hours. After the reaction is completed, the mixture is prepared in water or CH ₂ Cl ₂ And aqueous sodium bicarbonate, then in water, CH ₂ Cl ₂ And the organic solvent layer extracted from sodium thiosulfate was dried over magnesium sulfate, filtered and distilled under reduced pressure. The mixture obtained by distillation under reduced pressure was separated by column chromatography (Hex: ea=20:1) to obtain the desired compound (83 mg,0.15 mmol) in 80% yield.

¹ H NMR(CDCl ₃ )δ3.81-3.37(m,9H),1.65-1.51(m,4H),1.38-1.21(m,60H),0.88(t,J＝6.8Hz,6H)。

Implementation of the embodimentsExamples 1-9:3, 6-Dioxy-1-phenyl-2,8,11,14-tetraoxa-4, 5-diazahexadecane-16-oic acid _n (M＝600)

Poly (ethylene glycol) dicarboxymethyl ether (1 g,1.72 mmol) was dissolved in DMF (5.8 mL) and then CbzNHNH was taken up at 0deg.C ₂ (300 mg,1.81 mmol), hydroxybenzotriazole (244 mg,1.81 mmol) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (320. Mu.L, 1.81 mmol) were added dropwise to the resulting solution. The mixture was stirred at room temperature under argon for 18 hours. After the reaction is completed, the aqueous layer is coated with water and CH ₂ Cl ₂ And aqueous sodium bicarbonate, and extracting the organic layer with water, CH ₂ Cl ₂ And H ₃ PO ₄ And then distilled under reduced pressure to obtain a product with a yield of 40%.

¹ H NMR(400MHz,CD ₃ OD)δ7.39-7.30(m,5H),5.15(s,2H),4.13(s,2H),4.10(s,2H),3.73-3.60(m,70H)。

Examples 1 to 10:21- (tert-butyl) 16, 16-bis ((3- (tert-butoxy) -3-oxypropyloxy) methyl) -4, _n 14-Dioxy-6,9,12,18-tetraoxa-2,3,15-triazadi-undecanedioic acid 1-benzyl ester (m=600)

3, 6-Dioxy-1-phenyl-2,8,11,14-tetraoxa-4, 5-diazahexadecane-16-oic acid (3, 6-dioxo-1-phenyl-2,8,11,14-tetraoxa-4,5-diazahexadecan-16-oic acid) was combined (870 mg,1.16 mmol) with tris { [2- (tert-butoxycarbonyl) ethoxy]Methyl } methylamine (526 mg,1.04 mmol) was dissolved in DMF (4 mL), and then hydroxybenzotriazole (157 mg,1.16 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (248. Mu.L, 1.40 mmol) and diisopropylethylamine (243. Mu.L, 1.40 mmol). The mixture was stirred at room temperature under argon for 18 hours. After the reaction is completed, water and an organic solvent layer are added in CH ₂ Cl ₂ And phosphoric acid, then in water, CH ₂ Cl ₂ And aqueous sodium bicarbonate. The extracted organic solvent layer was dried over magnesium sulfate, filtered, and distilled under reduced pressure. The mixture obtained by distillation under reduced pressure was purified by column chromatography (CH ₂ Cl ₂ Meoh=15:1) to give the desired compound in 40% yield.

¹ H NMR(400MHz；CD ₃ OD)δ7.38-7.31(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＝6.0Hz,6H),1.46(s,27H)。

Examples 1 to 11:18, 18-bis ((2-carboxyethoxy) methyl) -3,6,16-trioxymethylene-1-2,8,11,14, _n 20-pentaoxa-4,5,17-triazatriacontane-23-oic acid (m=600)

1-benzyl 21- (tert-butyl) 16,16-bis ((3- (tert-butoxy) -3-oxypropyloxy) methyl) -4, 14-dioxa-6,9,12,18-tetraoxa-2,3,15-triazadi-undecanedioate (1-benzyl 21- (tert-butyl) 16,16-bis ((3- (tert-butyl) -3-oxoropoxy) methyl) -4, 14-diox-6,9,12,18-tetraoxa-2,3,15-triazahenicoside carboxylate) (Mn=600) (570 mg,0.46 mmol) was dissolved in 85% formic acid (4.0 mL) and then stirred at room temperature for 18 hours. After the completion of the reaction, the reaction product was distilled under reduced pressure at 50℃to obtain the desired compound in 99% yield.

¹ H NMR(CD ₃ OD)δ7.38-7.30(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)。

Examples 1 to 12: triacetylgalactose-PEG 600-Cbz hydrazide

18,18-bis ((2-carboxyethoxy) methyl) -3,6,16-trioxymethylene-1-2,8,11,14,20-pentaoxa-4,5,17-triazatriacontane-23-oic acid (18, 18-bis ((2-carboxyyloxy) methyl) -3,6,16-trioxo-1-2,8,11,14,20-pentaoxa-4,5,17-triazatricosan-23-oic acid) and tris { [2- (tert-butoxycarbonyl) ethoxy ] ethoxy]Methyl } methylamine (tris { [2- (tert-butoxin carboyl) method)]methyl } methyl amine) (1.58 mmol) was dissolved in DMF (4.4 mL), and then hydroxybenzotriazole (214 mg,1.58 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (280. Mu.L, 1.58 mmol) and diisopropylethylamine (550. Mu.L, 3.16 mmol) were added dropwise thereto at 0 ℃. The mixture was stirred at room temperature under argon for 18 hours. Further, diisopropylethylamine (550 μl,3.16 mmol) was further added dropwise at room temperature under argon, followed by stirring at room temperature for 24 hours. After the reaction is completed, in water, CH ₂ Cl ₂ And extracting the organic solvent layer from phosphoric acid. The extracted organic solvent layer is treated with water and CH again ₂ Cl ₂ And aqueous sodium bicarbonate, dried over magnesium sulfate, filtered, and distilled under reduced pressure. The remaining mixture was purified by column chromatography (CH ₂ Cl ₂ Meoh=10:1) to afford the desired compound in 23% yield.

¹ H NMR(400MHz,CD ₃ OD)δ7.40-7.31(m,5H),5.34(d,J＝3.0Hz,3H),5.07(dd,J＝11.2,3.3Hz,3H),5.16(s,2H),4.64(t,J＝8.5Hz,3H),4.16-4.02(m,16H),3.95-3.91(m,6H),3.77-3.61(m,106H),3.55(t,J＝5.6Hz,6H),3.38(t,J＝5.6Hz,6H),2.45(t,J＝6.0Hz,6H),2.14(s,9H),2.03(s,9H),1.95(s,9H),1.93(s,9H)。

Examples 1 to 13: triacetylgalactose-PEG 600-hydrazide

triacetylgalactose-PEG 600-Cbz hydrazide (373 mg,0.16 mmol) was dissolved in MeOH (2 mL), and Pd/C (13 mg) and acetic acid (28. Mu.L, 0.48 mmol) were then added dropwise thereto. The mixture was stirred under hydrogen at room temperature for 20 hours. After completion of the reaction, the mixture was filtered through Celite545 using methanol as a solvent, and then distilled under reduced pressure to obtain the desired compound (120 mg,0.05 mmol) in 99% yield.

¹ H NMR(400MHz,CD ₃ OD)δ1.94(s,9H),5.34(d,J＝3.0Hz,3H),5.07(dd,J＝11.2,3.4Hz,3H),4.64(d,J＝8.5Hz,3H),4.17-3.91(m,16H),3.77-3.61(m,106H),3.56(t,J＝5.5Hz,6H),3.39(t,J＝5.5Hz,6H),2.46(t,J＝5.9Hz,6H),2.15(s,9H),2.03(s,9H),1.95(s,9H),1.94(s,9H)。

Examples 1 to 14: dioctadecyl-propylene triacetyl galactose-PEG 600-acylhydrazone

Triacetylgalactose-PEG 600-hydrazide (0.05 mmol) and (R) -2, 3-bis (octadecyloxy) propanal (39 mg,0.06 mmol) were dissolved in EtOH (1 mL) and CH ₂ Cl ₂ (0.1 mL) and then stirred at room temperature under argon for 3 hours. After the completion of the reaction, the resultant was distilled under reduced pressure. The mixture obtained by distillation under reduced pressure was purified by column chromatography (CH ₂ Cl ₂ Meoh=10:1) to afford the desired compound in 26% yield.

¹ H NMR(CDCl ₃ )δ9.92(s,1H),7.43(d,J＝6.9Hz,1H),7.19(m,2H),7.03(s,1H),6.6(s,1H),5.32(d,J＝3.0Hz,3H),5.16(dd,J＝11.3,3.0Hz,3H),4.78(d,J＝8.5Hz,3H),4.15-3.40(m,134H),2.45(t,J＝5.9Hz,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＝6.8Hz,6H)。

Examples 1 to 15: dioctadecyloxy-propylidene tris-GalNAc-PEG 600-acylhydrazone (GalNAc-PEG 600-Hz-) DSG)

Dioctadecyloxy-propylidene triacetylgalacto-PEG 600-acylhydrazone (0.013 mmol) was dissolved in MeOH (1 mL) and CH ₂ Cl ₂ To the mixed solution of (0.1 mL), and sodium methoxide (0.7 mg,0.013 mmol) was added dropwise thereto. The mixture was stirred under argon at room temperature for 2 hours. After the completion of the reaction, the mixture was treated with Amberlite IR-120 ion exchange resin and distilled under reduced pressure to obtain the desired compound in 26% yield.

¹ H NMR(CDCl ₃ )δ9.97(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＝5.9Hz,6H),2.00(s,9H),1.55(m,4H),1.24(m,60H),0.87(t,J＝6.8Hz,6H)。

Examples 1 to 16:3, 6-Dioxy-1-phenyl-2,8,11,14-tetraoxa-4, 5-diazahexadecane-16- _n Acid (m=2000)

The desired compound was obtained in 40% yield in the same manner as in examples 1 to 9.

¹ H NMR(400MHz,CD ₃ OD)δ7.39-7.30(m,5H),5.15(s,2H),4.13(s,2H),4.10(s,2H),3.73-3.68(m,190H)。

Examples 1 to 17:21- (tert-butyl) 16, 16-bis ((3- (tert-butoxy) -3-oxypropyloxy) methyl) -4, _n 14-Dioxy-6,9,12,18-tetraoxa-2,3,15-triazadi-undecanedioic acid 1-benzyl ester (M=2000)

The desired compound was obtained in a yield of 30% in the same manner as in examples 1 to 10.

¹ H NMR(400MHz；CD ₃ OD)δ7.38-7.31(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＝6.0Hz,6H),1.46(s,27H)。

Examples 1 to 18:18, 18-bis ((2-carboxyethoxy) methyl) -3,6,16-trioxymethylene-1-2,8,11,14, 20-pentaoxa-4,5,17-triazatriacontane-23-oic acid (mn=2000)

The desired compound was obtained in 99% yield in the same manner as in examples 1 to 11.

¹ H NMR(CD ₃ OD)δ7.38-7.30(m,5H),5.15(s,2H),4.12(s,4H),3.78-3.96(m,190H),2.53(t,J＝6.0Hz,6H)。

Examples 1 to 19: triacetylgalactose-PEG 2K-Cbz-hydrazide

The desired compound was obtained in a yield of 30% in the same manner as in examples 1 to 12.

¹ H NMR(400MHz,CD ₃ OD)δ7.40-7.31(m,5H),5.34(d,J＝3.0Hz,3H),5.16(s,2H),5.07(dd,J＝11.2,3.3Hz,3H),4.64(t,J＝8.5Hz,3H),4.16-4.02(m,16H),3.95-3.91(m,6H),3.77-3.61(m,226H),3.55(t,J＝5.6Hz,6H),3.38(t,J＝5.6Hz,6H),2.45(t,J＝6.0Hz,6H),2.14(s,9H),2.03(s,9H),1.95(s,9H),1.93(s,9H)。

Examples 1 to 20: triacetylgalactose-PEG 2K-hydrazide

The desired compound was obtained in 99% yield in the same manner as in examples 1 to 13.

¹ H NMR(400MHz,CD ₃ OD)δ5.34(d,J＝3.0Hz,3H),5.07(dd,J＝11.2,3.4Hz 3H),4.64(d,J＝8.5Hz,3H),4.17-3.91(m,16H),3.77-3.60(m,226H),3.56(t,J＝5.5Hz,6H),3.39(t,J＝5.5Hz,6H),2.46(t,J＝5.9Hz,6H),2.15(s,9H),2.03(s,9H),1.95(s,9H),1.94(s,9H)。

Examples 1 to 21: dioctadecyl-propylene triacetyl galactose-PEG 2K-acylhydrazone

The desired compound was obtained in 37% yield in the same manner as in examples 1 to 14.

¹ H NMR(CDCl ₃ )δ9.95(s,1H),7.43(d,J＝6.9Hz,1H),7.19(m,2H),7.03(s,1H),6.6(s,1H),5.32(d,J＝3.0Hz,3H),5.16(dd,J＝11.3,3.0Hz,3H),4.78(d,J＝8.5Hz,3H),4.15-3.40(m,226H),2.45(t,J＝5.9Hz,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＝6.8Hz,6H)。

Examples 1 to 22: dioctadecyl-propylidene tri-GalNac-PEG 2K-acylhydrazone (GalNac-PEG 2K-Hz-) DSG)

The desired compound was obtained in 26% yield in the same manner as in examples 1 to 15.

¹ H NMR(CDCl ₃ )δ9.93(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＝5.9Hz,6H),2.00(s,9H),1.55(m,4H),1.24(m,60H),0.87(t,J＝6.8Hz,6H)。

Examples 1-23. 4-Oxiden-6, 9, 12-trioxa-2, 3-diazatridecanoic acid benzyl ester-PEG 600

2- (2- (2-methoxyethoxy) ethoxy) ethylAcid (150 mg,0.27 mmol) was dissolved in 2.7mL of DMF and CbzNHNH was added thereto at 0deg.C ₂ (50 mg,0.3 mmol), EDCI (58. Mu.L, 0.33 mmol) and HOBt (44 mg,0.33 mmol) followed by stirring at room temperature for one day. After completion of the reaction, the reaction product was transferred to DCM (40 mL) with diluted H ₃ PO ₄ (1M, 40 mL) and NaHCO ₃ Aqueous solution (1M, 40 mL) was washed and dried over magnesium sulfate. The reaction was separated on a silica gel column using DCM/methanol (10/1, v/v) to give 175mg of the compound in 91% yield.

¹ H NMR(400MHz,MeOH-d ₄ )δ7.39-7.30(m,5H),5.16(s,2H),4.20(s,2H),3.68-3.52(m,50H)。3.37(s,3H)

Examples 1 to 24:2- (2- (2-methoxyethoxy) ethoxy) acethydrazide-PEG 600

Cbz hydrazide (175 mg,0.25 mmol) was dissolved in methanol (2.8 mL), pd/C (18 mg,10 w/w%) was added thereto, and the mixture was stirred under hydrogen for 20 hours. After completion of the reaction, the mixture was filtered through Celite 545 to obtain 147mg of the compound in 95% yield.

¹ H NMR(400MHz,CDCl ₃ )δ4.14-4.01(m,2H),3.61(m,50H),3.34(s,3H)。

Examples 1 to 25: dioctadecyl-propylene PEG 600-acylhydrazone (PEG 600-Hz-DSG)

The resulting products of examples 1-24 (79 mg,0.127 mmol) were dissolved in 1.3mL of ethanol and aldehyde (113 mg,0.19 mmol) was added to the resulting products of examples 1-8. The mixture was stirred at 50 ℃ for 18 hours to terminate the reaction. The reaction product was purified by column to obtain 123mg of the compound in 85% yield.

¹ H NMR(400MHz,CDCl ₃ )δ9.97(s,1H),7.38(d,J＝7.0Hz,1H),4.15(m,3H),3.65-3.40(m,56H),3.36(s,3H),1.52(m,4H),1.23(m,60H),0.86(t,J＝6.4Hz,3H)。

Examples 1 to 26: 4-Oxiden-6, 9, 12-trioxa-2, 3-diazatridecanoic acid benzyl ester-PEG 2K

In the same manner as in examples 1 to 23, 160mg of the compound was obtained in a yield of 95%.

¹ H NMR(400MHz,MeOH-d ₄ )δ7.39-7.30(m,5H),5.16(s,2H),4.20(s,2H),3.68-3.52(m,190H)。3.36(s,3H)

Examples 1 to 27:2- (2- (2-methoxyethoxy) ethoxy) acethydrazide-PEG 2K

110mg of the compound was obtained in the same manner as in examples 1-24 in 75% yield.

¹ H NMR(400MHz,CDCl ₃ )δ4.14-4.01(m,2H),3.61(m,190H),3.34(s,3H)。

Examples 1 to 28: dioctadecyl-propylene PEG 2K-acylhydrazone (PEG 2K-Hz-DSG)

In the same manner as in examples 1 to 25, 57mg of the compound was obtained in 53% yield.

¹ H NMR(400MHz,CDCl ₃ )δ9.95(s,1H),7.36(d,J＝7.0Hz,1H),4.14(m,3H),3.60-3.40(m,200H),3.34(s,3H),1.51(m,4H),1.21(m,60H),0.84(t,J＝6.4Hz,3H)。

Examples 1 to 29:(R) -1- (4- (2, 3-bis (octadecyloxy) propoxy) phenyl) ethan-1-one

The resulting products of examples 1-7 (59.8 mg, 100. Mu. Mol) were dissolved in 2mL of THF, and 4-hydroxyacetophenone (15.0 mg, 110. Mu. Mol), PPh were added thereto at 0 ℃ ₃ (39.4 mg, 150. Mu. Mol) and DIAD (30 uL, 150. Mu. Mol). The mixture was stirred at room temperature for 12 hours and the reaction was terminated. The reaction product was extracted three times with dichloromethane and washed with brine. The organic layer was dried over anhydrous magnesium sulfate, and dried under reduced pressure. The resulting product was purified by silica chromatography to obtain 55.0mg of the compound in 77% yield.

¹ H-NMR(400MHz,CDCl ₃ )δ7.92(d,J＝8.8Hz,2H),6.95(d,J＝8.8Hz,2H),4.19-4.05(m,2H),3.79(quin,J＝5.1Hz,1H),3.64-3.57(m,4H),3.46(t,J＝6.6Hz,2H),2.55(s,3H),1.61-1.50(m,4H),1.32-1.21(m,60H),0.88(t,J＝6.8Hz)。

Examples 1 to 30: (R, E) -N' - (1- (4- (2, 3-bis (octadecyloxy) propoxy) phenyl) ethylene) -2- (2-methoxyethoxy) acethydrazide (PEG 600-PhHz-DSG)

m-PEG-hydrazide (mn= 550,Creative PEGworks,200mg,364mol) was dissolved in ethanol (4 mL), and the obtained products of examples 1 to 29 (260 mg, 264 mol) and acetic acid (100 ul,3.64 mmol) were added thereto. The mixture was stirred at 85 ℃ for 16 hours. The reaction product was purified by column purification to give 265mg of the product in 66% yield.

Examples 1 to 31:2- (methoxypolyethylene glycol 550) acetic acid (2R) -2, 3-bis (octadecyloxy) propyl ester (PEG 600-ester-DSG)

2- (methoxypolyethylene glycol 550) carboxylic acid (100 mg,0.156 mmol) was dissolved in DCM (30.0 mL), and (S) -2, 3-bis (octadecyloxy) propan-1-ol (87 mg,0.312 mmol), 4-dimethylaminopyridine (23 mg, 0.87 mmol) and dicyclohexylcarbodiimide (37 mg,0.180 mmol) were added dropwise thereto. The mixture was stirred under nitrogen at room temperature for 24 hours. After completion of the reaction, the reaction product was filtered through Celite545 and the organic layer was washed with water and CH ₂ Cl ₂ The organic solvent layer was dried over magnesium sulfate, filtered, and distilled under reduced pressure. The mixture obtained by distillation under reduced pressure was separated by column chromatography (DCM: meoh=20:1) to give the desired compound (68 mg,0.64 mmol) in 41% yield.

¹ H NMR(500MHz,CDCl ₃ )δ4.24(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)。

Example 2: preparation of lipid nanoparticles

Two types of nanoparticles were prepared to determine whether the PEG moiety-degradable functional group-lipid conjugates disclosed herein, i.e., PEG derivatives comprising functional groups that degrade under acidic conditions, can stably form nanoparticles under the acidic conditions in which the lipid nanoparticles were prepared. A lipid nanoparticle comprising PEG2K-Hz-DSG (formula 3) comprising hydrazone functional groups hydrolyzed under acidic conditions, another lipid nanoparticle comprising PEG2K-PE-DSG ((1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] -2000, avanti) which is not hydrolyzed under acidic conditions.

At the same time, siRNA was prepared at a concentration of 0.046mg/mL in a citrate buffer solution at pH 4. Lipid nanoparticles were prepared by mixing an ethanol solution with a buffer solution in a volume ratio of 3:1 using a micromixer chip or a T-line chip, and setting the flow rate of the mixed solution to 4 mL/min. The solution passing through the chip was further mixed with buffer in a volume ratio of 1:1 to change the pH to 7, and the resulting mixture was dialyzed in PBS buffer at pH 7.5 for 24 hours. The size of the lipid nanoparticles prepared was measured using a Dynamic Light Scattering (DLS) device. When the hydrazone of PEG2K-Hz-DSG is hydrolyzed into PEG and lipid (DSG) under acidic conditions to prepare lipid nanoparticles, the lipid component should be precipitated as a precipitate having a non-uniform size, like nanoparticles that do not include PEG moiety-degradable functional group-lipid conjugates. However, as can be seen from fig. 2, the nanoparticles including PEG2K-Hz-DSG have a uniform size, like the nanoparticles including PEG2K-PE-DSG, and the size of the nanoparticles decreases as the PEG content increases. This example shows that PEG moiety-degradable functional group-lipid conjugates comprising functional groups that degrade under acidic conditions can stably form nanoparticles instead of being degraded under acidic conditions in the preparation of lipid nanoparticles.

Example 3: comparison of efficacy between lipid nanoparticles comprising PEG-Hz-DSG or PEG-PE-DSG

To determine the efficacy of lipid nanoparticles comprising PEG-Hz-DSG or PEG-PE-DSG, lipid nanoparticles comprising sirnas targeting luciferase were prepared in the same manner as in example 2. Cell lines constitutively expressing luciferase were treated with lipid nanoparticles at different concentrations based on siRNA concentration, and the extent of inhibition of luciferase activity was evaluated.

To determine the effect of molecular weight and content of PEG in lipid nanoparticles on efficacy, 1, 2 and 5mol% PEG2000 (PEG 2K-Hz-DSG or PEG 2K-PE-DSG) or PEG1000 (PEG 1K-Hz-DSG or PEG 1K-PE-DSG) were prepared. Lipid nanoparticles comprising PEG600-Hz-DSG in more amounts of 1, 2, 5, 10, 20, 30 and 40mol% were also prepared and their efficacy was evaluated.

It can be seen that the lipid nanoparticle comprising PEG-PE-DSG had no efficacy (fig. 2A) or was degraded with increasing content (fig. 2B), regardless of the PEG content, whereas PEG-Hz-DSG was effective in inhibiting luciferase expression. In particular, lipid nanoparticles comprising PEG600-Hz-DSG more effectively inhibited luciferase expression until the content reached 20mol%, thereby improving efficacy.

Example 4: comparison in terms of cell uptake and targeted inhibition efficiency according to the PEG content in lipid nanoparticles Compared with the prior art.

It is well known that when lipid nanoparticles are prepared using PEG-DMG, which is generally used for lipid nanoparticle preparation, the efficiency of uptake into cells decreases when the PEG content increases. To confirm this, lipid nanoparticles containing 2, 5 and 10mol% PEG-DMG were prepared using 0.2mol% of the fluorescent dye Dil (1, 1'-dioctadecyl-3, 3' -tetramethyl indocarbocyanine ester (1, 1'-dioctadecyl-3, 3' -tetramethylindocarbocyanine perchlorate)) and then treated with HepG2 cell line for 24 hours. The cellular uptake efficiency of the nanoparticles was analyzed using a flow cytometer (FACS). The results showed that cellular uptake decreased with increasing PEG content (fig. 3, a).

The reduction in uptake results in a reduction in the efficacy of the lipid nanoparticle. Lipid nanoparticles comprising 0, 0.1, 0.2, 0.5, 1, 2, 5 and 10mol% PEG-DMG were prepared using sirnas targeting luciferases. Cells constitutively expressing luciferase were treated with the obtained lipid nanoparticle, and luciferase activity was measured. It can be seen from the graph of FIG. 3B that the efficiency is optimal when the PEG content falls within the range of 0.5 to 2 mol%. It can be seen that no efficacy was observed in the lipid nanoparticle containing 0 to 0.2mol% PEG, and as the PEG content increased in the range of 2mol% or more, the inhibition efficiency against luciferase activity decreased. This is because very low amounts of PEG may result in the formation of non-uniform particles, while exceeding a predetermined amount of PEG makes uptake into cells and escape from endosomes difficult.

To directly compare the efficacy between lipid nanoparticles comprising different amounts of PEG-DMG and PEG600-Hz-DSG, lipid nanoparticles comprising 1, 2, 5 and 10mol% PEG moiety-degradable functional group-lipid conjugates, respectively, and comprising sirnas targeting luciferases were prepared. Luciferase expression levels were measured 24 hours after treatment of constitutive luciferase-expressing HEPG2 and HEK293 cell lines with lipid nanoparticles. The results show that the efficiency of lipid nanoparticles comprising PEG-DMG decreases when the PEG content increases, whereas the efficiency of lipid nanoparticles comprising PEG600-Hz-DSG does not change or increases instead when the PEG content increases (C, D in fig. 3). EC (EC) ₅₀ Is calculated based on the data shown in fig. 3E and 3F. EC of lipid nanoparticles comprising 10% PEG-DMG and PEG600-Hz-DSG in HEPG2 cell line ₅₀ 8.9nM and 0.2nM, respectively, the difference between the two being about 45-fold, and the HEK293 cell line contained 10% of EC of lipid nanoparticles of PEG-DMG and PEG600-Hz-DSG ₅₀ 26nM and 0.72nM, respectively, the difference between the two being about 36-fold.

Example 5: demonstration of PEG removal of lipid nanoparticles

The removal of PEG from the endosome is advantageous in terms of efficiency in ensuring an effective interaction between the endosomal lipid membrane and the lipid nanoparticle after the lipid nanoparticle has entered the endosome of the cell. Lipid nanoparticles prepared using conventional PEG-lipid conjugates without degradable functional groups have a relatively stable structure under acidic conditions that mimic the internal environment of an endosome. On the other hand, lipid nanoparticles, such as PEG-Hz-DSG, comprising functional groups that degrade under acidic conditions become unstable as PEG is removed under acidic conditions. To confirm this, the change in size of the nanoparticles comprising 1.5mol% PEG-DMG (which is a typical lipid nanoparticle production ratio) and the lipid nanoparticles comprising 10mol% PEG600-Hz-DSG used in the present invention was measured in buffer solutions at pH 7.4, 5.5, 5.0 or 4.5.

No significant change was found when the change in particle size of nanoparticles prepared using PEG-DMG was observed over time at each pH condition (a in fig. 4). On the other hand, the size of the lipid nanoparticle comprising PEG600-Hz-DSG remained stable at pH 7.4 (neutral condition), but fusion between particles was activated in an acidic pH environment (B in fig. 4). Furthermore, it can be seen that over time, particles fuse continuously between each other under each acidic condition and gradually increase in size. In other words, it can be seen that PEG constituting the surface of the nanoparticle is effectively removed under acidic conditions.

Thus, PEG-Hz-DSG comprising functional groups such as hydrazone that hydrolyze under acidic conditions maintains structural stability under acidic conditions (pH 4, ethanol 20 to 40% buffer solution) when lipid nanoparticles are prepared (fig. 1), but after ethanol is removed, hydrazone rapidly hydrolyzes at acidic pH to separate PEG and lipid from each other.

Example 6: demonstration of hydrolysis time of degradable functional groups

To determine if there is a difference in efficacy depending on the time of removal of PEG from the surface of the lipid nanoparticle, lipid nanoparticles comprising 5 or 10mol% PEG600-Hz-DSG were prepared using siRNA targeting luciferase. The lipid nanoparticle was treated with a buffer solution of pH 7 or 4 for 24 hours, the constitutive luciferase-expressing cell line was treated with the lipid nanoparticle, and then the luciferase expression level was measured.

Lipid nanoparticles pretreated with pH 7 buffer solution can effectively inhibit luciferase expression, but lipid nanoparticles with PEG removed in pH 4 buffer solution were not effective at all (fig. 5). In other words, it was found that PEG must be removed in order to provide efficacy after the lipid nanoparticle enters the endosome of the cell, but that the nanoparticle loses its efficacy by removing PEG prior to ingestion into the cell.

Example 7: evaluation of the Effect of the chemical Structure of degradable functional groups on efficacy

The difference in hydrolysis half-life under acidic conditions occurs depending on the chemical structure of the degradable functional group in the PEG moiety-degradable functional group-lipid conjugate component. To determine the effect of hydrolysis sensitivity on the efficacy of lipid nanoparticles under acidic conditions, lipid nanoparticles were prepared comprising 1, 2, 5 and 10mol% of PEG derivatives having esters (PEG 600-ester-DSG, formula 8) or aryl hydrazones (PEG 600-PhHz-DSG, formula 9) as degradable functional groups.

[ 8]

[ 9]

Lipid nanoparticles were prepared by incorporating sirnas targeting luciferases in the lipid nanoparticles. The constitutive luciferase-expressing cell lines were treated with lipid nanoparticles for 24 hours, and then luciferase activity was measured (fig. 6, a).

It was demonstrated that the efficiency of lipid nanoparticles improved with increasing PEG600-Hz-DSG and PEG 600-ester-DSG content, and that the hydrazone functionality had better efficiency than the ester functionality.

To determine the correlation between these results and the sensitivity of each functional group to hydrolysis under acidic conditions, lipid nanoparticles comprising 10mol% of the corresponding PEG moiety-degradable functional group-lipid conjugate were transferred into PBS at pH 7.4 or citric acid buffer solution at pH 4.5 and the change in particle size was measured using dynamic light scattering (fig. 6, b). The particle size of the functional groups increases in the order hydrazone, ester and aryl hydrazone, which means that each functional group is easily hydrolyzed in that order under acidic conditions. In other words, as can be seen from fig. 6 a and B, as hydrolysis becomes more active, PEG is more effectively removed, thereby increasing efficiency.

Example 8: evaluation of lipid membrane fusion and hemolysis induction of lipid nanoparticles

To determine the extent of lipid nanoparticle fusion with the endosomal lipid membrane, the liposomal lipid membrane or red blood cells were mixed with the nanoparticle at a pH below the pKa of the ionizable lipid and lipid membrane fusion or hemolysis was assessed. The ionizable lipid used herein is DLin-MC3-DMA. The pKa of the amine groups of this material is about 6.5 and at pH 4 the amine groups are positively charged. The positive charge readily fuses with the negative charge of the endosomal lipid membrane by electrostatic attraction. However, when PEG is located on the surface of the nanoparticle, PEG has a shielding effect of reducing electrostatic attraction and also has an effect of preventing direct contact between the surface of the nanoparticle and the lipid membrane of the endosome.

To evaluate the fusion efficiency of lipid nanoparticles and endosomal lipid membranes, lipid nanoparticles comprising 10mol% PEG600-Hz-DSG or 1.5mol% PEG-DMG were prepared, which are typically used for the preparation of lipid nanoparticles. Each nanoparticle contained 18:1 of 0.5mol% NBD PE (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro-2-1, 3-benzooxadiazol-4-yl) (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro2-1, 3-benzoxadiazol-4-yl) (ammonium salt)) and 18:1 of 0.5mol% Liss rhodi PE ((1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (lissamine rhodamine B sulfonyl) (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (lissamine rhodamine B sulfonyl) (ammonium salt)).

Liposomes were prepared and used to mimic endosomal lipid membranes. Soy PS: DOPC: DOPE was dissolved in chloroform at a molar ratio of 25:25:50 to give a total concentration of 1mM, and chloroform was removed from the round bottom flask by drying under reduced pressure. To this was added 1mL of PBS and sonicated to prepare liposomes.

Each lipid nanoparticle was mixed with the liposomes in buffer solutions at pH 7.4, 6.5, 6.0, 5.5, 5.0 and 4.5. When NBD and Liss red contained in the lipid nanoparticle are close to each other, a fluorescence signal cannot be measured, but when the relative distance between fluorescent lipids increases as the nanoparticle fuses with the lipid membrane of the liposome, the fluorescence signal of NBD increases. Based on this, lipid membrane fusion efficiency can be measured. The lipid membrane fusion efficiency of the nanoparticles was determined by measuring the fluorescent signal of NBD at ex 475/em 540 wavelength.

By measuring the generation of fluorescent signals, it was confirmed that lipid nanoparticles comprising PEG-DMG or PEG600-Hz-DSG were fused with lipid membranes of liposomes under acidic conditions (FIG. 7, A). In particular, lipid nanoparticles comprising 10% PEG600-Hz-DSG (which have a higher lipid content than PEG-DMG lipid nanoparticles comprising 1.5% PEG) initiate fusion with the lipid membrane at a relatively higher pH and produce a stronger fluorescent signal. In other words, both nanoparticles were prepared using the same ionizable lipid, thus exhibiting the same effect of the positive charge of the ionizable lipid, but the PEG in the PEG-DMG lipid nanoparticle remained on the particle surface, whereas the PEG in the PEG600-Hz-DSG lipid nanoparticle was removed by hydrolysis of the hydrazone group, thus exhibiting relatively better lipid membrane fusion efficiency.

The efficiency of fusion of lipid nanoparticles and lipid membranes can also be determined by hemolysis experiments using erythrocytes. Fig. 7B shows the results of a haemolysis-induced reaction of lipid nanoparticles comprising 5 and 10mol% PEG-DMG, which is typically used for the preparation of lipid nanoparticles, at pH 4.5 or 7.4. It can be seen that when the PEG content was increased from 5% to 10%, the hemolysis reaction was inhibited. In other words, it can be seen that the lipid membrane fusion ability is inhibited due to the shielding effect of PEG as the PEG content increases. On the other hand, for lipid nanoparticles prepared using PEG600-Hz-DSG containing ligands, PEG was removed by hydrolysis under acidic conditions. Thus, lipid nanoparticles with an increased PEG derivative content of 10% were also effective in inducing hemolysis.

For reference, it can be seen that at neutral conditions of pH 7.4, neither type of lipid nanoparticle causes a hemolytic reaction, since the amine groups of the ionizable lipids are electrically neutral. In other words, other cells around the lipid nanoparticle do not interact when the pH is neutral, but when the lipid nanoparticle reaches the endosome, the lipid nanoparticle comprising the PEG derivative is relatively easy to fuse with the endosome lipid membrane.

Example 9:the length of the lipid constituting the PEG moiety-degradable functional group-lipid conjugate is linked to the nanoparticle Relationship of structural stability

From the above examples, it can be seen that PEG around the surface of lipid nanoparticles is necessary for the preparation process of lipid nanoparticles, but has drawbacks in terms of pharmacological efficacy. Thus, PEG-DMG, in which PEG is linked to a hydrocarbon lipid such as myristate having 14 carbon atoms, is used as PEG, so that PEG can be spontaneously removed from the lipid nanoparticle when the lipid nanoparticle circulates in the blood stream in vivo. Lipid nanoparticles prepared using substances with longer fatty acids such as palmitate (16 carbon atoms) and stearate (18 carbon atoms) cannot allow easy removal of PEG during circulation through the blood stream, thus cannot easily enter the endosome, and their efficacy is reduced.

However, the method of removing PEG using PEG-lipid conjugates with shorter fatty acids during circulation is very unstable in view of the structural stability of the lipid nanoparticle. When PEG-lipid conjugates begin to be separated and removed from the lipid nanoparticle, the stability of the particle itself becomes poor and other lipid components constituting the lipid nanoparticle also begin to be lost. To confirm this, the structural stability between lipid nanoparticles comprising 1.5% or 5% PEG-DMG (lipid carbon length 14) and lipid nanoparticles comprising 5% PEG moiety-degradable functional group-lipid conjugate (fatty acid length 18) (formula 4) was compared.

1mol% of fluorescent 18:1 NBD PE and 18:1 Lisrod (Liss Rhod) PE were each used to prepare lipid nanoparticles. Lipid nanoparticles were added to rat plasma at 37 ℃, samples were collected at predetermined times, and the fluorescence intensity of NBD was measured (fig. 8). When the particles remain stable, the fluorescent signal of the NBD fluorescent dye is weak because the two fluorescent dyes are relatively close within the nanoparticle. When the nanoparticle structure becomes unstable, it can cause the composition of the nanoparticle to leak out. In this case, the fatty acid labeled with each fluorescent dye is also released, resulting in an increase in fluorescence signal. As can be seen from the data, lipid nanoparticles prepared using PEG-DMG with short fatty acids have poor particle stability and a rapid increase in fluorescence signal, while PEG moiety-degradable functional groups with long fatty acids have a slow increase in fluorescence signal of lipid conjugates. In other words, in lipid nanoparticles prepared by deliberately removing PEG from the lipid nanoparticle using PEG-lipid conjugates with shorter fatty acids, PEG is removed and other lipid components are rapidly lost during circulation through the blood stream. On the other hand, lipid nanoparticles prepared using the PEG moiety-degradable functional group-lipid conjugates disclosed herein can maintain a relatively stable structure.

Example 10: measurement of immune response induction by lipid nanoparticles

The ionizable lipids that make up the lipid nanoparticle may trigger unwanted immune responses through interactions with immune cells. To reduce this side effect, PEG may be used. From the above examples, it can be seen that increasing PEG content can effectively alleviate immune responses by inhibiting unwanted interactions, but inevitably severely inhibits the efficiency of uptake of nanoparticles by cells and endosomal escape. Therefore, it is difficult to increase the PEG content beyond the minimum required amount.

However, the lipid nanoparticle containing the PEG moiety-degradable functional group-lipid conjugate of the present invention may use a ligand to increase cellular uptake efficiency and have a chemical functional group that can remove PEG in endosomes. Thus, when a PEG moiety-degradable functional group-lipid conjugate is used, even if the PEG content is increased, induction of immune response can be suppressed without decreasing the efficacy of the prepared lipid nanoparticle. Thus, lipid nanoparticles using 10mol% of PEG moiety-degradable functional group-lipid conjugate were mixed with Peripheral Blood Mononuclear Cells (PBMCs) extracted from rats, and the degree of cytokine induction by the lipid nanoparticles was measured. As can be seen from fig. 9, the extent to which the lipid nanoparticle using the PEG moiety-degradable functional group-lipid conjugate induced an immune response was similar to that of the group treated with PBS buffer as a negative control.

Example 11: evaluation of lipid nanoparticle efficacy using animals

Animal experiments were performed to determine in vivo effectiveness using lipid nanoparticles that were effective in cell experiments. To this end, lipid nanoparticles comprising 5mol% PEG2K-PE-DSG or PEG2K-Hz-DSG were prepared, and each lipid nanoparticle comprised siRNA targeting factor 7. Each lipid nanoparticle was injected into 8 week old C57BL/6 female mice (Raon Bio) by intravenous injection (i.v. injection) at a dose of 0.3 or 1mg/kg PBS or each siRNA based lipid nanoparticle. The volume of substance administered during injection was kept at 10mL/kg. The amount of factor 7 in serum was measured 48 hours after injection using factor 7 assay kit (Abcam) and the calculated amount was compared to negative control administered with PBS (fig. 10).

Similar to the experimental results using cells (fig. 2, a), in the lipid nanoparticle containing hydrazone (degradable functional group), the expression of factor 7 was inhibited in a dose-dependent manner, but no efficacy was found in the lipid nanoparticle containing no degradable functional group.

Example 12: assessment of efficacy comprising ligand lipid nanoparticles

To selectively deliver nanoparticles to tissues expressing a particular receptor, lipid nanoparticles can be prepared by incorporating ligands that specifically bind to the corresponding receptor. To determine the effect of lipid nanoparticles comprising ligands, lipid nanoparticles comprising 10mol% PEG600-Hz-DSG and lipid nanoparticles comprising a combination of 0.5mol% GalNac-PEG2K-Hz-DSG and 9.5mol% GalNac-PEG2K-Hz-DSG were prepared for specific delivery to liver tissue. Nanoparticles were administered to mice at a dose of 0.1mg/kg based on siRNA, and animal experiments were performed in the same manner as in example 11.

The results show that lipid nanoparticles comprising liver-specific ligands show better efficacy compared to lipid nanoparticles comprising no ligands (fig. 11).

Example 13: fine lipid nanoparticleCytotoxicity assessment

The relative toxicity of lipid nanoparticles comprising the usual PEG-DMG and lipid nanoparticles comprising the PEG moiety-degradable functional group-lipid conjugate for use in the present invention was evaluated. To this end, lipid nanoparticles comprising 1.5mol% PEG-DMG and lipid nanoparticles comprising a mixture of 1mol% GalNAc-PEG2K-Hz-DSG and 9mol% PEG600-Hz-DSG were prepared, respectively. Cell lines such as HepG2, HEK293, H460 and a549 were treated with lipid nanoparticles based on siRNA concentration in the lipid nanoparticles. For 72 hours, the activity of each cell line was measured using CellTiter-Glo kit (Promega) (fig. 12).

Treatment of cells with high concentrations of lipid nanoparticles comprising PEG-DMG resulted in many cell deaths, whereas lipid nanoparticles comprising 10mol% of PEG moiety-degradable functional group-lipid conjugates used in the present invention did not result in cell deaths, indicating that the lipid nanoparticles have very low toxicity.

The therapeutic agent using lipid nanoparticles according to the present invention minimizes in vivo side effects caused by nanoparticle components, effectively delivers nanoparticles to target cells, and effectively transports pharmacological substances such as nucleic acids from endosomes within target cells into cytoplasm.

The lipid nanoparticle according to the present invention can inhibit nonspecific interactions because the outside of the particle is coated with PEG. Furthermore, by attaching a ligand to the end of PEG, PEG can be delivered only to target cells. The lipid nanoparticle encapsulated in the endosome by the ligand and the cell receptor can increase the interaction between the surface of the lipid nanoparticle and the endosome lipid membrane because the degradable functional group is degraded in the endosome and PEG outside the nanoparticle is removed. Furthermore, the lipid nanoparticles may be stable during treatment with the particles, since relatively long chain fatty acids may be incorporated into the PEG-linked fat component, whereby the lipid nanoparticles are given structural stability.

While specific configurations of the invention have been described in detail, those skilled in the art will appreciate that the description is provided for illustrative purposes to illustrate the preferred embodiments and should not be construed as limiting the scope of the invention. Accordingly, the substantial scope of the present invention is defined by the appended claims and equivalents thereof.

Claims

1. A lipid nanoparticle comprising:

(a) Lipid formulations 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 in a lipid formulation.

2. The lipid nanoparticle of claim 1, wherein the PEG moiety-degradable functional group-lipid conjugate is represented by the following formula 1:

[ 1]

Wherein the method comprises the steps of

a is 0 or 1;

l is a targeting ligand;

m is H, OH, a single bond, O, S, C (O), NHC (O), C (O) NH, OC (O) or C (O) O; p is H ₂ O(CH ₂ CH ₂ O) _q CH ₂ Or CH (CH) ₂ CH ₂ O(CH ₂ CH ₂ O) _q CH ₂ Wherein 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、OC(O)、OC(O)O、O-N＝CR ₄ 、R ₄ c=n-O, S-S, S, trans-cyclooctene, or

Wherein R is ₄ Is H, C _1- C ₂₀ Alkyl, C _2- C ₂₀ Alkenyl, C _2- C ₂₀ Alkynyl group、C _3- C ₁₀ Cycloalkyl, C _6- C ₂₀ Aryl or heterocycle, which is a group comprising heteroatoms 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;

t is a single bond or 1,4-C ₆ H ₄ O-; and is also provided with

3. The lipid nanoparticle of claim 2, wherein the targeting ligand L is represented by the following formula 2:

[ 2]

Wherein the method comprises the steps of

a. b and c are 0 or 1, with the proviso that at least one of a, b or c is 1;

X ₁ 、X ₂ and X ₃ Is a targeting ligand;

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 Wherein 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 Wherein n is an integer from 1 to 20; and

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 _。

4. A lipid nanoparticle according to claim 3, wherein X ₁ 、X ₂ And X ₃ Selected from the group consisting of: N-acetyl-D-galactosamine (GalNAc), N-acetyl-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 antibodies, antigen binding fragments or antibody fragments, single chain variable fragments (scFv) and aptamers.

5. The lipid nanoparticle of claim 1, wherein the polyethylene glycol (PEG) moiety-degradable functional group-lipid conjugate is represented by the following formula 3:

[ 3]

Wherein n is an integer from 2 to 120.

6. The lipid nanoparticle of claim 1, wherein the polyethylene glycol (PEG) moiety-degradable functional group-lipid conjugate is represented by the following formula 4:

[ 4]

Wherein n is an integer from 2 to 120.

7. The lipid nanoparticle of claim 2, wherein the polyethylene glycol (PEG) moiety-degradable functional group-lipid conjugate is a mixture of a compound of formula 1 wherein a is 0 and a compound of formula 1 wherein a is 1.

8. The lipid nanoparticle of claim 7, wherein the molar ratio of the compound of formula 1 wherein a is 0 to the compound of formula 1 wherein a is 1 is 0.01 to 99.9:0.01 to 99.9.

9. The lipid nanoparticle according to claim 1, wherein the content of ligand-PEG moiety-degradable functional group-lipid conjugate in the lipid nanoparticle is 0.5 to 50mol% based on the total lipid constituting the lipid nanoparticle.

10. The lipid nanoparticle of claim 1, wherein the size of the lipid nanoparticle is 20 to 200nm.

11. The lipid nanoparticle of claim 1, wherein the ionizable lipid comprises at least one selected from the group consisting of: (6Z, 9Z,28Z, 31Z) -heptadecane-6,9,28,31-tetralin-19-yl ester (DLin-MC 3-DMA), [ (4-hydroxybutyl) azadiyl ] bis (2-hexyldecanoate) (ALC-0315), 8- [ (2-hydroxyethyl) [ 6-oxolano-6- (undecoxy) hexyl ] amino ] -octanoic acid (SM-102), 1-oleoyl-2-oleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-dioleoyl-3-dimethylaminopropane (DLin-DAP), 1, 2-dioleoyloxy-N, N-dimethylaminopropane (DLin-DMA), 1, 2-dioleoyl-4-dimethyl amino-1, 2-dioleoyl-1-dimethylpentane ] -2-dioleoyl-3-dioleoyl-2-dioleoyl-3-D-N, N-dimethylaminopropane (DLin-DAP), 1, 2-dioleoyl-3-N, N-dimethylaminopropane (DLin-DAP), n-dimethyl- (2, 3-dioleoyloxy) propylamine (DODMA), dioctadecyl amidoglycyl-spermine (DOGS), spermine cholesterol carbonyl amide (GL-67), biguanidino-spermidine-cholesterol (BGTC), 3 beta- (N- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol), 1'- (2- (4- (2- ((2- (bis (2-hydroxydecyl) amino) ethyl) piperazin-1-yl) ethylazadiyl) didodecyl-2-ol (C12-200), N-t-butyl-N' -tetradecylaminopropionamidine (bis C14 amidine), dimethyl Dioctadecyl Ammonium Bromide (DDAB), N- (1, 2-dimyristoxypropan-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide (IE), N-dioleoyl-N, N-dimethyl ammonium chloride (DODAC), dioleoyl-3-dioleylpropyl-1-yl) ethyldiamide (DMRn- (2-carboxypropyl) -2- (2-dicarboxylammonium bromide) (DMRN-2-dioleyl) carboxamide, n-dimethyl ammonium trifluoroacetate (DOSPA), 1, 2-dioleoyl trimethylpropane ammonium chloride (DOTAP), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTMA), and aminopropyl-dimethyl-bis (dodecyloxy) -propane ammonium bromide (GAP-DLRIE).

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

13. The lipid nanoparticle of claim 12, wherein the sterol lipid is cholesterol or cholesterol ester.

14. The lipid nanoparticle of claim 12, wherein the neutral lipid is a phospholipid or a sphingolipid.

15. The lipid nanoparticle of claim 14, wherein the phospholipid is selected from the group consisting of: DOPE (distearoyl phosphatidylethanolamine), DSPC (distearoyl phosphatidylcholine), POPC (palmitoyl oleoyl phosphatidylcholine), EPC (egg yolk phosphatidylcholine), DOPC (dioleoyl phosphatidylcholine), DPPC (dipalmitoyl phosphatidylcholine), DOPG (dioleoyl phosphatidylglycerol), DPPG (dipalmitoyl phosphatidylglyceride), DSPE (distearoyl phosphatidylethanolamine), PE (phosphatidylethanolamine), DPPE (dipalmitoyl phosphatidylethanolamine), DOPE (1, 2-dioleoyl-SN-glycero-3-phosphatidylethanolamine), 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 and sphingomyelin.

16. The lipid nanoparticle of claim 1, wherein the drug comprises at least one selected from the group consisting of: peptides, protein drugs, protein-nucleic acid structures, and anionic biopolymer-drug conjugates.

17. The lipid nanoparticle of claim 16, wherein the nucleic acid comprises 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.

18. A method of preparing the lipid nanoparticle of claim 1, comprising:

(a) Mixing an organic solution comprising a lipid formulation containing an ionizable lipid and a polyethylene glycol moiety (PEG moiety) -degradable functional group-lipid conjugate with a buffer solution comprising a drug, a nucleic acid, or a combination thereof to adjust pH; and

(b) The solvent is removed from the solution.

19. The method of claim 18, wherein the mixing ratio of the organic solution to the buffer solution is 1:1 to 1:100 on a volume basis.

20. The method nanoparticle of claim 18, wherein the PEG moiety-degradable functional group-lipid conjugate is represented by formula 1 below:

[ 1]

Wherein the method comprises the steps of

a is 0 or 1;

l is a targeting ligand;

m is H, OH, a single bond, O, S, C (O), NHC (O), C (O) NH, OC (O) or C (O) O;

p is H ₂ O(CH ₂ CH ₂ O) _q CH ₂ Or CH (CH) ₂ CH ₂ O(CH ₂ CH ₂ O) _q CH ₂ Wherein q is an integer from 2 to 120;

Wherein R is 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 Aryl or heterocycle, which is a group comprising heteroatoms 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;

t is a single bond or 1,4-C ₆ H ₄ O-; and is also provided with

21. The method of claim 20, wherein the targeting ligand L is represented by formula 2 below:

[ 2]

Wherein the method comprises the steps of

a. b and c are 0 or 1, with the proviso that at least one of a, b or c is 1;

X ₁ 、X ₂ and X ₃ Is a targeting ligand;

22. The method of claim 21, wherein X ₁ 、X ₂ And X ₃ Selected from the group consisting of: N-acetyl-D-galactosamine (GalNAc), N-acetyl-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 antibodies, antigen binding fragments or antibody fragments, single chain variable fragments (scFv) and aptamers.

23. The method of claim 18, wherein the polyethylene glycol (PEG) moiety-degradable functional group-lipid conjugate is represented by formula 3 below:

[ 3]

Wherein n is an integer from 2 to 120.

24. The method of claim 18, wherein the PEG moiety-degradable functional group-lipid conjugate is represented by formula 4 below:

[ 4]

Wherein n is an integer from 2 to 120.

25. The method of claim 20, wherein the PEG moiety-degradable functional group-lipid conjugate is a mixture of a compound of formula 1 wherein a is 0 and a compound of formula 1 wherein a is 1.

26. The method of claim 25, wherein the molar ratio of the compound of formula 1 wherein a is 0 to the compound of formula 1 wherein a is 1 is 0.01 to 99.9:0.01 to 99.9.

27. The method of claim 18, wherein the content of ligand-PEG moiety-degradable functional group-lipid conjugate in the lipid nanoparticle is 0.5 to 50mol% based on total lipids constituting the lipid nanoparticle.

28. The method of claim 18, wherein the lipid nanoparticle is 20 to 200nm in size.

29. The method of claim 18, wherein the ionizable lipid comprises at least one selected from the group consisting of: (6Z, 9Z,28Z, 31Z) -heptadecane-6,9,28,31-tetralin-19-yl ester (DLin-MC 3-DMA), [ (4-hydroxybutyl) azadiyl ] bis (2-hexyldecanoate) (ALC-0315), 8- [ (2-hydroxyethyl) [ 6-oxolano-6- (undecoxy) hexyl ] amino ] -octanoic acid (SM-102), 1-oleoyl-2-oleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-dioleoyl-3-dimethylaminopropane (DLin-DAP), 1, 2-dioleoyloxy-N, N-dimethylaminopropane (DLin-DMA), 1, 2-dioleoyl-4-dimethyl amino-1, 2-dioleoyl-1-dimethylpentane ] -2-dioleoyl-3-dioleoyl-2-dioleoyl-3-D-N, N-dimethylaminopropane (DLin-DAP), 1, 2-dioleoyl-3-N, N-dimethylaminopropane (DLin-DAP), n-dimethyl- (2, 3-dioleoyloxy) propylamine (DODMA), dioctadecyl amidoglycyl-spermine (DOGS), spermine cholesterol carbonyl amide (GL-67), biguanidino-spermidine-cholesterol (BGTC), 3 beta- (N- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol), 1'- (2- (4- (2- ((2- (bis (2-hydroxydecyl) amino) ethyl) piperazin-1-yl) ethylazadiyl) didodecyl-2-ol (C12-200), N-t-butyl-N' -tetradecylaminopropionamidine (bis C14 amidine), dimethyl Dioctadecyl Ammonium Bromide (DDAB), N- (1, 2-dimyristoxypropan-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide (IE), N-dioleoyl-N, N-dimethyl ammonium chloride (DODAC), dioleoyl-3-dioleylpropyl-1-yl) ethyldiamide (DMRn- (2-carboxypropyl) -2- (2-dicarboxylammonium bromide) (DMRN-2-dioleyl) carboxamide, n-dimethyl ammonium trifluoroacetate (DOSPA), 1, 2-dioleoyl trimethylpropane ammonium chloride (DOTAP), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTMA), and aminopropyl-dimethyl-bis (dodecyloxy) -propane ammonium bromide (GAP-DLRIE).

30. The method of claim 18, further comprising sterol lipids and neutral lipids.

31. The method of claim 30, wherein the sterol lipid is cholesterol or cholesterol ester.

32. The method of claim 30, wherein the neutral lipid is a phospholipid.

33. The method of claim 32, wherein the phospholipid is selected from the group consisting of: DOPE (distearoyl phosphatidylethanolamine), DSPC (distearoyl phosphatidylcholine), POPC (palmitoyl oleoyl phosphatidylcholine), EPC (egg yolk phosphatidylcholine), DOPC (dioleoyl phosphatidylcholine), DPPC (dipalmitoyl phosphatidylcholine), DOPG (dioleoyl phosphatidylglycerol), DPPG (dipalmitoyl phosphatidylglyceride), DSPE (distearoyl phosphatidylethanolamine), PE (phosphatidylethanolamine), DPPE (dipalmitoyl phosphatidylethanolamine), DOPE (1, 2-dioleoyl-SN-glycero-3-phosphatidylethanolamine), 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 and sphingomyelin.

34. The method of claim 18, wherein the drug comprises at least one selected from the group consisting of: peptides, protein drugs, protein-nucleic acid structures, and anionic biopolymer-drug conjugates.

35. The method of claim 34, wherein the nucleic acid comprises 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.