Cancer-targeting drug delivery system comprising heparin- and protamine-based self-assembled nanoparticles

Abstract

The present invention relates to a cancer-targeting drug delivery system comprising: a protamine-based self-assembling first nanoparticle including a negatively charged substance; and a heparin-based self-assembling second nanoparticle including a positively charged anticancer agent and Fe3+. Specifically, the present invention provides a drug delivery system that forms aggregates at a tumor site by a guidance effect upon administration of the second nanoparticle after administration of the first nanoparticle, and has an anticancer effect through induction of ferroptosis and immunogenic cell death without exhibiting the anticoagulant effect of heparin.

Description

Cancer-targeting drug delivery system comprising heparin- and protamine-based self-assembled nanoparticles

[0001] The present invention relates to a protamine-based self-assembled first nanoparticle comprising a negatively charged substance; and an anticancer agent and a positively charged anticancer agent and Fe 3+ The present invention relates to a cancer-targeting drug delivery system comprising a heparin-based self-assembled second nanoparticle, and a method for preventing or treating cancer using the same.

[0002] A drug delivery system (DDS) is a technology that optimizes drug therapy by designing a formulation that efficiently delivers the required amount of drug to the desired target site and minimizes side effects. Drug delivery systems are categorized into polymer-based drug delivery systems and fusion-based drug delivery systems.

[0003] Polymer-based drug delivery systems encapsulate drugs in polymeric materials to deliver them to their targets. These systems utilize synthetic polymers, proteins, micelles, liposomes, and antibodies. Polyethylene glycol (PEG) and hyaluronic acid are commonly used, but PEG is not broken down in the body and is excreted through the kidneys, which can lead to accumulation in the body. Hyaluronic acid, on the other hand, has a short half-life. Antibody-drug conjugates (ADCs) have the advantage of having fewer side effects and being widely available compared to other delivery systems. However, they can trigger an immune response if the body recognizes them as foreign.

[0004] Heparin is a type of acidic polysaccharide containing sulfate groups and possesses strong anticoagulant properties. Protamine, a strong base, binds strongly to the acidic heparin, forming a complex that neutralizes its anticoagulant effect. If this complex is formed, protamine's effectiveness is also lost.

[0005] According to patent document 1, a drug delivery system for treating lymphoma including an antibody with improved delivery capability compared to a single-target drug delivery system is disclosed, and according to non-patent document 1, an antibody-drug conjugate method using nanotechnology or controlling protein aggregation and drug binding ratio is disclosed. However, there is a lack of research on a nanoparticle-based drug delivery system that can kill cancer cells at the cancer site by utilizing a safe substance that is not recognized as a foreign substance in the body.

[0006] Under this background, the present invention has been completed by confirming an anticancer effect through the induction of ferroptosis and immunogenic cell death by forming heparin-protamine aggregates by the induction effect of the strong binding force between heparin and protamine when protamine self-assembled nanoparticles and heparin self-assembled nanoparticles are sequentially administered by utilizing the strong binding force between heparin and protamine, and accumulating at the tumor site.

[0007] [Prior Art Literature]

[0008] [Patent Document]

[0009] Republic of Korea Patent Publication No. 2023-0032980 (March 7, 2023)

[0010] [Non-patent literature]

[0011] Candice Maria Mackertish, Veysel Kayser, Biomedicines 2021, 9(8), 872

[0012] G. Lei, L. Zhuang, B. Gan, Nat. Rev. Cancer 22 (2022) 381-396.

[0013] Q. Cheng, L. Yue, J. Li, C. Gao, Y. Ding, C. Sun, M. Xu, Z. Yuan, R. Wang, Small 17 (2021) e2101332.

[0014] The object of the present invention is to provide a protamine-based self-assembled first nanoparticle comprising a negatively charged substance; and an anticancer agent and a positively charged anticancer agent and Fe 3+ A drug delivery system for targeting cancer is provided, comprising a heparin-based self-assembled second nanoparticle comprising:

[0015] Another object of the present invention is to provide a method for treating a non-human subject, comprising the steps of: (a) administering to a non-human subject a protamine-based first nanoparticle comprising a negatively charged substance; and (b) administering to a non-human subject a positively charged anticancer agent and Fe 3+ A method for preventing or treating cancer is provided, comprising administering to a non-human subject a heparin-based second nanoparticle comprising:

[0016] Another object of the present invention is to provide a protamine-based self-assembled first nanoparticle comprising a negatively charged substance; and an anticancer agent and a positively charged anticancer agent and Fe 3+ A cancer-targeting injection comprising a heparin-based self-assembled second nanoparticle comprising:

[0017] Another object of the present invention is to provide a protamine-based self-assembled first nanoparticle comprising a negatively charged substance; and an anticancer agent and a positively charged anticancer agent and Fe 3+ To provide a pharmaceutical composition for preventing or treating cancer, comprising heparin-based self-assembled second nanoparticles comprising:

[0018] In order to achieve the above object, the present invention provides a protamine-based self-assembled first nanoparticle comprising a negatively charged substance; and an anticancer agent and a positively charged anticancer agent and Fe 3+A drug delivery system for targeting cancer is provided, comprising a heparin-based self-assembled second nanoparticle comprising:

[0019] The present invention also comprises the steps of: (a) administering to a non-human subject a protamine-based first nanoparticle comprising a negatively charged substance; and (b) administering to a non-human subject a positively charged anticancer agent and Fe 3+ A method for preventing or treating cancer is provided, comprising administering to a non-human subject a heparin-based second nanoparticle comprising:

[0020] When the protamine-based self-assembling first nanoparticle and the heparin-based self-assembling second nanoparticle according to the present invention are sequentially administered to a subject, aggregates are formed by the heparin-induced guidance effect of protamine, and the nanoparticles accumulate at the tumor site, thereby having an anticancer effect of killing cancer through induction of ferroptosis and immunogenic cell death, and thus can be utilized as a novel drug delivery system for targeting cancer.

[0021] Figure 1a is a schematic diagram showing the formation process of self-assembled nanoparticles, Figure 1b is a schematic diagram showing the formation process of HP aggregates (aggregates of PPNC NPs and HDFe NPs) in a cancer site by the guidance effect after sequential administration of nanoparticles, and Figure 1c is a schematic diagram showing the immune activation process through induction of ferroptosis and immunogenic cell death (ICD) of HP aggregates in tumor tissue.

[0022] Figure 2a shows the formation process of palmitic acid-protamine (PalP) through chemical synthesis of palmitic acid-NHS and protamine conjugates, Figure 2b shows the particle size distribution of PalP nanoparticles, Figure 2c shows the results of comparing the zeta potential values ​​of protamine and PalP, Figures 2d and 2e show the fluorescence intensity changes of PalP-Cy5.5 according to DMSO and NaCl concentrations, Figures 2f and 2g show transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) images of PalP nanoparticles, Figure 2h shows the results of cytotoxicity evaluation according to PalP concentration, Figure 2i shows the fluorescence images of the in vivo distribution of protamine and PalP labeled with RITC in organs and tumor tissues, Figure 2j shows a quantitative graph of PalP-RITC in tumor tissues, and Figure 2k shows a fluorescence image of PalP-RITC in tumor tissues at 12 hours.

[0023] Figure 3 shows the results of comparing the chemical bonding process of Palp molecules analyzed by 1D proton NMR.

[0024] FIG. 4a shows the formation process of negatively charged curcumin (NCur), FIG. 4b shows the results of solubility evaluation of curcumin (Cur) and NCur, FIG. 4d shows the results of negative charge strength evaluation through zeta potential measurement of Cur and NCur, FIG. 4e shows the formation process of protamine-based nanoparticles (PPNC NPs), FIG. 4f shows the molecular dynamics simulation of self-assembled PPNC NPs, FIG. 4g shows the results of interaction analysis of PalP and NCur, FIG. 4h shows the results of stability measurement of PPNC NPs over time according to the ratio of PalP and NCur, FIGS. 4i to 4l show the results of particle size distribution, TEM image, stability, and zeta potential value measurement of PPNC NPs, FIG. 4m shows the NCur loading speed and image of PPNC NPs, and FIG. 4d shows the results of cytotoxicity evaluation of NCur and PPNC NPs according to the concentration.

[0025] Figure 5 shows a TEM image for verifying the particle shape of NCur.

[0026] Figure 6 shows an FE-SEM image for verifying the particle shape of PPNC NPs.

[0027] Figure 7a shows doxorubicin and Fe 3+ Heparin-based nanoparticles (HDFe NPs; heparin-based doxorubicin and Fe 3+(containing nanoparticle) self-assembly MD simulation, Fig. 7b shows the results of the analysis of molecular interactions within HDFe NPs, Fig. 7c shows the number of hydrogen bonds calculated from the molecular dynamics simulation of HDFe NPs, Fig. 7d shows the stability of HDox (HEP / DOX) mixtures according to the ratio of heparin (HEP) and doxorubicin (DOX), Fig. 7e shows the stability of HDFe NPs, Fig. 7f shows the results of particle size distribution analysis using FE-SEM images, Fig. 7i shows the elemental analysis quantification and images of HDFe NPs using SEM-EDS, Fig. 7j shows the stability of HDFe NPs, Fig. 7k shows the loading efficiency of DOX and HDFe NPs, and Figs. 7l and 7m show the results of cytotoxicity evaluation and anticoagulant activity according to the concentration of HDFe NPs, DOX, and HEP.

[0028] Figure 8 shows DOX and Fe 3+ It shows the combined structural formula of .

[0029] Figure 9 shows the particle size distribution of HDox using dynamic light scattering (DLS) under aqueous conditions.

[0030] Figure 10 shows elemental images of sulfur (S), oxygen (O), aluminum (Al), and silicon (Si) of HDFe NPs using EDS-SEM.

[0031] Figure 11 shows the theoretical structure of HDFe NP.

[0032] Fig. 12a is a schematic diagram of the aggregate formation process of PPNC NPs and HDFe NPs, Fig. 12b is an MD simulation of self-assembled HP aggregates, Fig. 12c is an intermolecular hydrophobic and electrostatic interactions within HP aggregates, Fig. 12d is an illustration of the number of hydrogen bonds calculated from the MD simulation of HP aggregates, Fig. 12e is an image of particle formation between PalP containing HEP and HDox and HP aggregate formation of PPNC NPs and HDFe NPs, Fig. 12f and Fig. 12g are TEM and FE-SEM images of HP aggregates, Fig. 12h is an illustration of the interaction between HEP ​​and protamine using biolayer interferometry (BLI), Fig. 12i is an image of a drug association experiment at 36 hours of PPNC NPs, HDFe NPs, and HP aggregates, and Fig. 12j is a result of measuring the drug binding effect of PPNC NPs, HDFe NPs, and HP aggregates.

[0033] Figure 13 shows the results of cytotoxicity evaluation according to the concentration of HP aggregates.

[0034] Figure 13a shows the results of the evaluation of intracellular lipid overload and lipid peroxidation, Figure 13b shows the results of the evaluation of LPO, Figure 13c shows the results of Western blot measurement of the expression levels of GPX4 and high mobility group box 1 (HMGB1), Figure 13d shows the results of comparing GPX4 expression, and Figure 13e shows the results of the cellular uptake of DOX and intracellular Fe 2+ The results of analyzing the presence of BODIFY, ROS generation, GPX4, SLC7A11 and lipid peroxidation levels by fluorescence images are shown in Figs. 13f to 14h, showing the BODIFY fluorescence intensity, ROS generation and intracellular Fe by substance. 2+ This shows the results of quantitative analysis of concentration.

[0035] Figure 14a shows an immunocytochemistry (ICC) image for comparing the expression levels of calreticulin (CRT) and HMGB1, Figure 14b shows the results of comparing the expression levels of CRT using FACS, Figure 14c shows a schematic diagram of co-culture of cancer cells and spleen cells, and Figures 14d to 14t show F4 / 80, respectively. + CD11b + cells, CD11b + CD80 + Cells, CD 3+ Cells, CD 3+ CD4 + Cells, CD 3+ CD8 + cells, CD8 + CD69 + cells, CD8 + CD25 + cells, CD8 + granzyme + cells, CD8 + IFN + Cells, CD48 + NK1. 1 + cells, CD49b + cells, CD49b + granulocytes + cells, CD49b + IFN + cells, CD49b + CD 3+ Cell, F4 / 80 + CD80 + Cell, F4 / 80 + CD206 + Figure 14u schematically illustrates the immune activation process of HP aggregates, comparing the results of cell and M1 / M2 ratios.

[0036] Figures 15a to 15c show the results of quantifying the fluorescence intensity over time after intravenous administration of HEP-Cy5.5. following subcutaneous administration of saline or PalP, and schematically illustrating the PalP-mediated induction effect and accumulation process of HEP-RITC. Figures 15d to 15f show the results of quantifying the fluorescence intensity in organs and tumor tissues after intravenous administration of HEP-RITC following intravenous administration of saline or PalP. Figures 15g to 15i show the results of quantifying the fluorescence intensity in organs extracted from mice intravenously administered with PalP and HEP-RITC, and the results of quantifying the fluorescence intensity of HEP-RITC at each time point after intravenous injection of HEP-RITC 12 hours later. Figure 15k shows a confocal image of a tumor administered HDFe NP-Cy5.5 12 hours after PalP-FITC administration.

[0037] Figure 16 shows the fluorescence distribution of PPNC NP and HDFe NP in mouse tumor tissue by administering HDFe NP-Cy5.5 12 hours after administering PPNC NP-FITC.

[0038] Figures 17a and 17b show the results of confirming the distribution in organs and tumor tissues of mice after administration of saline and HEP-RITC, respectively.

[0039] Figure 18a shows the results of comparison of anticancer efficacy through tumor volume measurement, and Figures 18b to 18e show tumor volumes, mouse body weights, tumor weights, and tumor images on the last day of the experiment for each substance. Figure 18f shows an image of tumor tissue stained with H&E and Ki-67, Figure 18g shows an image of tumor tissue stained with TUNEL, and Figure 18h shows the results of quantifying the positive area in the TUNEL image.

[0040] Hereinafter, the present invention will be described in detail.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0042] When the present invention is said to “include” a certain component or a certain step, this does not mean that other components or other steps are excluded, but rather that other components or other steps may be further included, unless specifically stated otherwise.

[0043] The term "administration" in the present invention means a method of causing at least partial localization to a desired site of an object or placing a given substance into an object. Administration may be carried out by any method known in the art.

[0044] As used herein, the term "prevention" may refer to any action that inhibits or delays the onset of cancer by administering the nanoparticles. Furthermore, the term "treatment" may refer to any action that improves or beneficially alters the symptoms of cancer by administering the nanoparticles.

[0045] In the present invention, the term “cancer” or “tumor” means cancer originating from a part or cell of the body.

[0046] The present invention relates to a protamine-based self-assembled first nanoparticle comprising a negatively charged substance; and a positively charged anticancer agent and Fe 3+ A drug delivery system for targeting cancer is provided, comprising a heparin-based self-assembled second nanoparticle comprising:

[0047] The above nanoparticles can form micelle, liposome or nanoaggregate structures by self-assembly, but are not limited thereto.

[0048] The above protamine-based first nanoparticles may further include a hydrophobic fatty group (fatty acid, etc.), and the fatty group may be a hydrophobic substance based on at least one fatty group selected from the group consisting of oleic acid, palmitic acid, palmitoleic acid, stearic acid, elaidic acid, linoleic acid, vaccenic acid, α-eleostearic acid, punicic acid, jacaric acid, arachidonic acid, paullinic acid, gondoic acid, and salts thereof, and may preferably be palmitic acid and oleic acid, but is not limited thereto.

[0049] The second nanoparticles may be administered simultaneously, separately, or sequentially with the first nanoparticles, and preferably sequentially, but are not limited thereto.

[0050] When the second nanoparticles are administered sequentially with the first nanoparticles, the second nanoparticles may be administered 1 to 30 hours after the administration of the first nanoparticles, preferably 5 to 20 hours after the administration of the first nanoparticles, and most preferably 10 to 15 hours after the administration of the first nanoparticles, but is not limited thereto.

[0051] The above administration may be at least one selected from the group consisting of intratumoral administration, oral administration, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration, and preferably may be intratumoral administration, subcutaneous administration, and intravenous administration, and most preferably, the first nanoparticle may be administered intratumorally and the second nanoparticle may be administered intravenously, but is not limited thereto.

[0052] The above negatively charged substance may be at least one selected from the group consisting of curcumin, nucleic acids, and negatively charged anticancer agents, and is preferably curcumin, but is not limited thereto.

[0053] The nucleic acid may be at least one selected from the group consisting of DNA, RNA, ASO (antisense oligonucleotide), microRNA (miRNA), small interfering RNA (siRNA), circular RNA, long noncoding RNA (lncRNA), small activating RNA (saRNA), messenger RNA (MRNA), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino, but is not limited thereto.

[0054] The above anticancer drugs are doxorubicin, cyclophosphamide, mecholrethamine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, duocarmycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, and triplatin. triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, camptothecin, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, Paclitaxel, docetaxel, izabepilone, vinblastine,It may be at least one selected from the group consisting of vincristine, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E, monomethyl auristatin F, daunorubicin, epirubicin, idarubicin, and valrubicin, and preferably doxorubicin, but is not limited thereto.

[0055] The first nanoparticle and the second nanoparticle can form aggregates by the guidance effect.

[0056] The above inductive effect refers to a phenomenon in which protamine interacts with heparin or heparin-based nanoparticles by attracting them and forming aggregates and accumulating upon administration of the first nanoparticle.

[0057] The above first nanoparticle and second nanoparticle can induce ferroptosis in cancer cells.

[0058] The weight ratio of the above negatively charged substance and protamine may be 20:1 to 1:20, preferably 3:1 to 1:3, but is not limited thereto.

[0059] The above anticancer agent, Fe 3+ And the weight ratio of heparin may be 50:1:10 to 1:1:1, preferably 25:1:5 to 5:2:1, but is not limited thereto.

[0060] The daily dosage of the first nanoparticle or the second nanoparticle may be 1 mg / kg to 50 mg / kg, preferably 5 mg / kg to 40 mg / kg, but is not limited thereto.

[0061] The cancer may be any one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, and is preferably colon cancer, but is not limited thereto.

[0062] The diameters of the first nanoparticle and the second nanoparticle may be 20 to 500 nm, preferably the diameter of the first nanoparticle may be 100 to 300 nm, and the diameter of the second nanoparticle may be 50 to 400 nm, most preferably the diameter of the first nanoparticle may be 150 to 200 nm, and the diameter of the second nanoparticle may be 200 to 250 nm, but is not limited thereto.

[0063]

[0064] The present invention also provides a method for treating a non-human subject, comprising: (a) administering to a non-human subject a protamine-based first nanoparticle comprising a negatively charged substance; and (b) administering to a non-human subject a positively charged anticancer agent and Fe 3+ A method for preventing or treating a tumor is provided, comprising administering to a subject a heparin-based second nanoparticle comprising:

[0065] The subject may be a mammal, such as a human, dog, cat, cow, horse, pig, sheep, goat, rabbit, mouse, hamster, hedgehog, ferret, or guinea pig, and may be an subject suffering from cancer or suspected of having cancer.

[0066]

[0067] The present invention also provides a protamine-based self-assembled first nanoparticle comprising a negatively charged substance; and a positively charged anticancer agent and Fe 3+ A tumor-targeting injection comprising a heparin-based self-assembled second nanoparticle comprising:

[0068]

[0069] The present invention also provides a protamine-based self-assembled first nanoparticle comprising a negatively charged substance; and a positively charged anticancer agent and Fe 3+ A pharmaceutical composition for preventing or treating a tumor is provided, comprising heparin-based self-assembled second nanoparticles comprising:

[0070]

[0071] The pharmaceutical composition of the present invention may be administered orally or parenterally in various dosage forms. When formulating the composition, it may be prepared using one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, or surfactants, diluents, or excipients.

[0072] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing one or more compounds with at least one excipient, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose or gelatin. For example, tablets or sugar-coated tablets can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture.

[0073] In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, or preservatives may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants in some cases, and anticoagulants, flavoring agents, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, packaging agents, pigments, and preservatives may be additionally included.

[0074] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, or suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0075] The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be formulated in the form of an injection for intratumoral, intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, topical, intranasal, intrapulmonary, and intrarectal injection according to a method known in the art.

[0076] In the case of the above injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for the injection include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. In addition, the injection may in most cases additionally include isotonic agents such as sugars or sodium chloride.

[0077] The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. That is, the total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered as a fractionated treatment protocol in which multiple doses are administered over a long period of time. It is important to consider all of the above factors and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0078] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0079] The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0080]

[0081] The description of the above treatment method, injection and pharmaceutical composition, its effects and all related descriptions are the same as those described above, so the description thereof is omitted to avoid excessive complexity of this specification due to duplicate description.

[0082]

[0083] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0084]

[0085] Example 1. Experimental materials and methods

[0086]

[0087] 1-1. Experimental materials

[0088]

[0089] Antibiotic-antimycotic solution (100X), dimethyl sulfoxide (DMSO), DMEM (Dulbecco's modified Eagle's medium, 2,7-dichlorodihydrofluorescein diacetate (DCF-DA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), methanol, protamine sulfate salt from salmon, palmitic acid, N-hydroxysuccinimide, N-hydroxysuccinimide (NHS), phosphate-buffered saline (PBS), rhodamine B isothiocyanate (RITC; rhodamine B isothiocyanate), fluorescein isothiocyanate isomer I (FITC; Fluorescein isothiocyanate isomer I), 2-(4-amidinophenyl)-6-indolecarbamidine hydrochloride (DAPI; 2-(4-amidi-nophenyl)-6-indolecarbamidine dihydrochloride), iron(III) chloride (Fe 3+ ), curcumin from turmeric (Curcuma longa, Tumeric) powder, tetramethylsilane (TMS), dimethyl sulfoxide-d6 (DMSO-d6), and deuterium oxide (D2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-calreticulin antibody, goat anti-rabbit IgG cross-adsorption ready probe TM Secondary antibody (Goat anti-Rabbit IgG Cross-Adsorbed Ready Probes)TM Secondary Antibody, Alexa Fluor TM 488), BODIPY 581 / 591 C11, Heparin sodium salt from porcine intestinal mucosa (UFH; average molecular weight: 15,000, 150 IU / mg), Paraformaldehyde solution 4% in PBS (PFA), Spectra / TM 7 Membrane tubing (Spectra / Por TM7 Membrane Tubing (dialysis membrane, 3.5 kD) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-glutathione peroxidase 4 antibody, anti-HMGB1 antibody, anti-xCT antibody, and Lipid Peroxidation (MDA) assay kit were purchased from Abcam (Cambridge, UK). Anti-mouse IgG HRP-linked antibodies and anti-rabbit IgG HRP-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Cyanine 5.5 (Cy5.5; Cyanine 5.5) amine was purchased from Lumiprobe (Hannover, Germany). Doxorubicin hydrochloride was purchased from FutureCHEM (Seoul, South Korea). The EZ-cytox kit was purchased from DoGenBio (Seoul, South Korea). Fetal bovine serum (FBS), Hanks' Balanced Salt Solution (HBSS; 1X), and 0.5% trypsin-EDTA (10X) were purchased from Gibco (Waltham, MA, USA). Neutral-buffered formalin (10%) was purchased from HuBenTech (Damyang, South Korea).

[0090]

[0091] 1-2. Preparation and Characterization of Palmitic Acid-Protamine (PalP)

[0092]

[0093] Palmitic acid-protamine (PalP) nanoparticles were synthesized by reacting protamine and palmitic acid-NHS in a mixture of formamide and anhydrous DMSO (99.8%). Fifty milligrams of protamine and 14 mg of palmitic acid-NHS were individually dissolved in 5 mL of formamide and 3 mL of anhydrous DMSO, respectively. The reaction was carried out at low speed (600 rpm) for 2 h. Before adding 14 mg of palmitic acid-NHS, 50 mg of protamine was pre-dissolved in 5 mL of formamide at room temperature. After the reaction, the material was gently precipitated with 30 mL of methanol, and the insoluble protamine was sequentially purified by methanol precipitation and centrifugation (2500 rpm, 4 min, room temperature). The removal of protamine and palmitic acid-NHS was confirmed by thin layer chromatography using methanol and chloroform (1:10) as solvents. The residual solvent was removed using a rotary evaporator, and the resulting precipitate was freeze-dried for 2 days to obtain a powder. The synthesis of the final compound was confirmed using a 1D proton 500 MHz NMR spectrometer at a concentration of 5 mg / mL in solvent mixtures of D2O and DMSO-d6 (2:8 and 8:2). NMR measurements were performed to observe the nanoassembly phenomenon based on the hydrophilic and hydrophobic properties of PalP. Protamine and palmitic acid-NHS were measured at the same concentrations as PalP in 100% D2O and DMSO-d6 solvents, respectively.

[0094]

[0095] 1-3. Preparation and characterization of PPNC NPs

[0096]

[0097] The reaction between curcumin (Cur) and sodium ions was carried out in 99.8% methanol. Negatively charged curcumin (NCur) was prepared by dissolving curcumin (Cur, 4.5 mg) in 990 μL of methanol and then adding 10 μL of NaOH (1 N). The reaction was carried out at room temperature with low speed (600 rpm) stirring for 2 h. After the solvent was evaporated using a rotary evaporator, 20 mL of distilled water was added, and the mixture was freeze-dried for 2 days. The resultant was precipitated with 5 mL of distilled water, sequentially purified with distilled water, and centrifuged (4,000 rpm, 4 min, room temperature). The collected supernatant was freeze-dried in a freeze dryer for 2 days to obtain a powder. The optimal ratio for a mixture of PalP and NCur (NCur) (PPNC NP) was determined through sedimentation evaluation experiments, and the mixture was prepared based on the determined ratio and experimental conditions. PalP and NCur were individually dissolved in appropriate solvents and used immediately after preparation without a separate freeze-drying process.

[0098]

[0099] 1-4. Preparation and characterization of HDFe NPs

[0100]

[0101] Unfractionated heparin (HEP), doxorubicin (DOX), and trivalent iron ions (Fe 3+ ) was carried out in distilled water (DW). HEP (50 mg) and DOX (9 mg) were each dissolved in 0.5 mL of distilled water. For each solution, sonication was performed for 3 min at room temperature using JAC Ultrasonic 3010 (KODO, Korea). The two solutions were then mixed and reacted at low speed (600 rpm) for 1 h. After freeze-drying for 2 days, HDox (2 mg) was dissolved again in 1 mL of distilled water and Fe 3+(0.24 mg) was added. The reaction was carried out at low speed (600 rpm) for 1 hour. After completion of the reaction, the solution containing HDFe NPs was freeze-dried for 2 days to obtain HDFe NP powder.

[0102]

[0103] 1-5. Stability optimization of PPNC NPs, HDox, and HDFe NPs

[0104]

[0105] To determine the most stable ratio of PPNC NPs, HDox, and HDFe NPs, a sedimentation evaluation was performed. 0.4 mg of NCur was dissolved in 1 mL of distilled water. The ratios of NCur to PalP were adjusted to 1:4, 1:2.5, 1:1, and 1:0.5 by mass, and the amount of PalP in distilled water was adjusted and mixed with the NCur solution. Similarly, HDox was prepared with HEP to DOX ratios of 25:1, 5:1, 2:1, 1.2:1, and 1:1 by mass, and then lyophilized for 2 days and dissolved in distilled water at a concentration of 0.5 mg / mL. The most stable HEP, DOX, and Fe 3+ To select the mixing ratio of Fe 3+ HDox mixture (DOX:HEP=5:1) containing HDox to Fe 3+ The ratios were adjusted to 5:1:0.1, 5:1:0.4, 5:1:1, and 5:1:1.4 by mass ratio, and after freeze-drying for 2 days, it was dissolved in distilled water at a concentration of 0.5 mg / mL. The supernatant was collected at various time points (0, 1, 3, 6, 12, and 24 h) and analyzed using a microplate reader (SPECTROstar Nano spectrophotometer, BMG Labtech, Germany) at 426 nm (NCur), 500 nm (DOX), and 334 nm (Fe). 3+ ) were measured (n = 3).

[0106]

[0107] 1-6. Characterization of Nanoparticles: Particle Size, Zeta Potential, Stability, and Structural Analysis

[0108]

[0109] The particle sizes of PalP, PPNC NPs, HDox, and HDFe NPs were measured in DW (1 mg / mL) using dynamic light scattering (DLS; Zeta sizer Nano, Malvern Instruments, Worcestershire, UK). The nanoparticles were prepared by dissolving 2 mg of PalP and PPNC NPs, and 0.4 mg of HDox, HDFe NPs, and HP aggregates in 2 mL of filtered DW through a simple process. Sonication was performed at RT for 5 min using a JAC Ultrasonic 3010 device (KODO, Korea), and the zeta potential of each particle was measured at a concentration of 1 mg / mL under the same conditions as the particle size measurement. The zeta potentials of particles including protamine, PalP, PPNC NPs, and HDFe NPs were also determined. Additionally, the zeta potentials of Cur and NCur were measured in MeOH using DLS. The two materials were prepared by dissolving 2 mg of each in MeOH (2 mL) through a simple process. The stability of PPNC NPs and HDFe NPs was evaluated using dynamic light scattering (DLS) at a concentration of 0.25 mg / mL in deionized water (DW). The nanoparticles were prepared by dissolving 1 mg in filtered DW (1 mL) through a simple dilution process. After gentle stirring, the mixture was sonicated for 5 min at room temperature (RT) using a JAC Ultrasonic 3010 device (KODO, Korea). The evaluation was performed using DLS (n=3) over various times (0, 1, 3, 6, 12, and 24 h). The spherical morphologies of NCur, PalP, PPNC NPs, and HDFe NPs were observed using TEM (EVO MA 10, Carl Zeiss, Germany) and field-effect scanning electron microscopy (FE-SEM) (MERLIN; Carl Zeiss, Germany). In particular, the nanostructure of PPNC NPs was examined using FE-SEM (JEOL; Tokyo, Japan).Elemental analysis of HDFe NP nanoparticles was performed using energy-dispersive X-ray spectroscopy (SEM-EDS; MERLIN; Carl Zeiss, Germany).

[0110] To investigate the structural properties of PalP nanoparticles, Cy5.5-labeled PalP-Cy5.5 was used. The nanostructure of PalP-Cy5.5 was evaluated based on the concentrations of dimethyl sulfoxide (DMSO) and sodium chloride (NaCl). PalP-Cy5.5 (0.5 mg / mL) was dissolved in DW mixed with DMSO (0–40%), and the change in fluorescence intensity of PalP-Cy5.5 according to the DMSO concentration was analyzed using a precision fluorescence spectrometer (FOBI, ​​CELLGENTEK, Republic of Korea) and a microplate reader (SpectraMax M2, Molecular Device, San Jose, CA, USA). Additionally, nanoformulation of PalP-Cy5.5 using NaCl involved dissolving PalP-Cy5.5 (1 mg / mL) in DW solution containing NaCl (0–2.1%). The fluorescence intensity of PalP-Cy5.5 based on NaCl concentration was analyzed similarly to the fluorescence analysis based on DMSO concentration.

[0111]

[0112] 1-7. Molecular Dynamics (MD) Simulation

[0113]

[0114] Heparin (PDB 3IRJ), curcumin (Cur), negatively charged curcumin (NCur), iron (Fe 3+), the molecular structure of doxorubicin (DOX) was described using Chemdraw Professional 20.1.1.125 (PerkinElmer Inc.). For molecular dynamics (MD) simulations, all molecules were transformed using the CHARMm (Chemistry at Harvard Molecular Mechanics) force field and optimized using the Prepare Ligand protocol in Discovery Studio 2022 software (BIOVIA; San Diego, CA, USA). MD simulations were performed using the standard dynamic cascade protocol, and all processes were observed within the NVE ensemble under the distance-dependent dielectric solvent model. The formation step of PPNC NPs was performed for 80 ps, ​​and the formation steps of HDFe NPs and HP aggregates were performed for 100 ps. Trajectory analysis for each formation step confirmed the presence and location of molecular interactions within the generated system. The analyzed hydrophobic, electrostatic, and hydrogen bonding interactions were graphically represented as column and line graphs using GraphPad Prism software. This study provides valuable insights into the dynamic behavior and molecular interactions of PPNC NPs, HDFe NPs, and HP aggregates, revealing their potential applications in various biomedical contexts.

[0115]

[0116] 1-8. In vitro test toxicity evaluation

[0117]

[0118] The cytotoxicity of PPNC NPs and HDFe NPs was evaluated using CT26.wt, a mouse colon cancer cell line. CT26.wt cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic / antimycotic. Cells were seeded at 1.0 × 10 in 96-well plates. 4Cells were seeded at a concentration of 10 cells / well and cultured for 1 h in a 5% CO2 atmosphere at 37°C. Afterwards, the cells were cultured with NCur and DOX at concentrations ranging from 0.1 to 500 μg / mL for 48 h.

[0119] Furthermore, the cytotoxicity of HP composites, which are mixtures of PPNC NPs and HDFe NPs, was evaluated. HP aggregates were prepared by mixing the two nanoparticles at a mass ratio of 1:1, and cells were cultured with HP aggregates at concentrations ranging from 0.1 to 500 μg / mL for 48 h. Cell viability (n = 6) was measured at 450 nm and 600 nm absorbance using an EZ-Cytox cell viability assay kit (Daeil Lab Service, South Korea) and a microplate reader (SPECTROstar Nano spectrophotometer, BMG Kabtech, Germany). Cell viability was calculated by comparing the absorbance values ​​of the measured samples with those of the control.

[0120]

[0121] 1-9. Bio-layer interferometry (BLI) analysis

[0122]

[0123] The protamine binding strength of HEP was evaluated using biolayer interferometry (BLI) experiments. Protamine binding kinetics were performed at 35°C and 1,000 rpm using a Gatorprime (European Headquarters, Germany). Prior to running, the amine-reactive sensor (Gator Probes, CA, USA) was hydrated in deionized water for at least 10 min and baselined with 200 μL of phosphate-buffered saline (PBS) buffer (pH 7.4) for 120 s. For amine binding, the sensor was activated with an EDC / NHS solution in MES buffer (pH 5–6) for 400 s. Protamine was bound to the amine-reactive sensor at a concentration of 10,000 μg / mL in MES buffer (pH 5–6). Equilibration was then performed in PBS buffer (pH 7.4) for 120 s. Subsequently, the binding of protamine and HEP was carried out in PBS buffer (pH 7.4) at a range of HEP concentrations (0.02–20 μM) for 60 s. Additionally, the dissociation of protamine and HEP occurred in PBS buffer (pH 7.4) for 60 s.

[0124]

[0125] 1-10. Analysis of coagulation rate using a dialysis membrane

[0126]

[0127] To compare and analyze the drug aggregation rates of HP aggregates, PPNC NPs, and HDFe NPs, evaluation was performed using a dialysis membrane (Spectra / Por™ 7 Membrane Tubing; Fisher Scientific, MA, USA). DOX was prepared at a concentration of 1 mM, HDFe NPs were also prepared at a concentration of 1 mM based on the DOX content, and PPNC NPs were prepared at a concentration of 1 mM based on the NCur content. HP aggregates were finally prepared by mixing PPNC NPs and HDFe NPs according to the concentrations of NCur and DOX contained in PPNC NPs and HDFe NPs. All prepared materials were gently stirred inside the dialysis membrane. The aggregation rates were measured at each time point (1, 2, 6, 18, and 36 h) after DOX and NCur were sufficiently dissolved in 70% DMSO solution, and the measurements were performed using a microplate reader (SPECTROstar Nano, BMG Labtech, Germany) (n = 3). Additionally, the DW solution used during dialysis was replaced every 2 hours, and the aggregation states of PPNC NPs, HDFe NPs, and HP aggregates during the 36-hour dialysis period were visually compared through captured images.

[0128]

[0129] 1-11. Synthesis of heparin-RITC (HEP-RITC), protamine-RITC, and palmitic-protamine-RITC

[0130]

[0131] Fluorescently labeled heparin (HEP), protamine, and palmitate-protamine (PalP) were prepared by dissolving 10 mg of HEP and 10 mg of PalP separately in 500 μl of distilled water (DW) using RITC. Each solution was then mixed with 500 μl of distilled water (DW) containing 1 mg of RITC. The reaction mixture was gently stirred at 600 rpm at room temperature for 1 day, after which 10 mL of DW was added to each. The resulting solutions were lyophilized for 2 days. After lyophilization, 20 mL of methanol was mixed and slowly precipitated. The precipitates were sequentially purified using methanol and separated using centrifugation (4000 rpm, 4 m, RT). The purification of PalP-RITC and HEP-RITC was confirmed by thin-layer chromatography (TLC) analysis using methanol, chloroform, and acetic acid (5:3:2). The remaining organic solvent residue was removed using a rotary evaporator, and the collected precipitate was freeze-dried for 2 days to obtain a powder.

[0132]

[0133] 1-12. Synthesis of Heparin-Cy5.5

[0134]

[0135] A heparin (HEP) molecule fluorescently labeled with Cy5.5 amine was synthesized by coupling the amine group of Cy5.5 to the carboxyl group of HEP. HEP (10 mg) and Cy5.5 amine (1 mg) were dissolved in 1 mL of distilled water and 200 μL of DMSO, respectively, and mixed. 0.6 mg of EDC and 0.3 mg of NHS were then added and reacted at room temperature for 24 h. The reaction mixture was gently stirred at 600 rpm for 1 day and the pH was maintained at 5–6 by monitoring with pH paper (DOO-SAN SCIENTIFIC, Korea) before and after the reaction. After the reaction, 10 mL of distilled water was added and lyophilized for 2 days. After lyophilization, the precipitate was slowly precipitated with 20 mL of methanol. Continuous purification of the precipitate was performed using methanol, and a centrifuge (2500 rpm, 4 m, RT) was used for separation. Purification of HEP-Cy5.5 was confirmed by thin-layer chromatography (TLC) using a developing solvent of methanol and chloroform (1:1). Organic solvent residue was removed using a rotary evaporator, and the collected precipitate was freeze-dried for 2 days to obtain a powder.

[0136]

[0137] 1-13. Solubility Evaluation and UV-Vis Spectroscopic Analysis of Curcumin and Negatively Charged Curcumin

[0138]

[0139] The solubility of curcumin (Cur) and negatively charged curcumin (NCur) was evaluated in various aqueous solvents. Curcumin and NCur (3 mg each) were dissolved in 1 mL of distilled water, and their solubility was observed. Additionally, UV-vis measurements were performed to compare and confirm the changes in absorbance of NCur and curcumin. 1 mg of curcumin and 1 mg of NCur were each dissolved in 1 mL of distilled water, and serial dilutions were performed to measure absorbance.

[0140]

[0141] 1-14. Active ingredient loading evaluation

[0142]

[0143] Protamine-based nanoparticles (PPNC NPs) with doxorubicin and Fe 3+ Heparin-based nanoparticles (HDFe NPs; heparin-based doxorubicin and Fe 3+ Drug loading of nanoparticles containing NCur was evaluated using a centrifugation-based method. Each set of nanoparticles (0.5 mg) was dissolved in 0.5 mL of DW. Unloaded NCur, DOX, and Fe were then separated using a centrifuge (12000 rpm, 4 m, RT). 3+ After centrifugation, the absorbance of the supernatant was measured at 426 nm and 500 nm using a microplate reader (SPECTROstar Nano spectrophotometer, BMG Labtech, Germany) (n=3).

[0144]

[0145] 1-15. Visual observation of particle formation

[0146]

[0147] Mixed images of PalP (1 mg / mL) and HEP (1 mg / mL), PalP (10 mg / mL) and HDox (10 mg / mL), and PalP (5 mg / mL) and PPNC NP (5 mg / mL) were prepared by dissolving each substance in distilled water at the above concentrations. These were then placed on a black or white background plate and the center was connected to facilitate mixing of the two substances. After mixing, photographs were taken at 5-second intervals to observe the rapid formation of particles.

[0148]

[0149] 1-16. Factor Xa analysis

[0150]

[0151] The anticoagulant activity of doxorubicin (DOX)-loaded heparin-containing nanoparticles (HEP; nanoparticle comprising heparin) HDox and heparin-based nanoparticles (HDFe NP; heparin-based nanoparticles) was evaluated using the COATEST anti-FXa chromogenic assay kit (Werfen, Bedford, MA). A mixture containing 100 μl of HDox and HDFe NP (0.4 IU), 100 μl of antithrombin (AT; antithrombin) reagent, and 800 μl of buffer solution was reacted at 37°C for 3 min. Then, 100 μl of factor Xa (FXa) solution was added to the HDox / AT and HDFe NP / AT complex solutions, and the reaction was performed at 37°C for 30 s. Subsequently, S-2222 substrate (200 μl) was added, and the reaction was continued at 37°C for 3 min. To terminate the reaction, 20% acetic acid solution (300 μl) was added. Anti-FXa activity was quantified using a microplate reader at absorbance at 405 nm (n=3).

[0152]

[0153] 1-17. Detection of malondialdehyde (MDA)

[0154]

[0155] To evaluate iron-dependent ferroptosis induction, malondialdehyde, the final product of lipid peroxidation, was quantified. CT26.wt cells were seeded at 1.0 × 10 in 60 mm dishes. 6Cells were seeded at a concentration of 1 cell / dish and cultured at 37°C in a 5% CO2 atmosphere for 1 day. Afterwards, the cells were treated with 0.1 μM NCur and DOX for 24 h. Changes in malondialdehyde levels were evaluated using an MDA assay kit (Abcam, UK) and measured at 532 nm with a microplate reader (SPECTROstar Nano, BMG Labtech, Germany) (n=3). Protein quantification for changes in malondialdehyde levels was consistently performed using a PierceTM BCA protein assay kit (Thermo Fisher Scientific, MA, USA).

[0156]

[0157] 1-18. Measurement of cellular reactive oxygen species

[0158]

[0159] To evaluate the production of reactive oxygen species (ROS) from PPNC NPs, HDFe NPs, and HP aggregates, a fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA; 2',7'-Dichlorodihydrofluorescein diacetate, Sigma-Aldrich, MO, USA) was used. Initially, 2.0 × 10 cells were seeded in a 35 mm black confocal dish (SPL Life Science, Korea) using high-glucose DMEM medium. 4 CT26wt cells were cultured at a density of 10 cells / dish for 3 h. The culture medium was then replaced, and the cells were treated with NCur and DOX at a concentration of 0.1 μM for 24 h. After culture, the DMEM medium was removed, and the cells were exposed to DCF-DA (20 μM) in a darkroom at 37°C for 30 min. After culture, fluorescence was captured using an ECLIPSE Ti2 series microscope (Nikon, Japan) equipped with a FITC filter.

[0160]

[0161] 1-19. Cellular uptake images of DOX

[0162]

[0163] CT26wt cells were cultured at 2.0 × 10 in 35 mm black confocal dishes (SPL Life Science, Korea) for 3 h using high glucose DMEM. 4 The cells were seeded at a density of 10 cells / dish. The culture medium was then replaced, and the cells were cultured for 24 h at a concentration of 0.1 μM based on NCur and DOX. After culture, the cells were fixed with paraformaldehyde (PFA), and images were taken using an ECLIPSE Ti2 series microscope (Nikon, Japan) equipped with a DOX fluorescence filter.

[0164]

[0165] 1-20. Analysis of intracellular iron ions

[0166]

[0167] Iron ions (Fe) within the cell 2+ ) were cultured in 35 mm black confocal dishes at 2.0 Х 10 cm in high glucose DMEM to determine the presence of 4 Cells were cultured at a density of 0.1 μM / dish for 3 hours. Afterwards, the existing medium was removed, and the cells were cultured with 0.1 μM concentrations of NCur and DOX for 24 hours. After culture, the DMEM medium was removed, and intracellular Fe was measured with a high-sensitivity diluted in Hank's Balanced Salt Solution (HBSS) for an additional 30 minutes at room temperature. 2+ The cells were further incubated in a dark room with 1 μM FerroOrange (Dojindo Laboratories, Japan), which specifically detects only 1 μg / mL of ...

[0168]

[0169] 1-21. In vivo distribution analysis of PalP and HEP-Cy5.5

[0170]

[0171] The in vivo translocation distribution of PalP and HEP was evaluated using 6-week-old male BALB / c nude mice (Orient Bio, South Korea). PalP (40 mg / kg) and saline were administered subcutaneously to each group of mice, followed by intravenous injection of HEP-Cy5.5 (a fluorescently labeled HEP derivative, 5 mg / kg). PalP and saline were both dissolved in 100 μL of saline, and HEP-Cy5.5 was dissolved in 200 μL of saline. The fluorescence intensities of the substances were measured 0, 1, 3, 6, 12, 24, and 48 h after intravenous administration of HEP-Cy5.5. Anesthesia for fluorescence intensity measurement was induced by intraperitoneal injection of 2,2,2-tribromoethanol (25 g) in tetra-amyl alcohol (15.5 ml) diluted 60-fold with saline. Mice were euthanized 48 hours later, and fluorescence intensity was measured and quantified using a precision fluorescence analyzer (POBI, CELLGENTEK, Korea).

[0172]

[0173] 1-22. In vivo distribution analysis of protamine and PalP

[0174]

[0175] To assess the in vivo distribution of protamine and PalP, 6-week-old male BALB / c nude mice (Orient Bio, South Korea) were used. Fluorescently labeled protamine-RITC or PalP-RITC (10 mg / kg) was injected intravenously to confirm the distribution. Each substance was dissolved in 200 μL of saline. The fluorescence intensity of the substance was measured 0, 1, 3, 6, 12, and 24 hours after intravenous injection. In addition, some mice were fasted for 18 hours before intravenous administration. Anesthesia for fluorescence intensity measurement was induced by intraperitoneal injection of 2,2,2-tribromoethanol (25 g) in tetraamyl alcohol (15.5 mL) diluted 60-fold with saline. Mice were euthanized 48 hours later, and fluorescence intensity was measured and quantified using a precision fluorescence analyzer (FOBI, ​​CELLGENTEK, South Korea). Additionally, cancer tissues from mice treated with saline and PalP-RITC for 12 hours were processed on DAPI-stained slides and analyzed for RITC fluorescence using a digital microscope ECLIPSE Ti2 Series (Nikon, Japan).

[0176]

[0177] 1-23. In vivo retention analysis of PPNC NPs and HDFe NPs in tumor tissues.

[0178]

[0179] To confirm the retention of PPNC NPs and HDFe NPs in tumor tissues, Cy5.5-labeled HDFe NPs were injected into the tail vein of BALB / c mice 12 hours after administration of FITC-labeled PPNC NPs. FITC-labeled PalP and Cy5.5-labeled HEP were used for visualization. The slides were analyzed in two groups: slides from mice administered PalP-FITC followed by HDFe NP-Cy5.5, and slides from mice administered PPNC NP-FITC followed by HDFe NP-Cy5.5. All mice were sacrificed 48 hours after administration of HDFe NPs, and the fluorescence on the slides was captured using a confocal laser scanning microscope (CLSM) LSM980 (Carl Zeiss, Germany).

[0180]

[0181] 1-24. Assessment of intracellular lipid peroxide (LPO) accumulation

[0182]

[0183] Intracellular accumulation of lipid peroxidation (LPO) in CT26.wt cells was assessed qualitatively and quantitatively using the BODIPY-C11 581 / 591 fluorescent probe. For imaging, cells were cultured in 35-mm black confocal dishes for 3 h, followed by the addition of NCur and DOX at a concentration of 0.1 μM for an additional 24 h. BODIPY-C11 581 / 591 (Invitrogen, MA, USA) was added and the cells were incubated at room temperature for 30 min. Fluorescence images were captured using an ECLIPSE Ti2 series (Nikon, Japan) with excitation and emission wavelengths of 488 / 510 nm. The fluorescence intensity of the fluorescence images was measured for quantitative analysis using ImageJ (US National Institutes of Health). Quantitative evaluation was performed using CT26.wt cells cultured for 3 h in black 96-well plates, followed by additional incubation with NCur and DOX at a concentration of 0.1 μM for 24 h. BODIPY-C11 581 / 591 (10 μM) was added and incubated for 30 min at room temperature, after which fluorescence was observed at excitation and emission wavelengths of 488 / 510 nm and 581 / 591 nm using a microplate reader (SpectraMax M2, Molecular Device, CA, USA).

[0184]

[0185] 1-25. Western blot analysis

[0186]

[0187] For GPX4 and HMGB1 analysis in CT26.wt cells, cells were cultured in 100 mm cell culture dishes at a concentration of 0.1 μM for 24 h until 70% confluency was reached. Cells were washed with DPBS and dispersed in RIPA lysis buffer. The lysate was centrifuged at 12,000 rpm for 10 min at 4°C to remove cell debris, and protein quantification was performed using a BCA protein assay kit (Thermo Fisher Scientific, MA, USA). The quantified protein from each sample was mixed with SDS loading buffer and heated for 5 min. Five μg of protein was separated on a 10% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 1.5 hours at 25°C in a 5% bovine serum albumin blocking solution containing Tris-Bis-T buffer. The membrane was then incubated with the primary antibody overnight at 4°C. The membrane was then washed three times with TBST buffer and incubated with anti-mouse immunoglobulin-horseradish peroxidase (IgG-HRP) antibody in blocking solution. After three additional washes, the bands were detected using an EZ-Western Lumi Femto (Daeil Lab Service, South Korea).

[0188]

[0189] 1-26. Immunocytochemistry (ICC)

[0190]

[0191] Immunocytochemistry (ICC) was performed to evaluate the expression regulation of ferroptosis-related factors (GPX4, SLC7A11) in CT26.wt cells and damage-associated molecular patterns (DAMPs) factors (CRT, HMGB1) in CT26.CL25 cells. 2.0 × 10 cells were seeded in 35 mm black confocal dishes (SPL Life Science, Korea) using high-glucose DMEM. 4 Cells were plated at a density of 10 cells / dish for 3 hours. Afterwards, the culture medium was replaced, and CT26.wt cells were cultured for an additional 24 hours with NCur and DOX at a concentration of 0.1 μM. CT26.CL25 cells were treated with 1 μM for 1 hour under the same conditions. After the incubation period, the DMEM medium was removed, and the cells were fixed using paraformaldehyde (PFA) in a dark environment. Both CT26.wt and CT26.CL25 cells were permeabilized for 10 minutes with 0.1% Triton X-100 in phosphate-buffered saline (PBS), except for HMGB1. To reduce nonspecific binding, 5% BSA in PBS was added for 10 minutes. After the blocking step, the primary antibodies against ferroptosis-related factors (GPX4, SLC7A11) or damage-associated molecular pattern (DAMP) factors (CRT, HMGB1) were treated overnight with primary antibodies at a ratio of 1:500 in 5% BSA. Afterwards, residual materials were removed with PBS-T, and secondary antibodies were treated for 1 h at a ratio of 1:500 diluted in 5% BSA. The nuclei of CT26.wt and CT26.CL25 cells were stained with DAPI, and images were taken using an ECLIPSE Ti2 series microscope (Nikon, Japan) equipped with a FITC filter (Ex, 460-500 / Em, 510-560).

[0192]

[0193] 1-27. In vitro flow cytometry

[0194]

[0195] CT26.CL25 cells were seeded at 0.5 x 10 in 6-well plates (SPL Life Science, Korea) using high-glucose DMEM medium. 5 Cells were initially seeded at a density of 10 cells / mL. Subsequently, cells were treated with a mixture of NCur and DOX at a concentration of 10 μM for 24 h. After treatment, cells were incubated with CRT antibody (NBP1-47518F, Novus Biologicals 10771 E Easter Ave, USA), H2DCF-DA (Thermo Fisher Scientific, MA, USA), and Ferro Orange reagent (Dojindo laboratories, Japan) at a 100:1 dilution ratio for 30 min at 37°C. CRT antibody staining was performed at 4°C for 30 min. After gentle washing with PBS, fluorescence was measured using a NovoCyte 3000 Flow Cytometer (Agilent Technologies, CA, USA).

[0196]

[0197] 1-28. Ex vivo immune cell analysis using flow cytometry

[0198]

[0199] Splenocytes isolated from C57BL / 6 mice were co-cultured with CT26.CL25 cells pretreated with NCur and DOX at a concentration of 10 μM for 2 hours at 37°C. To analyze immune cell activity, immune cell proliferation was assessed using flow cytometry. Cells were detached using trypsin / EDTA and then incubated with anti-mouse antibodies at a ratio of 1:100 in FACS buffer for 30 minutes at 4°C. Cells were then washed twice with FACS buffer. Fluorescence was analyzed using a NovoCyte 3000 flow cytometer (Agilent Technologies, CA, USA).

[0200]

[0201] 1-29. In vivo distribution analysis of PalP and HEP-RITC

[0202]

[0203] The biodistribution of PalP and HEP was evaluated using 6-week-old male BALB / c and BALB / c nude mice (Orient Bio, South Korea). Mice were treated with PalP at doses of 40 mg / kg (intratumorally) and 20 mg / kg (intravenously), respectively, followed by intravenous injection of RITC-labeled HEP at a dose of 40 mg / kg. Both PalP and HEP-RITC were dissolved in 100 μL. Mice receiving 20 mg / kg of PalP intravenously received an additional intravenous injection of HEP-RITC 12 hours later. The fluorescence intensity of the substances was measured at various time points: 6, 12, 24, and 48 hours after intravenous injection of HEP-RITC. In mice treated with 20 mg / kg of PalP, the HEP-RITC fluorescence intensity was measured for up to 72 hours. In addition, mice were fasted for 18 h before drug administration and anesthetized with Avastin (300 μL) intraperitoneally for fluorescence intensity measurement. Avastin was prepared by dissolving 2,2,2-tribromoethanol (25 g) in tetraamyl alcohol (15.5 mL), which was then diluted 60-fold with saline. After 8 h, the mice were euthanized. In addition, the fluorescence intensity of the mice was measured and quantified using a precision fluorescence analyzer (FOBI, ​​CELLGENTEK, Korea). In addition, the cancer tissues of mice treated with saline and PalP or HEP-RITC were processed into slides stained with DAPI after 72 h, and the RITC fluorescence was analyzed using a digital microscope ECLIPSE Ti2 series (Nikon, Japan).

[0204]

[0205] 1-30. In vivo antitumor efficacy

[0206]

[0207] Six-week-old male BALB / c mice were obtained from Orient Bio (Republic of Korea) for animal experiments. All animal experimental procedures were performed in compliance with the standard regulations of the Animal Experiment Ethics Committee of Konkuk University (Reference number: KU220748-1). Mice were placed dorsally (5.0 × 10 5 CT26.wt cells / 100 μL were subcutaneously inoculated. After one week, the tumor volume was approximately 50 to 100 mm 3 (largest diameter × smallest diameter 2 × 0.52), the mice were divided into six groups. The tumor-bearing mice received intravenous injection of saline, NCur (3 mg / kg), DOX (3 mg / kg), PPNC NP (3 mg / kg), HDFe NP (3 mg / kg), or the NP+NP combination. For the NP+NP combination, HDFe NP (3 mg / kg) was administered 12 hours after PPNC NP (3 mg / kg) for 24 days.

[0208] PPNC NPs and HDFe NPs were administered at 3 mg / kg each. The body weights and tumor volumes of mice were measured daily using digital calipers (ASIMETO, NC, USA). After 24 days of treatment, tumors were collected from sacrificed mice and fixed in 10% neutral-buffered formaldehyde (HuBENTec, South Korea). Tumor tissues were stained with hematoxylin and eosin (H&E), TUNEL, and Ki67. Tissue sections were then cut for histological observation using a multimedia video microscope (OS-33DPM, OSUN HITECH, South Korea) and an ECLIPSE Ti2 series (Nikon, Japan) digital microscope. TUNEL-stained tumor tissues were quantified using ImageJ (US National Institutes of Health) for further analysis.

[0209]

[0210] 1-31. Statistical Analysis

[0211]

[0212] Results are presented as mean ± standard deviation (SD). Error bars represent the standard deviation of the means obtained from independent samples. The statistical significance of differences between groups was analyzed using a one-way ANOVA test with a post hoc test. All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc.). Differences between groups were considered statistically significant if the p value was less than 0.05, 0.01, or 0.001 ( * p < 0.05, ** p < 0.01, *** p < 0.001).

[0213]

[0214] Example 2. Characterization and in vivo distribution analysis of PalP nanoparticles

[0215]

[0216] 2-1. Palp synthesis and characterization

[0217]

[0218] By leveraging the strong cationic properties of protamine, which readily interacts with negatively charged curcumin (NCur), and the self-assembly ability of palmitic-protamine (PalP), we developed nanoparticles with extended half-lives. A one-step synthetic protocol utilizing the amine group of protamine was performed at 26°C for a total of two days through organic synthesis of protamine and palmitic-NHS. For the organic synthesis, protamine was first dissolved in formamide for 24 h (Fig. 2a). The self-assembly of PalP to form nanoparticles was characterized using dynamic light scattering (DLS; Zeta sizer Nano, Malvern Instruments, Worcestershire, UK), and the measured particle size was 178.43 ± 18.80 nm (Fig. 2b). Furthermore, the particle stabilities of protamine and PalP were compared and analyzed using DLS. The zeta potential of protamine was measured as +7.35 ± 1.92 mV, whereas PalP exhibited a zeta potential of +25.69 ± 1.88 mV, indicating significant particle stability in aqueous solvents (Fig. 2c).

[0219]

[0220] 2-2. Fluorescence intensity analysis according to DMSO and NaCl concentration

[0221]

[0222] The fluorescence changes of Cy5.5-labeled PalP nanoparticles in response to various concentrations of dimethyl sulfoxide (DMSO) were investigated. PalP-Cy5.5 (0.5 mg / mL) showed an increase in fluorescence intensity as the DMSO concentration increased from 0 to 40%. The fluorescence intensity in 0% DMSO was 77.33 ± 5.78 au (arbitrary unit), and in 40% DMSO, the fluorescence intensity was 162.27 ± 3.55 au, which was 2.19 times higher. This suggests that the structure of PalP-Cy5.5 nanoparticles was destroyed by DMSO, resulting in an enhanced fluorescence intensity (Fig. 2d). In addition, the fluorescence change of PalP-Cy5.5 induced by NaCl was observed. The fluorescence intensity in 2.1% NaCl was 79.74 ± 1.36 au, which was a 1.49-fold decrease compared to 0% NaCl (116.58 ± 3.90 au). This indicates that the rapid structural rearrangement of nanoparticles occurs due to NaCl, and the fluorescence intensity of PalP-Cy5.5 decreases with increasing NaCl concentration (Fig. 2e). In summary, these results indicate that PalP nanoparticles self-assemble in aqueous solvents to form stable formulations.

[0223]

[0224] 2-3. Nanoparticle Characterization

[0225]

[0226] The distinct nanoscale morphology of the self-assembled PalP nanoparticles was observed by transmission electron microscopy (TEM) and field-effect scanning electron microscopy (FE-SEM) (MERLI, Carl Zeiss, German), showing a spherical shape with a size ranging from 50 to 500 nm (Fig. 2f and 2g). The chemical conjugation process of the PalP molecules was analyzed by 1D proton NMR. The main peaks of protamine appeared at 1.5–2.0 ppm and 4.0–5.0 ppm, and the main peak of palmitic acid-NHS appeared at 1.0–1.5 ppm. The main peak of protamine was observed under the solvent condition with excess D2O (D2O:DMSO-d6= 8:2), and the main peak of palmitic acid-NHS was observed under the condition with excess DMSO-d6 (D2O:DMSO-d6= 2:8). This means that nanoparticles were formed through the combination of protamine and palmitic acid-NHS (Fig. 3).

[0227]

[0228] 2-4. Cytotoxicity evaluation

[0229]

[0230] To evaluate the cytotoxicity of PalP, cell viability experiments were performed on CT26.wt cells treated with PalP at concentrations ranging from 0.01 to 100 μg / mL. At the highest concentration of 100 μg / mL, cell viability was 99.71 ± 5.0%. These results indicate that PalP exhibits minimal toxicity against CT26.wt cells at a concentration of 100 μg / mL (Fig. 2h).

[0231]

[0232] 2-5. Distribution analysis after in vivo administration

[0233]

[0234] To trace the distribution of the substance, BALB / c nude mice bearing CT26.wt tumors were administered RITC-labeled protamine and PalP at a dose of 20 mg / kg via the tail vein. Fluorescence intensity was measured 1 hour after administration using a fluorescence analyzer (FOBI, ​​Celgenetech, Korea). In addition, PalP-RITC (20 mg / kg) and saline were separately administered via the tail vein for accurate fluorescence intensity analysis. The heart, kidney, liver, lung, spleen, and tumor were extracted at 6, 12, 24, and 48 hours after administration, and fluorescence images were taken using a fluorescence analyzer (FOBI, ​​Celgenetech, Korea). As a result, PalP-RITC exhibited a significantly stronger fluorescence intensity than tumors administered with protamine-RITC. Furthermore, the strongest fluorescence intensity was observed 12 hours after administration in tumor tissues extracted from mice treated with PalP-RITC (Figure 2i). Quantification of PalP-RITC in tumor tissues showed that the fluorescence intensity was the highest at 3.3e+0.6 ± 3.7e+0.5 Area x IU 12 hours after administration (Fig. 2j). Here, “IU” stands for “intensity / min / gain.” This abbreviation is a unit of measurement used to quantify fluorescence intensity, and specifically, it represents the intensity and gain of the fluorescence signal obtained per unit time (minute). In addition, 12 hours after PalP-RITC administration to BALB / c mice, tumor tissues were sliced ​​and observed using a fluorescence microscope (ECLIPSE Ti2 series, Nikon, Japan) using the RITC fluorescence wavelength range. As a result, it was found that the group administered PalP-RITC had more RITC fluorescence in the tumor tissues 12 hours after administration than the group treated with saline (Fig. 2k). Based on the above results, in a follow-up study, HDFe NPs were sequentially administered at 12-hour intervals after administering PPNC NPs containing PalP as the main component.

[0235]

[0236] Example 3. Preparation and characterization of NCur and PPNC nanoparticles

[0237]

[0238] 3-1. Ncur manufacturing and water solubility analysis

[0239]

[0240] 1N NaOH (1% volume concentration) was added to methanol solvent at 26 °C for 2 h (Fig. 4a). The enhanced solubility of negatively charged curcumin (NCur) was visually confirmed by comparing the images of Ncur and curcumin (Cur) dissolved in distilled water (DW) (Fig. 4b). Furthermore, UV-vis spectra obtained using a microplate reader (SPECTROstar Nano spectrophotometer, BMG Labtech, Germany) showed that the absorption intensity of NCur decreased with the electron potential difference compared to Cur (Fig. 4c). Therefore, these results indicate that the formulation of NCur significantly improves the solubility of curcumin, although its solubility in organic solvents is limited.

[0241]

[0242] 3-2. Zeta potential measurement of Ncur

[0243]

[0244] In addition, the surface charge of NCur was compared with that of Cur by comparing and observing the zeta potential through dynamic light scattering (DLS) and the degree of molecular interaction on the material surface was investigated. The zeta potential value of NCur was -25.76 ± 10.57 mV, which was 11.9 times higher than that of Cur (-2.16 ± 0.24 mV). The significant increase in zeta potential indicates that the negative charge on the surface of NCur is stronger than that of Cur, suggesting that more negative charges exist on the surface of NCur (Fig. 4d).

[0245]

[0246] 3-3. Observation of NCur particles and preparation of PPNC NPs

[0247]

[0248] The particle morphology of NCur was observed using TEM imaging. No distinct particle shape was identified for NCur, and no identifiable particles were observed in the images, suggesting that a well-defined particle structure was not observed for NCur (Fig. 5).

[0249] Positively charged palmitic-protamine curcumin nanoparticles (PPNC NPs) were formed by exploiting the charge interaction between positively charged PalP nanoparticles and NCur (Fig. 4e). PPNC NPs were prepared by dissolving PalP and NCur individually in distilled water and then mixing the two solutions. We hypothesized that the amphiphilicity imparted by protamine binding to the lipophilic moiety would lead to self-assembly of the nanoparticles. Furthermore, we anticipated that the combination of the positive charge of protamine and the negative charge of NCur would lead to the formation of self-assembled nanoparticles.

[0250]

[0251] 3-4. Simulation of the formation and self-assembly of PPNC NPs

[0252]

[0253] To simulate the formation of PPNC NPs, molecular dynamics (MD) simulations were performed using BIOVIA Discovery Studio software from 0 to 80 ps. Using the Chemistry at Harvard Molecular Mechanics (CHARMm) force field, PPNC NP molecules exhibited stable self-assembly over time around the lipophilic groups (Fig. 4f). Furthermore, interactions related to hydrophobicity, electrostatics, and hydrogen bonding within PPNC NPs were compared from 0 to 80 ps. An increase in the number of interactions indicates more complex molecular interactions between PalP and NCur molecules within the simulated PPNC NPs (Fig. 4g).

[0254]

[0255] 3-5. Evaluation of structural stability according to Ncur and PalP ratios

[0256]

[0257] To select the most stable PPNC NPs, sedimentation was evaluated in distilled water for 24 h. NCur was mixed appropriately while keeping the mass of NCur constant and varying the mass of PalP. At specific time points, the absorbance of NCur remaining in the supernatant was measured at 426 nm using a microplate reader. The absorbance measurement at this wavelength indicates the relative level of sedimentation and stability. Based on the results obtained, it was observed that the most stable PPNC NPs were formed when the ratio of PalP to NCur was 1:1 by mass. The absorbance value of the most stable PPNC NPs at 24 h was measured to be 76.22 ± 2.54% (Fig. 4h).

[0258]

[0259] 3-6. Characteristics of PPNC NPs

[0260]

[0261] The self-assembly of PPNC NPs was characterized using DLS, and the particle size was found to be 233.5 ± 76.36 nm (Fig. 4i). The distinct nanoscale morphology of PPNC NPs was observed by TEM, showing a spherical shape in the range of 50–100 nm (Fig. 4j). The particle size was measured using DLS at specific time points (0, 1, 3, 6, 12, and 24 h) to evaluate the particle stability of PPNC NPs in distilled water, and the particle size range was found to be maintained at 200–400 nm (Fig. 4k). In addition, the particle stability of PPNC NPs was confirmed using DLS, and the zeta potential value was +21.36 ± 0.73 mV, indicating high particle stability in aqueous solvents (Fig. 4l).

[0262] To measure the loading efficiency of Ncur onto PalP, the material was precipitated using a centrifuge, and the absorbance of the supernatant was measured using a microplate reader to calculate the loading efficiency of Ncur. The loading ratio of NCur onto PPNC NPs was measured to be 70.11 ± 1.04%, while the loading ratio of NCur alone was measured to be 3.33 ± 2.66%, confirming the effective loading of PPNC NPs onto PalP (Fig. 4m). After centrifugation, the values ​​for all measured materials were calculated by subtracting the absorbance values ​​before and after centrifugation. Therefore, a larger difference in the absorbance values ​​indicates effective loading of the material and suggests that particles have been formed.

[0263] In addition, we observed a change in the color of the supernatant after precipitating Ncur and PPNC NPs through centrifugation. In the case of NCur, the difference in the color change of the supernatant before and after centrifugation was minimal due to inefficient particle formation, but in the case of PPNC NPs, a distinct decrease in the color of the supernatant was observed due to the interaction of PalP-bound NCur in the supernatant, indicating that particles were formed through the interaction between PalP and NCur after centrifugation. FE-SEM was used to further investigate the morphological structure of PPNC NPs through imaging. A unique nano-sized morphology of PPNC NPs was observed within the range of 300–500 nm (Fig. 6). To evaluate the cytotoxicity of NCur and PPNC NPs, a cell viability test was performed on CT26.wt cell line. At the highest concentration of NCur, 100 μg / mL, cell viability was confirmed to be 0.79 ± 0.27% (NCur) and 0.66 ± 0.86% (PPNC NP), and the IC50 of PPNC NP was found to be 43.63 μg / mL (Fig. 4n). The above results show that PPNC NP exhibits higher cytotoxicity than PalP.

[0264]

[0265] Example 4. Preparation and characterization of HDFe nanoparticles

[0266]

[0267] 4-1. HDFe NP self-assembly and MD simulation

[0268]

[0269] To develop a potent anticancer agent that can induce both apoptosis and ferroptosis of cancer cells, heparin (HEP), doxorubicin (DOX), and Fe 3+ We designed nanoparticles containing HDFe nanoparticles (HDFe NPs). The two-step procedure involves the preparation of negatively charged HEP and DOX and Fe 3+We utilized the charge-to-charge interaction between the components, and when the components were combined in the appropriate ratio, they self-assembled to form nanoparticles.

[0270] To reproduce the self-assembly characteristics of HDFe NPs in silico, MD simulations were performed from 0 to 100 ps using BIOBIA Discovery Studio software. HDFe NPs using the CHARMm force field showed stable self-assembly over time around hydrophobic DOX molecules (Fig. 7a). The number of hydrophobic and electrostatic interactions within HDFe NPs was calculated from 0 to 100 ps. The increase in interactions was attributed to the increase in HEP, DOX, and Fe within the simulated HDFe NPs. 3+ This indicates that the complexity of molecular interactions between HDFe NPs has increased (Fig. 7b). The number of hydrogen bonds in HDFe NPs was simulated between 0 and 100 ps. The number of hydrogen bonds increased from 37 to 120 for HDFe NPs, which is about a 3.2-fold increase, which is higher than that of HEP, DOX, and Fe NPs. 3+ This means that the molecular interactions between HEP ​​and DOX are more complex than those between HEP ​​alone (Fig. 7c). In addition, HEP, DOX, and Fe within HDFe NP particles 3+ The expected 2D array structure is described (Fig. 8).

[0271]

[0272] 4-2. Stability Evaluation According to the Composition Ratio of Hdox and HDFe NPs

[0273]

[0274] To select the most stable HDFe NPs, we performed a sedimentation evaluation for HEP / DOX (HDox). The HDox sedimentation evaluation was performed by measuring the absorbance of DOX remaining in the supernatant at 500 nm in distilled water at specific time points (0, 1, 3, 6, 12, and 24 h) using a microplate reader for 24 h. A higher absorbance indicates less rapid sedimentation and higher stability. As a result, the HEP to DOX ratios of 25:1 and 5:1 were found to be the most stable in terms of compound formation. In subsequent experiments, a higher DOX concentration ratio of 5:1 was used. The absorbance at 24 h for the selected ratio was confirmed to be 70.51 ± 5.83%, indicating excellent stability (Fig. 7d). DLS was used to investigate the particle size distribution of HDox. The particle size was observed to be 3638.33 ± 1296.85 nm, which is in the micro-size range rather than the nano-size. This suggests that HDox forms particles, but many of them are larger (Fig. 9).

[0275] The sedimentation evaluation of HDFe NPs was performed following the same protocol as HDox, and the most stable ratio (HEP:DOX:Fe = 5:1:0.4) among the four ratios was selected (Fig. 7e). The absorbance at 24 h for the selected ratio was measured to be 85.57 ± 1.48%, confirming excellent stability. The self-assembly of HDFe nanoparticles was characterized using DLS. The particle size was measured to be 190.17 ± 30.92 nm. In addition, the morphological image of the particle size of HDFe NPs showed the formation of spherical particles, as observed in the FE-SEM image (Fig. 7f). In addition, the zeta potential value of HDFe NPs was -37.41 ± 1.10 mV, indicating high particle stability in aqueous solvents (Fig. 7g). The distinct nanoscale morphology of the self-assembled HDFe NPs was observed by TEM, showing a spherical shape with a size ranging from 50 to 100 nm (Fig. 7h).

[0276]

[0277] 4-3. Evaluation of the molecular structure and DOX loading efficiency of HDFe NPs

[0278]

[0279] In addition, the Fe of HDFe NPs was characterized by scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM-EDS; MERLIN; Carl Zeiss, Germany). 3+ Elemental analysis was observed for the content of HDFe NPs. Fe 3+The elemental mass was measured to be 9.27 ± 6.30%, indicating that iron was the main element present in HDFe NPs, following oxygen and carbon (Figs. 7i and 10). In addition, the 3D molecular structure of HDFe NPs was predicted through MD simulation (Fig. 11). Particle stability was evaluated in an aqueous solvent such as distilled water, and DLS was measured at specific time points (0, 1, 3, 6, 12, and 24 h) for 24 h, maintaining the particle size at 100–400 nm (Fig. 7j). In addition, to confirm the stable loading of DOX in HEP, the material was precipitated using a centrifuge, and the absorbance of the supernatant was measured using a microplate reader to calculate the loading efficiency. The DOX loading efficiency of HDFe NPs was 53.13 ± 7.72%, and the loading efficiency of DOX alone was 0.80 ± 1.22%, and the difference in efficiency was 52.34 ± 7.19%, confirming successful loading (Fig. 7k). Overall, HEP, DOX, and Fe 3+ It was clearly demonstrated that the most stable ratio, HEP:DOX:Fe = 5:1:0.4, forms nanoparticles. In addition, the interactions within the particles were verified through simulation.

[0280]

[0281] 4-4. Evaluation of cytotoxicity and anticoagulant effects

[0282]

[0283] To evaluate the cytotoxicity of HEP, DOX, and HDFe NPs, cell viability experiments were performed on CT26.wt at concentrations ranging from 0 to 500 μg / mL, and HDFe NPs were evaluated based on DOX concentration. At the highest concentration of 500 μg / mL, the cell viability was 91.93 ± 6.67% (HEP), 0 ± 0% (DOX), and 0.39 ± 0.95% (HDFe NP), and the IC50 of HDFe NPs was 132.2 ng / mL, indicating high cytotoxicity (Fig. 7l).

[0284] Since HEP is an FDA-approved anticoagulant, it is used with DOX and Fe to prevent unintended side effects. 3+ It was necessary to evaluate whether the anticoagulant effect was reduced depending on the combination. The anticoagulant activity of HEP, HDox, and HDFe NPs was evaluated using an anti-FXa assay. The anti-FXa activities of the two complexes were measured as follows: HEP (100 ± 0.87%), HDox (59.67 ± 4.62%), and HDFe NP (66.67 ± 1.16%). The results show that the anticoagulant effect was significantly reduced compared to HEP (Fig. 7m). It was found that the two nanoparticles exhibited significantly reduced anticoagulant activity, suggesting that the undesirable anticoagulant effect could be alleviated. In conclusion, we successfully formulated HDFe NPs with minimized anticoagulant effect and significant cytotoxicity.

[0285]

[0286] Example 5. Formation of self-assembled aggregates of protamine and heparin-based nanoparticles.

[0287]

[0288] 5-1. Measurement of aggregate formation through self-assembly of PPNC NPs and HDFe NPs

[0289]

[0290] Due to the attraction between positive and negative charges, the two types of nanoparticles, PPNC NPs and HDFe NPs, were expected to self-assemble into aggregates by simultaneously attracting and entangling each other (Fig. 12a). To simulate the aggregation of the two nanoparticles, MD simulations were performed using BIOVIA Discovery Studio software over 0 to 100 ps. Based on the CHARMm force field, the HP aggregates were found to maintain their hydrophobic interiors over time, indicating the formation of stable self-assembled complexes. The MD simulations were performed with the PPNC NPs and HDFe NPs in a stable and self-assembled state (Fig. 12b). The number of hydrophobic and electrostatic interactions within the HP aggregates was compared from 0 to 100 ps. The increase in interactions indicates an increase in the complexity of the interparticle interactions between PPNC NPs and HDFe NPs within the HP aggregates (Fig. 12c). The number of hydrogen bonds in the simulated HP aggregates was quantified over 0 to 100 ps. The number of hydrogen bonds in the HP aggregates increased from 90 to 216, a 2.4-fold increase, confirming molecular interactions between PPNC NPs and HDFe NPs. This suggests that the interactions between PPNC NPs and HDFe NPs form a more cohesive structure within the HP aggregates (Fig. 12d).

[0291]

[0292] 5-2. Measurement of aggregate formation with PalP

[0293]

[0294] To observe the formation of new particles, we imaged mixtures of PalP with HEP or Hdox. To observe particle formation, all materials were mixed on a transparent acrylic plate and photographed. Particle formation with PalP using HEP or Hdox was observed as the materials were mixed over time, with a gradual increase in white or red particles, respectively. This indicates that new particles are formed through interactions between the materials.

[0295] HP aggregate formation was also evaluated using the same method described above. The results showed that HP aggregates were successfully formed through the interaction between PPNC NPs and HDFe NPs. As confirmed by image analysis, mixing NCur and DOX at a mass ratio of 1:1 facilitated aggregate formation. Aggregates are identified as black dots in the image, indicating cohesion and interaction between nanoparticles (Fig. 12e).

[0296]

[0297] 5-3. Cytotoxicity Evaluation and Measurement of the Interaction between HEP ​​and Protamine

[0298]

[0299] To evaluate the cytotoxicity of HP aggregates, a cell viability experiment was conducted on CT26.wt cells. At a concentration of 500 μg / mL, the cell viability was observed to be 2.21 ± 0.59%, and the IC50 was calculated to be 98.05 ng / mL, indicating the high cytotoxicity of HP aggregates (Figure 13). The distinct morphology of the self-assembled HP aggregates was observed through TEM and FE-SEM, forming clusters of multiple particles (Figures 12f and 12g). TEM and FE-SEM images of the HP aggregates revealed the presence of aggregated structures formed through the interaction between PPNC NPs and HDFe NP particles. The interaction between HEP ​​and protamine was further confirmed through bio-layer interferometry (BLI). Protamine was immobilized on an amine-responsive sensor, and its interaction with HEP was evaluated by the KD value. As a result of setting HEP at various concentrations (0.002 to 20 μM), the interaction between HEP ​​and protamine increased as the HEP concentration increased, showing a remarkable KD value of 474 nm, indicating a high affinity between HEP ​​and protamine (Fig. 12h).

[0300]

[0301] 5-4. Measuring drug retention efficiency

[0302]

[0303] To investigate the drug association between HP aggregates and HP aggregates, DOX, PPNC NPs, HDFe NPs, and HP aggregates were stirred using a dialysis membrane. Particle images at 36 h and drug association experiments for PPNC NPs, HDFe NPs, and HP aggregates are shown in Figure 12i. Subsequently, the remaining substances within the membrane were quantified using an absorbance spectrophotometer at each time point (1, 2, 6, 18, and 36 h). At 36 h, the drug retention efficiency of HP aggregates for NCur was 75.00 ± 0.54%, which was 7.6 times higher than the drug retention efficiency of Ncur using PPNC NPs (9.94 ± 0.00%).

[0304] In addition, the drug retention efficiency of the HP aggregates for DOX was 65.53 ± 0.31%, which showed a 1.5-fold higher association effect than the drug retention efficiency of DOX using HDFe NPs (42.44 ± 0.99%). The residual substance of DOX in the membrane was 0% at 36 h. The above results showed that the particles of PPNC NPs and HDFe NPs showed higher drug retention efficiency than individual substances such as DOX and NCur. In addition, the drug binding effect was highest when PPNC NPs and HDFe NPs were combined with the HP aggregates (Fig. 12j).

[0305]

[0306] Example 6. Evaluation of nanoparticles for ferroptosis and reactive oxygen species production.

[0307]

[0308] 6-1. Measurement of ferroptosis through MDA and LPO analysis

[0309]

[0310] We quantitatively evaluated changes in malondialdehyde (MDA), a representative product of lipid peroxidation associated with ferroptosis. In CT26.wt cells, HDFe NPs exhibited the highest MDA content (145.44 ± 5.23%), followed by HP aggregates (137.27 ± 4.47%) (Fig. 13a).

[0311] To assess the accumulation of intracellular lipid peroxidation (LPO), a key feature of ferroptosis, in CT26.wt cells, the BODIPY-C11 581 / 591 fluorescent probe was used. Red (reduced state) and green (oxidized state) fluorescence were observed at 581 / 591 nm (red fluorescence) and 488 / 510 nm (green fluorescence), respectively, using a microplate reader (SpectraMax M2). The intracellular LPO accumulation process can be visualized as a fluorescence transition from red to green. The groups treated with HDFe NPs and HP aggregates showed intensities of green fluorescence relative to the red fluorescence indicated by oxidized BODIPY-C11 of 1285.36 ± 27.67% and 1437.61 ± 238.95%, respectively, which were significantly increased compared to the control, DOX, and PPNC NP-treated groups. This indicates that the accumulation of LPO induced by strong ROS production in CT26.wt cells was enhanced by HDFe NPs and HP aggregates, and this accumulation is considered to be a hallmark of ferroptosis (Fig. 13b).

[0312]

[0313] 6-2. Measurement of ferroptosis through GPX4 analysis

[0314]

[0315] Western blot analysis was used to measure the expression changes of glutathione peroxidase 4 (GPX4), which plays an important role in the regulation of ferroptosis under each condition. GPX4 plays a role in blocking ferroptosis by repairing oxidative damage to lipids. When cells were treated with HDFe NPs, the concentration of GPX4 was significantly reduced. This was due to Fe 3+ When treated with a substance containing , there was a notable difference in GPX4 intensity. Following a similar trend, Western blot analysis showed that GPX4 expression was significantly reduced in the HDFe group compared to the other groups. In addition, an increase in HMGB1 expression was observed in the HDFe NP group as apoptosis was induced (Fig. 13c). In addition, the difference in GPX4 expression in each group was quantified using ImageJ (US National Institutes of Health) (Fig. 13d).

[0316]

[0317] 6-3. Measurement of ferroptosis using fluorescence imaging and immunocytochemical analysis

[0318]

[0319] The therapeutic mechanism was experimentally demonstrated in cells through fluorescence imaging. The study results showed that DOX and Fe 3+ It was found that internalization of HDFe NPs induced ferroptosis and ROS release, ultimately leading to cell death. The uptake of DOX was visualized using a DOX fluorescence filter. As expected, HDFe NPs and HP aggregates increased intracellular DOX levels.

[0320] In addition, intracellular Fe was measured using the Ferro-orange reagent. 2+We investigated whether HDFe NPs and HP aggregates directly affect intracellular ferroptosis by directly measuring the presence of iron. The results showed that the ferro-orange fluorescent probe was barely visible in the iron-free control and PPNC NP groups. In contrast, the iron-containing HDFe NPs and HP aggregate groups exhibited very strong fluorescent signals, indicating that iron directly induces ferroptosis in cancer cells.

[0321] H2DCF-DA was used to detect intracellular ROS levels. Cells treated with PPNC NPs showed weak fluorescence signals, whereas those treated with HDFe NPs and HP aggregates showed strong fluorescence signals. Immunocytochemistry (ICC) experiments were used to confirm whether ferroptosis was induced through the inactivation mechanism of GPX4 and SLC7A11 (Feng-Jiao Li et al, "System Xc- / GSH / GPX4 axis: An important antioxidant system for the ferroptosis in drug-resistant solid tumor therapy", Front Pharmacol. 2022; 13: 910292.). As a result, the fluorescence intensities of GPX4 and SLC7A11 were significantly reduced in the HDFe NP and HP aggregate groups compared to the control group. This suggests that HDFe NPs and HP aggregates significantly reduce the enzymatic activities of GPX4 and SLC7A11, thereby inducing ferroptosis. To observe intracellular LPO accumulation via fluorescence imaging, LPO was measured in CT26.wt cells using the BODIPY-C11 581 / 591 fluorescent probe. The LPO accumulation levels in the groups treated with HDFe NPs and HP aggregates showed stronger fluorescence values ​​in the oxidized form (green fluorescence) of the fluorescent probe compared to the control and PPNC NP-treated groups. This observation indicates that HP aggregates generated ROS, leading to strong LPO accumulation in CT26.wt cells (Fig. 13e).

[0322]

[0323] 6-4. Evaluation of iron accumulation using fluorescence imaging and flow cytometry

[0324]

[0325] To further validate the results, quantitative analysis was performed in CT26.CL25 cells by fluorescence or flow cytometry to determine BODIPY, ROS levels, and intracellular iron ions (Fe 2+ ) accumulation was evaluated. The fluorescence intensity evaluating BODIPY in the HP aggregate treatment group was 292.70 ± 46.53%, which was 2.9 times higher than that in the control group (Fig. 13f). Drug-induced ROS production and intracellular iron (Fe 2+ ) accumulation was quantitatively evaluated by flow cytometry (FACS). As a result, ROS levels increased in the HDFe NP and HP aggregate groups compared to the control group, and the HP aggregate group showed the highest increase rate of 314.13 ± 14.99% (Fig. 13g). Intracellular iron (Fe 2+ ) accumulation was significantly increased in the HDFe NP and HP aggregate groups compared to the control group, with the HP aggregate group showing the highest increase rate of 513.02 ± 6.46% (Fig. 13h).

[0326]

[0327] Example 7. Evaluation of immune cell activation and immunogenicity

[0328]

[0329] 7-1. ICD measurement through CRT and HMGB1 expression analysis

[0330]

[0331] Calreticulin (CRT) and high mobility group box 1 (HMGB1), which are representative markers of immunogenic cell death (ICD), were analyzed by fluorescence imaging after drug treatment for 1 hour. The expression of CRT and HMGB1 increased in the HDFe NP and HP aggregate groups compared to the control or PPNC NP groups (Fig. 14a). In addition, the cell surface relocation of CRT was quantitatively evaluated using flow cytometry (FACS) after drug treatment for 24 hours. The level of cell surface relocation of CRT was significantly increased in the HDFe NP and HP aggregate groups compared to the control and PPNC NP groups, and the HP aggregate group showed the highest CRT expression with a mean fluorescence intensity (MFI) of 12133.33 ± 287. In summary, we observed that ICD was significantly induced when treated with the HP aggregate group, as evidenced by the increase in CRT and HMGB1 (Fig. 14b).

[0332]

[0333] 7-2. Analysis of immune cell activation through dendritic cell measurement

[0334]

[0335] To investigate whether drug-treated cancer cells (CT26.CL25) activate immune cells, mouse spleen cells were extracted and co-cultured with drug-treated CT26.CL25 (Fig. 14c). As a result, dendritic cells (DCs) were observed to increase and be activated in the HP aggregate group. The number of dendritic cells increased 2.2-fold, and the measured values ​​for all groups were in the following order: control group (19.75 ± 0.91%), PPNC NP (24.93 ± 5.32%), and HP aggregate (44.13 ± 0.90%) (Fig. 14d). In addition, the activated dendritic cells increased 7.58-fold compared to the control group, and the measured values ​​for all groups were 29.79 ± 0.27% for the HP aggregate group, 23.06 ± 0.27% for the PPNC NP group, and 22.21 ± 0.12% for the control group (Fig. 14e).

[0336]

[0337] 7-3. Evaluation of T cell proliferation and activation

[0338]

[0339] CD, a total T cell marker 3+ As a result of evaluation, T cell proliferation was found to increase by approximately 11.40% in the HP aggregate group compared to the control group (Fig. 14f). In addition, CD4 + The T cell population was confirmed to have increased by 4.32% compared to the control group (Fig. 14g). In addition, compared to each control group, CD8 + Cells were approximately 12.76% (Fig. 14h), CD8 + CD69 + is 26.44% (Fig. 14i), CD8 + CD25 + was increased by 15.25% (Fig. 14j). In addition, granzyme + (granzyme + ) CD8 + T cells and interferon + (IFN + ; interferon + ) CD8 +An increase in T cells was observed in the granzyme treated with HP aggregates (45.49 ± 0.55%). + CD8 + T cells showed a significant increase compared to PPNC NPs (2.54 ± 0.13%) and control (2.06 ± 0.13%) (Fig. 14k). Similarly, IFN-γ treated with HP aggregates + CD8 + T cells (53.97 ± 0.82%) were significantly increased compared to cells treated with PPNC NPs (37.29 ± 0.62%) and the control group (36.84 ± 0.59%) (Fig. 14l).

[0340]

[0341] 7-4. Evaluation of NK cell proliferation and activation

[0342]

[0343] NK (Natural killer) cells, which play a role in directly killing cancer cells, also showed a higher CD48 level in the HP aggregate-administered group compared to the control group. + NK1.1 + is about 3.83% (Fig. 14m), CD49b + increased by 15.29% (Fig. 14n). NK cells and granzyme + and an increase in IFN-positive NK cells was also confirmed. granzyme + In the case of NK cells, the HP aggregate-treated group (6.87 ± 0.06%) showed a 38.18-fold increase compared to the control group (0.18 ± 0.05%) and an 89.65-fold increase compared to the PPNC NP-treated group (0.08 ± 0.02%) (Fig. 14o). Similarly, IFN +For NK cells, the HP aggregate-treated group (20.83 ± 0.75%) showed a 3.53-fold increase compared to the control group (5.9 ± 0.05%) and a 4.36-fold increase compared to the PPNC NP group (4.77 ± 0.06%) (Fig. 14p). In addition, NKT (Natural killer T) cells, which uniquely combine the characteristics of NK cells and T cells, were confirmed to increase approximately 3.39-fold compared to the control group (Fig. 14q).

[0344]

[0345] 7-5. Evaluation of macrophage proliferation and activation

[0346]

[0347] We also investigated the activation of macrophages, which influence both tumor growth (M2 macrophages) and suppression (M1 macrophages) in response to HP aggregate treatment. F4 / 80 + CD80 + As a result of gating for M1 macrophages defined as , the HP aggregate group showed 72.76 ± 3.64%, the PPNC NP group showed 36.85 ± 0.10%, and the control group showed 35.14 ± 0.48% (Fig. 14r). For M2 macrophages, the HP aggregate group showed 1.56 ± 0.10%, whereas the PPNC NP group showed 11.77 ± 0.45%, and the control group showed 10.72 ± 0.36% (Fig. 14s). In particular, when treated with HP aggregates, M1 increased and M2 decreased, confirming that the M1 / M2 ratio, an immunogenicity indicator, significantly increased. The M1 / M2 ratio was found to have increased by 3.35% compared to the control group (Fig. 14t). Overall, the HP aggregate group showed significant immune cell activation compared to the PPNC NP treatment and the control group. Therefore, as ICD is induced, DCs are activated, resulting in CD4 + and CD8 +Not only T cells but also NK cells were significantly activated, leading to a significant increase in the M1 / M2 ratio (Fig. 14u). Overall, the HP aggregate group demonstrated similar or superior immunogenicity and ICD induction compared to PPNC NPs, demonstrating potential as an immunotherapeutic agent.

[0348]

[0349] Example 8. Enhanced tumor targeting and aggregation formation through inductive effects

[0350]

[0351] 8-1. Measurement of the interaction between PalP and heparin through biodistribution tracking

[0352]

[0353] To investigate whether PalP, which carries a strong positive charge, and heparin, which carries a strong negative charge, are attracted to each other through charge-to-charge interaction at the tumor site as hypothesized, we tracked the biodistribution of fluorescently labeled heparin (HEP-Cy5.5 or HEP-RITC). First, tumor-free BALB / c nude mice were subcutaneously injected with saline or PalP (40 mg / kg), followed by immediate intravenous administration of HEP-Cy5.5 (5 mg / kg) via the tail vein. Fluorescence intensity was measured using a fluorescence analyzer (FOBI, ​​Celgenetech, Korea) at each time point (0, 1, 3, 6, 12, 24, and 48 h). No fluorescence was observed around the injection site in mice injected subcutaneously with saline. In contrast, mice injected with PalP showed high fluorescence intensity at and around the injection site from 1 h after administration, indicating that there is an interaction between PalP and HEP in vivo (Fig. 15a). At the initial time point, approximately 1 hour after subcutaneous injection of PalP, the fluorescence intensity of HEP-Cy5.5 reached its peak and gradually decreased over time. These observations suggest that PalP initially induces the accumulation of HEP-Cy5.5 at the injection site during subcutaneous injection. However, as PalP is absorbed and disperses at the injection site, HEP also disperses, resulting in a decrease in fluorescence intensity. These results indicate that HEP-RITC is dynamically distributed and eliminated depending on the distribution of PalP in vivo. In addition, the fluorescence intensities of the saline and PalP subcutaneous injection sites at 48 hours were measured to be 8.5e+06 ± 5.2e+05 Area x IU and 3.1e+07 ± 8.3e+05 Area x IU, respectively. As a result, the fluorescence intensity of mice subcutaneously administered PalP was found to be 2.05 times higher than that of mice subcutaneously administered saline (Fig. 15b).

[0354] The "guidance effect" refers to the phenomenon in which protamine present in each area due to PalP administration attracts and accumulates HEP or HEP-based nanoparticles. This occurs due to the strong interaction between HEP ​​and protamine. When PalP is administered subcutaneously and HEP is subsequently administered intravenously, the interaction between the two substances attracts circulating HEP to the site of protamine, resulting in the accumulation of HEP bound to protamine at that specific site (Fig. 15c). This phenomenon is referred to as the "HEP guidance effect by PalP," and it suggests that HEP can be specifically targeted and accumulated wherever PalP is present, demonstrating the potential of PalP as a guidance mechanism for HEP localization.

[0355]

[0356] 8-2. Measurement of tumor-specific accumulation through biodistribution tracking

[0357]

[0358] Additionally, CT26.wt xenograft mice were used to observe the distribution of HEP-RITC in the presence of tumors. Tumors were approximately 100–200 mm 3When the tumor size reached, the mice were divided into two groups: one group received intratumoral saline injection, and the other group received intratumoral PalP (40 mg / kg). Both groups were then intravenously injected with HEP-RITC (40 mg / kg) via the tail vein. The distribution of HEP-RITC was assessed by measuring fluorescence intensity. As expected, mice injected intratumorally with saline showed no fluorescence around the injection site. In contrast, mice treated with PalP showed high fluorescence intensity around the injection site (Fig. 15d). In addition, various organs (heart, kidney, liver, lung, and spleen) were extracted from mice administered intratumoral PalP and intravenous HEP-RITC at various time points (6, 12, 24, and 48 h). The fluorescence intensity of these organs was measured using a fluorescence spectrometer (FOBI) (Fig. 15e). Unlike the tumor tissues from saline-treated mice, the fluorescence of the tumor tissues extracted from PalP-treated mice gradually increased over time. The fluorescence intensity of the tumor tissue extracted at 48 hours was measured as 3.1e+07 ± 8.3e+05 Area x IU, which was 3.64-fold higher than the fluorescence intensity observed in the tumor tissue extracted from saline-treated mice (Fig. 15f). These results suggest that intratumoral injection of PalP leads to the uptake of HEP-RITC, resulting in a gradual increase in fluorescence intensity at the tumor site. This phenomenon is distinct from that in non-tumor mice, where the fluorescence intensity gradually decreased as PalP did not remain at the injection site for long and HEP-Cy5.5 dispersed throughout the body. These results highlight the tumor-specific accumulation and retention of HEP-RITC promoted by PalP, and demonstrate the potential of PalP as a targeted delivery system to enhance the localization of HEP-RITC in tumor tissues.

[0359]

[0360] 8-3. Measurement of drug accumulation through the interaction of PalP and heparin

[0361]

[0362] Finally, we evaluated the accumulation of HEP-RITC in tumor tissue over time after intravenous PalP administration. In a CT26.wt xenograft model, HEP-RITC was injected into the tail vein 12 hours after intravenous administration of PalP (20 mg / kg) (Fig. 15g). The results confirmed that the peak PalP accumulation in the tumor occurred at 12 hours (Fig. 2i). Based on these results, we selected a 12-hour interval between PalP and HEP-RITC administration for further analysis. Strong fluorescence intensity was observed in the tumor tissue starting 1 hour after administration, and this intensity persisted for up to 72 hours (Fig. 15g). Furthermore, the extracted tumor tissue consistently exhibited strong fluorescence signals from 12 to 72 hours after HEP-RITC administration (Fig. 15h). Quantitative analysis of fluorescence in the extracted tumor tissue confirmed that HEP-RITC accumulation gradually increased. In particular, at 72 hours, the fluorescence intensity of the tumor tissue was measured as 2.2e+07 ± 1.3e+06 Area x IU, showing a very strong fluorescence intensity (Fig. 15i). The above results indicate that intravenous administration of PalP effectively targets the tumor, and the subsequent interaction with HEP-RITC within the tumor tissue prolongs drug accumulation. In summary, it was found that the drug accumulated in the tumor tissue through sequential intravenous administration of PalP and HEP.

[0363]

[0364] 8-4. Measurement of accumulation in tumor tissue

[0365]

[0366] The fluorescence intensity of a fluorophore can be quenched by interactions with other molecules. Both DOX and curcumin possess ring-based structures that are susceptible to π-π stacking interactions. When these molecules are in close proximity or bind to other molecules, such as in dense or aggregated states, the fluorescence of the fluorophore can be significantly quenched. This fluorescence quenching has limited in vivo imaging in mice because it reduces the observed fluorescence signal. To overcome this limitation, we employed an alternative imaging approach by performing ex vivo imaging using extracted tissues. By imaging tissues outside the mouse, we minimized the quenching effect caused by the in vivo environment. Two groups of mice were used for tumor tissue imaging to assess drug biodistribution. The experimental group was intravenously injected with PalP, followed by intravenous injection of HEP-RITC 12 hours later. Tumor tissues were then harvested 72 hours after HEP-RITC injection for further analysis. Notably, significant RITC fluorescence was observed within the tumor tissues of the PalP-treated group. The above results suggest that pre-administration of PalP effectively accumulated HEP-RITC at the tumor site (Fig. 15j).

[0367]

[0368] 8-5. Cohesion Evaluation of PalP and HDFe NPs Using Confocal Imaging

[0369]

[0370] To evaluate the aggregation of PalP and HDFe NPs in tumor tissue due to the inductive effect, confocal imaging was performed in CT26.wt tumor-bearing mice. PalP-FITC was initially administered intravenously, followed by HDFe NP-Cy5.5 12 hours later. Tumors were excised for analysis 48 hours after the second administration (Fig. 15k). Confocal imaging confirmed the presence of both PalP and HDFe NPs within the tumor tissue. The merged imaging signals demonstrated colocalization of the nanoparticles and their simultaneous presence in the tumor microenvironment. This colocalization indicates effective aggregation promoted by the inductive effect. Furthermore, detection of DOX transported by HDFe NPs within tumor cells demonstrated the efficacy of targeted drug delivery. This suggests that the accumulation of PalP and HDFe NPs in tumor tissue due to the inductive effect has potential in targeted cancer therapy. Figure 16 shows confocal tissue imaging using PPNC NP-FITC and HDFe NP-Cy5.5. The experiment was performed in BALB / c mice implanted with CT26.wt tumors. PPNC NP-FITC was first intravenously administered to the mice, followed by HDFe NP-Cy5.5 intravenously 12 hours later. Forty-eight hours after HDFe NP-Cy5.5 administration, the tumors were excised for analysis. These results suggest that PPNC, as well as PalP, can promote guidance and aggregation, as demonstrated in Figure 15k. Figure 17 shows the results of a biodistribution study performed using tumor-bearing mice administered only saline. This figure provides a baseline for understanding the biodistribution in the absence of drug treatment, serving as the comparative control in Figure 2i. This figure also serves as the comparative control in Figure 15e. Notably, compared to Figure 15e, significantly lower fluorescence intensity was observed in the group administered HEP-RITC intravenously 12 hours after saline treatment, as shown in Figure 15f.The above results indicate that intratumorally administered PalP induces and promotes the accumulation of intravenously administered HEP-RITC.

[0371] In summary, the above results demonstrate that PPNC NPs can exert a "homing effect" that effectively guides HDFe NPs to tumor sites, thereby inducing aggregation of the two nanoparticles at the tumor site, thereby promoting sustained therapeutic action. The innovative approach utilized in the present invention holds the potential for sequential administration utilizing the "homing effect" as a promising strategy for enhancing therapeutic agent delivery to tumor sites. Utilizing this homing effect can enhance therapeutic agent targeting and accumulation, thereby enhancing the efficacy of cancer treatment.

[0372]

[0373] Example 9. Anticancer synergy effect

[0374]

[0375] 9-1. Anticancer evaluation through tumor volume measurement

[0376]

[0377] The in vivo anticancer activity of NCur, DOX, PPNC NP, HDFe NP, and sequential administration (NP + NP) groups of nanoparticles was evaluated. 5.0 x 10 5 CT26.wt tumor mouse models were created by subcutaneously injecting CT26.wt cells into the mice. When the transplanted tumors reached a size of 50 to 100 mm³, the mice were treated with drug concentrations equivalent to 3 mg / kg of DOX and NCur. The above substances were administered intravenously every 3 days for a total of 24 days. The NP + NP group was administered HDFe NP 12 hours after PPNC NP administration. The NP + NP group (559.01 ± 360.74 mm 3 ) is DOX (1437.25 ± 486.47 mm 3 ) or NCur(1656.85 ± 265.70 mm 3) treated group and control group (2743.27 ± 1143.52 mm 3 ) showed significantly lower tumor volume.

[0378]

[0379] 9-2. Anticancer evaluation through tumor volume measurement

[0380]

[0381] Tumor volume was also significantly higher in the NP + NP treatment group than in the PPNC NP (1852.68 ± 391.76 mm 3 ) and HDFe NP (2063.82 ± 890.04 mm 3 ) was significantly reduced compared to patients treated with PPNC NP and HDFe NP. The coefficient of drug interaction (CDI) value of the combination therapy of PPNC NP and HDFe NP was 0.4, indicating that the combination therapy had a synergistic effect (Fig. 18a). When the tumor volume for each group was individually measured, the NP + NP group showed a significantly increased tumor inhibition effect compared to the other groups (Fig. 18b). There was no noticeable change in the body weight of mice receiving each treatment during the 24-day treatment period, indicating no serious toxicity (Fig. 18c). After the tumor growth experiment, the mice were sacrificed and the tumor weights were measured. The tumor weight of mice treated with NP + NP (786.43 ± 626.59 mg) was significantly lower than that of DOX (1354.85 ± 258.47 mg), NCur (1542.38 ± 262.99 mg), PPNC NP (1607.9 ± 600.70 mg), HDFe NP (1855.18 ± 978.82 mg), and the untreated control group (2171.53 ± 713.48 mg). In addition, the relative size of the isolated tumor tissues was measured to compare the therapeutic effects (Fig. 18d). After 24 days of treatment, the tumors extracted from each group were sorted by size, and it was visually confirmed that the tumors in the NP + NP group were smaller than those in the other groups (Fig. 18e).

[0382]

[0383] 9-3. Anticancer evaluation using H&E and immunohistochemical staining

[0384]

[0385] H&E and Ki-67 staining analyses of isolated tumors showed that mice administered NP + NP had significantly destroyed tumor tissues and the lowest tumor proliferation rate (Fig. 18f). Cell death in tumor tissues was also observed as shown by TUNEL staining. Significantly higher fluorescence intensity was observed in tumor tissues treated with NP + NP, which was interpreted as active cell apoptosis in the tissues (Fig. 18g). The TUNEL fluorescence intensity of mice treated with NP + NP (276.21 ± 34.19%) was significantly higher than that of NCur (50.82 ± 16.02%), DOX (93.90 ± 6.02%), PPNC NP (114.91 ± 27.07%), HDFe NP (127.42 ± 8.58%), and the control group (31.36 ± 5.39%) (Fig. 18h). Results of anticancer efficacy tests demonstrated that the treatment with sequential administration of PPNC NPs and HDFe NPs exhibited potent anticancer effects by inducing cell death in CT26.wt tumors without causing toxicity.

[0386]

[0387] [National Research and Development Project Supporting This Invention]

[0388] [Project ID]1345370811

[0389] [Project Number] 2021RIS-001

[0390] [Ministry Name] Ministry of Education (P13)

[0391] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0392] [Research Project Name] Local Government-University Cooperation-Based Regional Innovation Project (086123605005223002301400)

[0393] [Research Project Name] Local Government-University Cooperation-Based Regional Innovation Project (Chungbuk Regional Innovation Platform)

[0394] [Name of Project Performing Organization] (Chungbuk Regional Innovation Platform) Chungbuk National University

[0395] [Research Period] March 1, 2024 - February 28, 2025

[0396] [Project ID]1711179563

[0397] [Assignment Number] 2020R1A2C1102831

[0398] [Ministry Name] Ministry of Science and ICT

[0399] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0400] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0401] [Research Project Name] Small molecule-based self-administered immunotherapy for optimal tumor-selective immunogenicity induction

[0402] Development of prefabricated nanomaterials

[0403] [Project Performing Organization Name] Konkuk University GLOCAL Campus

[0404] [Research Period] March 1, 2024 - February 28, 2025

[0405]

[0406] [Project ID]RS-2024-0042226830382106450001

[0407] [Ministry Name] Ministry of Science and ICT

[0408] [Research Project Name] IP Enhancement and Commercialization for UDCA Conjugate Commercialization

[0409] [Project Performing Organization Name] Konkuk University GLOCAL Campus

[0410] [Research Period] April 1, 2024 - December 31, 2024

Claims

1. Protamine-based self-assembled first nanoparticles containing a negatively charged substance; and Positively charged anticancer drugs and Fe 3+ Heparin-based self-assembled second nanoparticles comprising; A drug delivery system for targeting cancer, comprising:

2. A cancer-targeting drug delivery system, characterized in that the protamine-based self-assembled first nanoparticle in the first paragraph additionally includes a lipid group.

3. A cancer-targeting drug delivery system, characterized in that in the second paragraph, the fatty group is at least one acid selected from the group consisting of oleic acid, palmitic acid, palmitoleic acid, stearic acid, elaidic acid, linoleic acid, vaccenic acid, α-eleostearic acid, punicic acid, jacaric acid, arachidonic acid, paullinic acid, gondoic acid, and salts thereof.

4. A cancer-targeting drug delivery system, characterized in that the second nanoparticles are administered simultaneously, separately, or sequentially with the first nanoparticles in the first paragraph.

5. A cancer-targeting drug delivery system, characterized in that in the fourth paragraph, when the second nanoparticles are administered sequentially with the first nanoparticles, the second nanoparticles are administered 1 to 30 hours after the administration of the first nanoparticles.

6. A cancer-targeting drug delivery system, characterized in that in paragraph 4, the administration is at least one selected from the group consisting of intratumoral administration, oral administration, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, intradermal administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration.

7. A cancer-targeting drug delivery system, characterized in that the negatively charged substance in the first paragraph is at least one selected from the group consisting of curcumin, nucleic acid, and negatively charged anticancer agent.

8. A cancer-targeting drug delivery system according to claim 7, characterized in that the nucleic acid is at least one selected from the group consisting of DNA, RNA, ASO (antisense oligonucleotide), microRNA (miRNA), small interfering RNA (siRNA), circular RNA, long noncoding RNA (lncRNA), small activating RNA (saRNA), messenger RNA (Mrna), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino.

9. In paragraph 1 or paragraph 7, the anticancer agent is doxorubicin, cyclophosphamide, mecholrethamine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, duocarmycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, Satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, camptothecin, topotecan, irinotecan, etoposide, teniposide, Mitoxantrone, paclitaxel, docetaxel, izabepilone,A cancer-targeting drug delivery system characterized by comprising at least one selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E, monomethyl auristatin F, daunorubicin, epirubicin, idarubicin, and valrubicin.

10. A cancer-targeting drug delivery system, characterized in that the first nanoparticle and the second nanoparticle form aggregates by a guidance effect in the first paragraph.

11. A cancer-targeting drug delivery system according to claim 1, characterized in that the first nanoparticle and the second nanoparticle induce ferroptosis in cancer cells.

12. A cancer-targeting drug delivery system, characterized in that the weight ratio of the negatively charged substance and protamine in the first paragraph is 20:1 to 1:

20.

13. In the first paragraph, the anticancer agent, Fe 3+ A cancer-targeting drug delivery system, characterized in that the weight ratio of heparin to the drug is 50:1:10 to 1:1:

1.

14. A cancer-targeting drug delivery system, characterized in that the daily dosage of the first nanoparticle or the second nanoparticle is 1 mg / kg to 50 mg / kg in the first paragraph.

15. A cancer-targeting drug delivery system, characterized in that in paragraph 1, the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

16. A cancer-targeting drug delivery system, characterized in that the first nanoparticle and the second nanoparticle have a diameter of 20 to 500 nm in the first paragraph. 17.(a) administering to a non-human subject a protamine-based first nanoparticle comprising a negatively charged substance; and (b) Positively charged anticancer drugs and Fe 3+ A step of administering a heparin-based second nanoparticle comprising the same to a non-human subject; A method for preventing or treating cancer, comprising:

18. Protamine-based self-assembled first nanoparticles containing a negatively charged substance; and Positively charged anticancer drugs and Fe 3+ Heparin-based self-assembled second nanoparticles comprising; An injectable drug targeting cancer, comprising:

19. A cancer-targeting injection according to claim 18, characterized in that the second nanoparticles are administered simultaneously, separately, or sequentially with the first nanoparticles.

20. Protamine-based self-assembled first nanoparticles containing a negatively charged substance; and Positively charged anticancer drugs and Fe 3+ Heparin-based self-assembled second nanoparticles comprising; A pharmaceutical composition for preventing or treating cancer, comprising:

21. A pharmaceutical composition for preventing or treating cancer, characterized in that the second nanoparticles are administered simultaneously, separately, or sequentially with the first nanoparticles in the 20th paragraph.

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