Oxygen-carrying nanoliposome and use thereof

Abstract

The present invention relates to an oxygen-carrying nanoliposome in which oxygen is carried inside a shell of a phospholipid bilayer, and use thereof. The nanoliposome continuously releases oxygen, thereby maintaining an oxygen delivery effect for a long time, and increases anticancer efficacy without damaging the cells by delivering oxygen specifically to tumor cells.

Description

Oxygen-loaded nanoliposomes and their uses

[0001] The present invention relates to a nanoliposome containing oxygen and its use.

[0002] Macrophages are responsible for both adaptive and innate immunity in the human immune system. Macrophages exist in the blood as monocytes and differentiate into macrophages in response to environmental changes, such as disease onset or exposure to foreign substances, and in response to cell signaling molecules like cytokines. As the immune system is activated in a specific area, macrophages undergo two major polarizations. Among them, M1 macrophages (classically activated macrophages) are involved in pro-inflammatory responses and play a crucial role in suppressing the invasion of foreign substances into the body. In particular, M1 macrophages exhibit the characteristic of attacking and eliminating tumor cells. In contrast, M2 macrophages are involved in anti-inflammatory and immune suppression, playing a key role in wound healing and tissue remodeling. Some of the above M2 macrophages, unlike M1 macrophages, have the property of inducing tumor growth and tissue infiltration, which can affect tumor exacerbation. Since these macrophages have a significant impact on the body's immune activity as well as tumor death and growth, immunotherapies utilizing them are being studied. Among these macrophages, macrophages differentiated under the influence of the tumor microenvironment (hereinafter referred to as TME) are known as tumor-associated macrophages (hereinafter referred to as TAMs). In particular, M2 phenotype TAMs have the property of promoting tumor progression and reducing the effectiveness of drug therapy.In addition, during the migration and invasion stages of tumor cells, TAMs are differentiated into M2 macrophages by interleukin-4 (IL-4) expressed by CD4+ T cells and tumor cells, which plays a role in assisting the migration and metastasis of tumor cells (Cell. 2010;141(1)39-51, Curr Opin Immunol. 2010;22(2)231-7, Nature Reviews Immunol. 2015, 15:7386). Tumor cells induce these properties of macrophages to disrupt the human immune system and evade attacks from immune cells. Among the immune activities of macrophages, phagocytosis is also suppressed by the immune evasion function induced by tumor cell-derived expression factors. The immune evasion signal of tumor cells toward macrophages is called the 'Don't eat me signal', and this signal reduces the anti-tumor effect in the body by preventing the tumor cells from attacking macrophages by interfering with the recognition of cancer cells by macrophages.

[0003] The present invention was conceived to solve the above-described problem, and aims to provide a nanoliposome in which oxygen is loaded inside the shell of a phospholipid bilayer.

[0004] In addition, the present invention aims to provide a nanoliposome in which the surface of the nanoliposome is modified with a sugar compound, lipid, polymer, nucleic acid, protein, metal nanoparticle, metal oxide or metal nanocomposite.

[0005] In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating cancer, which comprises nanoliposomes or modified nanoliposomes as an active ingredient.

[0006] In addition, the present invention aims to provide a method for producing oxygen nanoliposomes.

[0007] In addition, the present invention aims to provide a method for preparing a metal nanocomposite-oxygen nanoliposome complex.

[0008] Furthermore, the present invention aims to provide a use of nanoliposomes for oxygen delivery to cancer cells.

[0009] Furthermore, the present invention aims to provide a use of a metal nanocomposite-oxygen nanoliposome complex for the treatment of cancer.

[0010] In addition, the present invention aims to provide a method for treating cancer.

[0011] The technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0012] To solve the above problem, the present invention provides a nanoliposome in which oxygen is loaded inside the shell of a phospholipid bilayer.

[0013] In addition, the present invention provides a nanoliposome in which the surface of the nanoliposome is modified with a sugar compound, lipid, polymer, nucleic acid, protein, metal nanoparticle, metal oxide, or metal nanocomposite.

[0014] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising nanoliposomes or modified nanoliposomes as an active ingredient.

[0015] In addition, the present invention provides a method for producing oxygen nanoliposomes.

[0016] In addition, the present invention provides a method for producing a metal nanocomposite.

[0017] In addition, the present invention provides a method for preparing a metal nanocomposite-oxygen nanoliposome complex.

[0018] The present invention also provides the use of nanoliposomes for oxygen delivery.

[0019] The present invention also provides the use of a metal nanocomposite-oxygen nanoliposome complex for the treatment of cancer.

[0020] The present invention also provides a method for treating cancer.

[0021] According to the present invention as described above, the oxygen-loaded nanoliposome of the present invention can maintain the oxygen delivery effect for a long time by continuously releasing oxygen, and has the effect of preventing problems such as ROS generation caused by excessive release of oxygen through continuous release of oxygen by diffusion.

[0022] The oxygen-loaded nanoliposome of the present invention is capable of activating the immune system of M1 macrophages and can be applied to macrophages under hypoxic conditions, thereby having the effect of enhancing the efficacy of tumor immunotherapy.

[0023] In addition, nanoliposomes modified with the antibody of the present invention can target a hypoxic tumor microenvironment and specifically deliver oxygen to tumor cells, thereby enhancing anticancer efficacy without damaging cells.

[0024] In addition, nanoliposomes whose surface is modified with the antibody of the present invention have the effect of improving the oxygen delivery ability to cells by inducing cell surface binding and intracellular incorporation by the antibody.

[0025] In addition, the metal nanocomposite-oxygen nanoliposome complex of the present invention has the effect of specifically targeting cancer cells and killing them.

[0026] In addition, the metal nanocomposite-oxygen nanoliposome complex of the present invention has the effect of blocking the immune evasion signal generated by the interaction between macrophages and cancer cells, thereby enhancing the phagocytosis of macrophages against cancer cells.

[0027] Figure 1 is a schematic diagram showing the synthesis process of oxygen nanoliposomes.

[0028] Figure 2 is a diagram (A) showing the size of oxygen nanoliposomes after synthesis and the change in size after 30 days, and a diagram (B) showing the double lipid membrane structure.

[0029] Figure 3 shows the results of confirming the release amount of oxygen contained inside the surface-modified oxygen nanoliposome over time.

[0030] Figure 4 is a diagram analyzing the change in particle size of surface-modified oxygen nanoliposomes.

[0031] Figure 5 is a diagram analyzing the cytotoxicity of oxygen nanoliposomes on blood cells.

[0032] Figure 6 is a diagram analyzing the characteristics of antibody-modified oxygen nanoliposomes and whether or not they are conjugated to antibodies:

[0033] oxygen nanosome: oxygen nanoliposome;

[0034] Immunonanosome: Antibody-modified oxygen nanoliposomes;

[0035] A: Particle size;

[0036] B: Zeta potential;

[0037] C: particle concentration;

[0038] D: Concentration of oxygen carried by the particles;

[0039] E; shape;

[0040] F: Protein concentration analysis results;

[0041] G: Nanodrop measurement results; and

[0042] H: Results of solid-phase bonding analysis.

[0043] Figure 7 shows the cytotoxicity of oxygen nanoliposomes and antibody-modified oxygen nanoliposomes:

[0044] oxygen nanosome: oxygen nanoliposome; and

[0045] Immunonanosome: Antibody-modified oxygen nanoliposomes.

[0046] Figure 8 is a diagram analyzing the cancer cell-specific binding ability of oxygen nanoliposomes and antibody-modified oxygen nanoliposomes:

[0047] oxygen nanosome: oxygen nanoliposome; and

[0048] Immunonanosome: Antibody-modified oxygen nanoliposomes.

[0049] Figure 9 is a diagram analyzing the adenosine reduction effect of oxygen nanoliposomes and antibody-modified oxygen nanoliposomes:

[0050] CNTL: hypoxic control group;

[0051] oxy-DPBS: non-liposomal oxygen;

[0052] oxygen nanosome: oxygen nanoliposome; and

[0053] Immunonanosome: Antibody-modified oxygen nanoliposomes.

[0054] Figure 10 is a diagram analyzing the M1 macrophage immune activation effect of oxygen nanoliposomes and antibody-modified oxygen nanoliposomes:

[0055] CNTL (Normoxia): Control group under normoxia conditions;

[0056] oxy-DPBS: non-liposomal oxygen;

[0057] oxygen nanosome: oxygen nanoliposome;

[0058] Immunonanosome: Antibody-modified oxygen nanoliposomes;

[0059] A: Schematic diagram of the macrophage immune activation assay experiment of adenosine converted by cells under hypoxic conditions;

[0060] B: Levels of TNF-α after 24 and 48 h of culture under normal and hypoxic conditions in various cells;

[0061] C: TNF-α levels in cells treated with oxygen nanoliposomes and antibody-modified oxygen nanoliposomes after 24 hours of culture under hypoxic conditions; and

[0062] D: TNF-α levels in cells treated with oxygen nanoliposomes and antibody-modified oxygen nanoliposomes after 48 hours of culture under hypoxic conditions.

[0063] Figure 11 is a diagram analyzing the manufacturing process (A) and its characteristics (B to G) of Man-AuNR-SNA, a metal nanomaterial that targets cancer cells and macrophages and can be applied to photothermal therapy.

[0064] Figure 12 is a diagram analyzing the cancer cell-specific binding ability of Man-AuNR-SNA:

[0065] A: Schematic diagram of cancer cell-specific binding mechanism of Man-AuNR-SNA;

[0066] B: Results of cancer cell-specific binding analysis of SNA using confocal microscopy;

[0067] C: Results of cancer cell-specific binding analysis of SNA using FACS; and

[0068] D and E: Cancer cell-specific binding assay results of Man-AuNR-SNA.

[0069] Figure 13 is a diagram analyzing the macrophage-specific binding ability of Man-AuNR-SNA:

[0070] A: Results of marker expression analysis of macrophages;

[0071] B: Results of specific binding ability analysis of mannose to M2 macrophages; and

[0072] C: Specific binding analysis results of Man-AuNR-SNA particles to M2 macrophages.

[0073] Figure 14 shows the cytotoxicity of Man-AuNR-SNA on various cells (A) and the cancer cell killing effect of Man-AuNR-SNA by photothermal therapy (B and C).

[0074] Figure 15 is a diagram confirming cancer cell death by combined treatment with Man-AuNR-SNA and photothermal therapy:

[0075] A: Expression analysis of cell death-related proteins; and

[0076] B and C: Annexin V / PI staining analysis results.

[0077] Figure 16 is a diagram confirming the immune evasion inhibition effect of cancer cells by combined treatment with Man-AuNR-SNA and photothermal therapy:

[0078] A: Cancer cell phagocytosis of macrophages by SNA treatment;

[0079] B: Macrophagic activity of the Man-AuNR-SNA particle-only treatment group; and

[0080] C: Macrophage activity of the combined treatment group of Man-AuNR-SNA treatment and photothermal therapy.

[0081] Figure 17 shows the results of cytotoxicity of the maximum concentration at which each of oxygen nanoliposomes and metal nanocomposites did not exhibit cytotoxicity, the results of cytotoxicity of the mixed particle-by-particle mixture of 10% oxygen nanoliposomes and metal nanocomposites (A), and the results of TEM analysis of the prepared Man-AuNR-SNA oxygen nanoliposome complex (B):

[0082] ONS: Oxygen nanoliposomes;

[0083] GNR: Metal nanocomposite; and

[0084] Mix: A mixture of oxygen nanoliposomes and metal nanocomplexes.

[0085] Figure 18 is a diagram showing the photothermal effect of an oxygen nanoliposome complex (Au-oxy) containing Man-AuNR-SNA under NIR irradiation.

[0086] Figure 19 is a diagram confirming the in vitro cancer-specific apoptotic effect of Man-AuNR-SNA oxygen nanoliposome complex and photothermal therapy.

[0087] Figure 20 is a diagram confirming the in vivo cancer-specific apoptotic effect of the Man-AuNR-SNA oxygen nanoliposome complex.

[0088] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. The advantages and features of the present invention, and methods for achieving them, will become clear with reference to the embodiments described in detail below together with the attached drawings. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms. These embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Like reference numerals refer to like elements throughout the specification.

[0089] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in a sense commonly understood by those of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise. The terminology used herein is for the purpose of describing embodiments and is not intended to limit the present invention. In this specification, singular forms also include plural forms, unless specifically stated otherwise.

[0090] The terms "comprises" and / or "comprising" as used in the specification do not exclude the presence or addition of one or more other components, steps, operations and / or elements.

[0091] The term "liposome" used in the specification refers to a hollow droplet-like structure formed by a phospholipid bilayer when phospholipid, a major component of biological membranes, is added to an aqueous solution.

[0092] The term "nucleic acid" as used herein refers to a 5-carbon sugar, a phosphate group, and a nitrogenous base as the basic backbone. When the sugar portion is ribose, it is referred to as RNA, and when it is deoxyribose, it is defined as DNA. The base portion of DNA is composed of four bases: adenine (A), thymine (T), cytosine (C), and guanine (G), and the base portion of RNA contains uridyl (U) instead of thymine (T) in DNA. The nucleic acid includes a nucleic acid derivative or analogue, and the nucleic acid derivative or analogue may be a nucleic acid that has changed any of the components of the nucleic acid defined above.

[0093] As used herein, the term "antibody" includes polyclonal antibodies and monoclonal antibodies, antigen-binding fragments of such antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, and the smallest recognition unit of an antibody, as well as single-chain derivatives of such antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies and related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin." The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0094] The terms "polypeptide," "peptide," "oligopeptide," and "protein," as used herein, are used interchangeably herein and refer to a polymeric form of amino acids of any length. The polymeric form may include encoded and non-encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having modified peptide backbones.

[0095] As used herein, the term "lectin" refers to a protein or peptide that binds to carbohydrate structures. Those skilled in the art will understand that a lectin is a protein or peptide that is highly specific for binding to sugar moieties.

[0096] As used herein, “anti-tumor effect” or “anticancer effect” refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in tumor cell count, a decrease in the number of metastases, an increase in lifespan, or an alleviation of various physiological symptoms associated with a cancerous condition.

[0097] As used herein, "targeting" refers to the ability to target a specific cell or site within a living organism, and refers to the property of a substance being specifically delivered to that site when distributed within the body. Targeting can be a property that is limited to a specific biological site, meaning that a significantly greater amount of the substance is delivered to that site compared to other sites.

[0098] As used herein, "treatment" refers to any action that improves or beneficially alters the symptoms of a disease by administering the composition of the present invention. Those skilled in the art to which the present invention pertains will be able to accurately determine the criteria for diseases for which the composition of the present invention is effective and determine the degree of improvement, enhancement, and treatment by referencing materials provided by the Korean Medical Association and other sources.

[0099] The term "therapeutically effective amount" used in conjunction with the active ingredient in the specification means an amount effective in preventing or treating the target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, the condition of the patient, etc. Therefore, the dosage for use in humans should be determined as an appropriate amount by taking both safety and efficacy into consideration. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.

[0100] The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, type of disease, cause of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0101] Throughout the specification, when we say that an element is "on" another element, this includes not only cases where the element is in contact with the other element, but also cases where another element exists between the two elements.

[0102]

[0103] In one aspect, the present invention relates to nanoliposomes in which oxygen is loaded inside the shell of a phospholipid bilayer.

[0104] In one embodiment, the diameter of the nanoliposome may be 100 to 200 nm.

[0105] In one embodiment, the concentration of oxygen loaded in the nanoliposome is 10 to 30 mg / L.

[0106] In one embodiment, the nanoliposome may include a polyethylene glycol (PEG) unconjugated phospholipid and a polyethylene glycol-conjugated phospholipid, wherein the polyethylene glycol unconjugated phospholipid is DSPC (Distearoylphosphatidylcholine), DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), SOPC (1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), DEPC (1,2-Dierucoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) or POPG (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol); and the polyethylene glycol-conjugated phospholipid is C6-C 20 A phospholipid may be a lipid comprising a poly(ethylene) glycol chain having a length of up to 5 kDa covalently bonded to an alkyl chain(s) of the length.

[0107] In one embodiment, the nanoliposome may comprise polyethylene glycol unconjugated phospholipids and polyethylene glycol conjugated phospholipids in a molar ratio of 4:1 to 7:1.

[0108] In one embodiment, the nanoliposome may additionally comprise a lipid or polymer in its liposomal membrane.

[0109] In one embodiment, the lipid may be cholesterol and the polymer may be polyoxyethylene or polyoxyethylene (100) stearyl ether.

[0110] In one embodiment, the nanoliposomes are capable of delivering oxygen to cells.

[0111] In one embodiment, the nanoliposomes can reduce adenosine increased by ATP accumulated outside the cell due to hypoxic conditions.

[0112] In one embodiment, the nanoliposome can induce immune activation of M1 macrophages, increase secretion of TNF-α decreased in M1 macrophages due to hypoxia, and decrease secretion of IL-10 increased in M1 macrophages due to hypoxia.

[0113] In one embodiment, the nanoliposome may additionally carry a lipid, a polymer, a nucleic acid, a protein, a metal nanoparticle, a metal oxide, an oxide nanoparticle, a drug, or a metal nanocomposition.

[0114] In one embodiment, the nanoliposome may be a drug delivery vehicle.

[0115] In one embodiment, the metal nanoparticle may be a metal nanoparticle comprising Au, Ag, Cu, ZnO, Co, CuO, Fe2O3, Mn2O3, Ni, Pd, Pt, TiO2 or Cr, and may be a multi-metal nanocomposite comprising two or more metals selected from the group consisting of Au, Ag, Cu, ZnO, Co, CuO, Fe2O3, Mn2O3, Ni, Pd, Pt, TiO2 and Cr.

[0116] In one embodiment, the oxide nanoparticles may be composed of zinc, titanium or a composite thereof.

[0117] In one embodiment, the drug can be a peptide drug, a protein drug, an anticancer agent, an antibacterial agent, an antiviral agent, an antidementia agent, an immunomodulator, a desensitizing agent, an antigen, a non-steroidal anti-inflammatory agent, an anti-inflammatory drug, an anesthetic, an antioxidant, an anti-infective agent, a chemotherapeutic agent, an anti-nociceptive agent, a DMOAD, an anabolic agent, an anti-catabolic agent, an autophagy modulator, an anti-osteoclast-mediated bone loss agent, a nutraceutical agent, an analgesic, a biologic, or a mixture thereof.

[0118] In one embodiment, the nanoliposome may further comprise a labeling agent, and the labeling agent may be at least one selected from the group consisting of a chromogenic enzyme, a radioisotope, a chromophore, a chromogenic substance, a luminescent substance, a fluorescent substance, a superparamagnetic particle, and an ultrasuper paramagnetic particle.

[0119] In one embodiment, the chromogenic enzyme can be a streptavidin-conjugated horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, luciferase, acetylcholinesterase, urease, catalase, asparginase, ribonuclease, malate dehydrogenase, staphylococcal nuclease, triose phosphate isomerase, glucose oxidase, cytochrome P450, and peroxidase compound.

[0120] In one embodiment, the fluorescent material may be a fluorescent protein, a photoprotein, luciferase, a fluorescent dye or a time-resolved fluorescence (TRF), and the fluorescent material may be Alexa Fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rubpy (tris(2,2-bipyridyl)ruthenium(Ⅱ)), FITC (fluoresein isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (5(6)-Carboxytetramethylrhodamine), Texas Red, It may be at least one selected from the group consisting of DAPI (4,6-diamidino-2-phenylindole) and coumarin.

[0121] In one aspect, the present invention relates to a nanoliposome, the surface of which is modified with a sugar, a sugar compound, a lipid, a polymer, a nucleic acid, a protein, a metal nanoparticle, a metal oxide or a metal nanocomposite.

[0122] In one embodiment, the metal nanoparticle may be a metal nanoparticle comprising Au, Ag, Cu, ZnO, Co, CuO, Fe2O3, Mn2O3, Ni, Pd, Pt, TiO2 or Cr, and may be a multi-metal nanocomposite comprising two or more metals selected from the group consisting of Au, Ag, Cu, ZnO, Co, CuO, Fe2O3, Mn2O3, Ni, Pd, Pt, TiO2 and Cr.

[0123] In one embodiment, the protein may be an antibody or antibody fragment, and the antibody may be an antibody to a protein whose expression is increased in the tumor microenvironment (TME), and may be an anti-CD73 antibody.

[0124] In one embodiment, nanoliposomes surface-modified with anti-CD73 antibodies or fragments thereof can deliver oxygen specifically to cancer cells.

[0125] In one embodiment, nanoliposomes surface-modified with an anti-CD73 antibody or fragment thereof can bind to cells expressing CD73 under hypoxic conditions, thereby enhancing oxygen delivery to the cells.

[0126] In one embodiment, nanoliposomes surface-modified with an anti-CD73 antibody or fragment thereof can reduce extracellular accumulated adenosine by inhibiting the conversion of AMP to adenosine present in a hypoxic environment by blocking CD73, the expression of which increases under hypoxic conditions.

[0127] In one embodiment, nanoliposomes surface-modified with an anti-CD73 antibody or a fragment thereof can induce immune activation of M1 macrophages, increase secretion of TNF-α decreased in M1 macrophages due to hypoxia, and decrease secretion of IL-10 increased in M1 macrophages due to hypoxia.

[0128] In one embodiment, the nanoliposomes modified with the metal oxide or metal nanocomposite can be used for photothermal therapy and photodynamic therapy by light irradiation.

[0129] In one embodiment, the light irradiation may be irradiating a near-infrared light source within an emission wavelength range of 700 to 1100 nm.

[0130] In one embodiment, the metal may be Au, Ag, Cu, ZnO, Co, CuO, Fe2O3, Mn2O3, Ni, Pd, Pt, TiO2, or Cr.

[0131] In one embodiment, the metal nanocomposite may comprise a cancer cell targeting moiety, an immune effector cell targeting moiety, and a metal moiety exhibiting a photothermal effect.

[0132] In one embodiment, the cancer cell targeting moiety can be a lectin that targets cancer cell glycans, such as a galectin, a siglec, a selectin, a C-type lectin, CD301, L-PHA (Phaseolus vulgaris leukocyte agglutinin), E-PHA (Phaseolus vulgaris erythroagglutinin), a tomato lectin (Lycopersicon esculentum lectin, LEA), a peanut lectin (Arachis hypogaea agglutinin, PNA), a potato lectin (Solanum tuberosum lectin), a pork mitogen (Phytolacca American lectin), a wheat germ agglutinin (Triticum Vulgaris lectin), an Artocarpus Artocarpus polyphemus lectin (Jacalin retin), Vicia villosa agglutinin (VVA), Helix pomatia agglutinin (HPA), Wisteria floribunda agglutinin (WFA), Sambucus nigra agglutinin (SNA), BC2LCM (lectin from the gram-negative bacterium Burkholderia cenocepacia), Maackia amurensis leukoglutinin (MAL), Psathyrella velutina (PVL), Sclerotium rolfsii lectin (SRL), Eucheuma serra agglutinin (ESA), CLEC17A (prolectin), Aleuria aurantia lectin, Sambucus sieboldiana lectin (SSA), Glechoma hederacea lectin (Gleheda), Morus nigra agglutinin (Morniga G),Salvia sclarea lectin, Salvia bogotensis lectin, Salvia horminum lectin, Clerodendrum trichotomum lectin, Moluccella laevis lectin, Griffonia simplicifolia (GsLA4), Psophocarpus tetragonolobus (acid WBAI), Abrus precatorius lectin, Molucella laevis lectin (MLL), Amaranthus caudatus lectin, Amaranthus leucocarpus It can be a lectin of Laelia autumnalis, a lectin of Artocarpus integrifolia, a lectin of Maclura pomifera, a lectin of Artocarpus lakoocha, agglutinin of Dolichos biflorusa, agglutinin of Dolichos biflorus, agglutinin of Glycine max or a lectin of Agaricus bisporus, with Sambucus nigra agglutinin being the most preferred.

[0133] In one embodiment, the immune effector cell may be a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a macrophage, a monocyte, a dendritic cell, or a neutrophil, most preferably a macrophage.

[0134] In one embodiment, the immune effector cell targeting moiety may be a sugar, and may be mannose (Man).

[0135] In one embodiment, the metal moiety may be one or more of gold, silver, copper, and iron of the metal series, (1) one or more of SiO2-metal and SiN-metal of the metal-insulator series, (2) one or more of TiO2-metal, ZnO-metal, and SnO2-metal of the metal-semiconductor series, or a composite thereof, and gold is most preferred.

[0136] In one embodiment, the metal nanocomposite may be in the form of a rod, particle or shell.

[0137] In one embodiment, the metal nanocomposite can be used for photothermal therapy and photodynamic therapy by light irradiation.

[0138] In one embodiment, the light irradiation may be irradiating a near-infrared light source within an emission wavelength range of 700 to 1100 nm.

[0139] In one aspect, the present invention relates to an oxygen nanoliposome complex modified with a metal nanocomposite comprising a cancer cell targeting moiety, an immune effector cell targeting moiety, and a metal moiety exhibiting a photothermal effect.

[0140] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising the oxygen nanoliposome or modified oxygen nanoliposome of the present invention as an active ingredient.

[0141] In one embodiment, the pharmaceutical composition for preventing or treating cancer may be a targeted anticancer agent.

[0142] In one embodiment, the cancer may be a solid cancer or a blood cancer, and may include colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, stomach cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, esophageal cancer, small intestine cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head and neck cancer, cutaneous melanoma, intraocular melanoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, glioblastoma multiforme, and pituitary adenoma. It can be any one selected from the group consisting of cancers, more preferably breast cancer, cervical cancer or pancreatic cancer.

[0143] In one embodiment, the composition may further comprise an anticancer agent.

[0144] In one embodiment, the anticancer agent is selected from the group consisting of acibaicin, aclarubicin, acodazole, acronicin, adozelesin, alanosine, aldesleukin, allopurinol sodium, altretamine, aminoglutethimide, amonafide, ampligen, amsacrine, androgens, anguidine, aphidicolin glycinate, asare, asparaginase, 5-azacytidine, azathioprine, Bacillus Calmette-Guerin (BCG), Baker's Antipol, beta-2-deoxythioguanosine, bisanthrene HCl, bleomycin sulfate, bulseophan, buthionine sulfoximine, BWA 773U82, BW 502U83 / HCl, BW 7U85 mesylate, cerasemide, carbetimer, carboplatin, carmustine, chlorambucil, Chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladribine, corticosteroids, Corynebacterium parvum, CPT-11, crisnatol, cyclocytidine, cyclophosphamide, cytarabine, Cytembena, Davis maleate, decarbazine, dactinomycin, daunorubicin HCl, diazauridine, dexrazoxane, dianhydrogalactitol, diaziquone, dibromodulcitol, didemine B, diethyldithiocarbamate, diclycoaldehyde, dihydro-5-azacytine, doxorubicin, echinomycin, dedatrexate, edelfosine, efloxacin, Elliot's solution, elsamitrucine, epirubicin, esorubicin, estramustine Phosphate, Estrogen, Etanidazole, Ethiophos, Etoposide, Padrazole, Fazarabine, Fenretinide, Filgrastim, Finasteride, Flavone Acetate, Floxuridine, Fludarabine Phosphate, 5'-Fluorouracil, Fluosol™, Flutamide, Gallium Nitrate, Gemcitabine, Goserelin Acetate, Hefsulfam, Hexamethylene Bisacetamide, Homoharringtonine, Hydrazine Sulfate, 4-Hydroxyandrostenedione, Hydrodiurea, Idarubicin HCl, Ifosfamide, 4-Ipomeanol, Iproplatin, Isotretinoin, Leucovorin Calcium, Leuprolide Acetate, Levamisole, Liposomal Daunorubicin, Liposomal Entrapped Doxorubicin, Lomustine,Lonidamine, Maytansine, Mechlorethamine Hydrochloride, Melphalan, Menogaril, Merbaron, 6-Mercaptopurine, Mesna, Methanol Extract of Bacillus calete-guerins, Methotrexate, N-Methylformamide, Mifepristone, Mitoguazone, Mitomycin-C, Mitotane, Mitoxantrone Hydrochloride, Monocyte / Macrophage Colony-Stimulating Factor, Nabilone, Nafoxidine, Neocarzinostatin, Octreotide Acetate, Ormaplatin, Oxaliplatin, Paclitaxel, Palla, Pentostatin, Piperazinedione, Pipobroman, Pirarubicin, Piritrexim, Piroxantrone Hydrochloride, PIXY-321, Plicamycin, Porfimer Sodium, Prednimustine, Procarbazine, progestins, pyrazole furin, razoxane, sargramostim, semustine, spirogermanium, spiromustine, streptonigrin, streptozocin, sulfenor, suramin sodium, tamoxifen, taxolere, tegafur, teniposide, terephthalamidine, teroxirone, thioguanine, thiotepa, thymidine injection, tiazofurin, topotecan, toremifene, tretinoin, trifluoperazine hydrochloride, trifluridine, trimetrexate, tumor necrosis factor (TNF), uracil mustard, vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinzolidine, Yoshi 864, zorubicin, cytosine arabinoside, etoposide, melphalan, taxol and their It may include a mixture. Preferably, it may be cisplatin, paclitaxel, 5-FU (5-fluorouracil), methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, melphalan, chlorambucil, cyclophosphamide, vindesine, mitomycin, bleomycin, tamoxifen and taxol, and more preferably, it may be cisplatin, paclitaxel or 5-FU (5-fluorouracil).

[0145] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount, and the pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations.

[0146] In one embodiment, the pharmaceutical composition may be in one or more dosage forms selected from the group consisting of oral dosage forms, topical preparations, suppositories, sterile injectable solutions and sprays, with oral or injectable dosage forms being more preferred.

[0147] The pharmaceutical composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or locally in the form of an injection) or orally, depending on the intended method. The dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives can be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. The pharmaceutical composition of the present invention can be administered by any device that allows the active ingredient to move to target cells. Preferred administration methods and preparations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Injectables can be manufactured using aqueous solvents such as saline solution and Ringer's solution, non-aqueous solvents such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0148] The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0149] In one aspect, the present invention relates to a cosmetic composition comprising the metal nanocomposite-oxygen nanoliposome complex of the present invention.

[0150] In one embodiment, the cosmetic composition of the present invention may be for skin improvement, and the skin improvement may be at least one selected from the group consisting of skin wrinkle improvement, skin moisturizing improvement, skin whitening, skin aging prevention, skin immunity or defense enhancement, pore reduction, skin barrier improvement, pigmentation inhibition, sebum inhibition or acne improvement, atopy improvement, or skin soothing.

[0151] In one embodiment, the cosmetic composition may be for removing sebum.

[0152] The cosmetic composition of the present invention can be manufactured by including a cosmetically effective amount of the above-described active ingredients of the present invention and a cosmetically acceptable carrier.

[0153] The cosmetic composition of the present invention can be manufactured by including a cosmetically effective amount of the above-described active ingredients of the present invention and a cosmetically acceptable carrier.

[0154] The cosmetic composition may be in any form suitable for topical application, including solutions, gels, solids, pastes, anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules, or vesicular dispersions of ionic (liposomes) and nonionic types, or in the form of creams, toners, lotions, powders, ointments, sprays, or concealer sticks. These compositions may be prepared according to methods conventional in the art. The composition according to the present invention may also be used in the form of a foam or an aerosol composition further containing a compressed propellant.

[0155] The cosmetic composition according to one embodiment of the present invention is not particularly limited in its formulation, and may be formulated as cosmetics such as, for example, a softening toner, an astringent toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, an eye essence, a cleansing cream, a cleansing foam, a cleansing water, a pack, a powder, a body lotion, a body cream, a body oil, and a body essence.

[0156] When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0157] When the cosmetic composition of the present invention is in the form of a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0158] When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0159] When the formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0160] When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0161] The cosmetic composition of the present invention can be applied to cosmetics such as skin, lotion, cream, essence, pack, foundation, color cosmetics, sunscreen, two-way cake, face powder, compact, makeup base, skin cover, eye shadow, lipstick, lip gloss, lip fix, eyebrow pencil, toner, and cleansing agents such as shampoo and soap.

[0162] A cosmetic composition according to one embodiment of the present invention may further include, in addition to the above-described effective ingredient, a functional additive and an ingredient included in a general cosmetic composition. The functional additive may include an ingredient selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extract.

[0163] The cosmetic composition of the present invention may also contain, as necessary, ingredients included in general cosmetic compositions in addition to the above-described effective ingredients. Examples of other ingredients to be contained include fat components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, sterilizers, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, and the like.

[0164] In one aspect, the present invention relates to a method for preparing oxygen nanoliposomes, comprising the steps of mixing polyethylene glycol unconjugated phospholipids and polyethylene glycol conjugated phospholipids; and the steps of sonicating while bubbling oxygen gas.

[0165] In one embodiment, after the step of mixing the phospholipids, a step of sonicating the prepared lipid solution at a temperature above the transition temperature may be additionally included.

[0166] In one aspect, the present invention relates to a method for preparing antibody-conjugated oxygen nanoliposomes, comprising the steps of modifying the surface of the oxygen nanoliposomes of the present invention with a crosslinker; and conjugating the antibody to the surface of the oxygen nanoliposomes by linking a primary amine group of the antibody with an NHS-ester functional group.

[0167] In one aspect, the present invention relates to a method for preparing a metal nanocomposite-oxygen nanoliposome complex, comprising the steps of: preparing an oxygen nanoliposome; preparing a metal nanocomposite comprising a cancer cell targeting moiety, an immune effector cell targeting moiety, and a metal moiety exhibiting a photothermal effect; mixing the oxygen nanoliposome and the metal nanocomposite; and sonicating.

[0168] In one aspect, the present invention relates to the use of the nanoliposomes of the present invention for oxygen delivery to cancer cells.

[0169] In one aspect, the present invention relates to a method of treating cancer, comprising administering to a subject suffering from cancer a nanoliposome or modified nanoliposome of the present invention.

[0170] Below, specific embodiments of the present invention will be described.

[0171]

[0172] Example 1: Preparation of oxygen nanoliposomes

[0173] 1-1. Preparation of oxygen nanoliposomes

[0174] To prepare a lipid solution, DSPC and DSPE-PEG (2000)-amine were mixed at a molar ratio of 85:15. Chloroform was evaporated in an oven at 70°C for 2–3 h to form a white thin film, and 8 mL of DPBS was added. The lipid solution thus prepared was sonicated for 10 min at 60°C, which is higher than the transition temperature (i.e., 55°C), using a bath sonicator (100 W) and bubbled with oxygen gas for 3 min. Tip sonication (on 2 s / off 2 s, 5 min RT, 26% amplitude) was used while directly supplying oxygen gas. The prepared oxygen nanoliposome solution was purified using a 1 μm PTFE filter and stored in the refrigerator (Fig. 1).

[0175]

[0176] 1-2. Characterization of oxygen nanoliposomes

[0177] The size and concentration of the oxygen nanoliposomes prepared in Example 1-1 were quantified. In addition, small angle neutron X-ray measurements and modeling thereof were performed.

[0178] As a result, the oxygen nanoliposomes were found to have a size of approximately 131.1 nm, and the particle size was maintained without significant changes even after 30 days (Fig. 2A). In addition, it was confirmed that the oxygen nanoliposomes were liposomes with a double lipid membrane (Fig. 2B).

[0179]

[0180] Example 2: Oxygen nanoliposomes with various membrane components

[0181] 2-1. Preparation of oxygen nanoliposomes containing various membrane components

[0182] To prepare oxygen nanoliposomes, DSPC, DSPE-PEG (2000)-amine and cholesterol were mixed in a 3-neck flask at a molecular ratio of 45:15:40, or DSPC, DSPE-PEG (2000)-amine, cholesterol and polymer (Polyoxyethylene (100) stearyl ether (Brij ® S100)) were mixed in a molecular ratio of 45:10:40:5. Chloroform was evaporated in an oven at 70°C for 2 to 3 hours to form a white thin film, and 8 mL of DPBS was added. The mixture thus prepared was sonicated for 10 minutes with a bath sonicator (100 W) at 60°C, which is higher than the transition temperature (55°C), and bubbled with oxygen gas for 3 minutes. While directly supplying oxygen gas, tip sonication (on 2 s / off 2 s, 5 min RT, 26% amplitude) was used for sonication. Each oxygen nanoliposome solution prepared was purified using a 1 μm PTFE filter and stored in the refrigerator.

[0183]

[0184] 2-2. Analysis of oxygen loading and oxygen secretion characteristics according to membrane composition

[0185] The oxygen loading capacity and oxygen secretion characteristics according to the membrane components of oxygen liposomes comprising DSPC and DSPE-PEG (2000)-amine (DSPC:DSPE-PEG (2000)-Amine) manufactured in Example 1-1, oxygen nanoliposomes comprising DSPC, DSPE-PEG (2000)-amine and cholesterol (DSPC:DSPE-PEG (2000)-Amine:Chol) manufactured in Example 2-1, and oxygen nanoliposomes comprising DSPC, DSPE-PEG (2000)-amine, cholesterol and polymer (DSPC:DSPE-PEG (2000)-Amine:Chol:Brij) were analyzed using a DO meter, which is a dissolved oxygen measuring device. Specifically, in order to analyze the amount of oxygen released from oxygen nanoliposomes, the dissolved gas of 18 mL of DPBS was removed using argon gas, 2 mL of the manufactured particles were added, and the chamber was sealed. Afterwards, the mixture was stirred using a magnetic bar and the oxygen level was measured every minute, and argon gas was continuously injected into the chamber to prevent external oxygen from entering.

[0186] By checking the oxygen concentration over time and comparing the amount of oxygen released from three oxygen nanoliposomes, the oxygen release rate was found to be the highest from DSPC:DSPE-PEG(2000)-Amine particles, followed by DSPC:DSPE-PEG(2000)-Amine:Chol and DSPC:DSPE-PEG(2000)-Amine:Chol:Brij (Fig. 3). Through this, it was confirmed that the oxygen release rate from oxygen liposomes was affected by the rigidity according to the membrane component of the liposome, and it was confirmed that oxygen release proceeded more slowly as the rigidity increased. Therefore, it was confirmed that when cholesterol or a polymer (Brij S100) was included during liposome synthesis, the release of oxygen loaded in the liposome was slightly more sustained than when it was not included (Fig. 3).

[0187]

[0188] 2-3. Analysis of particle size changes of oxygen nanoliposomes according to membrane composition

[0189] As a result of analyzing the change in particle size of oxygen nanoliposomes depending on the presence or absence of cholesterol or polymer (Brij S100), it was confirmed that even when cholesterol and polymer (Brij S100) were added, the nano size was between 100 and 200 nm (Fig. 4).

[0190]

[0191] Example 3: Drug-loaded oxygen nanoliposomes

[0192] 3-1. Drug loading of oxygen nanoliposomes

[0193] To prepare drug-loaded oxygen nanoliposomes, DSPC and DSPE-PEG-2000-amine were dissolved in chloroform at a molar ratio of 85:15, and then the immunomodulatory drug MPA was added to the solution. The chloroform was evaporated in an oven at 70°C for 2 to 3 hours to form a white thin film. The solution was rehydrated by adding 10 mL of DPBS, and then sonicated with a bath sonicator (100 W) at a temperature above 60°C to prepare a liposome suspension containing the dissolved MPA. The suspension was sonicated in pulse mode at 190 W for 5 minutes in the presence of oxygen using tip sonication to synthesize oxygen nanoliposomes loaded / loaded with MPA. To remove unloaded drugs, the solution was dialyzed against deionized water for 3 days using 1.4 kDa dialysis tubing. The amount of MPA in oxygen nanoliposomes was measured by observing the fluorescence intensity according to the standard curve of MPA (360 / 420 nm).

[0194]

[0195] 3-2. Cytotoxicity analysis

[0196] Oxygen nanoliposomes were administered to blood cells at various concentrations, and their cytotoxicity was analyzed. Furthermore, the cytotoxicity of oxygen nanoliposomes loaded with an immunomodulator (MPA) was also examined. Furthermore, hemolysis of red blood cells was analyzed according to the concentration of oxygen nanoliposomes and their lipid composition.

[0197] As a result, oxygen nanoliposomes were not cytotoxic to peripheral blood mononuclear cells (PBMCs) at all concentrations (Fig. 5A), and there was no significant change in cytotoxicity depending on its lipid composition (Fig. 5B). In addition, the immunomodulator (MPA) itself was found to have no cytotoxicity in the treated concentration group (Fig. 5C). The cell viability of MPA, PHA (phytohemagglutinin), oxygen nanoliposomes, MPA-loaded oxygen nanoliposomes, and MPA and PHA-loaded oxygen nanoliposomes were similar, and there was no significant difference in cytotoxicity (Fig. 5D). In addition, as a result of analyzing the hemolysis of red blood cells, hemolysis was less than 2% at all concentrations (Figs. 5E and F), confirming that no hemolytic reaction occurred due to oxygen nanoliposomes and liposome components. Through this, it was confirmed that oxygen nanoliposomes were not toxic to cells and red blood cells.

[0198]

[0199] Example 4: Preparation of antibody-modified oxygen nanoliposomes

[0200] 4-1. Conjugation of oxygen nanoliposomes and antibodies

[0201] The oxygen nanoliposomes prepared in Example 1, which have a carboxylic acid functional group, were surface-modified using EDC and NHS crosslinkers, and the NHS-ester functional group formed by combining with the primary amine group of the antibody to prepare antibody-conjugated oxygen nanoliposomes (immunonanosomes). Specifically, 2.2 mg of EDC and 1.1 mg of NHS were added to the oxygen-loaded nanoliposome solution prepared in Example 1, sealed, and gently stirred at 4°C for 3 hours. Thereafter, excess cross-linking reagents and byproducts were removed overnight using 6-8 kDa dialysis, and anti-human CD73 antibody was added to DSPE-PEG(2000)-carboxylic acid at a molar ratio of 1:2000. The mixture was gently stirred at 4°C for 9 hours, and then unreacted reagents were removed with oxygenated DPBS using a Sephadex G-25 column to produce oxygen nanoliposomes modified with antibodies and conjugated to the surface.

[0202]

[0203] 4-2. Confirmation of antibody conjugation

[0204] The conjugation / binding of CD73 antibody to the surface of oxygen nanoliposomes by antibody modification was confirmed using Micro BCA protein analysis and Nanodrop (Thermo Scientific, US) (A280). In addition, the dual fluorescence of the phospholipid shell stained with DiD lipophilic dye and FITC anti-human CD73 antibody was confirmed by a solid-phase binding assay. Specifically, the bilayer of oxygen nanoliposomes was stained with DiD lipophilic dye at 37°C for 30 min, and then the free dye was removed using a Sephadex G-25 column. The oxygen nanoliposomes (immunonanosomes) were modified with FITC-conjugated CD73 antibody to produce antibody-conjugated oxygen nanoliposomes. Afterwards, each nanoliposome was bound using a plate coated with protein G, which strongly binds to human IgG, and the fluorescence intensity (488 nm / 520 nm, 644 nm / 670 nm) was measured using a BioTek microplate reader Synergy H1 (VT, USA).

[0205] As a result, oxygen nanoliposomes were found to have a higher protein concentration than antibody-modified oxygen nanoliposomes (Fig. 6F), and Nanodrop measurement results confirmed that, unlike oxygen nanoliposomes, an IgG peak at 280 nm appeared in antibody-modified oxygen nanoliposomes (Fig. 6G). In addition, solid-phase binding analysis results showed that antibody-modified oxygen nanoliposomes stained with DiD specifically and strongly bound to the bottom of the plate by CD73 antibodies with IgG, showing a high DiD fluorescence level, whereas oxygen nanoliposomes showed a low fluorescence level (Fig. 6H), confirming that antibodies were conjugated to the surface of antibody-modified oxygen nanoliposomes.

[0206]

[0207] Example 5: Characterization of Antibody-Modified Oxygen Nanoliposomes

[0208] 5-1. Comparison of the characteristics of oxygen nanoliposomes and antibody-modified oxygen nanoliposomes.

[0209] The size and zeta potential of oxygen nanoliposomes and antibody-modified oxygen nanoliposomes were analyzed using a Zetasizer Pro (Malvern, UK), and particle concentration was measured using a NanoSight LM10 nanoparticle tracking analysis (Malvern, UK). Furthermore, the oxygen concentration of the particles was measured using a polarographic DO meter. Furthermore, their morphology was analyzed using TEM.

[0210] The average size of oxygen nanoliposomes was 124.4 nm (± 3.870 nm), and the average size of antibody-modified oxygen nanoliposomes was 142.5 nm (± 1.286 nm) (Fig. 6A). It was confirmed that the size increased after antibody conjugation but maintained the nano-size. In addition, the zeta potential measurement results showed that the zeta potential increased due to antibody binding (Fig. 6B). In addition, the particle concentrations of oxygen nanoliposomes and antibody-modified oxygen nanoliposomes were approximately 1.38 E+13 particles / mL (± 6.04 E+11 particles / mL) and approximately 6.25 E+12 particles / mL (± 9.91 E+11 particles / mL), respectively (Fig. 6C). The oxygen concentrations of the oxygen nanoliposomes and antibody-modified oxygen nanoliposomes were approximately 22.52 mg / L (± 0.661 mg / L) and approximately 20.05 mg / L (± 0.391 mg / L), respectively, and 6.21 mg / L (± 0.098 mg / L) in the DPBS buffer without oxygen (Fig. 6D). In addition, when the particle shapes were analyzed by TEM, both particles exhibited a spherical liposome shape and a nano-sized size of 100-200 nm (Fig. 6E).

[0211]

[0212] 5-2. Cytotoxicity analysis

[0213] 1×10 cells 4 Cells / 100 μL were dispensed into a 96-well plate, and the cytotoxicity of oxygen nanoliposomes and antibody-modified oxygen nanoliposomes at various concentrations was confirmed at 450 nm using a cell counting kit-8 analysis.

[0214] As a result, compared to the untreated control group, cell viability varied depending on the concentration, but it was commonly found that no cytotoxicity was observed at concentrations below 500 μg / mL (Fig. 7). In addition, it was found that cell proliferation increased due to oxygen released by the diffusion mechanism from both nanoliposomes. Based on this, 500 μg / mL was selected as the concentration that could deliver oxygen without showing cytotoxicity such as ROS generation due to excessive oxygen.

[0215]

[0216] 5-3. Cancer cell binding ability analysis

[0217] The cancer cell-specific binding ability of antibody-modified oxygen nanoliposomes was compared with that of oxygen nanoliposomes. Specifically, oxygen nanoliposomes and antibody-modified oxygen nanoliposomes were diluted to the same particle concentration (7.41 E+12 and 7.65 E+12, respectively), and the phospholipid bilayer was stained with DiO dye. Two pancreatic cancer cell lines (AsPC-1 and MIA PaCa-2) and one pancreatic duct epithelial cell (H6C7) were seeded in 12-well plates at a density of 8 × 10 4 DiD lipophilic dye solution was added to the cells / 2 mL aliquots to stain the cell membrane, and then cultured for 24 h under normoxic and hypoxic conditions (cultured in a chamber purged with a 1% O2 / 4.99% CO2 / 94.01% N2 gas mixture), and oxygen nanoliposomes dyed with the above DiO dye and antibody-modified oxygen nanoliposomes were treated, respectively. After that, the cells were fixed with 4% paraformaldehyde, fluorescence images were taken using a confocal microscope, and flow cytometry was performed to quantify the degree of nanoliposome binding.

[0218] As a result, antibody-free oxygen nanoliposomes showed low binding to cancer cells, and oxygen nanoliposomes (green fluorescence) were hardly observed on the cell surface and inside the cytoplasm regardless of hypoxia culture (Fig. 8A). In contrast, antibody-modified oxygen nanoliposomes showed high binding to cancer cells under both normal and hypoxic conditions (Fig. 8A), confirming antibody-mediated cell-specific binding. In particular, the fluorescence intensity of antibody-modified oxygen nanoliposomes increased in the cancer cell group in which surface CD73 expression increased through hypoxia culture, and MIA PaCa-2, which had the highest CD73 expression, showed higher particle binding than other cells, and some of them were also observed inside the cytoplasm. In addition, the degree of binding of nanoliposomes to cells was confirmed by flow cytometry, and the degree of binding of antibody-modified oxygen nanoliposomes to cells was higher than that of oxygen nanoliposomes under both normal and hypoxic conditions (AsPC-1 N ON: 66.9%, AsPC-1 N IN: 89.4%, MIA N ON: 17.6%, MIA N IN: 80.5%, H6C7 N ON: 22.8%, H6C7 N IN: 77.3%, AsPC-1 H ON: 58.6%, AsPC-1 H IN: 89.0%, MIA H ON: 16.1%, MIA H IN: 79.8%, H6C7 H ON: 30.6%, and H6C7 H IN: 67.1%) (Fig. 8B). Specifically, for AsPC-1, antibody-modified oxygen nanoliposomes showed a binding value 22.5% higher than that of oxygen nanoliposomes under normal culture conditions, and about 30.4% higher under hypoxic culture conditions. For MIA PaCa-2, which has relatively high CD73 expression compared to other cells, antibody-modified oxygen nanoliposomes showed a binding value 62.9% higher than that of oxygen nanoliposomes under normal culture conditions, and 63.7% higher under hypoxic culture conditions.Under normal culture conditions for H6C7, antibody-modified oxygen nanoliposomes showed a binding value 54.5% higher than that of oxygen nanoliposomes, and under hypoxic culture conditions, it was 36.5% higher.

[0219] Regardless of cell culture conditions, antibody-modified oxygen nanoliposomes showed higher binding affinity to all three cell types than oxygen nanoliposomes, confirming that antibody-induced cell surface binding and intracellular incorporation enhanced oxygen delivery to cells. This means that such specific binding affinity can more effectively deliver oxygen to cells with higher CD73 expression under hypoxic conditions and achieve CD73 blockade in cells with higher hypoxic levels.

[0220]

[0221] Example 6: Analysis of the adenosine-reducing effects of oxygen nanoliposomes and antibody-conjugated oxygen nanoliposomes.

[0222] When oxygen is delivered to cells, ATP accumulation outside the cells is suppressed, thereby reducing adenosine. When antibodies bind to CD73, the binding of AMP to CD73 is blocked, thereby inhibiting the conversion of AMP to adenosine in the hypoxic environment. Therefore, the effects of oxygen delivery to cells by antibody-modified oxygen nanoliposomes and adenosine reduction by CD73 blockade were analyzed. Specifically, cells were seeded in 6-well plates at 8 × 10 5 Cells were dispensed at a concentration of 2 mL / cells and cultured under hypoxic conditions for 24 or 48 h. Then, non-liposomal oxy-DPBS, oxygen nanoliposomes, and antibody-modified oxygen nanoliposomes were treated at a concentration of 500 μg / mL for 4 h, respectively, and the change in the amount of adenosine converted was analyzed using HPLC 1260 Infinity II (Agilent, CA, USA). Subsequently, the amount of adenosine converted was normalized to that of the control group (CNTL) that was not treated with nanoliposomes and quantitatively analyzed.

[0223] As a result, oxygen liposomes capable of continuously releasing oxygen significantly reduced adenosine compared to oxy-DPBS (Fig. 9A), and antibody-modified oxygen nanoliposomes capable of blocking CD73 exhibited the greatest adenosine-reducing effect, which was particularly pronounced when the hypoxic incubation time increased (Figs. 9A and B). This trend was more evident in cancer cells.

[0224] Through this, it was confirmed that antibody-modified oxygen nanoliposomes with oxygen delivery and CD73 blocking effects can suppress the extracellular accumulation of adenosine caused by up-expressed CD73 and its activity in a hypoxic environment.

[0225]

[0226] Example 7: Analysis of M1 Macrophage Immune Activation by Oxygen Nanoliposomes and Antibody-Modified Oxygen Nanoliposomes

[0227] To confirm the change in immune activity by oxygen nanoliposomes, adenosine converted by cells under hypoxic conditions was designed to move through the pores of the membrane of a transwell insert, stimulate M1 macrophages, and induce changes in the immune response, and then hypoxic co-culture was performed (Fig. 10A). The level of TNF-α, a representative pro-inflammatory cytokine of macrophages, was analyzed as an indicator of immune activity. Specifically, 1 × 10 THP-1 cells were seeded on the bottom of a 24-well plate. 5 Cells were seeded at 1 mL and differentiated into M0 macrophages by treating with 320 nmol / L PMA for 24 hours, and then polarized into M1 macrophages by treating with 100 ng / mL LPS and 20 ng / mL IFN-γ for 24 hours. The medium was replaced with 100 ng / mL LPS and 20 ng / mL IFN-γ, and a transwell insert was inserted into each well. Normal pancreatic cells and pancreatic cancer cells were seeded into the inserts at a density of 1 × 10 4Cells / 100uL were dispensed and cultured under normal and hypoxic conditions for 24 and 48 hours, respectively. Subsequently, non-liposomal oxy-DPBS, oxygen nanoliposomes, and antibody-modified oxygen nanoliposomes were treated at a concentration of 500μg / mL for 4 hours each, and the medium containing 100ng / mL LPS and 20ng / mL IFN-γ was replaced, and the insert was replaced with medium containing 1 mM AMP. The culture was further incubated for 6 hours to allow adenosine to stimulate M1 macrophages. After stimulation, the M1 macrophage culture medium was collected and centrifuged, and the levels of TNF-α and IL-10 (increased production due to the inhibitory effect caused by adenosine binding to M1 macrophages) in the supernatant were analyzed by ELISA.

[0228] As a result, hypoxic conditions increased CD73 expression, which was then converted to adenosine, and this decreased the immune activity of M1 macrophages. The TNF-α levels of macrophages co-cultured with each cell type decreased as the hypoxic co-culture time increased, and the decrease was greatest in macrophages co-cultured with MIA PaCa-2 (Figure 10B), confirming that this was due to the high CD73 expression and the resulting greatest degree of adenosine conversion. In addition, co-cultured M1 macrophages increased the secretion of IL-10 compared to M1 macrophages cultured alone, and this increased when cultured under hypoxic conditions, and the secretion increased as the hypoxic culture time increased (data not shown). Through this, we confirmed that adenosine accumulated excessively under hypoxic conditions induces a decrease in the immune activity of M1 macrophages. In this situation, when the oxygen nanoliposomes and antibody-modified oxygen nanoliposomes of the present invention were treated, the secretion of TNF-α tended to increase and recover in all cell lines, and the level was found to increase in the order of oxy-DPBS (transient oxygen delivery), oxygen nanoliposomes (stable oxygen delivery), and antibody-modified oxygen nanoliposomes (oxygen delivery and CD73 blockade) (Fig. 10C and D). In particular, in the case of M1 macrophages co-cultured with cancer cell lines, the level of TNF-α secreted was found to be recovered to a similar degree to that secreted by the control group cultured normally alone.

[0229] Therefore, it was confirmed that adenosine accumulation in the hypoxic tumor microenvironment (TME) can be prevented and restored by oxygen delivery and CD73 blockade using the nanoliposomes of the present invention, thereby inducing immune reactivation of M1 macrophages whose activity was inhibited.

[0230]

[0231] Example 8: Preparation of metal nanocomposites

[0232] 8-1. Manufacture of Man-AuNR-SNA

[0233] A metal nanomaterial, Man-AuNR-SNA, containing Sambucus nigra agglutinin (SNA) lectin as a targeting factor for cancer cell glycans, mannose (Man) as a targeting factor for the surface of macrophages, and gold nanorods (AuNR) as a targeting factor for photothermal therapy was prepared. First, gold nanorods (AuNR) were synthesized using the 'seed-mediated growth method'. Specifically, CTAB (5 mL, 0.2 M) and HAuCl4 (2.5 mL, 1 mM) were mixed with stirring, and then NaBH4 (600 mL, 0.01 M) was added. A seed solution was formed at 30 °C and stirred until the color changed from yellow to dark brown. To prepare the growth solution, 9 g of CTAB, 1.23 g of NaOL, and 24 mL of AgNO34 M were mixed in 250 mL of triple-distilled water, HAuCl4 (250 mL, 1 mM) was added, and the mixture was kept without stirring for 15 min and then stirred for 90 min. 3 mL of 37 wt% HCl was injected and stirred for 15 min, and ascorbic acid (1.25 mL, 64 mM) was added and stirred until the solution became transparent. 800 mL of the seed solution prepared above was injected into the growth solution, stirred for 30 s, and maintained at 30 °C for 12 h. The finally synthesized AuNRs were concentrated by centrifugation to remove CTAB and stored in a refrigerator. To conjugate SNA lectin to the prepared AuNR, SH-PEG-COOH (heterobifunctional polyethylene glycol) was added to the prepared AuNR and stirred. DW containing 0.02% SDS was added and stirred overnight. To remove unbound SH-PEG-COOH, the AuNR was centrifuged twice and adjusted to pH 5.AuNRs were functionalized with carboxyl groups by redispersing in MES buffer at pH 5. To conjugate SNA lectin protein to carboxylated AuNRs, EDC / NHS cross-linker was added, stirred for 20 min, centrifuged to remove unbound cross-linker, and redispersed in MES buffer at pH 7. SNA was then injected, stirred overnight, and unbound SNA was removed by centrifugation and redispersed in DW (AuNR-SNA). SH-PEG-Mannose was added and stirred overnight to conjugate mannose to AuNR-SNA, preparing Man-AuNR-SNA conjugates (Figure 11A).

[0234]

[0235] 8-2. Characteristics of Man-AuNR-SNA

[0236] The optical properties of the Man-AuNR-SNA conjugates prepared above were confirmed by analyzing the absorption spectrum from 300 to 999 nm using a microplate reader (BioTek / Agilent, USA). The particle size and surface charge were measured using a Zetasizer Pro (Malvern, UK). In addition, the particle concentration was measured using a NanoSight LM10 nanoparticle tracking analysis (Malvern, UK), and the particle number was calculated using this. The shape and morphology of AuNR and Man-AuNR-SNA were confirmed by TEM analysis using a JEM-F200 Transmission Electron Microscope (JEOL Ltd, Japan). In addition, for the qualitative analysis of the conjugates, AuNR, Man-AuNR-SNA, and SH-PEG-Mannose were freeze-dried, and FTIR (Fourier transform infrared spectrum) analysis was performed using an FTIR Spectrophotometer (ALPHA II, Germany). For the qualitative analysis,1 H Nuclear Magnetic Resonance ( 1 H NMR) 600 MHz (VNS, Varian) was used to measure the spectra. In addition, the concentration of SNA in the Man-AuNR-SNA conjugate was measured using a BCA protein assay kit, and the concentration of mannose was measured by the thiol group quantification method of Ellman's assay using HPLC 1260 Infinity II (Agilent, CA, USA) on a Poreshell 120 EC-C18 column (Agilent, CA, USA). In addition, to confirm whether Man-AuNR-SNA has the photothermal effect, which is an inherent characteristic of gold-based materials, Man-AuNR-SNA was exposed to an 850 nm NIR laser within the 700-1100 nm NIR wavelength range, which is optimal for tissue penetration through photothermal therapy, and its photothermal effect was analyzed. In addition, the photothermal stability of Man-AuNR-SNA was evaluated through three heating and cooling cycles under the laser exposure.

[0237] As a result, it was observed that the surface of AuNR changed due to the binding of SNA and mannose, and it was confirmed that the rod shape and nano size were maintained even after binding (Fig. 11B). As a result of surface charge analysis, the surface charge value decreased (-20.29 mV) due to the introduction of a carboxyl group, and the surface charge value of AuNR-SNA increased due to the amine group present in the lectin protein by the binding of SNA (-6.77 mV), and the surface charge value decreased again due to the binding of mannose (-15.99 mV), and finally, it was confirmed that the nonspecific binding to cells would be less due to the characteristic of the Man-AuNR-SNA particles having a negative surface charge (Fig. 11C). 1H NMR spectral analysis revealed that the characteristic peak of mannose was not observed in AuNR, whereas a peak was observed at 3.55 ppm in AuNR-Mannose (Fig. 11D). FTIR spectra of SH-PEG-Mannose, AuNR, and Man-AuNR-SNA also revealed that the characteristic peak of PEG was not observed in AuNR, whereas C=O (1709 cm -1 ) and -CH2(2942 cm -1 ) and SH-PEG-Mannose, all characteristic peaks were consistent (Fig. 11E). As a result of quantitative analysis of all substances contained in the manufactured Man-AuNR-SNA by NTA, the particle concentration was approximately 4x10 11 particles / mL, and the SNA concentration was measured by BCA analysis, and approximately 1.25 mg / mL was bound, and the mannose concentration was analyzed by Ellman's assay, and approximately 1 mg / mL was bound. In addition, in the case of DW and cell culture medium, the temperature slightly increased to approximately 32 °C upon exposure to 850 nm NIR laser, whereas Man-AuNR-SNA increased to approximately 55 °C (Figs. 11F to G), and it was confirmed that it had excellent photothermal stability (Fig. 11H). Therefore, it was confirmed that Man-AuNR-SNA is suitable as a material for inducing photothermal therapy by 850 nm NIR irradiation because it absorbs maximum light at approximately 840 nm.

[0238]

[0239] 8-3. Analysis of cancer cell-specific binding ability of Man-AuNR-SNA

[0240] In general, cancer cells overexpress sialic acid on their surface, whereas normal cells do not (Fig. 12A). Therefore, the binding ability of Man-AuNR-SNA particles to normal H6c7 cells and three cancer cells, MIA PaCa-2, PANC-1, and AsPC-1, was analyzed using SNA, a sialic acid-binding lectin. Specifically, H6c7, MIA PaCa-2, PANC-1, and AsPC-1 cells were seeded in a 96-well plate at a density of 1 × 10 , respectively, with medium. 4 After seeding at a density of 10 cells / well, the cells were cultured for 24 hours. The culture medium was removed, fixed with 2% paraformaldehyde, and Man-AuNR-SNA particles were added to each well at a concentration of 5 μg / mL based on the SNA concentration, and reacted at room temperature for 2 hours. Unbound particles were removed by washing three times with 1XPBS, and the fluorescence of particles bound to cells was measured using a microreader (BioTek / Agilent, USA) at λ Em 490 nm, λ ExIt was confirmed at 520 nm. In addition, a control AuNR-BSA particle treatment condition (BSA concentration standard 5 μg / mL) was also added to MIA PaCa-2 pancreatic cancer cells for comparative analysis. In addition, to confirm whether the immune evasion signal generated by the interaction between macrophages and cancer cells is blocked by SNA treatment, resulting in phagocytosis of cancer cells by macrophages, M0, M1, and M2 phenotype macrophages were prepared using a macrophage polarization method, stained with calcein red-AM, and cancer cells reacted with SNA were stained with calcein-AM and fluorescently labeled, and then macrophages and cancer cells were co-cultured and observed under a confocal microscope. As a result, SNA specifically bound only to cancer cells compared to normal cells (Fig. 12B). To confirm whether this binding is mediated by sialic acid, sialidase (i.e., neuraminidase) was treated, and the degree of SNA binding was analyzed by FACS. As a result, the population of normal cells bound by SNA under the non-sialidase treatment condition was 28.9%, whereas the three cancer cell types showed high binding ability of 68.5%, 82.5%, and 98.3%, respectively. Unlike the normal cells, which showed no change in binding ability when treated with sialidase, the binding ability decreased to 29.4%, 45.4%, and 44.8%, respectively (Fig. 12C), confirming that SNA can specifically bind to pancreatic cancer cells and that this is mediated by sialic acid. To confirm whether such cancer cell-specific binding effect of SNA also occurs by Man-AuNR-SNA, the binding ability to cells was evaluated using fluorescently labeled Man-AuNR-SNA particles. As a result, a higher fluorescence signal was detected in the three cancer cell types than in the normal cells, and the results showed a particle concentration-dependent result (Fig. 12D and E). In contrast, when particles conjugated with BSA rather than SNA were used as a control, a very low signal was detected in pancreatic cancer cells MIA PaCa-2.

[0241] Therefore, it can be seen that Man-AuNR-SNA particles can specifically bind to cancer cells via SNA.

[0242]

[0243] 8-4. Analysis of Macrophage-Specific Binding Ability of Man-AuNR-SNA

[0244] Since Siglec-10, which forms an immune checkpoint by interacting with sialic acid of cancer cells, is most highly expressed in the M2 macrophage population (M0 34.9%, M1 46.4%, and M2 58.6%), the expression of CD11b, a macrophage-wide marker, CD80, an M1-specific marker, and CD206, an M2-specific marker, was analyzed by FACS in M0, M1, and M2 phenotype macrophages. As a result, CD11b was found to be over 99% of the population in all phenotypes, CD80 was found to be 19.2% in M0, 89.2% in M1, and 23.5% in M2, and CD206 was found to be 27.9% in M0, 29.9% in M1, and 46.0% in M2 (Fig. 13A). Since CD206, which is an M2 marker and mannose receptor, is overexpressed only in the M2 phenotype, it was confirmed that mannose can target M2 phenotype macrophages. Accordingly, to confirm the specific binding ability of mannose to M2 macrophages, confocal microscopy analysis was performed using SH-PEG-Mannose and Maleimide-FITC. As a result, mannose was bound to the surface of M2 macrophages, and FITC was labeled by the binding between the terminal thiol (-SH) and maleimide, and fluorescence was detected (Fig. 13B). To determine whether the specific binding of mannose to M2 macrophages was also observed in Man-AuNR-SNA particles, fluorescently labeled Man-AuNR-SNA particles were treated with M0, M1, and M2 phenotype macrophages, respectively, and the fluorescence of the particles bound to the cells was measured using a microplate reader (BioTek / Agilent, USA) at λ Em490 nm, λ Ex It was confirmed at 520 nm. In addition, the control group treated with AuNR-SNA particles without mannose conjugation to M2 phenotype macrophages was also analyzed. As a result, a significantly higher fluorescence signal was detected in M2 macrophages compared to M0 and M1 macrophages, and this was dependent on the concentration of Man-AuNR-SNA particles treated (Fig. 13C). In contrast, in the control group treated with particles without mannose conjugation, a significantly lower signal was detected in M2 macrophages, confirming that Man-AuNR-SNA particles can specifically bind to M2 macrophages via mannose.

[0245]

[0246] 8-5. Cytotoxicity analysis of Man-AuNR-SNA

[0247] Human skin fibroblasts HDF, pancreatic ductal epithelial cells H6c7, M0, M1 and M2 macrophages, and pancreatic cancer cells MIA PaCa-2, PANC-1 and AsPC-1 were treated with Man-AuNR-SNA at concentrations of 2-10 μg / mL for 24 h, and then cytotoxicity was evaluated using the CCK-8 (cell counting kit-8) assay.

[0248] As a result, Man-AuNR-SNA showed a cell viability of more than 80% at all concentrations (Fig. 14A), confirming that it was safe with almost no cytotoxicity in all cells, and that the use of the particle alone did not cause cell death.

[0249]

[0250] 8-6. Analysis of the cancer cell apoptosis-inducing effect of Man-AuNR-SNA by photothermal therapy

[0251] To confirm the cancer cell death induction effect of Man-AuNR-SNA by photothermal therapy, Man-AuNR-SNA particles were added at low concentrations (SNA: 5 μg / mL, NP: 4x10) as in Example 8-5. 9particles / mL) and high concentration (SNA: 10 μg / mL, NP: 8x10 9 particles / mL) were treated for 2 h in each cancer cell line, and the cells were exposed to NIR laser (850 nm) for 0 to 10 min to perform photothermal therapy, followed by CCK-8 assay. In addition, 5 μg / mL of Man-AuNR-SNA particles (NP: 4x10 9 particles / mL) for 2 h, and the cells were exposed to NIR laser (850 nm) for 0–10 min. The expression of apoptosis-related markers was analyzed by RT-qPCR. Cells were stained using Annexin V-FITC Apoptosis kit (Invitrogen, V13242) and analyzed by flow cytometry using FACS Aria II (BD Biosciences, CA, USA) to analyze apoptosis and necrosis.

[0252] CCK-8 analysis results showed that in the case of normal H6c7 cells, both low- and high-concentration treatment conditions of Man-AuNR-SNA showed similar results to the untreated condition, and even when irradiated with NIR for 10 minutes, cell viability was over 80%. On the other hand, in the three cancer cell types, cell viability decreased as the NIR irradiation time increased under low-concentration treatment with Man-AuNR-SNA, and decreased to less than about 60% after 10 minutes of treatment, and under high-concentration conditions, it reached less than 60% even after 2 minutes of NIR irradiation, and decreased to less than 40% after 10 minutes (Fig. 14B and C). In addition, RT-qPCR analysis results showed that, compared to the untreated control group, the expression of survival-related protein Bcl-2 decreased under the photothermal therapy application condition, and the expression of apoptosis-related proteins p53, Bax, and Caspase-3 significantly increased (Fig. 15A). Therefore, when this strategy was applied, it was confirmed that apoptosis was effectively induced in cancer cells MIA PaCa-2. In addition, Annexin V / PI staining analysis results showed that while cell death was hardly induced when Man-AuNR-SNA treatment and NIR irradiation were used alone, cell death was significantly increased when combined (Fig. 15B and C).

[0253] Through this, it was confirmed that the combined use of Man-AuNR-SNA particle treatment and NIR irradiation had an excellent anticancer effect.

[0254]

[0255] 8-7. Analysis of the immune evasion inhibition effect of cancer cells by the combination of photothermal therapy and Man-AuNR-SNA.

[0256] Pancreatic cancer cells were treated with SNA, and cancer cells labeled with different fluorescence and macrophages of each phenotype were co-cultured for 6 hours. The cancer cell phagocytosis was evaluated by observing the cancer cell phagocytosis with or without SNA treatment using a confocal microscope. In addition, in order to analyze the phagocytosis and the resulting anticancer effect by the combined treatment of Man-AuNR-SNA (5 μg / mL concentration based on SNA) and NIR irradiation (10 minutes), Man-AuNR-SNA particle-bound and fluorescently labeled cancer cells and fluorescently labeled macrophages were co-cultured as in Example 8-3, and the cell behavior was photographed at 1-minute intervals from 0 hour to 6 hours later using a STELLARIS 5 confocal microscope (LEICA, Germany) and an Incubator T Series (LCI, Korea).

[0257] SNA treatment alone detected high fluorescence in the co-localized region of cancer cells and macrophages, and 3D z-stack image analysis confirmed that the region was completely occupied by cancer cells within macrophages (Fig. 16A), confirming that SNA blocked the sialic acid of cancer cells and enhanced the phagocytic activity of macrophages. Imaging analysis showed that under the condition of Man-AuNR-SNA particle treatment alone, phagocytosis occurred at approximately 4 hours after imaging (Fig. 16B), confirming that it was phagocytosis induced by SNA. In addition, under the condition of combined treatment with Man-AuNR-SNA and NIR irradiation, phagocytosis occurred at approximately 1 hour after imaging, and relatively more cancer cells were killed (Fig. 16C). This suggests that the combined use of Man-AuNR-SNA treatment and photothermal therapy induces immune activation while blocking a specific immune checkpoint.

[0258]

[0259] Example 9: Preparation of a metal nanocomposite-oxygen nanoliposome complex and analysis of its effects.

[0260] 9-1. Preparation of Man-AuNR-SNA oxygen nanoliposome complex

[0261] Oxygen nanoliposomes were prepared by the method of Example 1, and metal nanocomposites were prepared by the method of Example 8. Afterwards, referring to the cytotoxicity results of oxygen nanoliposomes and metal nanocomposites, the particles of metal nanocomposites mixed in 10% oxygen nanoliposomes (up to 1 x 10) were prepared. 10 After confirming the cytotoxicity results (particles / mL) (Fig. 17A), a mixed complex solution was prepared by mixing at various concentration ratios that did not show cytotoxicity. The mixed solution was constantly stirred using a magnetic bar and reacted, and then ultrasonicated using a bath sonicator to prepare a homogeneous metal nanocomposite-oxygen nanoliposome complex. Finally, the morphology and mixing of the complex were confirmed through TEM analysis (Fig. 17B).

[0262]

[0263] 9-2. Photothermal effect of Man-AuNR-SNA oxygen nanoliposome complex

[0264] To confirm the photothermal effect of the oxygen nanoliposome complex (Au-oxy) containing the Man-AuNR-SNA prepared in Example 9-1 above under NIR irradiation, the temperature change of the complex was measured using an NIR laser and a thermal imaging camera. At this time, distilled water was used as a negative control for comparison with the nanocomposite.

[0265] As a result, the control group, distilled water, showed a temperature change of about 34°C under NIR irradiation for up to 420 seconds, while the mixed complex showed a temperature of about 48°C (Fig. 18), confirming an NIR-specific heating effect of about 14°C.

[0266]

[0267] 9-3. In vitro cancer-specific apoptosis effect of Man-AuNR-SNA oxygen nanoliposome complex and photothermal therapy

[0268] The oxygen nanoliposome complex (Au-oxy) containing Man-AuNR-SNA prepared in Example 9-1 was treated to cervical cancer cell line HeLa, breast cancer cell line MDA-MB-231, and HDF (normal epithelial cell line), respectively, and irradiated with NIR for 10 minutes, after which cell death was confirmed by Live Cell / Dead Cell analysis.

[0269] As a result, it was found that cancer cells were specifically killed, unlike normal cells (Fig. 19), and it can be inferred that this is an effect through cancer cell targeting by Man-AuNR-SNA.

[0270]

[0271] 9-4. In vivo cancer-specific apoptotic effect of Man-AuNR-SNA oxygen nanoliposome complexes

[0272] After forming spheroids of malignant melanoma cell line B16F1 or transplanting them onto the backs of mice, the oxygen nanoliposome complex containing Man-AuNR-SNA was treated and NIR was irradiated to analyze the size of the spheroids and the size of cancer tissue in the mice.

[0273] As a result, the combined treatment of Man-AuNR-SNA oxygen nanoliposome complex and photothermal therapy resulted in a decrease in cancer spheroids, and a significant decrease in the size of cancer tissue transplanted into mice (Fig. 20).

Claims

1. Nanoliposomes with oxygen loaded inside the shell of a phospholipid bilayer.

2. In paragraph 1, Nanoliposomes having a diameter of 100 to 200 nm.

3. In paragraph 1, Nanoliposomes having a concentration of oxygen loaded in the nanoliposomes of 10 to 30 mg / L.

4. In paragraph 1, Nanoliposomes comprising polyethylene glycol (PEG) unconjugated phospholipids and polyethylene glycol conjugated phospholipids.

5. In paragraph 4, Polyethylene glycol-unconjugated phospholipids include DSPC (Distearoylphosphatidylcholine), DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), SOPC (1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), Nanoliposomes, which are DEPC (1,2-Dierucoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) or POPG (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol).

6. In paragraph 4, Polyethylene glycol conjugated phospholipids are C6-C 20 Nanoliposomes, which are phospholipids comprising poly(ethylene) glycol chains having a length of up to 5 kDa covalently linked to lipids having alkyl chain(s) of the same length.

7. In paragraph 4, Nanoliposomes having a molar ratio of polyethylene glycol unconjugated phospholipids and polyethylene glycol conjugated phospholipids of 4:1 to 7:

1.

8. In paragraph 1, Nanoliposomes further loaded with lipids, polymers, nucleic acids, proteins, metal nanoparticles, metal oxides, drugs or metal nanocomposites.

9. In paragraph 8, The drug is a nanoliposome, which is a peptide drug, a protein drug, an anticancer agent, an antibacterial agent, an antiviral agent, an antidementia agent, an immunomodulator, a desensitizer, an antigen, a non-steroidal anti-inflammatory drug, an anti-inflammatory drug, an anesthetic, an antioxidant, an anti-infective agent, a chemotherapeutic agent, an anti-nociceptive agent, a DMOAD, an anabolic agent, an anti-catabolic agent, an autophagy regulator, an anti-osteoclast-mediated bone loss agent, a nutraceutical agent, an analgesic, a biologic, or a mixture thereof.

10. A nanoliposome whose surface is modified with a sugar, a sugar compound, a lipid, a polymer, a nucleic acid, a protein, a metal nanoparticle, a metal oxide, or a metal nanocomposite of the first paragraph.

11. In paragraph 10, Modified nanoliposomes, wherein the protein is an antibody or a fragment thereof.

12. In paragraph 11, Modified nanoliposomes, where the antibody is directed against a protein whose expression increases in the tumor microenvironment (TME).

13. In paragraph 10, Modified nanoliposomes that deliver oxygen specifically to cancer cells.

14. In paragraph 10, Modified nanoliposomes for photothermal and photodynamic therapy by light irradiation.

15. In paragraph 10, Modified nanoliposomes wherein the metal is Au, Ag, Cu, ZnO, Co, CuO, Fe2O3, Mn2O3, Ni, Pd, Pt, TiO2 or Cr.

16. In paragraph 10, A modified nanoliposome comprising a metal nanocomposite, a cancer cell targeting moiety, an immune effector cell targeting moiety and a metal moiety exhibiting a photothermal effect.

17. A pharmaceutical composition for preventing or treating cancer, comprising the nanoliposome of paragraph 1 or the modified nanoliposome of paragraph 10 as an active ingredient. 18.1) A step of mixing polyethylene glycol unconjugated phospholipids and polyethylene glycol conjugated phospholipids; and 2) A method for producing oxygen nanoliposomes, comprising a step of sonication while bubbling oxygen gas. 19.1) Step of manufacturing oxygen nanoliposomes; 2) A step of preparing a metal nanocomposite comprising a cancer cell targeting moiety, an immune effector cell targeting moiety, and a metal moiety exhibiting a photothermal effect; 3) Step of mixing oxygen nanoliposomes and metal nanocomplexes; 4) A method for producing a metal nanocomposite-oxygen nanoliposome complex, comprising a step of ultrasonic treatment.

20. Use of nanoliposomes for oxygen delivery to cancer cells.

21. A method for treating cancer, comprising administering to a subject suffering from cancer the nanoliposome of claim 1 or the modified nanoliposome of claim 10.

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