Nano-drug delivery system that targets cancer cells in hypoxic region of tumor microenvironment and method for preparing same

Abstract

The present invention relates to a nano-drug delivery system that targets cancer cells in the hypoxic region of tumor microenvironment and a method for preparing same. The novel nano-drug delivery system according to the present invention can capture an extracellular matrix-degrading enzyme and encapsulate an anticancer drug by binding SO3-PEG-amine as a bifunctional ligand to manganese dioxide nanoparticles, thereby significantly improving drug permeability. In addition, through such high drug permeability, cancer cells in the hypoxic region of tumor microenvironment can be effectively killed.

Description

Nano drug delivery system targeting cancer cells in hypoxic regions of tumor microenvironments and method for manufacturing the same

[0001] The present invention relates to a nano drug delivery system targeting cancer cells in a hypoxic region of a tumor microenvironment and a method for manufacturing the same.

[0002]

[0003] Despite being a leading cause of death worldwide, most cancers rely on conventional radiotherapy, chemotherapy, and other surgical interventions to treat them. Cancer cells exhibit abnormal metabolism compared to normal cells, creating a microenvironment conducive to tumor formation and progression. They evolve within the tumor microenvironment (TME) by communicating with adjacent stromal cells and orchestrating cellular and molecular events in surrounding tissues to promote survival, growth, and metastasis.

[0004] Tumor cells that survive in hypoxic conditions are known to be more active and resistant to anticancer drugs than tumor cells with larger volumes in the tumor microenvironment. Furthermore, blood vessels in the tumor microenvironment are influenced by the interstitial fluid pressure (IFP) exerted by cancer cells, allowing cancer cells to proliferate at a rate exceeding the rate of angiogenesis. The dense extracellular matrix (ECM) produced by cancer-associated fibroblasts and the poor drainage of tumor fluids due to defective lymphatic function can further increase the IFP in the tumor microenvironment. Anticancer nanomaterials released from intratumoral blood vessels are hindered by the IFP and thus cannot diffuse throughout the tumor. Under these circumstances, it is extremely difficult for nanomaterials to penetrate the tumor fluid and reach hypoxic tumor cells.

[0005] Meanwhile, collagen protein is a major component of the extracellular matrix. Its viscoelastic properties play a crucial role in maintaining healthy tissue architecture. However, collagen overproduction can contribute to drug resistance in the tumor microenvironment pathologically. The extracellular matrix stroma of solid tumors generates high interstitial fluid pressure, leading to vascular collapse and limiting drug absorption from the circulatory system.

[0006] To date, various anticancer drugs have been developed that target biomarkers specific to hypoxic tumor cells. However, these treatments suffer from the limitations of the aforementioned tumor microenvironment, which limits their escape distance from intratumoral blood vessels. Therefore, the development of drug delivery systems capable of overcoming interstitial fluid pressure within the tumor microenvironment and killing cancer cells in hypoxic regions is urgently needed.

[0007] As a previous study on nano-drug complexes targeting cancer cells in the hypoxic region of the tumor microenvironment, YANG, Guangbao, et al. reported the apoptotic effect on cancer cells in the tumor microenvironment using nano-drug complexes loaded with photosensitizer Ce6 and anticancer drug doxorubicin, i.e., silicon dioxide nanoparticles coated with manganese dioxide (YANG, Guangbao, et al. Hollow MnO2 as a tumor-microenvironment-responsive biodegradable nano-platform for combination therapy favoring antitumor immune responses. Nature communications, 2017, 8.1: 902.), and LI, Juanjuan, et al. revealed the apoptotic effect on cancer cells in the tumor microenvironment using nano-drug complexes loaded with anticancer drug doxorubicin, bound collagenase to the surface of the nanoparticles, and then surrounded by a fusion membrane of macrophage cell membrane and liposomes (LI, Juanjuan, et al. Biomimetic liposomal nanozymes improve breast cancer chemotherapy with enhanced penetration and alleviated hypoxia. Journal of Nanobiotechnology, 2023, 21.1: 1-16.). In addition, Chinese patent publication CN111150853A disclosed the use of a nano drug carrier, which encapsulates the photosensitizer Ce6 and the anticancer drug doxorubicin in silicon dioxide nanoparticles coated with manganese dioxide and binds oligonucleotides, for the targeted treatment of non-small lung cancer.

[0008] However, there has been no literature describing nanoparticles that can overcome the short escape distance from blood vessels due to interstitial fluid pressure in the tumor microenvironment and reach hypoxic cancer cells. In the present invention, a novel dual-functional ligand, SO3-PEG-amine, is bound to mesoporous nanoparticles through surface modification, and then an anticancer drug is encapsulated inside the nanoparticles, and an extracellular matrix-degrading enzyme can be captured through electrostatic interaction with the dual-functional ligand.

[0009] Herein, the inventors of the present invention synthesized a novel dual-functional ligand SO3-PEG-amine, and synthesized a novel nano drug carrier by binding it to the surface of manganese dioxide nanoparticles, and confirmed that the novel functional ligand and collagenase were very well bound, and through collagenase bound to the novel functional ligand on the surface of manganese dioxide nanoparticles, the drug permeability of the novel nano drug carrier and the existing anticancer drug encapsulated in the nano drug carrier into the hypoxic region of the tumor was greatly increased, and the anticancer effect on cancer cells in the hypoxic region was greatly improved, and through tumor transparency imaging technology, the maximum drug penetration depth (DPDmax), half of DPDmax (DPD 50 ), DPD in intratumoral blood vessels 50 The number of cancer cells containing anticancer drugs (drug penetration or DPA) 50 ) was measured and quantitative analysis was performed to confirm the remarkable anticancer effect of the nano drug delivery system of the present invention, thereby completing the present invention.

[0010]

[0011] The purpose of the present invention is to provide a dual-functional ligand capable of capturing an enzyme capable of degrading the tumor extracellular matrix. Furthermore, it is also to provide a dual-functional ligand capable of capturing a protein or antibody that can bind well to tumor cells for targeting purposes.

[0012] Another object of the present invention is to provide a novel nano drug delivery system that targets cancer cells in a hypoxic region in a tumor microenvironment, comprising the above-described dual-functional ligand.

[0013] Another object of the present invention is to provide a method for producing the dual functional ligand and nano drug delivery system.

[0014] Another object of the present invention is to provide an anticancer pharmaceutical composition comprising the nano drug delivery system.

[0015] Another object of the present invention is to provide a method for evaluating drug distribution after injection of the nano drug carrier using tumor transparency imaging technology.

[0016] Another object of the present invention is to provide a method for evaluating the distribution of apoptotic cancer cells after injection of the nano drug carrier using tumor transparency imaging technology.

[0017]

[0018] To achieve the above purpose,

[0019] One aspect of the present invention provides a nano drug delivery system comprising a porous nanoparticle having at least a portion of a surface coated with manganese dioxide, wherein the surface of the nanoparticle is modified with a dual-functional ligand, wherein the dual-functional ligand comprises a polyethylene glycol moiety and a sulfate moiety.

[0020] Another aspect of the present invention provides a dual functional ligand represented by the following chemical formula 1.

[0021] [Chemical Formula 1]

[0022] PEG-L1-NH-L2

[0023] (In the above chemical formula 1,

[0024] PEG is polyethylene glycol, Including,

[0025] n is an integer greater than or equal to 2, preferably an integer from 2 to 5000, or from 2 to 1000,

[0026] R is hydrogen or alkylene having C1 to C3,

[0027] L1 is C1 to C 10 is alkylene,

[0028] L2 has one or more carbons -OSO3 - C1 to C substituted with 10 ) is alkylene.

[0029] Another aspect of the present invention is a method for manufacturing the nano drug delivery system,

[0030] (a) A step of synthesizing SiO2 nanoparticles;

[0031] (b) a step of adding a potassium permanganate (KMnO4) solution to the synthesized SiO2 nanoparticle suspension and performing ultrasonic treatment to obtain nanoparticles coated with manganese oxide on SiO2;

[0032] (c) a step of dispersing the above nanoparticles in a Na2CO3 solution, stirring them, and then centrifuging them to obtain porous nanoparticles;

[0033] (d) a step of adding polyallylamine hydrochloride (PAH) to the aqueous dispersion of the porous nanoparticles to obtain a first reaction product, and then adding polyacrylic acid (PAA) to the solution in which the first reaction product is suspended to obtain a second reaction product; and

[0034] (e) A method for producing a nano drug carrier is provided, comprising the step of producing a nano drug carrier by binding a functional ligand to the second reaction product.

[0035] Another aspect of the present invention provides an anticancer pharmaceutical composition comprising the nano drug delivery system.

[0036] Another aspect of the present invention is a method for evaluating drug distribution in a tissue after injection of the nano drug carrier,

[0037] (a') A step of forming cancer tissue by transplanting a culture medium containing cancer cells into an animal;

[0038] (b') The volume of the cancer tissue is 100 to 2000 mm 2 When this is done, a step of injecting a nano drug delivery system manufactured according to the present invention;

[0039] (c') A step of collecting cancer tissue injected with the nano drug delivery system and making it transparent;

[0040] (d') A method for evaluating the distribution of a nano drug delivery system is provided, including a step of evaluating the distribution of a drug in the above-mentioned transparent cancer tissue sample.

[0041] Another aspect of the present invention is a method for evaluating the distribution of apoptotic cancer cells in a tissue after injection of the nano drug carrier,

[0042] (a'') A step of forming cancer tissue by transplanting a culture medium containing cancer cells into an animal;

[0043] (b'') The volume of the cancer tissue is 100 to 2000 mm 2 When this is done, a step of injecting a nano drug delivery system manufactured according to the present invention;

[0044] (c'') A step of collecting cancer tissue injected with the nano drug delivery system and making it transparent;

[0045] (d'') A method for evaluating the distribution of apoptotic cancer cells is provided, including a step of evaluating the distribution of apoptotic cancer cells in the above-mentioned transparent cancer tissue sample.

[0046]

[0047] The novel nano drug delivery system according to the present invention can significantly enhance drug permeability by binding SO3-PEG-amine as a dual-functional ligand to manganese dioxide nanoparticles, thereby capturing extracellular matrix-degrading enzymes and encapsulating anticancer drugs. Furthermore, this high drug permeability can effectively kill cancer cells in hypoxic regions within the tumor microenvironment. The SO3-PEG-amine ligand can bind not only extracellular matrix-degrading enzymes but also proteins and antibodies.

[0048]

[0049] Figure 1 illustrates the synthetic steps of the dual functional ligand -SO3-PEG-amine.

[0050] Figure 2 shows a schematic diagram of the synthesis of H-MnO2-PEG-SO3 nanoparticles according to the present invention.

[0051] Figures 3 a, 3 b, 3 c, 3 d, and 3 e are EF-TEM images of nanoparticles obtained in Example 2 and nanoparticles obtained in Comparative Example 1. Figures 3 a, 3 b, and 3 e show EF-TEM images of SiO 2 NP, SiO 2 @ MnO 2 NP, and H-MnO 2 NP obtained in Example 2, and Figures 3 c and 3 d show EF-TEM images of sSiO 2 NP, SiO 2 @ MnO 2 NP obtained in Comparative Example 1.

[0052] Figures 4a and 4b are UV-vis absorption spectra of nanoparticles obtained in Examples 2 and 3.

[0053] Figure 5 is a reaction equation showing the reactivity of MnO2 toward H2O2 for O2 production and proton removal.

[0054] Figure 6 is a graph showing the results of confirming the hydrogen peroxide scavenging effect by H-MnO2-PEG-SO3 nanoparticles. Figure 6a shows the quenching of H2O2 by H-MnO2-PEG-SO3NP (90 μM) as a function of time in cell medium containing 10% FBS, and Figure 6b shows the reaction between H2O2 and H2O2 substrate as a function of time in cell medium containing 10% FBS in the absence of H-MnO2-PEG-SO3NP.

[0055] Figure 7 is a graph showing the results of O2 production by adding H2O2 (1 mM) to H-MnO2-PEG-SO3 nanoparticles at various concentrations (20, 60, 100 μM).

[0056] Figure 8 is a graph measuring the enzyme activity of collagenase. Figure 8a shows the enzyme activity of collagenase encapsulated in Clg@H-MnO2-PEG-SO3 nanoparticles, and Figure 8b shows the enzyme activity of free collagenase (control).

[0057] Figure 9 shows the results of analyzing the diffusion of nanoparticles in an extracellular matrix-mimicking gel. Figure 9a shows the diffusion of Clg-Ru@H-MnO2-PEG-SO3 nanoparticles, and Figure 9b shows the diffusion of Ru@H-MnO2-PEG-SO3 nanoparticles.

[0058] Figure 10 shows the results of analyzing drug distribution according to particle size.

[0059] Figures 10a and 10b show 3D images of tumor tissues injected with Ru@H-MnO2-PEG-SO3 nanoparticles (69 nm) and Ru@H-MnO2-PEG-SO3 nanoparticles (283 nm), respectively. I1 of Figure 10a and (A) of Figure 10b show blood vessels (red channel), I2 of Figure 10a and (B) of Figure 10b show released Ru (green channel), I3 of Figure 10a and (C) of Figure 10b are combined images of I1 and I2 of Figure 10a and (A) and (B) of Figure 10b, respectively, I4 of Figure 10a is an enlarged image of the white box area of ​​I3, and (D) of Figure 10b is an image showing tumor tissues with nuclei labeled with Hoechst. Figures K of Figure 10a and (E) of Figure 10b represent 3D reconstructed images of Figure 10a I3 and Figure 10b (C), respectively, where spots within the images are represented in spectral colors based on the distance to the nearest blood vessel. Figure 10c is a graph showing the number of cells occupying Ru according to the distance from the blood vessel.

[0060] Figure 11 shows the results of analyzing the distribution of drugs and apoptotic cancer cells by chemotherapy.

[0061] Figure 11a shows a 3D image of tumor tissue injected with DOX@H-MnO2-PEG-SO3 nanoparticles. J1 represents a blood vessel (red channel), J2 represents the released Ru (green channel), J3 is the combined image of J1 and J2, and J4 represents the tumor tissue with Hoechst-labeled nuclei. M represents a 3D reconstructed image, and spots within the image are colored in spectrum based on the distance to the nearest blood vessel.

[0062] Figure 11b is a graph showing the number of apoptotic cells according to the distance from the blood vessel.

[0063] Figure 12 shows the results of analyzing the distribution of drugs and apoptotic cancer cells by photodynamic therapy.

[0064] Figure 12a shows a 3D image of tumor tissue injected with Ce6-Ru@H-MnO2-PEG-SO3 nanoparticles. C1 represents a blood vessel (red channel), C2 represents the released Ru (green channel), C3 is the combined image of C1 and C2, and C4 is a magnified image of the white box area in C3. D represents a 3D reconstructed image, and spots in the image are colored in spectrum based on the distance to the nearest blood vessel.

[0065] Figure 12b is a graph showing the number of apoptotic cells according to the distance from the blood vessel.

[0066] Figure 13 shows the results of comparing the distribution of drugs and apoptotic cancer cells by chemotherapy and photodynamic therapy.

[0067] Figure 13a shows a 3D image of tumor tissue injected with Ce6@H-MnO2-PEG-SO3 nanoparticles. C1 represents a blood vessel (red channel), C2 represents the released Ru (green channel), C3 is the combined image of C1 and C2, and C4 is an image showing tumor tissue with Hoechst-labeled nuclei. D represents a 3D reconstructed image, and spots in the image are colored in spectrum based on the distance to the nearest blood vessel.

[0068] Figure 13b is a graph showing the number of apoptotic cells according to the distance from the blood vessel.

[0069] Figure 14 shows the results of analyzing the distribution of drugs and apoptotic cancer cells by antiangiogenic agents.

[0070] Figure 14a shows a 3D image of tumor tissue injected with Soraf-Ru@H-MnO2-PEG-SO3 nanoparticles. G1 represents blood vessels (red channel), G2 represents released Ru (green channel), G3 is a combined image of G1 and G2, and G4 is a magnified image of the white box area in G3. H represents a 3D reconstructed image, and spots in the image are colored in spectrum based on the distance to the nearest blood vessel.

[0071] Figure 14b is a graph showing the number of apoptotic cells according to the distance from the blood vessel.

[0072] Figure 15 shows the results of comparing the distribution of drugs and apoptotic cancer cells by photodynamic therapy and antiangiogenic agents.

[0073] Figure 15a shows a 3D image of tumor tissue injected with Soraf@H-MnO2-PEG-SO3 nanoparticles. B1 represents a blood vessel (red channel), B2 represents the released Ru (green channel), B3 is the combined image of B1 and B2, and B4 is an image showing tumor tissue with Hoechst-labeled nuclei. C represents a 3D reconstructed image, and spots in the image are colored in spectrum based on the distance to the nearest blood vessel.

[0074] Figure 15b is a graph showing the number of apoptotic cells according to the distance from the blood vessel.

[0075] Figure 16 shows the results of analyzing the distribution of drugs and apoptotic cancer cells by combined use of antiangiogenic agents and photodynamic therapy.

[0076] Figure 16a shows a 3D image of tumor tissue injected with Soraf-Ce6-Ru@H-MnO2-PEG-SO3 nanoparticles. B1 represents a blood vessel (red channel), B2 represents the released Ru (green channel), B3 is a combined image of B1 and B2, and B4 is a magnified image of the white box area in B3. C represents a 3D reconstructed image, and spots in the image are colored in spectrum based on the distance to the nearest blood vessel.

[0077] Figure 16b is a graph showing the number of apoptotic cells according to the distance from the blood vessel.

[0078] Figure 17 shows the results of comparing the distribution of drugs and apoptotic cancer cells by antiangiogenic agents alone and by combined use of antiangiogenic agents and photodynamic therapy.

[0079] Figure 17a shows a 3D image of tumor tissue injected with Soraf-Ce6@H-MnO2-PEG-SO3 nanoparticles. F1 represents a blood vessel (red channel), F2 represents the released Ru (green channel), F3 is the combined image of F1 and F2, and F4 represents the tumor tissue with Hoechst-labeled nuclei. G represents a 3D reconstructed image, and spots in the image are colored in spectrum based on the distance to the nearest blood vessel.

[0080] Figure 17b is a graph showing the number of apoptotic cells according to the distance from the blood vessel.

[0081] Figures 18a to 18d are images comparing vascular structural damage according to nanoparticles. The top image shows a 3D vascular image of a 1 mm tumor tissue prepared from a mouse that received an intravenous injection. The bottom image is an enlarged image of the orange boxed area in the right image.

[0082] Figure 19 shows the results of analyzing the distribution of drugs and apoptotic cancer cells by collagenase binding.

[0083] Figures 19a and 19b show 3D images of tumor tissues injected with Clg-Ru@H-MnO2-PEG-SO3 and Soraf-Clg-Ru@H-MnO2-PEG-SO3 nanoparticles, respectively. The left image shows blood vessels (red channel), the middle image shows released Ru (green channel), and the right image is a composite image of the left and middle images, respectively.

[0084] Figures 19c and 19d show 3D reconstructed images, where spots within the images are colored spectralally based on their distance to the nearest blood vessel.

[0085] Figures 19e, 19f, 19g, 19h, 19i, and 19j show heatmap images of Ru release from nanoparticles. The number of cancer cells containing Ru is indicated by spectral color based on the presence of the nearest blood vessel (shown in the panels). All nanoparticles were tested in five different groups (n = 5), and the corresponding data are shown in Set IV.

[0086] Figure 19k is a graph showing the number of apoptotic cells according to the distance from the blood vessel.

[0087] Figure 20 shows the results of comparing the distribution of drugs and apoptotic cancer cells by combined use of an antiangiogenic agent and photodynamic therapy and by combined use of an antiangiogenic agent, photodynamic therapy, and collagenase.

[0088] Figure 20a shows a 3D image of tumor tissue injected with Soraf-Clg-Ce6@H-MnO2-PEG-SO3 nanoparticles. H1 represents a blood vessel (red channel), H2 represents the released Ru (green channel), H3 is the combined image of H1 and H2, and H4 is an image showing tumor tissue with Hoechst-labeled nuclei. I represents a 3D reconstructed image, and spots in the image are colored in spectrum based on the distance to the nearest blood vessel.

[0089] Figure 20b is a graph showing the number of apoptotic cells according to the distance from the blood vessel.

[0090] Figure 21 is a heatmap image of Ru release by nanoparticles. The number of cancer cells containing Ru is indicated by spectral color based on the presence of the nearest blood vessel (shown in the panel).

[0091] Figure 22 shows the results of analyzing the distribution of drugs and apoptotic cancer cells by combined use of antiangiogenic agents, photodynamic therapy, and collagenase in a hypoxic region.

[0092] Figure 22a shows a 3D image of tumor tissue injected with Soraf-Clg-Ce6@H-MnO2-PEG-SO3 nanoparticles. B shows blood vessels (red channel), C shows released Ru (green channel), D shows HIF1α cells (magenta channel), and E shows the combined image of B-D.

[0093] Figure 22b is an enlarged image of the white box area in E of Figure 22a, showing the distribution of blood vessels (F1), apoptotic cells (F2), and HIF1α cells (F3), respectively. F4 is a combined image of F2 and F3, and F5 is a combined image of all F1-F3 with nuclear staining.

[0094] G1 of Fig. 22c is a combined image of B and C of Fig. 22a, and G2 of Fig. 22c represents a 3D reconstructed image of G1. H1 of Fig. 22c is a combined image of B and D of Fig. 22a, and H2 of Fig. 22c represents a 3D reconstructed image of H1.

[0095] K1 in Fig. 22d represents a 3D reconstructed image of G1 in Fig. 22c, K2 in Fig. 22d represents a 3D reconstructed image of G2 in Fig. 22c, and spots in the images are displayed in spectral colors based on the distance to the nearest blood vessel.

[0096] Figure 22e is a graph showing the number of apoptotic cells according to the distance from the blood vessel, and Figure 22f is a graph showing the number of HIF1α cells according to the distance from the blood vessel.

[0097] Figure 23 compares Ru penetration with and without oxygen-generating nanoparticles. The graph shows the number of cells containing Ru distributed according to distance from the blood vessel.

[0098]

[0099] Hereinafter, the present invention will be described in detail.

[0100] One aspect of the present invention provides a nano drug delivery system comprising a porous nanoparticle having at least a portion of a surface coated with manganese dioxide, wherein the surface of the nanoparticle is modified with a dual-functional ligand, wherein the dual-functional ligand comprises a polyethylene glycol moiety and a sulfate moiety.

[0101] The porous nanoparticles may include mesoporous nanoparticles.

[0102] The porous nanoparticles may include silica nanoparticles.

[0103] The dual-functional ligand is covalently bonded to the particle surface, including an amine group, and the covalent bond may include an amide bond. The amide bond may be formed between the acrylic acid of the polyacrylic acid and the amine of the dual-functional ligand.

[0104] The above porous nanoparticles include a surface modifier, and the surface modifier may be a cationic polymer and an anionic polymer sequentially coated.

[0105] The cationic polymer may contain an ammonium cation, and preferably may contain polyallylamine. The amine of the polyallylamine is cationized with ammonium.

[0106] The anionic polymer may contain a carboxylate anion, and preferably may contain polyacrylic acid. The acrylic acid of the polyacrylic acid is anionized with a carboxylate.

[0107] The above nano drug delivery system can be manufactured into a nano drug containing an anticancer drug and a protein by encapsulating an anticancer drug inside and binding a protein to the outside, and thus can be used for delivering an anticancer drug or protein, and can also be used as a composition for delivering an anticancer drug.

[0108] In the present invention, any material that can be encapsulated or bound to a nano drug carrier can be applied without limitation, and the material includes an anticancer drug and a protein, and the anticancer drug may include an anticancer agent, a photosensitizer, and an antiangiogenic agent, and the protein may include an enzyme and an antibody.

[0109] The above anticancer agent is not limited to any drug that is effective in treating cancer, and may include cytotoxic anticancer agents, targeted anticancer agents, immuno-oncology agents, metal-based anticancer agents such as platinum and ruthenium, low-molecular-weight compounds, and antibodies. The protein may include extracellular matrix-degrading enzymes and antibodies. In the present invention, doxorubicin, Ce6, sorafenib, ruthenium, and collagenase are used, but these are only examples and are not limiting.

[0110]

[0111] Another aspect of the present invention provides a dual-functional ligand represented by the following chemical formula 1, comprising an amine moiety for binding to porous nanoparticles and a sulfate moiety for capturing an enzyme capable of degrading tumor extracellular matrix.

[0112] [Chemical Formula 1]

[0113] PEG-L1-NH-L2

[0114] In the above chemical formula 1,

[0115] PEG is polyethylene glycol, Including,

[0116] n is an integer from 1 to 50000,

[0117] R is hydrogen or alkylene having C1 to C3,

[0118] L1 is C1 to C 10 is an alkylene, preferably an alkylene of C1 to C6,

[0119] One carbon of the above alkylene is or can be replaced with,

[0120] L2 has one or more carbons -OSO3 - C1 to C substituted with 10 alkylene, preferably having 1 to 3 carbon atoms -OSO3 - C1 to C substituted with 10 is an alkylene.

[0121] The number of ethoxy repeating units (n) of polyethylene glycol may be 1 to 50,000, 1 to 40,000, 1 to 30,000, 3 to 20,000, 3 to 10,000, 2 to 1,000, 2 to 100, or 3 to 50. In the present invention, the number of ethoxy repeating units is 5, but this is only an example and is not limited thereto.

[0122] The dual-functional ligand according to the present invention improves the biocompatibility, water solubility, physiological stability, and circulation time of H-MnO2-PEG-SO3 nanoparticles, and exhibits effects such as enhanced permeation and retention (EPR) effect and increased tumor accumulation through intracellular drug delivery. In addition, the sulfate group (-SO3) present in the PEGylated amine can capture extracellular matrix-degrading enzymes through electrostatic interactions.

[0123] The dual-functional ligand can bind to a protein, and the protein can be an enzyme or antibody capable of degrading the tumor extracellular matrix. The enzyme can be any substance capable of degrading the extracellular matrix, and can be introduced without limitation. The antibody can be introduced without limitation as long as it is a substance suitable for targeting tumor cells.

[0124] The above extracellular matrix may be selected from the group consisting of proteoglycans, elastin, fibronectin, vitronectin, laminin, gelatin, and collagen. In the present invention, collagen is bound to the functional ligand, but this is only an example and is not limited thereto.

[0125]

[0126] Another aspect of the present invention is a method for manufacturing a nano drug delivery system according to the present invention,

[0127] (a) A step of synthesizing SiO2 nanoparticles;

[0128] (b) a step of adding a potassium permanganate (KMnO4) solution to the synthesized SiO2 nanoparticle suspension and performing ultrasonic treatment to obtain nanoparticles coated with manganese oxide on SiO2;

[0129] (c) a step of dispersing the above nanoparticles in a Na2CO3 solution, stirring them, and then centrifuging them to obtain porous nanoparticles;

[0130] (d) a step of adding polyallylamine hydrochloride (PAH) to the aqueous dispersion of the porous nanoparticles to obtain a first reaction product, and then adding polyacrylic acid (PAA) to the solution in which the first reaction product is suspended to obtain a second reaction product; and

[0131] (e) A method for producing a nano drug carrier is provided, comprising the step of producing a nano drug carrier by binding a functional ligand to the second reaction product.

[0132] The method for manufacturing the above nano drug delivery system may additionally include a step of encapsulating an anticancer drug inside the nano drug delivery system and binding an extracellular matrix decomposing enzyme to the outside.

[0133]

[0134] Another aspect of the present invention provides an anticancer pharmaceutical composition comprising a nano drug delivery system according to the present invention.

[0135] Another aspect of the present invention provides a method for treating cancer, comprising administering to a subject in need of cancer treatment a pharmaceutically effective amount of the nano drug delivery system of claim 1, wherein an anticancer drug is encapsulated therein.

[0136] Another aspect of the present invention provides use of the nano drug carrier of claim 1, wherein an anticancer drug is encapsulated therein, in the manufacture of a drug for treating cancer.

[0137] The nano drug delivery system according to the present invention encapsulates an anticancer drug inside to selectively and specifically deliver the anticancer drug to cancer cells, binds an extracellular matrix-degrading enzyme to the outside, and can reach hypoxic regions by degrading the extracellular matrix in the tumor microenvironment, and has excellent drug permeability, so that it can be used as an anticancer pharmaceutical composition.

[0138] The above pharmaceutical composition induces apoptosis of cancer cells in the hypoxic region of the tumor microenvironment.

[0139] In the present invention, nanoparticles encapsulating photosensitizers exhibit a cancer cell death effect through the destruction of intratumoral blood vessels induced by photodynamic therapy. This is because reactive oxygen species generated upon activation of the photosensitizer cause severe oxidative damage to tumor cells, ultimately leading to the apoptosis of endothelial cells in the tumor vasculature. In the present invention, it was confirmed that Ce6 was released from the destroyed blood vessel walls and accumulated in surrounding tissues, increasing cell death.

[0140] While chemotherapy drugs like doxorubicin induce cell death by interfering with DNA during cell division, sorafenib is a multikinase inhibitor with antiproliferative, antiangiogenic, and apoptotic effects on tumor cells and tumor vasculature. Sorafenib can selectively slow tumor growth and spread by effectively reducing blood supply.

[0141] Furthermore, photodynamic therapy combined with antiangiogenic agents for cancer treatment may provide synergistic effects. While photodynamic therapy destroys tumor cells by generating reactive oxygen species, antiangiogenic therapy blocks the formation of new blood vessels, depriving tumor cells of essential resources. This suggests that targeting blood vessels using photosensitizers and antiangiogenic agents could be a useful strategy for enhancing drug penetration and efficacy in the tumor microenvironment.

[0142] The present invention takes into account the advantages of O2-generating MnO2 nanoparticles and uses nanoparticles combined with collagenase to decompose dense extracellular matrix and improve drug delivery.

[0143] In the present invention, the cancer may be a cancer that has developed resistance to conventional anticancer drugs, and may be a solid cancer, for example, a brain tumor / glioma, a pituitary adenoma, an acoustic neuroma, a uveal malignant melanoma, a meningioma, a pharyngeal cancer, a laryngeal cancer, a tongue cancer, a thyroid cancer, a breast cancer, a lung cancer, a thymoma, a thymic cancer, a mesothelioma, an esophageal cancer, a stomach cancer, a colon cancer, a hepatocellular cancer, a biliary tract cancer, a pancreatic cancer, a renal cell cancer, a bladder cancer, a prostate cancer, a renal pelvis / ureter cancer, a penile cancer, a testicular tumor, a uterine cancer, an ovarian cancer, a vulvar cancer, a skin cancer, a malignant melanoma (skin), a basal cell carcinoma, a precursor of a skin cancer, an intraepidermal carcinoma, a spinous cell carcinoma, a mycosis fungoides, a malignant bone tumor (osteosarcoma), a soft tissue sarcoma, a chondrosarcoma, a malignant fibrous histiocytoma, or a metastatic cancer thereof, but is not limited thereto.

[0144] In the pharmaceutical composition according to the present invention, the pharmaceutical composition can be directly applied to the skin during clinical administration, or administered in various oral and parenteral dosage forms. When formulated, it can be manufactured using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0145] Oral dosage forms include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsions, syrups, granules, elixirs, and troches, which contain, in addition to the active ingredient, diluents (for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine), lubricants (for example, silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidine, and, if desired, disintegrating agents or effervescent mixtures such as starch, agar, alginic acid or its sodium salt, and / or absorbents, coloring agents, flavoring agents, and sweetening agents.

[0146] The pharmaceutical composition according to the present invention can be administered parenterally, and parenteral administration can be by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. In addition, it can be administered by a method of direct application to the skin, and when applied, it can be manufactured and applied in the form of ointment, spray, toner, lotion, cream, massage cream, essence, cleansing product, pack, powder, patch, gel, etc., and any conventional application method can be used without particular limitation on the above types.

[0147] At this time, in order to formulate the pharmaceutical composition into a dosage form for parenteral administration, the pharmaceutical composition may be mixed with water together with a stabilizer or buffer to prepare a solution or suspension, which may be prepared into a unit dosage form in an ampoule or vial. The composition may be sterilized and / or contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying promoters, salts for osmotic pressure control, and / or buffers, and other therapeutically useful substances, and may be formulated according to conventional mixing, granulation, or coating methods.

[0148]

[0149] Another aspect of the present invention is a method for evaluating drug distribution in a tissue after injection of a nano drug carrier according to the present invention,

[0150] (a') A step of forming cancer tissue by transplanting a culture medium containing cancer cells into an animal;

[0151] (b') The volume of the cancer tissue is 100 to 2000 mm 2 When this is done, the step of injecting the nano drug delivery system of clause 4;

[0152] (c') A step of collecting cancer tissue injected with the nano drug delivery system and making it transparent;

[0153] (d') A method for evaluating drug distribution is provided, comprising a step of evaluating drug distribution in the above transparent cancer tissue sample.

[0154] The cancer described above may be a cancer resistant to conventional anticancer drugs, a hematological cancer, a solid cancer, or a metastatic cancer thereof. In the present invention, the distribution of the nano drug carrier was evaluated using breast cancer tissue samples, but this is merely an example and is not intended to be limiting.

[0155] The animal of step (a') above may be, but is not limited to, a human or non-human mammal, for example, a non-human primate such as a monkey, a rat, a dog, a cat, a rabbit, a horse, or a cow. The animal may be a rat, but this is by way of example only and is not limited thereto.

[0156] The nano drug delivery system of the above step (b') may be encapsulated with one or more substances selected from the group consisting of ruthenium (Ru) and anticancer drugs, and may have an extracellular matrix decomposing enzyme bound to the outside.

[0157] The step of making the tissue transparent in the above step (c') can be used without limitation with any known tissue making method or system, and is a step of making the tissue transparent according to a known method using a composition for making the tissue transparent. In this case, the ruthenium is characterized in that it induces apoptosis of cancer cells and is not removed in the step of making the tumor tissue transparent.

[0158] Transparency of the organization can be performed using a commercialized system or a laboratory method, and in the present invention, a commercialized system is selected as an example.

[0159] A method is known that employs a method of tissue structuring using hydrogel and lipid removal using electrophoresis as a system for tissue transparency, and the X-CLARITY™ tissue transparency system can be used as an example.

[0160] Methods for tissue transparency using hydrogels and electrophoresis include, for example,

[0161] A step of fixing the tumor tissue with a fixative solution (step 1);

[0162] A step of infiltrating the hydrogel into the tumor tissue (step 2); and

[0163] The step of removing lipids by electrophoresis of the tumor tissue in a solution for transparency (step 3) may be included.

[0164] The above fixing solution may be paraformaldehyde, and the step of fixing the tumor tissue may be performed at 0 to 10°C for 12 to 36 hours.

[0165] The above hydrogel can be used without limitation as long as it can maintain the structure of the tissue, and in order to facilitate penetration of the hydrogel, it can be penetrated by a method of polymerizing or crosslinking after penetration in a monomer or oligomer, or in a non-crosslinked state.

[0166] The step of removing lipids by electrophoresis can be performed under a current of 0.5 to 1.5 A in a composition containing a surfactant for removing lipids.

[0167] The above step (d') is a step for evaluating the distribution of the drug in the transparent cancer tissue sample. For this evaluation, the transparent cancer tissue sample can be imaged in three dimensions. The three-dimensional imaging is performed by reconstructing the initially acquired confocal Z-stack images into a three-dimensional image, and software for three-dimensional imaging can be used for this reconstruction.

[0168] The 3D reconstructed image can be used to quantify drug distribution within the tissue using software. The software used for this quantification is not particularly limited, but the Imaris program is an example. To quantify drug distribution, nuclei, blood vessels, and drug-containing cancer cells can be detected within the 3D image, and the distance from the nearest blood vessel to the drug-containing cancer cells can be quantified.

[0169] The maximum distance that a nano drug carrier loaded with an anticancer drug penetrates from the nearest blood vessel can be expressed as maximum drug penetration depth (DPDmax), and half of this distance is DPD 50 can be expressed as DPD in intratumoral blood vessels. 50 The number of cancer cells containing the drug (drug penetration or DPA) 50 ) can be used to quantify drug distribution.

[0170] Quantification of the above drug distribution allows for the evaluation of the extent to which the encapsulated drug is delivered when the nano drug carrier is injected, as the location and amount of the drug are displayed within a three-dimensional image.

[0171] The above evaluation method can count drug-containing cells at a level of 100 to 1000 μm around the blood vessel.

[0172] Another aspect of the present invention is a method for evaluating the distribution of apoptotic cancer cells in a tissue after injection of a nano drug carrier according to the present invention,

[0173] (a'') A step of forming cancer tissue by transplanting a culture medium containing cancer cells into an animal;

[0174] (b'') The volume of the cancer tissue is 100 to 2000 mm 2 When this is done, the step of injecting the nano drug delivery system of clause 4;

[0175] (c'') A step of collecting cancer tissue injected with the nano drug delivery system and making it transparent;

[0176] (d'') A method for evaluating the distribution of apoptotic cancer cells is provided, comprising a step of evaluating the distribution of apoptotic cancer cells in the above-mentioned transparent cancer tissue sample.

[0177] In the above method for evaluating the distribution of apoptotic cancer cells, the description of the above method for evaluating the distribution of drugs applies equally unless they are contradictory.

[0178] However, in the above method for evaluating the distribution of apoptotic cancer cells, instead of the maximum drug penetration depth (DPDmax), the maximum distance at which cells killed by a nano drug delivery vehicle loaded with an anticancer drug from the nearest blood vessel exist can be expressed as the maximum cell death depth (CDDmax).

[0179] In specific embodiments and experimental examples of the present invention, the inventors prepared SO3-PEG-amine as a dual-functional ligand (Fig. 1), and combined it with manganese dioxide nanoparticles to prepare a novel nano drug carrier (H-MnO2-PEG-SO3) (Fig. 2), and analyzed the characteristics of the nanoparticles using EF-TEM to confirm that the nanoparticles were successfully synthesized at each step (Figs. 3a to 3e and Table 1). After preparing a nano drug carrier containing anticancer agents (Ce6, doxorubicin, sorafenib, ruthenium, and collagenase) in the nano drug carrier, the optical absorption characteristics were measured using UV-Vis spectroscopy (Figs. 4a and 4b), confirming that the anticancer agents were successfully loaded.

[0180] In addition, the nano drug carrier (H-MnO2-PEG-SO3) was confirmed to be able to improve a hypoxic environment by extinguishing hydrogen peroxide (Figs. 5 and 6) and generating oxygen (Fig. 7). In addition, the collagenase enzyme activity and nanoparticle diffusion effect were measured in the nano drug carrier (Clg@H-MnO2-PEG-SO3) bound to collagenase, and it was confirmed that the enzyme activity of the nanoparticles was maintained even after binding to collagenase (Fig. 8), and that the degradation of collagen by the enzyme occurred, resulting in improved diffusion of the nanoparticles (Figs. 9a and 9b).

[0181] The present invention analyzed the distribution of the drug and apoptotic cancer cells within tumor tissue by injecting nanoparticles containing an anticancer drug manufactured according to the present invention into mice injected with the 4T1 murine mammary carcinoma cell line. Quantitative analysis was performed by tissue-clearing tumor samples, followed by three-dimensional imaging using TUNEL staining and immunohistochemical staining.

[0182] As a result of analyzing the drug distribution according to particle size, it was confirmed that the drug penetration amount of Ru@H-MnO2-PEG-SO3 (62 nm) was significantly higher than that of Ru@H-MnO2-PEG-SO3 (283 nm) (Fig. 10).

[0183] Analysis of the cancer cell killing effects of Ru@H-MnO2-PEG-SO3 and DOX@H-MnO2-PEG-SO3 showed that chemotherapy (DOX) had the highest cell killing depth (CDD) max ) value was higher, which showed excellent drug permeability, but the number of apoptotic cells was much lower (Fig. 11). As a result of drug distribution analysis of Ru@H-MnO2-PEG-SO3 and Ce6-Ru@H-MnO2-PEG-SO3, the maximum drug penetration depth (DPP) of photodynamic therapy (Ce6) was max ) value was higher, and the number of cells containing Ru was more than twice as high, confirming excellent drug delivery effect (Fig. 12).

[0184] Analysis of the cancer cell killing effects of DOX@H-MnO2-PEG-SO3 and Ce6@H-MnO2-PEG-SO3 showed that the spatial distribution of apoptotic cells was DPD max Although similar in aspect, the number of apoptotic cells induced by photodynamic therapy (Ce6) was significantly higher than that induced by chemotherapy (DOX), confirming that the cancer cell killing effect of photodynamic therapy was superior (Fig. 13).

[0185] Drug distribution analysis of Ce6-Ru@H-MnO2-PEG-SO3 and Soraf-Ru@H-MnO2-PEG-SO3 showed that DPP of antiangiogenic agent (Soraf) max The number of cells containing Ru and Ru was significantly higher, confirming excellent drug delivery effect in the presence of antiangiogenic agents (Fig. 14). Analysis of the cancer cell killing effect of Ce6@H-MnO2-PEG-SO3 and Soraf@H-MnO2-PEG-SO3 showed that the number of apoptotic cells induced by the antiangiogenic agent (Soraf) was significantly higher than that induced by photodynamic therapy (Ce6), confirming that the cancer cell killing effect of the antiangiogenic agent was superior (Fig. 15).

[0186] Drug distribution analysis of Soraf-Ru@H-MnO2-PEG-SO3 and Soraf-Ce6-Ru@H-MnO2-PEG-SO3, DPP of antiangiogenic agent-photodynamic therapy (Soraf-Ce6) max The number of cells containing Ru and Ru was significantly high, confirming the excellent drug delivery effect due to the synergistic effect of antiangiogenic agents and photodynamic therapy (Fig. 16). As a result of analyzing the cancer cell killing effect of Soraf@H-MnO2-PEG-SO3 and Soraf-Ce6@H-MnO2-PEG-SO3, extensive destruction of the blood vessel wall was observed due to the synergistic effect of antiangiogenic agent-photodynamic therapy (Soraf-Ce6), and the number of apoptotic cells induced by antiangiogenic agent-photodynamic therapy (Soraf-Ce6) was significantly higher than that induced by antiangiogenic agent (Soraf) alone, confirming the superior cancer cell killing effect of antiangiogenic agents (Fig. 17).

[0187] In addition, in order to analyze the drug release effect when treated with a combination of antiangiogenic agents and photodynamic therapy, Dox@H-MnO2-PEG-SO3, Ce6@H-MnO2-PEG-SO3, Soraf-Ru@H-MnO2-PEG-SO3, and Soraf-Ce6@H-MnO2-PEG-SO3 were injected and the blood vessel structure was observed. As a result, it was confirmed that the drug release effect was increased when treated with a combination of antiangiogenic agents and photodynamic therapy, as the damaged blood vessel part after blood vessel staining was more evident than when only sorafenib was administered (Figs. 18a to 18d).

[0188] Drug distribution analysis of Clg-Ru@H-MnO2-PEG-SO3 and Soraf-Clg-Ru@H-MnO2-PEG-SO3 showed that DPP was activated in the presence of extracellular matrix (ECM) degrading enzyme (Clg). maxThe number of cells containing Ru and Ru was incomparably higher than that of the nano drug carrier in the presence of an antiangiogenic agent (Soraf-Ru@H-MnO2-PEG-SO3), confirming its superior drug delivery effect in the presence of an extracellular matrix-degrading enzyme (Clg) (Figs. 19 to 19d). These results confirmed the significant influence of Clg-Ru@H-MnO2-PEG-SO3 in enhancing drug delivery to hypoxic areas by targeting tumor extracellular matrix (Figs. 19e to 19j).

[0189] Additionally, the drug delivery effect was analyzed when treating with a combination of antiangiogenic agents and ECM-degrading enzymes. The results showed that the combination of antiangiogenic agents and ECM-degrading enzymes had a higher effect on DPP than the ECM-degrading enzyme alone. max It was confirmed that the number of cells containing values ​​and Ru was significantly high (Figs. 19a to 19d and 19k).

[0190] Distribution analysis of apoptotic cells induced by Soraf-Ce6@H-MnO2-PEG-SO3 and Soraf-Clg-Ce6@H-MnO2-PEG-SO3 showed that the extracellular matrix degradation of ECM-degrading enzyme (Clg) combined with antiangiogenic agent (Soraf) and photodynamic therapy (Ce6) generated more widely dispersed apoptotic cells than the antiangiogenic agent-photodynamic therapy combination (Fig. 20a), and the number of apoptotic cells was significantly higher with increasing distance from blood vessels (Fig. 20b). These results suggest that H-MnO2-PEG-SO3 with collagenase enhances drug penetration into the tumor microenvironment, thereby enhancing the degree of apoptosis compared to other treatments without collagenase (Fig. 21).

[0191] In addition, the distribution analysis of apoptotic cells and HIF1α-positive cells in tumor tissues treated with Soraf-Clg-Ce6@H-MnO2-PEG-SO3 revealed the coexistence of apoptotic cells with HIF1α-positive cells in the tumor microenvironment (Figs. 22a to 22c), and apoptotic cells were observed in blood vessels at 380 μm DPD. max , and HIF1α-positive cells were confirmed to be distributed at a distance of 40–320 μm from the blood vessels (Fig. 22d, Fig. 22e, and Fig. 22f). These results confirmed that the nano drug carrier could reach the hypoxic region by combining antiangiogenic agent-photodynamic therapy and ECM-degrading enzyme. This suggests that the catalytic properties of MnO2 and the rapid degradation of collagen, a major extracellular matrix component in the tumor microenvironment, of the ECM-degrading enzyme helped reduce the interstitial fluid pressure and enhance drug release deep into the hypoxic tumor microenvironment. In addition, it suggests that the co-delivery of ECM-degrading enzyme with photosensitizer and antiangiogenic agent can effectively kill hypoxic cells in solid tumors.

[0192] In addition, in order to confirm the effect of the oxygen generating ability of the nano drug delivery system according to the present invention on drug delivery, Ru released from Ce6-Ru@H-MnO2-PEG-SO3 and Ce6-Ru@Lip was analyzed, and it was confirmed that the Ru release amount of Ce6-Ru@H-MnO2-PEG-SO3 was twice as high, confirming that the drug release effect was significantly higher than that of liposomes (Fig. 23).

[0193]

[0194] Hereinafter, the present invention will be described in detail through examples and experimental examples.

[0195] However, the following examples and experimental examples are only intended to specifically illustrate one aspect of the present invention, and the present invention is not limited to the following examples and experimental examples.

[0196]

[0197] <Example 1> Synthesis of a dual-functional ligand

[0198] To improve the biocompatibility of the H-MnO2-based drug delivery system, a novel dual-functional -SO3-PEG-amine (5) was synthesized through the synthetic steps shown in Fig. 1.

[0199] Specifically, starting from commercially available ethylenediamine, the first monoprotection of ethylenediamine was performed in the presence of di-tert-butyl dicarbonate and triethylamine in dichloromethane (DCM) at 0°C to produce monosubstituted tert-butyl(2-aminoethyl)carbanate (1). Next, 2, 5, 8, 11, 14, 17-hexaoxicosan-20-oic acid was reacted with compound (1) in the presence of EDC·HCl / HOBt (EDC=1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole hydrate; TEA, triethylamine) to produce tert-butyl (20-oxo-2,5,8,11,14,17-hexaoxa-21-azatricosan-23-yl)carbamate (2). Next, tert-butyl deprotection was performed using a solution of HCl (4 M) in anhydrous dioxane at 0°C to generate the intermediate N-(2-aminoethyl)-2,5,8,11,14,17-hexaoxaicosan-20-amide (3), to which glycidol was added to synthesize N-(2-((2,3-dihydroxypropyl)amino)ethyl)-2,5,8,11,14,17-hexaoxaicosan-20-amide (4). Finally, the compound (4) was treated with SO3.pyr dissolved in dry DMF for 30 h at room temperature, and the pH was basified to 9.0 using a saturated NaHCO3 solution to synthesize the final compound -SO3-PEG-amine (5) as a bis-sodium salt.

[0200] <Example 2> Synthesis of H-MnO2-PEG-SO3 nanoparticles

[0201] A schematic diagram of the synthesis steps of H-MnO2-PEG-SO3 nanoparticles is shown in Fig. 2.

[0202] ingredient

[0203] Tetraethyl orthosilicate (TEOS), (3-aminopropyl)-triethoxysilane (APTES), poly(allylamine hydrochloride) (PAH, MW 15,000), polyacrylic acid (PAA, MW 1,800), ammonium hydroxide (NH3·H2O), cyclohexane, and Triton X-100 were purchased from Sigma-Aldrich, and potassium permanganate (KMnO4), 1-(3-Diaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), sodium carbonate (Na2CO3), and n-hexanol were purchased from Alfa Aesar Chemical Ltd. (Korea). All reagents were used without further purification, and ultrapure water (18.2 MΩ cm, Milli-Q water, Millipore) was used in the chemical synthesis and aqueous solution preparation steps.

[0204] Step 1: Synthesis of mesoporous H-MnO2 nanoparticles (NPs)

[0205] H-MnO2NPs were synthesized using solid SiO2 (sSiO2) NPs as a template using the reverse microemulsion method. Specifically, 5.3 mL of Triton X-100, 22.5 mL of cyclohexane, and 5.4 mL of n-hexanol were mixed in a 100-mL round-bottom flask and stirred uniformly for 5 minutes. Subsequently, 1.0 mL of water and 0.75 mL of ammonia were added, and the mixture was stirred for 30 minutes. Then, 100 μL of APTES and 500 μL of TEOS were mixed and slowly added to the flask, and the reaction was continued at 25°C for 24 hours. sSiO2NPs were collected by centrifugation (14,800 rpm, 10 min) and washed three times with ethanol and water. Then, KMnO4 aqueous solution (15 mL, 10 mg mL-1) was added to the sSiO2NPs (20 mL, 1 mg / mL) suspension under ultrasonic treatment, and the reaction was stirred for 8 h. Then, mesoporous sSiO2@MnO2NPs were collected by centrifugation at 14,800 rpm for 10 min. Finally, sSiO2@MnO2NPs were dispersed in Na2CO3 (2 M) solution, stirred at 60 °C for 12 h, and centrifuged at 14,800 rpm to obtain mesoporous H-MnO2NPs, which were washed three times for further use.

[0206] Step 2: Surface modification of H-MnO2

[0207] For surface modification, 25 mg of PAH and 20 mL of an aqueous dispersion of H-MnO2 (2.5 mg / mL) were placed in a 100 mL flask and stirred for 2.5 h. Then, H-MnO2-PAH was collected by high-speed centrifugation and washed three times with water. After that, H-MnO2-PAH was resuspended in 20 mL of water, and 25 mg of PAA was added. The reaction mixture was stirred for an additional 2.5 h, and finally, the product H-MnO2-PAH / PAA was collected by centrifugation and washed three times with water.

[0208] The prepared H-MnO2-PAH / PAA was mixed with 14.3 mg of EDC and 10.8 mg of NHS in 10 mL of water, and 12.5 mg of the functional ligand -SO3-PEG-amine(5) obtained in Example 1 was added, followed by stirring overnight. Surface-modified H-MnO2-PEG-SO3 nanoparticles (NP, 62.19 ± 0.22 nm) were then obtained by centrifugation and washed with ultrapure water for further use.

[0209] <Example 3> Synthesis of H-MnO2-PEG-SO3 nanoparticles (NPs) loaded with anticancer drugs

[0210] 1) Dox@H-MnO2-PEG-SO3

[0211] For doxorubicin (Dox) loading, H-MnO2-PEG-SO3NPs (5 mg) were added to 5 mL of phosphate-buffered saline (PBS, pH 7.4) containing Dox (5 mg), stirred overnight in the dark to allow drug absorption, and Dox-loaded nanoparticles were obtained, which were then purified by centrifugation at 6500 rpm for 10 minutes.

[0212] 2) Ru@H-MnO2-PEG-SO3

[0213] In the same manner as loading doxorubicin in 1) above, 5 mg of ruthenium (Ru) was loaded onto H-MnO2-PEG-SO3NPs (5 mg).

[0214] 3) Ce6@H-MnO2-PEG-SO3

[0215] In the same manner as loading doxorubicin in 1) above, 5 mg of Ce6 was loaded onto H-MnO2-PEG-SO3NPs (5 mg).

[0216] 4) Soraf@H-MnO2-PEG-SO3

[0217] In the same manner as loading doxorubicin in 1) above, 5 mg of sorafenib (Soraf) was loaded onto H-MnO2-PEG-SO3NPs (5 mg).

[0218] The encapsulation efficiency (EE) of the nanoparticles manufactured in 1) to 4) above was calculated using the following formula. The EE (%) of Dox, Ru, Soraf, and Ce6 were confirmed to be 86.0%, 83.6%, 81%, and 88.4%, respectively, and the obtained nanoparticles were used in further experiments.

[0219] [Mathematical Formula 1]

[0220] Encapsulation efficiency (%) = {(total drug added - free non-entrapped drug) / (total drug added)} Х 100

[0221] 5) Soraf-Ru@H-MnO2-PEG-SO3

[0222] To simultaneously load sorafenib-ruthenium, 5 mg of H-MnO2-PEG-SO3NPs were dispersed in 5 mL of PBS / DMSO (1:1) solution containing sorafenib (2.5 mg) and ruthenium (2.5 mg). The mixture was stirred in the dark for 24 h and then centrifuged at 6500 rpm for 10 min to collect Soraf-Ru@H-MnO2-PEG-SO3 nanoparticles.

[0223] 6) Soraf-Ce6@H-MnO2-PEG-SO3

[0224] Soraf-Ce6 was loaded onto H-MnO2-PEG-SO3NP (5 mg) in the same manner as the method of simultaneously loading sorafenib-ruthenium in 5) above.

[0225] 7) Ce6-Ru@H-MnO2-PEG-SO3

[0226] Ce6-Ru was loaded into H-MnO2-PEG-SO3NP (5 mg) in the same manner as the method of simultaneously loading sorafenib-ruthenium in 5) above.

[0227] 8) Soraf-Ce6-Ru@H-MnO2-PEG-SO3

[0228] Sorafenib (1.67 mg), Ce6 (1.67 mg), and ruthenium (1.67 mg) were loaded into H-MnO2-PEG-SO3NP (5 mg) in the same manner as the method of simultaneously loading sorafenib-ruthenium, except that PBS was used instead of the PBS / DMSO (1:1) solution of 5) above.

[0229] 9) Clg@H-MnO2-PEG-SO3

[0230] 5 mg of H-MnO2-PEG-SO3NPs were added to 5 mL of PBS (pH 7.4) containing 2.5 mg of collagenase, and the mixture was stirred at 4°C for 24 h. Then, Clg@H-MnO2-PEG-SO3NP (Clg = collagenase) was collected by centrifugation at 6500 rpm for 10 min and washed three times.

[0231] Collagenase captured in H-MnO2-PEG-SO3 was purified by centrifugation using a 10 kDa cut-off filter (AMICON Ultra-2 mL 10 kDa) and then washed with 1X PBS (pH 7.4).

[0232] 10) Clg-Ru@H-MnO2-PEG-SO3

[0233] For ruthenium loading, 5 mg of Clg@H-MnO2-PEG-SO3NP was added to 5 mL of PBS (pH 7.4) containing ruthenium (5 mg), and the mixture was stirred overnight at 4°C and then centrifuged at 6500 rpm for 10 min to obtain ruthenium-loaded Clg-Ru@H-MnO2-PEG-SO3NP.

[0234] 11) Clg-Soraf@H-MnO2-PEG-SO3

[0235] Clg-Soraf@H-MnO2-PEG-SO3 was obtained by the same method as the method of loading ruthenium into Clg@H-MnO2-PEG-SO3NP in 10) above.

[0236] 12) Soraf-Clg-Ce6@H-MnO2-PEG-SO3

[0237] For simultaneous loading of sorafenib-Ce6, Clg@H-MnO2-PEG-SO3NP (5 mg) was dispersed in 5 mL of PBS containing sorafenib (2.5 mg) and Ce6 (2.5 mg) and stirred at 4°C for 24 h. Then, centrifugation at 6500 rpm for 10 min yielded Sorafen-Clg-Ce6@H-MnO2-PEG-SO3NP.

[0238] 13) Soraf-Clg-Ru@H-MnO2-PEG-SO3

[0239] In the same manner as in the method of simultaneously loading sorafenib-Ce6 into Clg@H-MnO2-PEG-SO3NP in 12) above, sorafenib (2.5 mg) and ruthenium (2.5 mg) were co-loaded into a solution of Clg@H-MnO2-PEG-SO3NPs (5 mg) in PBS to obtain Soraf-Clg-Ru@H-MnO2-PEG-SO3NP.

[0240] Collagenase bound to the above H-MnO2-PEG-SO3 (Clg-Ru@H-MnO2-PEG-SO3, Clg-Soraf@H-MnO2-PEG-SO3, and Soraf-Clg-Ru@H-MnO2-PEG-SO3) was purified by centrifugation using a 10 kDa cutoff filter (AMICON Ultra-2 mL 10 kDa) and washed with 1×PBS (pH 7.4). The synthesized NPs were used immediately for further experiments or stored at 4°C until use.

[0241] <Comparative Example 1> Synthesis of mesoporous H-MnO2 nanoparticles (283.5 ± 7.17 nm)

[0242] To analyze drug penetration characteristics according to particle size, H-MnO2-PEG-SO3 (283.5 ± 7.17 nm) nanoparticles were synthesized.

[0243] First, 28 mL of ethanol, 4 mL of deionized water, and 1 mL of NH3·H2O were added to a 100 mL round-bottom flask, and the mixture was stirred at 60°C for 5 min. Then, 1 mL of TEOS was added to the flask, and the mixture was reacted at 60°C for 2.5 h. Then, sSiO2NPs were collected by centrifugation (14,800 rpm, 10 min), washed three times with ethanol and water, and stored in water until further use.

[0244] Afterwards, to prepare sSiO2@MnO2NP, the sSiO2NP suspension was added to water (40 mL) containing 1.2 g of KMnO4 dissolved therein while sonicating. The reaction mixture was sonicated for 2 h and stirred at 25°C for 18 h. Then, sSiO2@MnO2NP was obtained by centrifugation (14,800 rpm, 15 min).

[0245] Finally, sSiO2@MnO2NP was dispersed in Na2CO3 (2 M) solution and stirred at 60°C for 12 h, followed by centrifugation (14,800 rpm) to obtain H-MnO2NP, which was washed three times. The particle size of H-MnO2NP was confirmed to be 283.5 ± 7.17 nm using TEM.

[0246] <Comparative Example 2> Ce6-Ru encapsulated in liposomes

[0247] To compare the penetration effect of ruthenium with and without oxygen generation, Ce6-Ru@Lip was prepared, and liposomes containing Ce6 and ruthenium were prepared using the thin-film hydration method.

[0248] Specifically, DSPC / DSPE-PEG2000 / cholesterol (10:0.2:5 μmoles) was dissolved in chloroform, and then Ce6 solution dissolved in DMF and ruthenium solution dissolved in methanol were added to the mixture at a molar ratio of 1:1. The solvent in the mixture was slowly removed using rotary evaporation until a thin film was formed. The lipid layer was then hydrated with PBS, and the PBS dispersion of liposomes was briefly sonicated. Unilamellar vesicles were prepared by liposome extrusion using a 0.2 μm pore size polycarbonate membrane (Avanti polar Lipids, USA). Ce6 and ruthenium not encapsulated in the liposomes were removed by centrifugation at 14,000 × g for 30 min using an Amicon Ultra centrifugal filter. The formed liposomes were characterized using a dynamic light scattering spectrophotometer (DLS-7000, Ohtsuka Electric Co., Ltd., Japan).

[0249] <Experimental Example 1> Characteristics of H-MnO2-PEG-SO3 nanoparticles

[0250] The particle size and surface morphology of the nanoparticles manufactured in Example 2 were measured using energy-filtering TEM (EF-TEM).

[0251] First, before measurement, nanoparticles were suspended in water and samples were prepared on a perforated carbon film mounted on a copper grid. A drop of the diluted nanoparticle solution was placed on the grid and dried overnight at 25°C. The zeta potential of the nanoparticles was determined using dynamic light scattering (DLS) with a 633 nm laser (Zetasizer Nano ZS, Malvern Instruments Ltd., UK). All measurements were equilibrated in water at 25°C for 120 s. The number of runs and duration for each measurement were set automatically.

[0252] The UV-Vis spectra of nanoparticles were measured using an Evolution™ 60 UV-Vis Spectrophotometer (Thermo Fischer Scientific, USA).

[0253] As shown in Figures 3a to 3e, sSiO2NPs exhibited a very uniform and monodisperse morphology, whereas the rough surface of MnO2@SiO2 was clearly observed upon MnO2 coating. The uniform hollow structure and spherical morphology of H-MnO2NPs with an average diameter of approximately 62 nm were clearly visualized using EF-TEM.

[0254] SampleMean particle size(nm)Zeta potential(mV)MnO2NPs62.19±0.22-32.9MnO2-PEG NPs69.0±0.66-17.6

[0255]

[0256] As shown in Table 1, the stepwise changes in the ζ potential during the synthesis of H-MnO2-PEG-SO3 in Example 2 indicate that the nanoparticles were successfully modified at each step. After coating MnO2 (MnO2@sSiO2), the ζ potential of sSiO2 NPs changed from -38.0 to -21.6 mV. In addition, after etching the silica, the ζ potential of H-MnO2 NPs showed -32.9 mV. It was confirmed that the final product, H-MnO2-PEG-SO3, had a cathode surface with a ζ potential of -17.6 mV.

[0257] <Experimental Example 2> Confirmation of the production of H-MnO2-PEG-SO3 nanoparticles loaded with anticancer drugs.

[0258] To confirm whether the nanoparticles according to Example 3 of the present invention were formed normally, the light absorption characteristics were measured using UV-Vis spectroscopy.

[0259] As shown in Fig. 4a and Fig. 4b, when Ce6 was loaded into H-MnO2-PEG-SO3NP, characteristic absorption peaks were observed at 400 and 690 nm, for nanoparticles loaded with Dox, an absorption peak was observed at approximately 495 nm, for nanoparticles loaded with Ru, absorption peaks were observed at 290 and 464 nm, and for nanoparticles bound with collagenase, an absorption peak representing the characteristic peak of collagenase was observed at 260 nm (Fig. 4a).

[0260] When Ru and Ce6 were co-loaded into H-MnO2-PEG-SO3NP, absorption peaks were observed at approximately 290, 420, 464, and 690 nm, and for nanoparticles loaded with Soraf and Ru, absorption peaks were observed at approximately 250, 290, and 470 nm (Fig. 4b).

[0261] Collagenase-bound Clg-Ru@H-MnO2-PEG-SO3NPs showed characteristic absorption peaks at 260, 290, and 464 nm, and Soraf-Ce6-Ru@MnO2-PEG NPs loaded with Soraf, Ce6, and Ru together showed characteristic absorption peaks at 250, 294, 424, and 680 nm (Fig. 4b).

[0262] The above results suggest that anticancer drugs and collagenase were successfully loaded into H-MnO2-PEG-SO3 nanoparticles.

[0263] <Experimental Example 3> Decomposition of hydrogen peroxide by nanoparticles

[0264] MnO2 (90 μM) was added to cell medium containing 10% FBS at 37°C, and various concentrations of H2O2 were added to react. The H2O2 decay by MnO2 was measured over time using a fluorescence-measuring H2O2 assay kit (catalog no. MAK165, Sigma). This kit utilizes a peroxidase substrate that generates a red fluorescent product (λex = 540 nm / λem = 590 nm) after reacting with H2O2. Meanwhile, cell medium containing 10% FBS was used as a control. Finally, the fluorescence signal (the decay of H2O2 in the presence and absence of MnO2) was collected and analyzed using a fluorescence microplate reader (Multi-Mode Microplate Reader; Molecular Devices, iD3).

[0265] Since MnO2 catalyzes the conversion of H2O2 to O2, MnO2NPs have been widely used to alleviate tumor hypoxia and sensitize photodynamic therapy cells by providing an O2 substrate. As shown in Figure 5, the reaction between MnO2 and H2O2 is a complex reaction that generates O2 by consuming H+ ions and decomposing H2O2.

[0266] As shown in Fig. 6a, when H-MnO2-PEG-SO3 (90 μM) was added to various concentrations of H2O2 (0–100 mM), the fluorescence of the peroxidase substrate continuously decreased. On the other hand, when only H2O2 was sequentially added, the fluorescence of the peroxidase substrate continuously increased (Fig. 6b). These results indicate that H-MnO2-PEG-SO3 nanoparticles quenched H2O2 to generate a red fluorescent product.

[0267] <Experimental Example 4> In vitro oxygen production using nanoparticles

[0268] Various concentrations of H-MnO2-PEG-SO3 were added to H2O2 (1 mM), and O2 production was monitored using a portable dissolved oxygen meter (catalog no. HI9146, Hanna Instruments). The probe was calibrated according to the manufacturer's instructions.

[0269] As a result, as shown in Fig. 7, as the concentration of H-MnO2-PEG-SO3 increased, the production of oxygen increased, and since H2O2 generates more oxygen as the reaction time increases, it was observed that a significant amount of O2 was generated by the reaction of MnO2 (20, 60, 100 μM) and H2O2 (1 mM). These results suggest that H-MnO2-PEG-SO3 can improve hypoxia in hypoxic regions by converting high concentrations of H2O2 in the tumor microenvironment into oxygen.

[0270] <Experimental Example 5> Enzyme activity of Clg@H-MnO2-PEG-SO3

[0271] The enzyme activity of free and encapsulated collagenases was assayed using the Enzchek Gelatinase / Collagenase Assay Kit (catalog no. D-12060, Molecular Probes). 80 μL of collagenase buffer (1 × 10), 20 μL of collagen-FITC, and 100 μL of sample (free Clg / Clg@H-MnO2-PEG-SO3) were used in this experiment. The reaction mixture was incubated at 25°C, and the enzyme activity was measured over time, and the fluorescence was measured using a microplate reader (Multi-Mode Microplate Reader; Molecular Devices, iD3) at λex = 485 ± 10 nm / λem = 530 nm ± 15 nm.

[0272] The increase in fluorescence is proportional to the proteolytic activity, and the proteolytic activity was confirmed by the increase in fluorescence intensity in the presence of Clg@H-MnO2-PEG-SO3 (Fig. 8a) and free collagenase (Fig. 8b). In the absence of Clg@H-MnO2-PEG-SO3 and free collagenase, the fluorescence intensity remained constant throughout the reaction period. These results indicate that the enzymatic activity of the nanoparticles is maintained even after collagenase binding, and that the enzyme retains its enzymatic properties.

[0273] <Experimental Example 6> Diffusion of Clg-Ru@H-MnO2-PEG-SO3 in an ECM-mimicking gel

[0274] The MaxGel™ ECM mixture was thoroughly mixed, pipetted onto a confocal dish, and incubated overnight at 37°C to promote gelation. Once the gel was formed, Clg-Ru@H-MnO2-PEG-SO3 / Ru@H-MnO2-PEG-SO3 was slowly added to the ECM-mimicking gel surface and incubated at 37°C for 24 h. NP diffusion in the ECM gel was imaged using a Leica TCS SP8 DMI8-CS system equipped with a HC PL APO CS 10x / 0.40 DRY objective (Leica Microsystems GmbH, Wetzlar, Germany). 3D fluorescence images were obtained using the Z-stacking function of the system, which consisted of sequential images captured at 2 μm intervals (Ru: 488 nm excitation). The X / Y resolution was 1024 × 1024 pixels.

[0275] As a result, the diffusion of collagenase into the gel matrix was indirectly confirmed through fluorescence imaging of ruthenium II complex (Ru) encapsulated in MnO2-PEG-SO3NP penetrating the ECM gel. As shown in G1 (Clg-Ru@H-MnO2-PEG-SO3) in Fig. 9a and G2 (Ru@H-MnO2-PEG-SO3) in Fig. 9b, the collagenase-bound Clg-Ru@H-MnO2-PEG-SO3NP showed a 3-fold increase in the degradation of the extracellular matrix (ECM) compared to Ru@H-MnO2-PEG-SO3NP.

[0276] This suggests that when collagenase was bound to the H-MnO2-PEG-SO3 surface, enzymatic degradation of collagen occurred in the H-MnO2-PEG-SO3NP diffusion path, thereby enhancing the diffusion of nanoparticles.

[0277] <Experimental Example 7> Analysis of drug and apoptotic cancer cell distribution in tumor tissue

[0278] 1. Cell culture

[0279] Experiments were performed using the 4T1 murine mammary carcinoma cell line. 4T1 cells were purchased from ATCC (catalog no. CRL-2539). The cell line was maintained as a monolayer in ATCC formulated RPMI-1640 medium (catalog no. ATCC 30-2001) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic-antimycotic solution (catalog no. 15240062) at 37°C under 5% CO2. Fetal bovine serum (catalog no. 16000044) was purchased from Gibco Life Technologies (Carlsbad, CA).

[0280] 2. Injection of nanoparticles loaded with anticancer drugs

[0281] Before injecting nanoparticles, 4T1 cells (2.5 x 10 6) was mixed with 100 μL Geltrex Matrix and injected into the right flank of 7-week-old female athymic nude mice (BALB / c nu / nu; each weighing approximately 20 g). Mice were purchased from SLC, Inc. (Hamamatsu, Japan). The formed tumors were measured using a caliper, and the tumor volume was calculated using the formula V = 1 / 2 (width × length).

[0282] 2-1. Injection of nanoparticles loaded with fluorescent probe (Ru)

[0283] Experiments to determine the distribution of ruthenium-containing nanoparticles were performed in a tumor volume of approximately 1500 mm 3 When reached, MnO2 (Ru@H-MnO2-PEG-SO3) was intravenously injected into 4T1 xenograft-bearing mice (MnO2 dose = 5 mg kg -1 and Ru = 4.18 mg kg -1 ; n = 5). Tumors were resected approximately 48 hours after injection.

[0284] 2-2. Injection of nanoparticles loaded with photosensitizers

[0285] Experiments to determine the distribution of nanoparticles containing photosensitizers were performed in tumors with a volume of approximately 150 mm 3 When the tumor reached 0, Ce6@H-MnO2-PEG-SO3 (MnO2 dose = 5 mg / kg, Ce6 = 4.42 mg / kg) and Ce6-Ru@H-MnO2-PEG-SO3 (MnO2 dose = 5 mg / kg, Ru = 2.12 mg / kg, Ce6 = 2.33 mg / kg) were intravenously injected into 4T1 xenograft-bearing mice. Twenty-four hours after injection was designated as day 0. For laser treatment, the tumor was exposed to a 660 nm laser for 15 min (200 mW / cm) after day 0. 2) was investigated. The photosensitizing effect of Ce6@H-MnO2-PEG-SO3 / Ce6-Ru@H-MnO2-PEG-SO3 was detected on the second day. The tumor was resected approximately 48 hours (on the second day) after radiation exposure.

[0286] 2-3. Injection of nanoparticles loaded with antiangiogenic agents

[0287] Experiments to determine the distribution of nanoparticles containing antiangiogenic agents were performed in tumors with a volume of approximately 150 mm 3 When the dose reached 5 mg / kg, Soraf@MnO2-PEG (MnO2 dose = 5 mg / kg, Soraf = 4.05 mg / kg) and Soraf-Ru@H-MnO2-PEG-SO3 (MnO2 dose = 5 mg / kg, Ru = 2.12 mg / kg, Soraf = 1.99 mg / kg) were intravenously injected into 4T1 xenograft-bearing mice. Nanoparticles were administered twice weekly for 3 weeks. Tumors were resected approximately 48 hours after the last treatment.

[0288] 2-4. Injection of nanoparticles loaded with photosensitizers and antiangiogenic agents

[0289] Experiments to determine the distribution of nanoparticles containing photosensitizers and antiangiogenic agents were performed in tumors with a volume of approximately 150 mm 3 When the dose reached 5 mg / kg, Ce6 was started. Soraf-Ce6@H-MnO2-PEG-SO3 (MnO2 dose = 5 mg / kg, Ce6 = 2.33 mg / kg, and Soraf = 1.99 mg / kg) and Soraf-Ce6-Ru@H-MnO2-PEG-SO3 (MnO2 dose = 5 mg / kg, Ru = 1.41 mg / kg, Ce = 1.55 mg / kg, and Soraf = 1.32 mg / kg) were intravenously injected into 4T1 xenograft-bearing mice. Twenty-four hours after injection was designated as day 0. For laser treatment, the tumors were exposed to a 660 nm laser for 15 min (200 W / cm) after day 0. 2) were investigated. The photosensitizing effects of Soraf-Ce6@H-MnO2-PEG-SO3 and Soraf-Ce6-Ru@H-MnO2-PEG-SO3 were detected on the second day. The tumors were resected approximately 48 hours (day 2) after radiation exposure.

[0290] 2-5. Injection of nanoparticles conjugated with extracellular matrix (ECM)-degrading enzymes

[0291] Experiments to determine the distribution of collagenase-conjugated nanoparticles were performed in a tumor volume of approximately 1500 mm 3 When the dose reached 5 mg / kg, Clg@H-MnO2-PEG-SO3 (MnO2 dose = 5 mg / kg, Clg = 2.0 mg / kg) and Clg-Ru@H-MnO2-PEG-SO3 (MnO2 = 5 mg / kg, Ru = 4.18 mg / kg, Clg = 2.0 mg / kg) were intravenously injected into 4T1 xenograft-bearing mice. Five days after injection, the tumors were resected.

[0292] 2-6. Injection of nanoparticles containing extracellular matrix degrading enzymes, photosensitizers, and antiangiogenic agents.

[0293] Experiments to determine the distribution of nanoparticles loaded with collagenase, Ce6 and Soraf were performed in a tumor volume of approximately 1500 mm 3 When the tumor reached 0, Clg-Soraf-Ce6@H-MnO2-PEG-SO3 (MnO2 dose = 5 mg / kg, sorafenib = 1.99 mg / kg, Clg = 2.0 mg / kg, Ce6 = 2.33 mg / kg) was intravenously injected into 4T1 xenograft-bearing mice. The time point 24 hours after injection was designated as day 0. For laser treatment, the tumor was exposed to a 660 nm laser for 15 minutes (200 mW / cm) after day 0. 2 ) was investigated. The hypoxic tumor microenvironment targeting effect of Clg-soraf-Ce6@H-MnO2-PEG-SO3 was detected on day 5. The tumor was resected 5 days after treatment.

[0294] To visualize vascular structures in all experimental groups, mice were injected with Dylight 649 Lycopersicon Esculentum (tomato) lectin (100 μg / 100 μL). All animals were euthanized by CO2 asphyxiation, and tumors were resected 10 minutes later. Tumors were rinsed with 1x PBS and immediately fixed in 4% paraformaldehyde for 24 hours at 4°C.

[0295] 3. Organizational Transparency

[0296] Fixed tumor samples were washed with PBS at 4°C for 24 h. The samples were then transferred to 15 mL light-resistant Falcon tubes containing 3 mL of freshly prepared A4PO monomer solution and 100 mg of VA-044 initiator and incubated at 4°C for 24 h. The samples were then degassed for 15 min using partial vacuum, flushed with nitrogen, and incubated in a hot water bath at 37°C for 4 h to induce polymerization. The samples were then transferred to an X-Clarity tissue sample holder, lowered into the XClarity ETC chamber, and cleared with 4% SDS buffer for 2 days (flow rate: 40 rpm, temperature: 37°C; current: 1.0 A). After clearing, the samples were thoroughly washed in PBST at 37°C for 24 h and then stored in 1x PBS at 4°C.

[0297] 4. Staining and imaging of transparent tissues

[0298] Staining and imaging were performed to analyze drug distribution and the distribution of apoptotic cancer cells or HIF1α-positive cells within the transparent tissue.

[0299] 4-1. Drug distribution analysis

[0300] 4-1-1. Staining and 3D Imaging

[0301] For tumor samples injected with Ru@H-MnO2-PEG-SO3 / Ce6-Ru@H-MnO2-PEG-SO3 / Soraf-Ru@H-MnO2-PEG-SO3 / Soraf-Ce6-Ru@H-MnO2-PEG-SO3 / Clg-Ru@H-MnO2-PEG-SO3 / Clg-Soraf-Ru@H-MnO2-PEG-SO3, drug distribution was confirmed by the following method.

[0302] Tumor tissues, 1 mm thick, were stained with Hoechst 33342 (Life Technologies Ltd., Carlsbad, CA; ex / em 350 / 461 nm) diluted 1:100 and cultured in a shaking water bath at 37°C for 2 days, then washed with 1x PBS containing 0.2% (v / v) Triton X-100 (PBST) in a staining buffer at 37°C for 2 days. Next, the samples were rinsed with DDW for 10 min (repeated twice more) and immersed in X-CLARITY™ mounting solution (RI=1.46, Logos Biosystems, Inc., Gyeonggi-do, Korea) at room temperature before confocal imaging. Three-dimensional fluorescence images were captured using the Z-stacking function of the system (Hoechst 33342: 405 nm, Ru: 488 nm, and blood vessel: 633 nm excitation). The X / Y resolution was 1024 × 1024 pixels (scan depth, 500 μm). Three-dimensional images were acquired using a Leica TCS SP8 DMI8-CS system equipped with a 20× / 0.75 IMM objective, and the data were analyzed using IMARIS software.

[0303] 3D images were analyzed using the Bitplane Imaris CL and XT software packages. Segmentation and distance transformation were performed in Imaris software.

[0304] First, the nucleus size was determined using the 'spots' application. Channel 1 (blue) was selected with the 'Segment only region of interest (ROI)' option selected in the algorithm step of the 'spots' application, and the entire image was processed. The 'estimated diameter' for spot detection was set to 5 μm. The 5 μm value for nuclei was determined using the 'Apply Measurement Points' function in the slice view. The quality filter value was then manually set to 10 or greater only in the lower threshold control box.

[0305] Second, the "Surface" application was used for vessel segmentation. The ROI was set to the entire image, and Channel 2 (vessels - red) was selected as the source channel. After smoothing with the optimal filter value, vessels were segmented using a manually selected threshold (background subtraction).

[0306] Third, the 'Surface' technique was used once more to segment the Ru-bound cancer cell membrane. The ROI was set to the entire image, and the source channel was selected as Channel 3 (Ru-containing cells—green channel). The Ru-containing cell signals were smoothed using the optimal filter value and then segmented using a manually selected threshold (background subtraction).

[0307] Finally, the 'Find Spots Near Surface' application was used to identify nuclei surrounded by Ru-containing cells and automatically perform a 3D Euclidean distance transform outside the segmented vessels with spots close to the Ru-containing cell surface.

[0308] 4-1-2. Quantitative Analysis

[0309] After injecting nano drug carriers containing ruthenium (Ru), the maximum drug penetration depth (DPDmax) and half of DPDmax (DPD) were determined based on 3D images of the transparent tumor samples. 50), and drug penetration amount (DPA) were measured. The maximum distance that the nano drug carrier loaded with anticancer drugs penetrated from the nearest blood vessel was expressed as maximum drug penetration depth (DPDmax), and half of this distance was expressed as DPD 50 It was expressed as DPD in blood vessels in tumor tissue. 50 The number of apoptotic cells containing nano drug carriers loaded with anticancer drugs is determined by the drug penetration or DPA 50 It was expressed as .

[0310] 4-2. Analysis of the distribution of apoptotic cancer cells

[0311] 4-2-1. TUNEL staining and 3D imaging

[0312] For tumor samples injected with DOX@H-MnO2-PEG-SO3 / Ce6@H-MnO2-PEG-SO3 / Soraf@H-MnO2-PEG-SO3 / Soraf-Ce6@H-MnO2-PEG-SO3 / Clg-Soraf-Ce6@H-MnO2-PEG-SO3, the distribution of apoptotic cancer cells was confirmed through TUNEL staining.

[0313] Tumor tissues of 1 mm thickness were stained using a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining kit. First, the tumor tissues were incubated in permeabilization buffer for 30 min at 4°C and then washed with 1x PBS for 30 min at 4°C. Then, the tissues were placed in staining solution (TdT enzyme Hoechst 33342 labeling safe buffer) and incubated in a table-top incubator at 37°C for 16 h, and then washed with 1x PBS at 37°C for 1 h. Finally, the stained samples were rinsed in DDW for 10 min (repeated twice more) and immersed in X-CLARITY™ mounting solution (RI=1.46, Logos Biosystems, Inc., Gyeonggi-do, Korea) at room temperature before confocal imaging. Tumor tissues were imaged using a Leica TCS SP8 DMI8-CS system equipped with a HC PLAPO CS 10x / 0.40 DRY objective (Leica Microsystems GmbH, Wetzlar, Germany). Three-dimensional fluorescence images were captured using the Zstacking function of the system (Hoechst 33342: 405 nm, apoptotic cells: 488 nm, blood vessels: 633 nm excitation). The X / Y resolution was 1024 × 1024 pixels (scan depth, 300 μm).

[0314] 3D images were analyzed using the Bitplane Imaris CL and XT software packages. Segmentation and distance transformation were performed in Imaris software.

[0315] First, the nucleus size was determined using the 'spots' application. Channel 1 (blue) was selected with the 'Segment only region of interest (ROI)' option selected in the algorithm step of the 'spots' application, and the entire image was processed. The 'estimated diameter' for spot detection was set to 5 μm. The 5 μm value for nuclei was determined using the 'Apply Measurement Points' function in the slice view. The quality filter value was then manually set to 10 or greater only in the lower threshold control box.

[0316] Second, the "Surface" application was used for vessel segmentation. The ROI was set to the entire image, and Channel 2 (vessels - red) was selected as the source channel. After smoothing with the optimal filter value, vessels were segmented using a manually selected threshold (background subtraction).

[0317] Third, the 'Surface' technique was used once more to segment the FITC-conjugated TdT dye. The ROI was set to the entire image, and the source channel was selected as Channel 3 (apoptotic cells - green channel). The apoptotic cell signal was smoothed using the optimal filter value and then segmented using a manually selected threshold (background subtraction).

[0318] Finally, we identified nuclei surrounded by apoptotic cells using the 'Find Spots Near Surface' application and automatically performed a 3D Euclidean distance transform outside the segmented vessels with spots close to the surface of apoptotic cells.

[0319] 4-2-2. Immunostaining and 3D imaging

[0320] For tumor samples injected with Clg-Soraf-Ce6@H-MnO2-PEG-SO3, the distribution of HIF1α-positive cells, a hypoxia-inducible factor, and apoptotic cancer cells in the tumor microenvironment was confirmed through immunostaining.

[0321] Tumor tissues, 1 mm thick, were cultured with a 1:100 dilution of HIF1α Alexa Fluor® 594-conjugated antibody (R&D Systems, Minneapolis, MN, USA; ex / em 594 / 615 nm) and staining buffer (6% (vol / vol) BSA, 0.2% (vol / vol) Triton X-100, 0.01% (vol / vol) sodium azide, 1× PBS) in a shaking incubator at 37°C for 5 days. The tissues were then cultured in a staining solution (TdT enzyme Hoechst 33342 labeling safe buffer) in a tabletop incubator at 37°C for 24 h, and then washed with 1× PBS at 37°C for 24 h. Finally, the stained samples were rinsed in DDW for 10 min (repeated twice more) and immersed in X-CLARITY™ Mounting Solution (RI=1.46, Logos Biosystems, Inc., Gyeonggi-do, Korea) at room temperature before confocal imaging. Tumor tissues were imaged using a Leica TCS SP8 DMI8-CS system equipped with an HC PL APO CS 10x / 0.40 DRY objective (Leica Microsystems GmbH, Wetzlar, Germany). 3D fluorescence images were captured using the Z-stacking function of the system (Hoechst 33342: 405 nm, Apoptotic cells: 488 nm, HIF1α cells: 594 nm, Blood vessels: 633 nm excitation). The X / Y resolution was 1024 × 1024 pixels.

[0322] 3D images were analyzed using the Bitplane Imaris CL and XT software packages. Segmentation and distance transformation were performed in Imaris software. The first, second, and third (except Ru) steps mentioned in 4-2-1 above were repeated.

[0323] Next, the 'Surface' application was used once again to segment Alexa 594-bound HIF1α. The ROI was set to the entire image, and the source channel was selected as Channel 4 (HIF1α cells: magenta channel). HIF1α cell signals were smoothed with the optimal filter value and then segmented using a manually selected threshold (background subtraction).

[0324] Finally, we identified nuclei surrounded by HIF1α cells using the 'Find Near Surface Spots' application and automatically performed 3D distance transformation outside the segmented vessels with spots close to the HIF1α cell surface.

[0325] 4-2-3. Quantitative Analysis

[0326] Based on 3D images of TUNEL-stained and immunohistochemically stained tumor samples, the maximum distance at which cells killed by the nano drug delivery vehicle loaded with anticancer drugs exist from the nearest blood vessel was expressed as maximum cell death depth (CDDmax).

[0327] 5. Experimental Results and Analysis

[0328] Drug distribution analysis by particle size

[0329] Drug penetration was analyzed through drug distribution according to particle size. Through 3D reconstructed images showing the penetration gradients of Ru@H-MnO2-PEG-SO3 (69 nm) and Ru@H-MnO2-PEG-SO3 (283 nm, Comparative Example 1) in relation to blood vessels (Figs. 10a and 10b), Ru@H-MnO2-PEG-SO3 (69 nm) and Ru@H-MnO2-PEG-SO3 (280 nm) showed 100 and 92 μm DPD in blood vessels, respectively. max It was confirmed that it was distributed up to (Fig. 10c). Although DPD maxAlthough they showed similar distribution patterns, the DPA (drug penetration amount) of the released Ru was confirmed to be significantly higher for Ru@H-MnO2-PEG-SO3 (62 nm) than for Ru@H-MnO2-PEG-SO3 (283 nm).

[0330]

[0331] Analysis of the cancer cell killing effects of Ru@H-MnO2-PEG-SO3NP and DOX@H-MnO2-PEG-SO3NP

[0332] To investigate the apoptotic effects induced by chemotherapy, DOX and Ru were loaded onto H-MnO2-PEG-SO3. Figure 11a (J1) shows the vascular structure of a 4T1-derived tumor, and Figure 11a (J2) shows the distribution of apoptotic cells in the tumor microenvironment. Figure 11a (J3) shows the distribution of apoptotic cells along blood vessels (overlay images of Figure 11a (J1) and (J2)).

[0333] As shown in Figures 11a and 11b, the 3D distribution of apoptotic cell expression in tumor tissues treated with Dox@H-MnO2-PEG-SO3 was 0-120 μm CDD max It shows that apoptotic cells are in the range of 100 μm DPD of Ru fluorescent probe max It was confirmed that Ru exists at a longer distance from the blood vessels compared to Ru@H-MnO2-PEG-SO3 (69 nm). The 3D reconstructed image shows that Ru released from tumor tissue treated with Ru@H-MnO2-PEG-SO3 (69 nm) is present at a distance of 100 μm DPD from the blood vessels. max In contrast, apoptotic cells induced by chemotherapy (doxorubicin) were detected in blood vessels up to 120 μm CDDmax (Fig. 11a). However, the number of apoptotic cells was much lower than the number of cancer cells that took up Ru at the same DPD (Fig. 11b).

[0334]

[0335] Drug distribution analysis of Ru@H-MnO2-PEG-SO3NP and Ce6-Ru@H-MnO2-PEG-SO3NP

[0336] To investigate the role of photodynamic therapy, Ce6 and Ru were loaded onto H-MnO2-PEG-SO3. Through 3D reconstruction images, Ru@H-MnO2-PEG-SO3 and Ru of Ce6-Ru@H-MnO2-PEG-SO3 was 100 μm and 115 μm DPD in blood vessels, respectively max It was confirmed that the penetration depth of Ce6-Ru@H-MnO2-PEG-SO3 was distributed up to (Fig. 10a and Fig. 12a). Ce6-Ru@H-MnO2-PEG-SO3 showed a greater penetration depth than Ru@H-MnO2-PEG-SO3, and the amount of Ru released from Ce6-Ru@H-MnO2-PEG-SO3 was more than twice that released from Ru@H-MnO2-PEG-SO3 (Fig. 12a and Fig. 12b). This confirmed that photodynamic therapy enhances the drug delivery effect.

[0337]

[0338] Analysis of the cancer cell killing effects of chemotherapy and photodynamic therapy

[0339] Comparison of the cancer cell killing effects of DOX@H-MnO2-PEG-SO3NP (Fig. 11a) and Ce6@H-MnO2-PEG-SO3NP (Fig. 13a) revealed that the spatial distribution of apoptotic cells was DPD max The results were similar in terms of the side effects. However, the number of apoptotic cells induced by photodynamic therapy (Ce6@H-MnO2-PEG-SO3NP) was significantly higher than that induced by chemotherapy (DOX@H-MnO2-PEG-SO3NP) (Fig. 13b). This confirmed the superior cancer cell killing effect of photodynamic therapy.

[0340]

[0341] Drug distribution analysis of Ce6-Ru@H-MnO2-PEG-SO3NP and Soraf-Ru@H-MnO2-PEG-SO3NP

[0342] To investigate the role of antiangiogenic agents in drug delivery, Soraf and Ru were loaded into H-MnO2-PEG-SO3. As shown in Fig. 14a, merging the vascular image and Ru distribution reveals a remarkable penetration distance of Ru in the tumor microenvironment. Comparing Fig. 14a with Fig. 13a, the difference in drug delivery efficiency between Soraf-Ru@H-MnO2-PEG-SO3 and Ce6-Ru@H-MnO2-PEG-SO3 was clearly observed. The Ru released from Ce6-Ru@H-MnO2-PEG-SO3 and Soraf-Ru@H-MnO2-PEG-SO3 was 115 μm and 156 μm, respectively, in the blood vessels. max It was confirmed that the Ru was distributed up to (Fig. 14b). DPD of Ru released from Soraf-Ru@H-MnO2-PEG-SO3 max DPD of Ru released from Ce6-Ru@H-MnO2-PEG-SO3 max Compared to the photodynamic therapy effect on drug delivery, a 40 μm increase in drug distribution was observed in the presence of antiangiogenic agents.

[0343]

[0344] Analysis of the cancer cell killing effects of photodynamic therapy and antiangiogenic agents

[0345] Comparison of the cancer cell killing effects of Ce6@H-MnO2-PEG-SO3NP (Fig. 13a) and Soraf@H-MnO2-PEG-SO3NP (Fig. 15a) revealed that the increase in the proportion of apoptotic cells due to the action of the anti-angiogenic agent (sorafenib) was evidenced by the increase in green fluorescent spots, which showed a wider distribution compared to the photodynamic therapy effect. In addition, the spatial distribution of apoptotic cells induced by sorafenib was confirmed to be larger than that induced by photodynamic therapy with Ce6@H-MnO2-PEG-SO3 (Fig. 15b). This suggests that H-MnO2-PEG-SO3 with sorafenib significantly enhanced the degree of intratumoral cell killing compared to Ce6@H-MnO2-PEG-SO3.

[0346]

[0347] Drug distribution analysis of Soraf-Ru@H-MnO2-PEG-SO3NP and Soraf-Ce6-Ru@H-MnO2-PEG-SO3NP

[0348] The combined effect of antiangiogenic and photosensitizer on drug delivery was analyzed. Comparing Fig. 14a and Fig. 16a, the 3D reconstructed images show that the Ru released from Soraf-Ru@H-MnO2-PEG-SO3 and Soraf-Ce6-Ru@H-MnO2-PEG-SO3 are 156 μm and 215 μm DPD, respectively. max It shows that the maximum amount of Ru released from Soraf-Ce6-@H-MnO2-PEG-SO3 was significantly higher than that released from Soraf-Ru@H-MnO2-PEG-SO3 (Fig. 16b). This is due to the synergistic effect of antiangiogenic and photosensitizer on drug delivery compared to the antiangiogenic effect of DPD. max It suggests that there was an increase of 60 μm in .

[0349]

[0350] Analysis of the cancer cell killing effect of antiangiogenic agents alone and in combination with antiangiogenic agents and photodynamic therapy.

[0351] Comparison of the apoptotic effects of Soraf@H-MnO2-PEG-SO3NP (Fig. 15a) and Soraf-Ce6@H-MnO2-PEG-SO3NP (Fig. 17a) revealed that the proportion of apoptotic cells due to the combined action of sorafenib and photodynamic therapy was more widely distributed than that induced by sorafenib alone. This was evidenced by the increase in green fluorescent spots. In particular, in the Soraf-Ce6@H-MnO2-PEG-SO3 treatment group, extensive destruction of the blood vessel wall due to the synergistic effect of the antiangiogenic agent-photodynamic therapy was observed (F1 in Fig. 17a), which was confirmed by the TUNEL staining results. However, in the Ce6 treatment group and the sorafenib treatment group (Figs. 13a and 15a), no noticeable destruction of the blood vessel wall was observed.

[0352] As shown in Fig. 17a, we confirmed that the signal of blood vessels overlapped with the signal of apoptotic cells (TUNEL), and the overlapping part of the two signals was marked in yellow (F3 in Fig. 17a). In addition, we confirmed that the 3D distribution of apoptotic cells (spectral spots) was in the range of 0-186 μm CPDmax. As shown in Fig. 17b, Soraf-Ce6@H-MnO2-PEG-SO3 induced a larger spatial distribution of apoptotic cells than Soraf@H-MnO2-PEG-SO3 in terms of CPD. The total number of apoptotic cells induced by sorafenib combined with photodynamic therapy was confirmed to be higher than that induced by sorafenib alone.

[0353] Analysis of drug release effects during combined treatment with antiangiogenic agents and photodynamic therapy

[0354] After injection of Dox@H-MnO2-PEG-SO3, Ce6@H-MnO2-PEG-SO3, Soraf-Ru@H-MnO2-PEG-SO3, and Soraf-Ce6@H-MnO2-PEG-SO3, the vascular structure was observed. As shown in Figures 18a to 18d, when Dox-containing MnO2 was administered, no noticeable vascular damage was observed, and the overall vascular structure remained intact and clear. However, when Ce6-containing MnO2 was administered, certain parts within the entire vascular structure were perforated and separated due to vascular destruction induced by photodynamic therapy. Although this effect did not spread throughout the entire vascular network, damaged vascular structures that were not visible in the Dox@MnO2 treatment were observed. In Soraf@MnO2, sorafenib inhibited angiogenesis by inducing apoptosis by targeting VEGF-dependent angiogenic sprouting. Areas of disruption of vascular structure and suppression of microvascular morphology were observed.

[0355] These results suggest that antiangiogenic agents can enhance drug release in the tumor microenvironment. Furthermore, the significantly lower proportion of small vessels observed at the same magnification compared to vessels not treated with sorafenib indirectly supports the efficacy of sorafenib.

[0356] When treated with the combination of sorafenib and photodynamic therapy, the area of ​​damaged blood vessels became more apparent after vascular staining. This increased level of damage was more pronounced than when treated with sorafenib alone, suggesting an enhanced drug release effect.

[0357]

[0358] Drug distribution analysis of Clg-Ru@H-MnO2-PEG-SO3NP and Soraf-Clg-Ru@H-MnO2-PEG-SO3NP

[0359] As shown in the right image of Fig. 19a, merging the blood vessel and Ru distribution images revealed that a large amount of Ru was released in the tumor microenvironment. Soraf-Ru@H-MnO2-PEG-SO3 and Clg-Ru@H-MnO2-PEG-SO3 clearly showed a significant difference in drug delivery effect (Figs. 14a and 19a). The 3D reconstructed image showed that the Ru distribution of Clg-Ru@H-MnO2-PEG-SO3 was 327 μm DPD in the blood vessel. max It shows that the maximum amount of Ru released from Clg-Ru@H-MnO2-PEG-SO3 was incomparably higher than that released from Soraf-Ru@H-MnO2-PEG-SO3, and compared to the antiangiogenic effect on drug delivery, the DPD in the presence of collagenase, an ECM-degrading bioenzyme max increased exponentially (161 μm longer).

[0360] As shown in Figures 19e to 19j, the observed differences in drug distribution confirmed the significant impact of Clg-Ru@H-MnO2-PEG-SO3NP in enhancing drug delivery to hypoxic regions by targeting the tumor extracellular matrix.

[0361] The enhanced drug delivery effect of the combination of antiangiogenic agents and ECM-degrading bioenzymes was further analyzed (Fig. 19b). The Ru distribution of Soraf-Clg-Ru@H-MnO2-PEG-SO3 in blood vessels was 392 μm DPD max was extended to (Fig. 19d). Compared to the effect of the bioenzyme alone on drug delivery, combining antiangiogenic agents with collagenase resulted in an increase in DPD max The total amount of Ru released from Soraf-Clg-Ru@H-MnO2-PEG-SO3 was higher than that released from Clg-Ru@H-MnO2-PEG-SO3 due to the synergistic effect (Fig. 19k).

[0362]

[0363] Analysis of the cancer cell killing effects of Soraf-Ce6@H-MnO2-PEG-SO3NP and Soraf-Clg-Ce6@H-MnO2-PEG-SO3NP

[0364] The distribution of apoptotic cells induced by Soraf-Ce6@H-MnO2-PEG-SO3 and Soraf-Clg-Ce6@H-MnO2-PEG-SO3 in the tumor microenvironment was compared. The extracellular matrix degradation of collagenase combined with sorafenib and photodynamic therapy generated more widely dispersed apoptotic cells compared to the combination of sorafenib and photodynamic therapy (Figs. 17a and 20a). The 3D distribution of apoptotic cell expression (spectral spots) showed a range of 0–369 μm CDDmax. The number of apoptotic cells induced by Soraf-Clg-Ce6@H-MnO2-PEG-SO3 was significantly higher than that induced by Soraf-Ce6@H-MnO2-PEG-SO3 as the distance from the blood vessel increased (Fig. 20b).

[0365] These results suggest that collagenase-encapsulated H-MnO2-PEG-SO3 enhances drug penetration into the tumor microenvironment, thereby enhancing cell death compared to other treatments without collagenase. The heatmap image in Figure 21 demonstrates that collagenase-encapsulated NPs can significantly enhance drug delivery to hypoxic regions compared to antiangiogenic agents and photodynamic therapy.

[0366] In addition, the distribution of apoptotic cells and HIF1α-positive cells in tumor tissues treated with Soraf-Clg-Ce6@H-MnO2-PEG-SO3 was analyzed, and as shown in Fig. 22a, Fig. 22b, and Fig. 22c, the images of the distribution of blood vessels (red), HIF1α-positive cells (magenta), and apoptotic cells (green) were merged to reveal the coexistence of apoptotic cells with HIF1α-positive cells in the tumor microenvironment. Based on the 3D reconstructed images, graphic data were plotted to specify the number of cells (apoptotic cells and HIF1α-positive cells) according to the distance from blood vessels, and in the 3D reconstructed images, apoptotic cells were found to be located within 380 μm DPD of blood vessels. max , and HIF1α-positive cells were distributed at a distance of 40–320 μm from the blood vessels (Fig. 22d, Fig. 22e, and Fig. 22f). This quantitative verification indicates that Soraf-Clg-Ce6@H-MnO2-PEG-SO3 reached the hypoxic region of the tumor model. This was indirectly confirmed by the apoptosis of hypoxic tumor cells, which was manifested by the coexistence of HIF1α-positive cells and apoptotic cells in the tumor microenvironment.

[0367] Consequently, the combination of the catalytic properties of MnO2 and the tumor microenvironment-responsive bioenzyme entrapped in nanocarriers suggests that the rapid degradation of collagen, a major extracellular matrix component in the tumor microenvironment, reduces interstitial fluid pressure and enhances drug release deep into the hypoxic tumor microenvironment. Furthermore, the co-delivery of photosensitizers and antiangiogenic agents with the nanocarrier-responsive bioenzyme entrapped in the tumor microenvironment suggests that the co-delivery of photosensitizers and antiangiogenic agents can effectively kill hypoxic cells in solid tumors.

[0368]

[0369] <Experimental Example 8> Effect of Oxygen Generation Capacity on Drug Delivery

[0370] To determine the effect of the oxygen generating ability of H-MnO2-PEG-SO3 on drug delivery, the Ru released from Ce6-Ru@H-MnO2-PEG-SO3 was compared with the Ru released from the liposome nanoformulation (Ce6-Ru@Lip, Comparative Example 2) through TUNEL staining and imaging in the same manner as in Experimental Example 7.

[0371] As shown in Fig. 23, when drug penetration into Ce6-Ru@Lip-treated tumor tissue was evaluated, Ru released from Ce6-Ru@H-MnO2-PEG-SO3 and Ce6-Ru@Lip was detected at 115 μm and 100 μm DPD in blood vessels, respectively. max It was confirmed that the Ru released from Ce6-Ru@H-MnO2-PEG-SO3 was twice as high as that released from Ce6-Ru@Lip, and this difference indicates that the proportion of Ru released through H-MnO2-PEG-SO3 decomposition is significantly higher than that of liposomal drug release along with the photodynamic therapy efficacy.

Claims

1. Comprising porous nanoparticles having at least a portion of the surface coated with manganese dioxide, The surface of the above nanoparticles is modified with a dual functional ligand, A nano drug carrier wherein the above dual functional ligand comprises a polyethylene glycol moiety and a sulfate moiety.

2. In paragraph 1, The above porous nanoparticles are nano drug carriers comprising mesoporous nanoparticles.

3. In paragraph 1, The above dual functional ligand is a nano drug carrier covalently bonded to the particle surface, including an amine group.

4. In paragraph 3, A nano drug delivery system wherein the covalent bond comprises an amide bond.

5. In paragraph 1, The above porous nanoparticles are nano drug carriers containing a surface modifier.

6. In paragraph 5, A nano drug delivery system in which the surface modifier is sequentially coated with a cationic polymer and an anionic polymer.

7. In paragraph 1, A nano drug delivery system characterized in that the drug delivery system is for delivering anticancer drugs or proteins.

8. In paragraph 7, A nano drug delivery system, characterized in that the anticancer drug is encapsulated inside the drug delivery system and the protein is bound to the outside of the drug delivery system.

9. A dual functional ligand represented by the following chemical formula 1, comprising an amine moiety and a sulfate moiety: [Chemical Formula 1] PEG-L1-NH-L2 (In the above chemical formula 1, PEG is polyethylene glycol, Including, n is an integer between 2 and 1000, R is hydrogen or alkylene having C1 to C3, L1 is C1 to C 10 is alkylene, L2 has one or more carbons -OSO3 - C1 to C substituted with 10 ) is alkylene.

10. In paragraph 9, A dual functional ligand characterized by improving at least one selected from the group consisting of biocompatibility, water solubility, physiological stability, circulation time and tumor accumulation of a drug delivery system.

11. In paragraph 9, A dual-functional ligand, characterized in that the tumor extracellular matrix is ​​selected from the group consisting of proteoglycan, elastin, fibronectin, vitronectin, laminin, gelatin and collagen.

12. A method for manufacturing a nano drug delivery system according to paragraph 1, (a) A step of synthesizing SiO2 nanoparticles; (b) a step of adding a potassium permanganate (KMnO4) solution to the synthesized SiO2 nanoparticle suspension and performing ultrasonic treatment to obtain nanoparticles coated with manganese oxide on SiO2; (c) a step of dispersing the above nanoparticles in a Na2CO3 solution, stirring them, and then centrifuging them to obtain porous nanoparticles; (d) a step of adding polyallylamine hydrochloride (PAH) to the aqueous dispersion of the porous nanoparticles to obtain a first reaction product, and then adding polyacrylic acid (PAA) to the solution in which the first reaction product is suspended to obtain a second reaction product; and (e) A method for producing a nano drug carrier, comprising the step of producing a nano drug carrier by binding a dual functional ligand to the second reaction product.

13. An anticancer pharmaceutical composition comprising the nano drug delivery system of claim 1, wherein an anticancer drug is encapsulated therein.

14. In paragraph 13, An anticancer pharmaceutical composition characterized in that the pharmaceutical composition induces cell death of cancer cells in a hypoxic region of a tumor microenvironment. 15.(a') A step of forming cancer tissue by transplanting a culture medium containing cancer cells into an animal other than a human; (b') The volume of the cancer tissue is 100 to 2000 mm 2 When this is done, the step of injecting the nano drug delivery system of the first clause; (c') A step of collecting cancer tissue injected with the nano drug delivery system and making it transparent; (d') A method for evaluating drug distribution, comprising a step of evaluating drug distribution in the above transparent cancer tissue sample.

16. In paragraph 15, The above (d') step is the maximum drug penetration depth (DPDmax), half of DPDmax (DPD 50 ), DPD in intratumoral blood vessels 50 The number of cancer cells containing anticancer drugs (drug penetration or DPA) 50 ) is characterized by measuring and quantitatively analyzing.

17. In paragraph 15, The above evaluation method is characterized by counting cells containing an anticancer drug at a level of 100 to 1000 μm around a blood vessel. 18.(a') A step of forming cancer tissue by transplanting a culture medium containing cancer cells into an animal other than a human; (b') The volume of the cancer tissue is 100 to 2000 mm 2 When this is done, the step of injecting the nano drug delivery system of the first clause; (c') A step of collecting cancer tissue injected with the nano drug delivery system and making it transparent; (d') A method for evaluating the distribution of apoptotic cancer cells, comprising a step of evaluating the distribution of apoptotic cancer cells in the above-mentioned transparent cancer tissue sample.

19. In paragraph 18, The above evaluation method is characterized by quantitative analysis by measuring the maximum depth of cell death (CDDmax).

20. In paragraph 18, The above evaluation method is characterized by counting apoptotic cancer cells at a level of 100 to 1000 μm around the blood vessel.

21. A method for treating cancer, comprising a step of administering a pharmaceutically effective amount of the nano drug delivery system of claim 1, in which an anticancer drug is encapsulated therein, to a subject in need of cancer treatment.

22. Use of the nano drug carrier of claim 1 with an anticancer drug encapsulated therein in the manufacture of a drug for treating cancer.

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