WO2025063807A1 - Lipid nanoparticles comprising tumor-targeting ligands, method for preparing same, and pharmaceutical composition comprising same - Google Patents

Abstract

Disclosed in the present application are: lipid nanoparticles comprising a tumor-targeting ligand; a method for preparing same; and a pharmaceutical composition for preventing or treating tumors, comprising same. The lipid nanoparticles include a nucleic acid, an ionizable lipid, a phospholipid, a sterol, a first conjugate, and a second conjugate in which a tumor-targeting ligand is linked. The first conjugate is a conjugate of a phospholipid and a polymer, and the second conjugate is a conjugate in which a tumor-targeting ligands is linked to the first conjugate. The lipid nanoparticles have a high targeting ability to solid tumors expressing PD-L1.

Description

Lipid nanoparticles containing tumor-targeting ligands, methods for preparing the same, and pharmaceutical compositions containing the same

[Cross-reference to related applications]

This application claims priority to Republic of Korea Patent Application No. 10-2023-0125377, filed September 20, 2023, the entire contents of which are incorporated herein by reference.

[Description of Nationally Supported Research and Development]

This study was conducted at the Korea Institute of Science and Technology (KIST) under the management of the Korea Institute of Science and Technology (KIST) under the Ministry of Science and ICT. The research project name is the Korea Institute of Science and Technology Research Operation Expense Support (Main Project Expense), and the research project name is Customized Diagnosis and Treatment, Regeneration, Rehabilitation, and New Drug Development (Project Unique Number: 1711196534, Project Number: 2E32330).

In addition, this study was conducted at the Korea Institute of Science and Technology under the management of the National Research Foundation of Korea under the Ministry of Science and ICT, and the research project name is Individual Basic Research (MSIT), and the research project name is Development of an Anticancer Immunotherapy Strategy Based on a Cancer/Macrophage Dual-Targeting MiRNA Delivery System (Project Unique Number: 1711192380, Project Number: 2022R1A2C2006861).

The present disclosure discloses a lipid nanoparticle comprising a tumor-targeting ligand, a method for preparing the same, and a pharmaceutical composition comprising the same for preventing or treating a tumor.

Effective delivery of small molecule drugs, proteins, nucleic acids, etc. to target cells requires continuous development. In particular, delivery of nucleic acids to target cells is technically difficult due to relative instability and low cell permeability, and there are still technical limitations in targeting and delivering to desired cells. For example, most existing lipid nanoparticle (LNP)-based therapeutics accumulate in specific organs (liver, kidney, etc.), so their therapeutic effects are minimal when used as cancer-targeting therapeutics.

In one aspect, the present disclosure aims to provide lipid nanoparticles comprising a tumor targeting ligand.

In another aspect, the present disclosure aims to provide a method for producing the lipid nanoparticle.

In another aspect, the present disclosure aims at providing a pharmaceutical composition for preventing or treating tumors comprising the lipid nanoparticles.

In one aspect, the present disclosure provides a lipid nanoparticle comprising: a nucleic acid; an ionizable lipid; a phospholipid; a sterol; a first conjugate; and a second conjugate conjugated with a tumor targeting ligand, wherein the first conjugate is a conjugate of a phospholipid and a polymer, and the second conjugate is a tumor targeting ligand conjugated to the first conjugate.

In an exemplary embodiment, the nucleic acid may comprise at least one selected from the group consisting of RNA, messenger RNA (mRNA), antisense oligonucleotide (ASO), DNA, plasmid, ribosomal RNA (rRNA), micro RNA (miRNA), trans RNA (tRNA), small interfering RNA (siRNA), and small nuclear RNA (snRNA).

In an exemplary embodiment, the ionizable lipid is (6Z, 9Z, 28Z, 31Z)-heptathriaconta 6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid (SM-102), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyl carbamoyloxy-3-dimethylaminopropane (DLin-CDAP), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy) propylamine (DODMA), dioctadecylamidoglycylspermine (DOGS), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl) amino) ethyl)(2-hydroxydecyl) amino) ethyl) piperazin-1-yl) ethylazanediyl) didodecan-2-ol (C12-200), dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyl oxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyl oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxyl) propyl)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyl trimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy) The composition may include at least one selected from the group consisting of N,N,N-trimethylammonium chloride (DOTMA) and aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE).

In an exemplary embodiment, the phospholipid is selected from the group consisting of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DiPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), Consisting of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), ceramide and sphingomyelin. It may include one or more selected from the military.

In an exemplary embodiment, the sterol may include at least one selected from the group consisting of cholesterol, ergosterol, campesterol, oxysterol, desmosterol, sitosterol, and stigmasterol.

In an exemplary embodiment, the first conjugate can include at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol (DSG-PEG), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG).

In an exemplary embodiment, the tumor targeting ligand may be a PD-L1 binding peptide that targets a solid tumor expressing PD-L1.

In an exemplary embodiment, the solid tumor may be at least one selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanoma, bladder cancer, brain tumor, CNS cancer, cervical cancer, esophageal cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, colon cancer, neuroblastoma, and sarcoma.

In an exemplary embodiment, the second conjugate may be a first conjugate functionalized with dibenzocyclooctyne (DBCO) and a tumor targeting ligand functionalized with an azide group linked by a click reaction.

In an exemplary embodiment, the ionized lipid, phospholipid, sterol, first conjugate and second conjugate may be mixed in a molar ratio of 40 to 60:5 to 15:30 to 45:1 to 2:0.1 to 1, respectively.

In an exemplary embodiment, the average size of the lipid nanoparticles may be from 50 nm to 200 nm.

In another aspect, the present disclosure provides a method for preparing a lipid nanoparticle, comprising the steps of: (1) functionalizing a tumor-targeting ligand with an azide group; (2) functionalizing a first conjugate with dibenzocyclooctyne (DBCO); (3) reacting the first conjugate functionalized with DBCO with the tumor-targeting ligand functionalized with the azide group through a click reaction to prepare a second conjugate in which the tumor-targeting ligand is conjugated to the first conjugate; and (4) mixing a lipid solution containing an ionized lipid, a phospholipid, a sterol, the first conjugate, and the second conjugate, and a nucleic acid solution, wherein the first conjugate is a conjugate of a phospholipid and a polymer, and the second conjugate is a tumor-targeting ligand conjugated to the first conjugate.

In another aspect, the present disclosure provides a pharmaceutical composition for preventing or treating a tumor comprising the lipid nanoparticle.

In one aspect, the technology disclosed in the present disclosure is effective in providing lipid nanoparticles comprising a tumor targeting ligand.

In another aspect, the technology disclosed in the present disclosure has the effect of providing a method for producing the lipid nanoparticle.

In another aspect, the technology disclosed in the present disclosure is effective in providing a pharmaceutical composition for preventing or treating tumors comprising the lipid nanoparticles.

Figure 1 is a schematic diagram of a synthetic reaction of a PEG-lipid introduced with a tumor-targeting ligand according to one embodiment, and a structure and in vivo behavior of a lipid nanoparticle including the same.

FIGS. 2A, 2B, and 2C are 1H-NMR graphs of the synthetic reactants and products of a tumor-targeting ligand and PEG-lipid according to one embodiment.

FIG. 3 is a Cryo-TEM (cryo-transmission electron microscopy) image of lipid nanoparticles manufactured according to one embodiment (bar: 100 nm).

Figure 4 shows the results of comparing the surface coating rate of a tumor-targeting ligand according to the addition ratio of the second conjugate according to one experimental example.

Figure 5 shows the results of analyzing the size of lipid nanoparticles according to the addition ratio of the second conjugate using DLS (Dynamic Light Scattering) according to one experimental example.

Figure 6 shows the results of analyzing the change in particle size over time using DLS (Dynamic Light Scattering) of lipid nanoparticles manufactured in Example 2 (Ligand LNP) and Comparative Example 1 (Con LNP) after storage at 4°C according to one experimental example.

Figure 7 shows the results of analyzing the mRNA encapsulation efficiency of lipid nanoparticles manufactured in Example 2 (Ligand LNP) and Comparative Example 1 (Con LNP) according to one experimental example.

Figure 8 is a fluorescence image comparing the expression efficiency of mRNA according to the storage period (storage temperature 4°C) of lipid nanoparticles encapsulating EGFP mRNA according to one experimental example (bar: 100 μm).

Figure 9 shows the results of evaluating the binding ability of a His-binding pair protein and lipid nanoparticles according to one experimental example. a is an outline of the experiment evaluating the binding ability of a His-binding pair protein and lipid nanoparticles, and b and c are fluorescence image data and graphs evaluated according to a of Figure 9 (***: p < 0.001).

Figure 10 is a fluorescence image and graph showing the binding ability of lipid nanoparticles to the tumor cell surface according to one experimental example (bar: 100 μm, **: p < 0.01).

Figure 11 is a graph showing the photographs and fluorescence intensity of intratumoral fluorescence expression at different time points taken using IVIS equipment after lipid nanoparticles were injected into the tail vein of a cancer animal model (*: p < 0.05, **: p < 0.01, #: p < 0.05).

Figure 12 is a photograph and graph showing intratumoral fluorescence and Luciferase protein expression 24 hours after injection of lipid nanoparticles loaded with Luciferase mRNA into a cancer animal model via the tail vein, captured using IVIS equipment (*: p < 0.05, ***: p < 0.001, #: p < 0.05, ###: p < 0.001).

Hereinafter, the present disclosure is described in detail.

In one aspect, the present disclosure provides a lipid nanoparticle comprising: a nucleic acid; an ionizable lipid; a phospholipid; a sterol; a first conjugate; and a second conjugate conjugated with a tumor targeting ligand, wherein the first conjugate is a conjugate of a phospholipid and a polymer, and the second conjugate is a tumor targeting ligand conjugated to the first conjugate.

PD-L1 (Programmed death-ligand 1) is a protein on the surface of cancer cells or hematopoietic cells. PD-L1 on the surface of cancer binds to PD-1 on the surface of T cells, which inhibits immune cell activity. Among the functions of PD-L1 is a major regulatory role in T cell activity. Antibodies that bind to PD-1 or PD-L1 and block the interaction can allow T cells to attack tumors.

PD-L1 is expressed in a wide range of cancers, with a high frequency of up to 88% in some types of cancer. In many cancers, including lung, kidney, pancreatic, and ovarian cancers, PD-L1 expression is associated with decreased survival and poor prognosis.

To treat tumors through nucleic acid delivery via lipid nanoparticles, tumor targeting is essential. The present disclosure provides lipid nanoparticles with significantly improved targeting ability for tumors expressing PD-L1.

In the present invention, the lipid nanoparticle (LNP) has a size in the nanometer (nm) scale in terms of morphology and has a core-shell structure including one phospholipid layer. For example, it can be structurally configured to entrap an insoluble drug and/or nucleic acid in the center (core) or surface (shell). The lipid nanoparticle according to the present disclosure not only increases the in vivo delivery efficiency of nucleic acids, but also has the effect of significantly increasing the delivery power to target cells.

In this application, the term “peptide” refers to a linear molecule formed by amino acid residues joining together via peptide bonds.

Representative amino acids and their respective abbreviations are: alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), tryptophan (Trp, W), valine (Val, V), asparagine (Asn, N), cysteine (Cys, C), glutamine (Gln, Q), glycine (Gly, G), serine (Ser, S), threonine (Thr, T), tyrosine (Try, Y), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine (His, H), and lysine (Lys, K).

The individual, subject or patient herein may be a mammal, such as a human, cow, horse, pig, dog, sheep, deer, rabbit, goat or cat, and preferably a human.

A pharmaceutically acceptable carrier herein refers to an ingredient in a pharmaceutical composition or formulation other than an active ingredient, and is a non-toxic substance to the subject. A pharmaceutically acceptable carrier includes, for example, a buffer, an excipient, a stabilizer, or a preservative.

The therapeutically effective amount or pharmaceutically effective amount herein refers to an amount of a compound or composition that is effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and does not cause side effects. The level of the effective amount may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method of administration, the time of administration, the route of administration, and the excretion rate, the duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field.

In an exemplary embodiment, the nucleic acid may comprise at least one selected from the group consisting of RNA, messenger RNA (mRNA), antisense oligonucleotide (ASO), DNA, plasmid, ribosomal RNA (rRNA), micro RNA (miRNA), trans RNA (tRNA), small interfering RNA (siRNA), and small nuclear RNA (snRNA).

In an exemplary embodiment, the nucleic acid may have an anticancer effect.

In an exemplary embodiment, the nucleic acid may comprise one or more mRNA molecules.

In an exemplary embodiment, the nucleic acid may comprise one or more mRNA molecules having an anti-cancer effect.

In an exemplary embodiment, the lipid nanoparticle may be loaded with the nucleic acid.

The above ionized lipids have the characteristic of being in a cationic state at acidic pH but maintaining an electrically neutral state at neutral pH. By forming an ionic bond with mRNA, which has an anionic property, the ionized lipids can encapsulate mRNA at a high rate and deliver the mRNA intact to target cells.

In an exemplary embodiment, the ionizable lipid is (6Z, 9Z, 28Z, 31Z)-heptathriaconta 6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid (SM-102), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyl carbamoyloxy-3-dimethylaminopropane (DLin-CDAP), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy) propylamine (DODMA), dioctadecylamidoglycylspermine (DOGS), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl) amino) ethyl)(2-hydroxydecyl) amino) ethyl) piperazin-1-yl) ethylazanediyl) didodecan-2-ol (C12-200), dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyl oxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyl oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxyl) propyl)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyl trimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy) The composition may include at least one selected from the group consisting of N,N,N-trimethylammonium chloride (DOTMA) and aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE).

The above phospholipid refers to a lipid molecule consisting of two hydrophobic fatty acid tails and a hydrophilic head consisting of a phosphate group. The two components are mostly joined together by a glycerol molecule. In addition, the phosphate group is often modified with a simple organic molecule such as choline (i.e., converted to phosphocholine) or ethanolamine (i.e., converted to phosphoethanolamine).

In an exemplary embodiment, the phospholipid is selected from the group consisting of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DiPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), Consisting of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), ceramide and sphingomyelin. It may include one or more selected from the military.

The term sterol, also known as steroid alcohols, is a subgroup of steroids that occur naturally in plants, animals, and fungi, or can be produced by some bacteria.

In an exemplary embodiment, the sterol may include at least one selected from the group consisting of cholesterol, ergosterol, campesterol, oxysterol, desmosterol, sitosterol, and stigmasterol.

In an exemplary embodiment, the first conjugate may be a PEGylated phospholipid, which may be a conjugate of a phospholipid and polyethylene glycol.

In an exemplary embodiment, the first conjugate can include at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol (DSG-PEG), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG).

In an exemplary embodiment, the first conjugate is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-5000 (DSPE-PEG5K), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000 (DSPE-PEG2K), 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DPG-PEG5K), 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DPG-PEG2K), 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DSG-PEG5K), It may include at least one selected from the group consisting of 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DSG-PEG2K), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DMG-PEG5K), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

In an exemplary embodiment, the tumor targeting ligand may be a PD-L1 binding peptide.

In an exemplary embodiment, the tumor targeting ligand may be a PD-L1 binding peptide that targets a solid tumor expressing PD-L1.

The above PD-L1 binding peptide is a peptide that binds to PD-L1 expressed on the surface of a solid tumor and may have a sequence of sequence number 1.

In an exemplary embodiment, the solid tumor may express or overexpress PD-L1 on its surface.

In an exemplary embodiment, the solid tumor may be at least one selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanoma, bladder cancer, brain tumor, CNS cancer, cervical cancer, esophageal cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, colon cancer, neuroblastoma, and sarcoma.

In an exemplary embodiment, the lipid nanoparticle may have improved targeting ability for a solid tumor expressing PD-L1. That is, the lipid nanoparticle may contain a tumor-targeting ligand and bind to PD-L1 of a solid tumor with high targeting ability, thereby efficiently killing and/or inhibiting the growth of tumor cells through nucleic acid delivery.

The second conjugate may be a tumor-targeting ligand conjugated to the first conjugate by a method known in the art that is appropriately selected and/or modified by a person skilled in the art. For example, the second conjugate may be a tumor-targeting ligand conjugated to the first conjugate through a DBCO-azide (dibenzocyclooctyne-azide) bond reaction, a maleimide-thiol (maleimide-thiol) bond reaction, an N-hydroxysuccinimide ester-amine (NHS ester-amine) bond reaction, a BCN-azide (bicyclononyne-azide) bond reaction, or an ADIBO-DBCO (aza-dibenzocyclooctyne-dibenzocyclooctyne) bond reaction.

In an exemplary embodiment, the second conjugate may be a click reaction-linked first conjugate functionalized with dibenzocyclooctyne (DBCO) and a tumor targeting ligand functionalized with an azide group. The DBCO covalently binds to the N-terminal azide moiety of the tumor targeting ligand.

In an exemplary embodiment, the second conjugate may be a first conjugate functionalized with dibenzocyclooctyne (DBCO) and a PD-L1 binding peptide functionalized with an azide group, linked by a click reaction.

In an exemplary embodiment, the second conjugate may have a structure represented by the following chemical formula 1.

[Chemical Formula 1]

In an exemplary embodiment, the second conjugate may be a first conjugate (PEG-lipid) functionalized with dibenzocyclooctyne (DBCO) represented by Chemical Formula 3 and a PD-L1 binding peptide functionalized with an azide group represented by Chemical Formula 2.

[Chemical formula 2]

[Chemical Formula 3]

In the above chemical formulas 1 to 3,

R ₁ to R ₃ are each independently hydrogen; a C ₁ -C ₁₃ alkyl group; a C ₁ -C ₆ alkoxy group; a C ₆ -C ₁₀ aryl group; a C ₃ -C ₁₀ cyclyl group; a C ₃ -C ₁₀ heteroaryl group; a C ₃ -C ₁₀ heterocyclyl group; -C(O)-(C ₁ -C ₁₃ alkyl); -C(O)-(C ₆ -C ₁₀ aryl group); or -C(O)-(C ₃ -C ₁₀ heteroaryl group); and

m, n, p, q or r are each independently 0, 1, 2, 3 or 4,

W ₁ to W ₃ are each independently -NR ₃ -; -NR ₃ CH ₂ -; -NR ₃ -C(O)-; -NR ₃ -C(O)-NR4-; -NR ₃ -C(S)-NR ₄ -; -C(O)-; -C(O)CH ₂ -; -C(O)O-; -C(O)NR ₃ -; -(CH ₂ )-; -(CR ₃ R ₄ )-; -(CH ₂ )(CR ₃ R ₄ )-; -S(O) ₂ -; -NR ₃ S(O) ₂ -; or -S(O) ₂ NR ₃ -;

The above C ₁ -C ₁₃ alkyl group, C ₃ -C ₁₀ cyclyl group, C ₁ -C ₆ alkoxy group are hydrogen; hydroxy group; halogen group; C ₁ -C ₁₃ alkyl group; C ₁ -C ₆ alkoxy group; amino group (-NR ₃ R ₄ ); nitro group (-N(O) ₂ ); amide group (-(C=O)NR ₃ R4); carboxylic acid group (-C(O)OH); nitrile group (-CN); urea group (-NR ₃ (C=O)NR ₄ -); sulfonamide group (-NHS(O) ₂ -); sulfide group (-S-); sulfone group (-S(O) ₂ -); phosphyryl group (-P(O)R ₃ R ₄ ); C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; and comprises at least one substituent selected from the group consisting of C ₃ -C ₁₀ heterocyclyl groups,

The above C ₆ -C ₁₀ aryl group, C ₃ -C ₁₀ heteroaryl group or C ₃ -C ₁₀ heterocyclyl group is selected from the group consisting of hydrogen; a hydroxy group; a halogen group; a carbonyl group (-(C=O)R ₃ R ₄ ); a C _{1 -C 3} alkyl group substituted or unsubstituted with a halogen or a C ₃ -C ₁₀ heterocyclyl group; a C 1 -C ₃ alkoxy group substituted or unsubstituted with a halogen or a C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; an amino group (-NR ₃ R ₄ ); a nitro group (-N(O) ₂ ); an amide group (-(C=O)NR ₃ R ₄ ); a carboxylic acid group (-C(O)OH); a nitrile group (-CN); a urea group (-NR ₃ (C=O)NR ₄ -); Containing at least one substituent selected from the group consisting of a sulfonamide group (-NHS(O) ₂ -); a sulfide group (-S-); a sulfone group (-S(O) ₂ -); a phosphyryl group (-P(O)R ₃ R ₄ ); a C _{6 -C 10} aryl group; a C 3 -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

wherein R ₃ and R ₄ are each independently hydrogen; a C ₁ -C ₆ alkyl group; a C ₁ -C ₆ alkenyl group; a C ₁ -C ₆ alkynyl group; a C ₆ -C ₁₀ aryl group; a C ₃ -C ₁₀ heteroaryl group; or a C ₃ -C ₁₀ heterocyclyl group; , and R ₃ may optionally include at least one _of N, O, S, NH, C=N, C=O, -NHC(O)-, -NHC(O)NH-, -NHS(O) ₂ -, or SO ₂ together with the nitrogen or carbon atom connected to R 4, and R ₃ and R ₄ may form a 3 to 7 membered saturated ring which may be optionally substituted with at least one of hydrogen, a C ₁ -C ₁₃ alkyl group, a C ₆ -C ₁₀ aryl group, a C ₃ -C ₁₀ heteroaryl group, a hydroxyl group, a halide group and a cyano group, and the C ₃ -C ₁₀ heteroaryl group and the C ₃ -C ₁₀ heterocyclyl group include at least one heteroatom selected from the group consisting of N, O, and S.

In an exemplary embodiment, R ₁ or R ₂ is each independently hydrogen; or a C ₁ -C ₁₃ alkyl group;, m or p is each independently 0, 1, 2, 3 or 4; R ₃ is hydrogen; or a C ₁ -C ₃ alkyl group;, W ₁ is -(CH ₂ )-; -(CR ₃ R ₄ )-; or -(CH ₂ )(CR ₃ R ₄ )-;, r is 0, 1 or 2, W ₂ is -(CH ₂ )-; or -(CR ₃ R ₄ )-;, n is 1 or 2, W ₃ is -(CH ₂ )-; or -(CR ₃ R ₄ )-;, and q can be 0, 1 or 2.

In an exemplary embodiment, the Peptide in Chemical Formula 1 may be a PD-L1 binding peptide.

In an exemplary embodiment, the ionized lipid, phospholipid, sterol, first conjugate and second conjugate may be mixed in a molar ratio of 40 to 60:5 to 15:30 to 45:1 to 2:0.1 to 1, respectively.

In another exemplary embodiment, the ionized lipid, the phospholipid, the sterol, the first conjugate and the second conjugate can be mixed in a molar ratio of 45 to 55:7 to 13:34 to 42:1 to 1.5:0.2 to 0.5, a molar ratio of 48 to 52:8 to 12:36 to 40:1 to 1.4:0.2 to 0.4, a molar ratio of 49 to 51:9 to 11:38 to 39:1.1 to 1.3:0.2 to 0.3, a molar ratio of 49 to 51:9 to 11:38 to 39:1.1 to 1.3:0.3 to 0.4 or a molar ratio of 50:10:38.5:1.2:0.3.

In an exemplary embodiment, the lipid nanoparticle may preferably contain 0.01 to 0.5 mol % or 0.1 to 0.5 mol % of the second conjugate relative to 100 mol % of the total of the ionized lipid, phospholipid, sterol, first conjugate and second conjugate, in terms of the manufacturing efficiency of the lipid nanoparticle and the improvement of the tumor targeting ability of the lipid nanoparticle.

In an exemplary embodiment, the average size of the lipid nanoparticles may be from 50 nm to 200 nm.

In another aspect, the present disclosure provides a method for preparing a lipid nanoparticle, comprising the steps of: (1) functionalizing a tumor-targeting ligand with an azide group; (2) functionalizing a first conjugate with dibenzocyclooctyne (DBCO); (3) reacting the first conjugate functionalized with DBCO with the tumor-targeting ligand functionalized with the azide group through a click reaction to prepare a second conjugate in which the tumor-targeting ligand is conjugated to the first conjugate; and (4) mixing a lipid solution containing an ionized lipid, a phospholipid, a sterol, the first conjugate, and the second conjugate, and a nucleic acid solution, wherein the first conjugate is a conjugate of a phospholipid and a polymer, and the second conjugate is a tumor-targeting ligand conjugated to the first conjugate.

Conventionally, lipid nanoparticles capable of targeting specific cancers were manufactured by first manufacturing lipid nanoparticles and then introducing targeting ligands (post-conjugation), which requires an additional process of removing unsynthesized targeting ligands. On the other hand, the lipid nanoparticles according to the present disclosure are manufactured by first manufacturing lipids into which a low-molecular-weight (about 5 kDa or less) targeting ligand is preferentially introduced, and then manufacturing the lipid nanoparticles using the lipids, thereby enabling more efficient production.

In another aspect, the present disclosure provides a pharmaceutical composition for preventing or treating a tumor comprising the lipid nanoparticle.

In another aspect, the present disclosure provides a method for preventing or treating a tumor, comprising administering or applying to a subject in need thereof a pharmaceutical composition comprising an amount of the lipid nanoparticle effective for preventing or treating a tumor.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the lipid nanoparticle for use in preventing or treating tumors.

In another aspect, the present disclosure provides a use of a pharmaceutical composition comprising the lipid nanoparticle for preventing or treating a tumor.

In another aspect, the present disclosure provides use of the lipid nanoparticles in preparing a pharmaceutical composition for preventing or treating a tumor.

Administration of the pharmaceutical composition to a subject delivers the lipid nanoparticles to target cells of the subject, and the lipid nanoparticles can kill or delay the growth of the target cells of the subject.

In an exemplary embodiment, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

In an exemplary embodiment, the pharmaceutical composition can provide therapeutic outcomes such as a reduction in tumor size, a delay in tumor growth, an extension of patient survival, and remission.

In an exemplary embodiment, the pharmaceutical composition may be administered intravenously or directly into the tumor.

In an exemplary embodiment, prior to administering the pharmaceutical composition to the subject, a step may be taken to determine whether the tumor to be treated expresses PD-L1. For example, any suitable test known in the art may be selected and used by a person skilled in the art. For example, immunohistochemistry may be used to determine whether PD-L1 is expressed on the cell surface of tumor cells.

The sequences provided by our center are as follows.

Sequence number 1

PD-L1 binding peptide: NYSKPTDRQYHF

Sequence number 2

Cy5-labeled oligo DNA: 5'-AGCTCTGTTTACGTCCCAGC-3'

Hereinafter, the present disclosure will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are intended only to illustrate the present disclosure, and that the scope of the present disclosure is not to be construed as being limited by these examples.

Example 1. Preparation of PEG-lipid (second conjugate) with tumor targeting ligand introduced

In this example, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000 (DSPE-PEG2K) was used as a conjugate of a phospholipid and a polymer (also referred to herein as a first conjugate or PEG-lipid), and a second conjugate was prepared using a PD-L1 binding peptide as a tumor targeting ligand.

Specifically, the N-terminal azide-modified PD-L1 binding peptide was purchased from Peptron Company (Daejeon, Republic of Korea). The specific sequence of the azide-functionalized PD-L1 binding peptide (Peptide-Azide) is as follows.

Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe (N3-NYSKPTDRQYHF, d-form)

Additionally, DSPE-PEG2K-DBCO, which is functionalized with dibenzocyclooctyne (DBCO), was purchased from Avanti Polar Lipids (AL, USA).

50 nmol (10 μL) of the above DSPE-PEG2K-DBCO (5 mM in 50% EtOH) and 50 nmol (8.18 μL) of Peptide-Azide (10 mg/mL in Rnased-free water) were placed in a 1.5 mL microcentrifuge tube and reacted at 1100 rpm, 37 °C for 1 hour to prepare a second conjugate (DSPE-PEG2K-Peptide) in which a PD-L1 binding peptide is conjugated to the PEG-lipid, i.e., the first conjugate, to which a tumor-targeting ligand has been introduced.

As a result, as shown in FIGS. 1, 2a, 2b, and 2c, when X and Y are functional group pairs of the click reaction, it was confirmed that X-PEG-lipid (DBCO-PEG2K-DSPE) and Y-ligand (Azide-Peptide) were synthesized into a PEG-lipid with a tumor-targeting ligand introduced after 1 hour of reaction at 37°C at the same molar ratio.

Example 2. Preparation of lipid nanoparticles comprising PEG-lipid (second conjugate) introduced with tumor-targeting ligand

By adding DSPE-PEG2K-Peptide obtained in Example 1, lipid nanoparticles were manufactured as follows.

- Preparation of lipid solution: SM-102, cholesterol, DSPC, DMG-PEG2K, and DSPE-PEG2K-Peptide were placed in a glass vial at a molar ratio of 50:38.5:10:1.2:0.3, dissolved in 100% (v/v) EtOH, and then prepared as a lipid film using an evaporator. Afterwards, the solution was resuspended in 100% (v/v) EtOH so that the lipid concentration became 50 mM.

- Preparation of mRNA solution: mRNA was purchased as EGFP (model name; L-7201) mRNA from TriLink biotechnologies (CA, USA). The amount of mRNA was prepared so that the charge ratio of SM-102: mRNA was 6, and the volume of the solution was prepared twice that of the lipid solution using 10 mM sodium citrate buffer (pH 3).

- LNP synthesis was performed according to the manufacturer's protocol using a Precision Nanosystems NanoAssemblr ^® Spark ^TM device.

- Two solvent exchanges were performed using a 0.5 mL 10 kDa MWCO centrifugal filter with PBS at 14,000 ×g for 10 minutes.

The cryo-TEM image of the lipid nanoparticles containing the second conjugate at a molar ratio of 0.3% manufactured as described above, observed using a low-temperature transmission electron microscope (Tecnai F20 G2, FEI Company, USA), is shown in Figure 3.

Comparative example 1. Not including the second conjugate Lipid nanoparticle manufacturing

Lipid nanoparticles not containing a second conjugate conjugated with a tumor-targeting ligand were prepared as follows.

- Preparation of lipid solution: SM-102, cholesterol, DSPC, and DMG-PEG2K were placed in a glass vial at a molar ratio of 50:38.5:10:1.5, dissolved in 100% (v/v) EtOH, and then prepared as a lipid film using an evaporator. Afterwards, the solution was resuspended in 100% (v/v) EtOH so that the lipid concentration became 50 mM.

- Preparation of mRNA solution: mRNA was purchased as EGFP (model name; L-7201) mRNA from TriLink biotechnologies (CA, USA). The amount of mRNA was prepared so that the charge ratio of SM-102: mRNA was 6, and the volume of the solution was prepared twice that of the lipid solution using 10 mM sodium citrate buffer (pH 3).

- LNP synthesis was performed according to the manufacturer's protocol using a Precision Nanosystems NanoAssemblr ^® Spark ^TM device.

- Two solvent exchanges were performed using a 0.5 mL 10 kDa MWCO centrifugal filter with PBS at 14,000 ×g for 10 minutes.

Experimental Example 1. Analysis of the surface coating rate of tumor targeting ligands

- Lipid nanoparticles containing 0.01, 0.1, 0.3, and 0.5% of DSPE-PEG2K-Peptide were synthesized so that the combined molar ratio of DMG-PEG2K and DSPE-PEG2K-Peptide was 1.5%. The synthesis method was the same as in Example 2 except for the molar ratio of DMG-PEG2K and DSPE-PEG2K-Peptide.

- Particle size and number were measured using Nanoparticle Tracking Analysis (NTA, Nanosight NS300, Malvern, UK).

- As there was no significant difference in particle size between the groups, particles were assumed to have the same size of approximately 115 nm, and the concentration of PD-L1 binding peptide per 1.2×10 ¹⁰ particles was measured using the Pierce BCA protein measurement Kit.

- The amount of measured PD-L1 binding peptide was divided by the number of LNP particles to calculate the amount of protein expressed on the surface per particle.

As a result, as shown in Fig. 4, it was confirmed that the amount of tumor-targeting ligand expressed on the surface of lipid nanoparticles increased as the amount of the second conjugate added increased.

Experimental Example 2. DLS (Dynamic Light Scattering) Analysis

- Lipid nanoparticles containing 0.01, 0.1, 0.5, 0.7, and 1.0% DSPE-PEG2K-Peptide were synthesized so that the combined molar ratio of DMG-PEG2K and DSPE-PEG2K-Peptide was 1.5%. The synthesis method was the same as in Example 2 except for the molar ratio of DMG-PEG2K and DSPE-PEG2K-Peptide.

- The lipid nanoparticles manufactured as described above were analyzed by DLS (Zetasizer Nano ZS, Malvern Instruments, UK).

As a result, it was confirmed that lipid nanoparticles having a size of 1,000 nm or more were produced when the second conjugate was added at a molar ratio of 0.7% or more (see Figure 5).

Meanwhile, the lipid nanoparticles manufactured in Example 2 and Comparative Example 1 were stored at 4°C and the change in particle size over time was analyzed by DLS (Zetasizer Nano ZS, Malvern Instruments, UK), and the results are shown in Fig. 6. Both the lipid nanoparticles (Ligand LNP) of Example 2 and the lipid nanoparticles (Con LNP) manufactured in Comparative Example 1 showed no significant change in particle size even after 28 days. The PDI (polydispersity index), which indicates the distribution width of the particle size, also showed a value of 0.1 or less even after 28 days, indicating that uniform particles were maintained.

Experimental Example 3. Analysis of encapsulation efficiency

The mRNA encapsulation efficiency of the lipid nanoparticles manufactured in Example 2 and Comparative Example 1 was analyzed as follows and shown in Figure 7.

- Encapsulation efficiency was measured using Quant-iT ^TM RiboGreen RNA assay Kit (Invitrogen Corporation Carlsbad, California, USA).

- The same amount of LNP was incubated in 1XTE or 1XTE with 1% Triton X-100 solution for 20 minutes, and the fluorescence intensity of the stained RNA was measured according to the manufacturer's protocol.

- RNA measured in 1XTE was considered as non-encapsulated external RNA, and RNA measured in 1XTE with 1% Triton X-100 was considered as total RNA, and the calculation was performed as follows.

1) Total RNA - External RNA = Encapsulated RNA

2) Encapsulated RNA/Total RNA × 100 = Encapsulation efficiency (%)

As a result, there was no significant difference in the encapsulation efficiency of mRNA when manufacturing lipid nanoparticles of Example 2 (Ligand LNP) and Comparative Example 1 (Con LNP), confirming that lipid nanoparticles can be manufactured without affecting the encapsulation of mRNA even when the second conjugate is added.

Experimental Example 4. Analysis of mRNA expression efficiency according to the storage period of lipid nanoparticles

The lipid nanoparticles encapsulated with EGFP (enhanced green fluorescent protein) mRNA prepared in Example 2 were stored at 4°C for 4 weeks, and the expression efficiency was measured once a week using the following method.

- CT26.CL25 cells expressing PD-L1 were cultured in 6 wells according to the conventional method. After cell attachment, the cell culture medium was replaced with 1 mL and the lipid nanoparticles (Ligand LNP) of Example 2 were treated based on 1 μg of EGFP mRNA.

- EGFP expression was photographed 24 hours after ligand LNP treatment using a fluorescence microscope (EVOS M5000 fluorescence microscope, Thermo Fishers, USA).

As a result, as shown in Fig. 8, regardless of the storage period, mRNA was well expressed, confirming that the lipid nanoparticle according to the present disclosure stably encapsulates and expresses nucleic acids.

Experimental Example 5. Analysis of Tumor Target Binding Ability of Lipid Nanoparticles

- Tumor-targeting fluorescent lipid nanoparticles (Ligand NLP) and non-targeting fluorescent lipid nanoparticles (Con LNP) were prepared by encapsulating 20% Cy5-labeled oligo DNA and 80% EGFP mRNA in the same manner as in Example 2 and Comparative Example 1 except for the nucleic acid to be loaded. The Cy5-labeled oligo DNA (5'-AGCTCTGTTTACGTCCCAGC-3') used in this experimental example was purchased from Bioneer (Daejeon, Korea).

- The Ligand LNP or Con LNP (300 ng based on mRNA) prepared above was added to His-binding pair protein (2 μg) in 1 mL of PBS, and the binding reaction was induced at 4°C for 10 minutes.

- Afterwards, 1 mg of magnetic beads capable of binding to His tag were added and incubated at 25°C for 10 minutes.

- Images and fluorescence intensity of fluorescent LNPs attracted by magnetism and bound to His-binding pair proteins were measured (see Figure 9).

As a result, it was confirmed that only the tumor-targeting fluorescent lipid nanoparticle (Ligand NLP) could significantly increase tumor targeting through the second conjugate by binding to His-binding pair protein.

Experimental Example 6. Analysis of Tumor Target Binding Ability of Lipid Nanoparticles

The tumor-targeting binding ability of the tumor-targeting fluorescent lipid nanoparticles (Ligand NLP) and non-targeting fluorescent lipid nanoparticles (Con LNP) manufactured in the above Experimental Example 5 was analyzed as follows and is shown in Figure 10.

- According to the conventional method, PD-L1 antibody was added to CT26.CL25 cells expressing PD-L1 and incubated at 37°C for 1 hour to block PD-L1 protein on the cell surface.

- Afterwards, 1 μg of the manufactured tumor-targeting fluorescent lipid nanoparticles (Ligand NLP) and non-targeting fluorescent lipid nanoparticles (Con LNP) were treated based on mRNA, incubated at 25°C for 30 minutes, washed with PBS, and photographed using a fluorescence microscope (EVOS M5000 fluorescence microscope, Thermo Fishers, USA).

As a result, it was confirmed that the cancer cell surface binding ability of tumor-targeting fluorescent lipid nanoparticles (Ligand LNP) increased compared to non-targeting fluorescent lipid nanoparticles (Con LNP), and that the effect was inhibited when treated with antibodies that interfere with cancer cell targeting.

Experimental Example 7. Analysis of Tumor Targeting Ability of Lipid Nanoparticles

Luciferase mRNA was encapsulated in the same manner as in Example 2 and Comparative Example 1 except for the nucleic acid to be loaded, thereby producing tumor-targeting fluorescent lipid nanoparticles (Ligand NLP) and non-targeting fluorescent lipid nanoparticles (Con LNP). Thereafter, each lipid nanoparticle was injected into a cancer animal model, and the tumor-targeting ability of the lipid nanoparticles was analyzed by the following method. The mRNA was purchased as Luciferase (model name; L-7202) mRNA from TriLink biotechnologies (CA, USA).

- The tumor-targeting fluorescent lipid nanoparticles (Ligand LNP) and non-targeting fluorescent lipid nanoparticles (Con LNP) manufactured above were intravenously injected into a cancer animal model at 0.4 mg/kg, and the fluorescence intensity was measured at each time point using an IVIS Lumina Series Ⅲ system (PerkinElmer, USA).

- The cancer animal model was used by subcutaneously injecting 2 × 10 ⁶ CT26.CL25 cells (in 100 μL) into the left leg of 7-week-old Balb/c nude mice and growing them to 80 mm ³ .

As a result, as shown in Fig. 11, the accumulation of tumor-targeting fluorescent lipid nanoparticles (Ligand LNP) in tumors increased over time compared to non-targeting fluorescent lipid nanoparticles (Con LNP), confirming that the lipid nanoparticles according to the present disclosure have remarkably excellent tumor-targeting ability.

Experimental Example 8. Analysis of Tumor Targeting Ability of Lipid Nanoparticles

The tumor-targeting fluorescent lipid nanoparticles (Ligand NLP) and non-targeting fluorescent lipid nanoparticles (Con LNP) manufactured in the above Experimental Example 7 were injected into a cancer animal model, and the tumor-targeting ability of the lipid nanoparticles was measured using the following method.

- The tumor-targeting fluorescent lipid nanoparticles (Ligand LNP) and non-targeting fluorescent lipid nanoparticles (Con LNP) manufactured above were intravenously injected into a cancer animal model at 0.4 mg/kg, and the tumors were extracted 24 hours later.

- The cancer animal model was used by subcutaneously injecting 2 × 10 ⁶ CT26.CL25 cells (in 100 μL) into the left leg of 7-week-old Balb/c nude mice and growing them to 80 mm ³ .

- Luciferin (30 mg/mL in PBS) was simultaneously sprayed onto the extracted tumor, and the fluorescence intensity was measured using an IVIS Lumina Series Ⅲ system (PerkinElmer, USA).

As a result, as shown in Fig. 12, it was confirmed that the tumor-targeted fluorescent lipid nanoparticles (Ligand LNP) showed increased accumulation in the tumor and luciferase protein expression compared to the non-targeted fluorescent lipid nanoparticles (Con LNP).

Meanwhile, when lipid nanoparticles were manufactured according to the methods of Example 2 and Comparative Example 1, but with 1 mol% of DiR (DiIC ₁₈ (7); 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide), a lipophilic near-infrared fluorescent cyanine dye, added to the entire lipid composition of the lipid nanoparticles, it was confirmed that the accumulation of tumor-targeted fluorescent lipid nanoparticles (Ligand LNP) in tumors was significantly increased compared to non-targeted fluorescent lipid nanoparticles (Con LNP).

While the specific parts of the present disclosure have been described in detail, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present disclosure. Accordingly, the substantial scope of the present disclosure is defined by the appended claims and their equivalents.

Sequence number 1 (PD-L1 binding peptide): NYSKPTDRQYHF

Sequence number 2 (Cy5-labeled oligo DNA): 5'-AGCTCTGTTTACGTCCCAGC-3'

Claims (14)

Nucleic acid; ionized lipids; phospholipids; sterol; first conjugate; and A second conjugate comprising a tumor targeting ligand conjugated thereto, The above first conjugate is a conjugate of a phospholipid and a polymer, The second conjugate is a lipid nanoparticle in which a tumor-targeting ligand is conjugated to the first conjugate. In paragraph 1, A lipid nanoparticle, wherein the nucleic acid comprises at least one selected from the group consisting of RNA, messenger RNA (mRNA), antisense oligonucleotide (ASO), DNA, plasmid, ribosomal RNA (rRNA), micro RNA (miRNA), trans RNA (tRNA), small interfering RNA (siRNA), and small nuclear RNA (snRNA). In paragraph 1, The above ionized lipids are (6Z, 9Z, 28Z, 31Z)-heptathriaconta 6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid (SM-102), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyl carbamoyloxy-3-dimethylaminopropane (DLin-CDAP), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy) propylamine (DODMA), dioctadecylamidoglycylspermine (DOGS), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl) amino) ethyl)(2-hydroxydecyl) amino) ethyl) piperazin-1-yl) ethylazanediyl) didodecan-2-ol (C12-200), dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyl oxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyl oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxyl) propyl)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyl trimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy) A lipid nanoparticle comprising at least one selected from the group consisting of N,N,N-trimethylammonium chloride (DOTMA) and aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE). In paragraph 1, The above phospholipids are 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DiPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), A lipid nanoparticle comprising at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), ceramide, and sphingomyelin. In paragraph 1, A lipid nanoparticle, wherein the above sterol comprises at least one selected from the group consisting of cholesterol, ergosterol, campesterol, oxysterol, desmosterol, sitosterol, and stigmasterol. In paragraph 1, A lipid nanoparticle, wherein the first conjugate comprises at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol (DSG-PEG), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG). In paragraph 1, The above tumor targeting ligand is a lipid nanoparticle that is a PD-L1 binding peptide that targets solid tumors expressing PD-L1. In Article 7, The above solid tumor is at least one selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanoma, bladder cancer, brain tumor, CNS cancer, cervical cancer, esophageal cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, colon cancer, neuroblastoma, and sarcoma. In paragraph 1, A lipid nanoparticle in which a tumor-targeting ligand is conjugated to the first conjugate via a DBCO-azide (dibenzocyclooctyne-azide) bond, a maleimide-thiol (maleimide-thiol) bond, an NHS ester-amine (N-hydroxysuccinimide ester-amine) bond, a BCN-azide (bicyclononyne-azide) bond, or an ADIBO-DBCO (aza-dibenzocyclooctyne-dibenzocyclooctyne) bond reaction. In paragraph 1, A lipid nanoparticle wherein the second conjugate is a first conjugate functionalized with dibenzocyclooctyne (DBCO) and a tumor-targeting ligand functionalized with an azide group, which are linked by a click reaction. In paragraph 1, A lipid nanoparticle wherein the ionized lipid, phospholipid, sterol, first conjugate and second conjugate are mixed in a molar ratio of 40 to 60:5 to 15:30 to 45:1 to 2:0.1 to 1, respectively. In paragraph 1, Lipid nanoparticles having an average size of 50 nm to 200 nm. A method for producing lipid nanoparticles according to any one of claims 1 to 12, (1) A step of functionalizing a tumor targeting ligand with an azide group; (2) a step of functionalizing the first conjugate with dibenzocyclooctyne (DBCO); (3) a step of reacting the first conjugate functionalized with the DBCO with the tumor-targeting ligand functionalized with the azide group through a click reaction to produce a second conjugate in which the tumor-targeting ligand is conjugated to the first conjugate; and (4) a step of mixing a lipid solution containing ionized lipids, phospholipids, sterols, first conjugates and second conjugates and a nucleic acid solution; The above first conjugate is a conjugate of a phospholipid and a polymer, A method for producing a lipid nanoparticle, wherein the second conjugate is a tumor-targeting ligand conjugated to the first conjugate. A pharmaceutical composition for preventing or treating tumors, comprising a lipid nanoparticle according to any one of claims 1 to 12.

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