Self-assembled nanoparticles comprising choline-polyester conjugate, preparation method therefor, and drug delivery composition comprising same

Abstract

The present invention relates to self-assembled nanoparticles comprising a novel choline-poly(ε-caprolactone) conjugate, in which, by using the specific interaction between choline and choline transporters, chemically modifying the surface of nanoparticles with choline to temporarily prevent choline transporters from transporting choline into cells, and self-assembling an amphiphilic conjugate consisting of hydrophilic choline and hydrophobic poly(ε-caprolactone) in an aqueous phase, nanostructures are prepared, and choline modified on the surface of the nanoparticles is specifically bound to choline transporters of cancer cells, wherein this binding reduces the intracellular uptake of choline by choline transporters, which may result in delayed or suppressed cancer cell growth and lead to cancer cell death.

Description

Self-assembled nanoparticles comprising a polymer conjugate of choline and polyester, a method for producing the same, and a drug delivery composition comprising the same

[0001] The present invention provides self-assembled nanoparticles comprising a polymer conjugate of choline and polyester, a method for producing the same, and a drug delivery composition comprising the same.

[0002] Choline, which is called vitamin B4 due to its structural similarity to the vitamin B complex, is a water-soluble, bioactive substance with a quaternary ammonium positive charge. Choline's water-solubility limits its entry into the cytoplasm through the cell membrane, so it can only enter the cell with the help of a protein called a choline transporter and bioenergetics. Choline that has entered the cell acts as a precursor that can be transformed into chemical and biological compounds that perform important functions within the cell, such as maintaining cell composition and homeostasis, and regulating and mediating physiological activities. Specifically, all cells must synthesize phospholipids within the cell to form the cell membrane and organelle membranes. During this process, choline is converted into phosphatidylcholine or sphingomyelin within the cell, which become components of the biological membrane. In addition, all cells require gene amplification or conversion into proteins in order to grow or maintain homeostasis. At this time, choline is converted into betaine within the cell and supplies methyl groups (-CH3) to the homocysteine-methionine conversion process, helping DNA methylation and ultimately influencing gene amplification and protein expression. In addition, choline is converted into acetylcholine, a neurotransmitter in nerve cells, and helps regulate functions such as neurotransmission, cognition, memory, muscle activity, excitement, and development in the central nervous system, autonomic nervous system, and peripheral nervous system. Therefore, limited or deficient intracellular supply of choline can cause cell growth retardation and death, and functional decline or loss of tissues, organs, and nervous systems, which can cause various diseases such as degenerative brain diseases, non-alcoholic liver disease, and cognitive and developmental disorders. Conversely, if the choline requirement in diseased cells is greater than that in normal cells, it may lead to worsening of the disease.In particular, cancer cells grow faster than normal cells, and for this rapid growth, a supply of cell membrane and organelle membrane components, as well as DNA and proteins, which regulate cellular genetics and homeostasis, is essential. Specifically, cancer cells overexpress choline transporter proteins more than normal cells, promoting the influx of choline into the cell, which is then utilized for cell growth and function maintenance.

[0003] An object of the present invention is to provide a novel choline-poly(ε-caprolactone) conjugate.

[0004] Another object of the present invention is to provide self-assembled nanoparticles comprising the above choline-poly(ε-caprolactone) conjugate and a method for producing the same.

[0005] Another object of the present invention is to provide a drug delivery composition comprising the self-assembled nanoparticles as an active ingredient.

[0006] Another object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer, comprising the self-assembled nanoparticles as an active ingredient.

[0007] Another object of the present invention is to provide a health functional food composition for improving or preventing cancer, which contains the self-assembled nanoparticles as an active ingredient.

[0008] To achieve the above object, the present invention provides a choline-poly(ε-caprolactone) conjugate represented by the following chemical formula 1.

[0009] [Chemical Formula 1]

[0010]

[0011] In the above chemical formula 1, n and m may be the same or different from each other, and may be 1 to 100.

[0012] In addition, the present invention provides self-assembled nanoparticles comprising the above choline-poly(ε-caprolactone) conjugate.

[0013] In addition, the present invention provides a drug delivery composition comprising the self-assembled nanoparticles as an active ingredient.

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

[0015] In addition, the present invention provides a health functional food composition for improving or preventing cancer, which comprises the self-assembled nanoparticles as an active ingredient.

[0016] In addition, the present invention provides a method for manufacturing self-assembled nanoparticles, characterized by comprising the steps of: a) dissolving the choline-poly(ε-caprolactone) conjugate of claim 1 in an organic solvent; and b) concentrating the solution dissolved in the organic solvent of step a) to form a thin film.

[0017] The present invention relates to self-assembled nanoparticles comprising a novel choline-poly(ε-caprolactone) conjugate and uses thereof, which can reduce the influx of choline into cells by a choline transporter, thereby delaying or inhibiting the growth of cancer cells and leading to their death, and thus can be used as a cancer treatment agent.

[0018] Figure 1 shows a schematic diagram of the synthesis of a choline-poly(epsilon-caprolactone) conjugate (hereinafter referred to as CholPCL-P).

[0019] Figure 2 is for confirming the chemical structure of CholPCL-P. 1 H-NMR spectroscopy spectrum (solvent: DMSO-d6) is shown.

[0020] Figure 3 is a method for confirming whether choline exists on the surface of choline-polycaprolactone nanoparticles (hereinafter referred to as CholPCL-NP:UC) manufactured using chloroform. 1 H-NMR spectroscopy spectrum (solvent: D2O) is shown.

[0021] Figure 4 shows the results of cytotoxicity evaluation (n=12) of CholPCL-NP:UC against (a) MRC-5 cells, (b) Jurkat cells, and (c) Raw 264.7 cells.

[0022] Figure 5 shows the results of cancer cell killing evaluation (n=12) of CholPCL-NP:UC on (a) HCT116 cells, (b) HT29 cells, and (c) MCF-7 cells.

[0023] Figure 6 shows the results of an evaluation of HCT116 colon cancer cell death (n=12) of choline-poly(epsilon-caprolactone) nanoparticles (hereinafter referred to as DOX@CholPCL-NP:UC) manufactured using chloroform loaded with doxorubicin (hereinafter referred to as DOX), a chemotherapy anticancer drug.

[0024] Figure 7 shows (a) cellular uptake (CU) (n=3), (b) nuclear uptake (NU) (n=3), (c) mitochondrial uptake (MU) (n=3), and (d) the mitochondria-to-nucleus preference (MNP) (n=3) of DOX@CholPCL-NP:UC in HCT116 colon cancer cells.

[0025] Figure 8 shows DOX@CholPCL-NP:UC or DOX@PCL in the presence of choline. diOH-NP: Shows changes in the uptake capacity of HCT116 cells, MRC-5 cells, or Jurkat cells (n=3).

[0026] Figure 9 shows the change in the amount of choline in HCT116 cells after treatment with DOX@CholPCL-NP:UC for 0.5 or 4 hours (n=3).

[0027] Figure 10 shows changes in mitochondrial membrane potential of HCT116 cells treated with CholPCL-NP:UC as measured by (a) microplate fluorometry (n=8) or (b) flow cytometry (n=3).

[0028] Figure 11 shows (a) dot plots or (b) bar plots of the necrotic and apoptotic states of HCT116 cells (n=4) 24 hours after treatment with DOX, CholPCL-NP:UC, or DOX@CholPCL-NP:UC.

[0029] Figure 12 shows (a) changes in tumor volume over time in a cancer-induced mouse model intravenously administered with CholPCL-NP:UC or DOX@CholPCL-NP:UC. * p< 0.05 and ** p< 0.01 versus DPBS-administered mice control group) and (b) the change in mouse weight over time.

[0030] Hereinafter, the present invention will be described in more detail.

[0031]

[0032] The present invention provides a choline-poly(ε-caprolactone) conjugate represented by the following chemical formula 1.

[0033] [Chemical Formula 1]

[0034]

[0035] In the above chemical formula 1, n and m may be the same as or different from each other, and may be 1 to 100, preferably 20 to 30.

[0036] The molecular weight of the above choline-poly(epsilon-caprolactone) conjugate may be 500 to 10,000 Da.

[0037]

[0038] In addition, the present invention provides self-assembled nanoparticles comprising the above choline-poly(ε-caprolactone) conjugate.

[0039] The average particle size of the self-assembled nanoparticles may be 10 to 300 nm.

[0040] The self-assembled nanoparticles may further include a drug. Specifically, the self-assembled nanoparticles may be formed by self-assembling a choline-polymer conjugate, wherein the drug is encapsulated in the nanoparticles.

[0041] The above drug may be a chemical drug, a protein drug, or a genetic drug.

[0042] The above chemical drugs are selected from the group consisting of doxorubicin, pheophorbide a, paclitaxel, dexamethasone, curcumin, epirubicin, gemcitabine, cilolimus, etoposide, vinblastine, vinca alkaloids, docetaxel, Gleevec, cyclophosphamide, teniposide, 5-fluorouracil, camptocecin, tamoxifen, anasterozole, floxuridine, leuprolide, flotamide, zoledronate, vincristine, streptozotocin, ifosfamide, topotecan, belotecan, irinotecan, vinorelbine, hydroxyurea, valrubicin, methotrexate, mechlorethamine, chlorambucil, busulfan, doxifluridine, prednisone, testosterone, mitoxantrone and prednisolone. It may contain more than one type.

[0043] The above protein drug may include at least one selected from the group consisting of bovine serum albumin (BSA), immunoglobulin G, insulin, erythropoietin, interferon, interleukin, growth hormone, beta-glucocerebrosidase, alpha-glucosidase, lactase, lipase, adenosine deaminase, human serum albumin, oprelvekin, aldesleukin, alteplase, reteplase, tenecteplase, urokinase, teriparatide, exenatide, octreotide, glucagon, papain, and lepirudin.

[0044] The above genetic drug may include at least one selected from the group consisting of plasmid DNA, mRNA, siRNA, miRNA, shRNA, antisense oligonucleotide (ASO), and oligonucleotide.

[0045]

[0046] In addition, the present invention provides a drug delivery composition comprising the self-assembled nanoparticles as an active ingredient.

[0047] The above drug delivery composition can be absorbed from the gastrointestinal tract.

[0048] The above drug may be, but is not limited to, a chemical drug, a protein drug, or a genetic drug.

[0049] The above chemical drugs are selected from the group consisting of doxorubicin, pheophorbide a, paclitaxel, dexamethasone, curcumin, epirubicin, gemcitabine, cilolimus, etoposide, vinblastine, vinca alkaloids, docetaxel, Gleevec, cyclophosphamide, teniposide, 5-fluorouracil, camptocecin, tamoxifen, anasterozole, floxuridine, leuprolide, flotamide, zoledronate, vincristine, streptozotocin, ifosfamide, topotecan, belotecan, irinotecan, vinorelbine, hydroxyurea, valrubicin, methotrexate, mechlorethamine, chlorambucil, busulfan, doxifluridine, prednisone, testosterone, mitoxantrone and prednisolone. It may contain more than one type.

[0050] The above protein drug may include at least one selected from the group consisting of bovine serum albumin (BSA), immunoglobulin G, insulin, erythropoietin, interferon, interleukin, growth hormone, beta-glucocerebrosidase, alpha-glucosidase, lactase, lipase, adenosine deaminase, human serum albumin, oprelvekin, aldesleukin, alteplase, reteplase, tenecteplase, urokinase, teriparatide, exenatide, octreotide, glucagon, papain, and lepirudin.

[0051] The above genetic drug may include at least one selected from the group consisting of plasmid mRNA, siRNA, miRNA, shRNA, antisense oligonucleotide (ASO), and oligonucleotide.

[0052]

[0053] In addition, the present invention provides a pharmaceutical composition for treating or preventing cancer, comprising the self-assembled nanoparticles as an active ingredient.

[0054] The above cancer disease may be any one selected from the group consisting of colorectal cancer, breast cancer, triple-negative breast cancer (TNBC), cervical cancer, lung cancer, pancreatic cancer, colon cancer, bone cancer, skin cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but is not limited thereto.

[0055] In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0056] Specifically, carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like, into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0057] According to one embodiment of the present invention, the pharmaceutical composition can be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes.

[0058] The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and degree of the disease, the drug form, the route and period of administration, and may be appropriately selected by a person skilled in the art, and the daily dosage may be 0.01 mg / kg to 200 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, and more preferably 0.1 mg / kg to 100 mg / kg. Administration may be once a day or divided into several times, and the scope of the present invention is not limited thereby.

[0059]

[0060] In addition, the present invention provides a health functional food composition for improving or preventing cancer, which comprises the self-assembled nanoparticles as an active ingredient.

[0061] The above health functional food may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0062] In addition, it may contain fruit pulp for the production of natural fruit juice, synthetic fruit juice, and vegetable drinks. These ingredients may be used independently or in combination. Furthermore, the health functional food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol, and vitamin complex.

[0063] In addition, the above health functional food may additionally contain food additives, and its suitability as a “food additive” is determined by the specifications and standards for the relevant item in accordance with the general provisions and general testing methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise provided.

[0064] Examples of items listed in the above "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kohlrabi pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

[0065] At this time, the content of the effective ingredient added to the food during the process of manufacturing the health functional food can be appropriately increased or decreased as needed, and preferably, it can be added so that it is included in an amount of 1 to 90 parts by weight per 100 parts by weight of the food.

[0066]

[0067] In addition, the present invention provides a method for producing self-assembled nanoparticles, characterized by comprising the steps of: a) dissolving the choline-poly(ε-caprolactone) conjugate of claim 1 in an organic solvent; and b) concentrating the solution dissolved in the organic solvent of step a) to form a thin film; and preferably, after step b), the method may further comprise a step c) of adding water to the formed thin film and then applying ultrasonic waves to break it up.

[0068] The above organic solvent may be any one of ethanol, methanol, and chloroform, but is not limited thereto.

[0069] In step a), additional chemicals may be added.

[0070] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the following examples and other embodiments merely illustrate the content of the present invention and are not intended to limit the scope of the present invention. The examples and other embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0071]

[0072] <Example 1> Synthesis and analysis of choline-poly(ε-caprolactone) conjugate [Choline-Poly(ε-caprolactone) conjugate;CholPCL-P]

[0073] As shown in Fig. 1, the choline-poly(epsilon-caprolactone) conjugate (CholPCL-P) having the chemical structure of choline-poly(ε-caprolactone)-choline [Choline-poly(ε-caprolactone)-Choline] is poly(ε-caprolactone) dicarboxylate [Poly(ε-caprolactone) dicarboxylate; hereinafter, PCL]. diCOOH The carboxyl group of [Raham] and the hydroxyl group of choline chloride (Chol) were formed by forming an ester bond using EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine). For example, PCL having a molecular weight of 1460 daltons, 2000 daltons, or 5000 daltons diCOOH Dissolve 0.137 mmol, 0.1 mmol, and 0.04 mmol of PCL in chloroform (CHCl3) at a concentration of 50 mg / mL, respectively. diCOOH EDC and DMAP, which are equivalent to 4 times the molar number, were prepared by dissolving them in CHCl3 at a concentration of 50 mg / mL. After that, the dissolved PCL diCOOH , EDC and DMAP solutions were mixed and then PCL diCOOHCholine chloride equivalent to 4 times the molar number was added. 100 μL of triethylamine (TEA) was added, and CholPCL-P was synthesized by stirring at room temperature under nitrogen for 48 hours. To obtain the synthesized CholPCL-P, the polymer reaction product was precipitated in cold ether to obtain CholPCL-P. The precipitated CholPCL-P was re-dissolved in DMSO, placed in purified water, and dialyzed through a dialysis membrane with a molecular weight cut-off (MWCO) of 3500 daltons to remove unreacted substances, and the nanoparticles or precipitated CholPCL-P were purified. Finally, the purified CholPCL-P solution was lyophilized to obtain CholPCL-P. After dissolving the synthesized CholPCL-P in DMSO-d6 solvent, as shown in Figure 2 1 PCL using H-NMR spectroscopy diCOOH PCL with a molecular weight of 1460 daltons and choline attached to each end diCOOH PCL with 2.03 cholines per molecule and a molecular weight of 2000 daltons diCOOH PCL with 1.93 cholines per molecule and a molecular weight of 5000 daltons diCOOH The chemical structure of choline-poly(epsilon-caprolactone)-choline was confirmed by introducing 2.00 cholines per molecule. PCL with molecular weights of 1460, 2000, or 5000 daltons diCOOH Each CholPCL-P synthesized using 1.5 -P, CholPCL2-P, and CholPCL5-P were named.

[0074]

[0075] <Example 2> Preparation of self-assembled nanoparticles (CholPCL-NP:U) of CholPCL-P using thin film hydration and their particle size, zeta potential and particle stability

[0076] Freeze-dried CholPCL-P is dissolved in a volatile solvent [e.g., ethanol (EtOH), methanol (MeOH), chloroform (CHCl3)], and then nanoparticles are prepared using the thin film hydration method. For example, 2 mg of CholPCL-P is dissolved in a volatile solvent at a concentration of 2 mg / mL to prepare a CholPCL-P solution, which is then transferred to a glass vial. The solvent is evaporated using a rotary evaporator to form a thin film, and 2 mL of purified water (polymer concentration 1 mg / mL) is added. Then, a probe ultrasonicator is used to prepare nanoparticles of uniform size. When nanoparticles are prepared by ultrasonication, the codes are U, and when the volatile solvent is ethanol, methanol, or chloroform, the codes are E, M, and C, respectively. That is, CholPCL1 in ethanol, methanol, and chloroform .5 -P was melted and prepared as a thin film, and then nanoparticles manufactured by ultrasound were hydrated and CholPCL1 was prepared. .5 -NP:UE, CholPCL1 .5 -NP:UM and CholPCL1 .5 -NP:UC, and the average particle sizes of these nanoparticles were 58.4 ± 16.2 nm, 71.9 ± 19.5 nm, and 50.7 ± 7.9 nm, respectively, and the zeta potentials were measured as +54.47 ± 0.4 mV, +41.97 ± 0.1 mV, and +67.7 ± 2.2 mV, respectively. In addition, the nanoparticles prepared by dissolving CholPCL2-P or CholPCL5-P in chloroform, hydrating the thin film, and then ultrasonically manufacturing the thin film were named CholPCL2-NP:UC and CholPCL5-NP:UC, respectively, and the average particle sizes of these nanoparticles were 66.6 ± 15.6 nm and 86.1 ± 23.2 nm, respectively, and the zeta potentials were measured as +40.07 ± 1.9 mV and +36.73 ± 2.2 mV, respectively.

[0077] CholPCL-NP:UC was prepared using D2O instead of water, as shown in Fig. 3. 1The presence of choline on the nanoparticle surface was confirmed using H-NMR spectroscopy. In addition, particle size changes over time were observed to evaluate the particle stability of CholPCL-NP:UC under aqueous conditions. As shown in Table 1, CholPCL1 .5 -NP:UC and CholPCL2-NP:UC did not show significant changes in particle size for 48 hours, but CholPCL5-NP:UC was stable for up to 24 hours after preparation, and aggregation was observed for 48 hours thereafter, confirming that the particle stability of nanoparticles may be reduced when the molecular weight of PCL used is large.

[0078] NPSize (nm)0 hours12 hours24 hours48 hoursCholPCL1 .5 -NP:UC57.6±15.750.2±14.154.6±15.157.2±12.4CholPCL2-NP:UC66.6±15.667.2±15 .268.6±14.467.7±18.4CholPCL5-NP:UC86.1±23.281.1±21.1119.5±24.6aggregated

[0079] In particular, in the following examples, CholPCL1 with good particle stability .5 -NP:UC was used for the experiment, and for convenience, it was referred to as CholPCL-NP:UC.

[0080]

[0081] <Example 3> Evaluation of cytotoxicity and cancer cell death of CholPCL-NP:U manufactured by thin-film hydration method

[0082] The cytotoxicity of CholPCL-NP:U itself was evaluated using 5,000 normal lung cells (hereinafter referred to as MRC-5), 10,000 T lymphocyte cells (hereinafter referred to as Jurkat), or 2,000 macrophages (hereinafter referred to as Raw 264.7), and the cancer cell killing ability of CholPCL-NP:U itself was evaluated using 2,000 colon cancer cells (hereinafter referred to as HCT116), 3,000 colon cancer cells (hereinafter referred to as HT29), or 5,000 breast cancer cells (hereinafter referred to as MCF-7).

[0083] The aforementioned cells were plated on a 96-well plate and cultured for 24 hours. Then, the cells were treated with various concentrations of CholPCL-NP:UC, and the cytotoxicity or cancer cell killing ability was evaluated by the MTT method after 48 hours. As shown in Figure 4, CholPCL-NP:UC at a concentration of 100 μg / mL or less showed IC against MRC-5 cells, Jurkat cells, and Raw 264.7 cells. 50 No cytotoxicity of CholPCL-NP:UC itself was observed, with cell viability exceeding 90%. However, as shown in Figure 5, CholPCL-NP:UC had an IC of 5.8 μg / mL in HCT116 cells. 50 The value was 42.9 μg / mL in HT-29 cells. 50 The values ​​were shown. For MCF-7 cells, approximately 45% of cancer cell killing ability was confirmed at 100 μg / mL.

[0084]

[0085] <Example 4> Preparation of Drug@CholPCL-NP:U loaded with hydrophobic chemical agent using thin film hydration method and its physicochemical properties

[0086] Drug@CholPCL-NP:U, which encapsulates a hydrophobic chemical drug, was prepared using a thin film hydration method, and doxorubicin (DOX) was used as a model drug. 2 mg of CholPCL-P was dissolved in 0.649 mL of a volatile solvent (chloroform) to prepare a CholPCL-P solution. 0.351 mL of a drug solution prepared at a concentration of 0.3 mg / mL was added to the CholPCL-P solution, and the solvent was evaporated using a rotary evaporator to form a thin film. 2 mL of purified water was added to the thin film formed at a CholPCL-P concentration of 1 mg / mL, and then an ultrasonicator was used to prepare DOX@CholPCL-NP:UC, in which a drug was encapsulated with a uniform size. The average particle size and zeta potential of the manufactured DOX@CholPCL-NP:UC were 67.8 ± 9.0 nm and +54.8 ± 1.3 mV (n=3), respectively. When the target drug loading content was 5 wt%, the actual drug loading content and loading efficiency were 4.8 ± 0.04 wt% and 95.5 ± 2.7% (n=3), respectively.

[0087]

[0088] <Example 5> Evaluation of cancer cell killing ability of DOX@CholPCL-NP loaded with doxorubicin, a hydrophobic anticancer drug.

[0089] To evaluate the cancer cell killing ability of DOX@CholPCL-NP:UC prepared from the above Example 4, 2,000 HCT116 colon cancer cells were plated in a 96-well plate and cultured for 24 hours, and then DOX@CholPCL-NP:UC with various DOX drug concentrations were treated to the HCT116 colon cancer cells. After 48 hours, the cell viability of HCT116 colon cancer cells was evaluated using the MTT method to evaluate the cancer cell killing ability of DOX@CholPCL-NP:UC. As shown in Fig. 6, the IC of DOX and DOX@CholPCL-NP:UC for HCT116 cells 50The values ​​were approximately 0.96 μg / mL and 0.096 μg / mL, respectively, and these results indicated that DOX@CholPCL-NP:UC showed 10 times better cancer cell killing ability than DOX in HCT116 colon cancer cells.

[0090]

[0091] <Example 6> Evaluation of the influx and distribution of CholPCL-NP into cells and organelles

[0092] To evaluate the uptake and distribution of nanoparticles into cells and organelles, DOX@CholPCL-NP:UC, which was loaded with a fluorescent model drug DOX, was prepared and used in Example 4. 200,000 HCT116 colon cancer cells were plated on a 6-well plate and cultured in 2 mL of cell culture medium for 24 h, and then treated with DOX@CholPCL-NP:UC at a DOX concentration of 2.0 μg / mL. After 4 h, the cells were washed twice with phosphate buffered saline (DPBS) and detached with trypsin-EDTA to evaluate the cellular uptake (CU) of DOX@CholPCL-NP:UC. Nuclear uptake (NU) and mitochondrial uptake (MU) were determined by isolating organelles according to the protocols of the Nuclei PURE Prep kit and Mitochondrial Fractionation kit, respectively. The DOX fluorescence intensity in cells and organelles was measured using a flow cytometer, and each was analyzed and evaluated as the DOX@CholPCL-NP:UC uptake. At this time, DOX, known to prefer accumulation in mitochondria, and doxorubicin hydrochloride (DOX HCl), a hydrophilic drug known to prefer accumulation in the nucleus, were used as controls. As shown in Figs. 7(a), 7(b), and 7(c), when the CU, NU, and MU values ​​of DOX are each set to 1, the CU values ​​of DOX·HCl and DOX@CholPCL-NP:UC were approximately 4.5 and approximately 4.2, the NU values ​​were approximately 6.5 and approximately 2.4, and the MU values ​​were approximately 0.9 and approximately 2.2, respectively. In addition, as shown in Fig. 7(d), when the mitochondrial uptake (MU) was calculated relative to the nuclear uptake (NU) to calculate the mitochondria preference (MU / NU), when the MU / NU value of DOX was set to 1, the MU / NU values ​​of DOX·HCl and DOX@CholPCL-NP:UC were approximately 0.14 and 0, respectively.It was calculated as 9. This result means that DOX@CholPCL-NP:UC has 4.2 times better intracellular uptake, 2.4 times better nuclear uptake, and 2.2 times better mitochondrial uptake than DOX, and it also has a preference for mitochondria over the nucleus similar to DOX. In addition, the nuclear and mitochondria uptake abilities of DOX@CholPCL-NP:UC were reduced to 52-57% compared to the intracellular uptake ability of DOX@CholPCL-NP:UC. This is because, firstly, unlike DOX, which has cell membrane penetration ability, DOX@CholPCL-NP:UC enters cells through endocytosis, and the amount reaching the cytoplasm may be affected by the timing and presence of endosomal escape, and secondly, the cytoplasmic movement rate of DOX@CholPCL-NP:UC, which has nano-sized particles, is relatively slow compared to the cytoplasmic diffusion rate of DOX, which has low molecular weight.

[0093]

[0094] <Example 7> Evaluation of the intracellular uptake pathway of CholPCL-NP

[0095] To evaluate whether CholPCL-NP utilizes the choline transporter overexpressed in cancer cells when entering cancer cells, choline and CholPCL-NP were allowed to compete, and then the CU change of CholPCL-NP was analyzed by flow cytometry. Specifically, 200,000 HCT116 colon cancer cells were plated on a 6-well plate and cultured for 24 hours, and DOX@CholPCL-NP:UC ([DOX] = 2.0 ㎍ / mL) prepared in Example 4 and choline ([Chol] = 30 mg / mL or 60 mg / mL) were simultaneously treated to the cells. The amount of DOX@CholPCL-NP:UC that entered the cells 30 minutes after nanoparticle treatment was evaluated by the fluorescence intensity of DOX, and the procedure followed the method of Example 6. MRC-5 cells (300,000 cells) which are normal lung cells and Jurkat cells (200,000 cells) which are T lymphocyte cells were used as control cells for cancer cells, and DOX@PCL was used as control nanoparticles of DOX@CholPCL-NP:UC according to the method of Example 4. diOH- NP:UC was manufactured.

[0096] As shown in Fig. 8, when HCT116 colon cancer cells were challenged with choline at a concentration of 30 mg / mL or 60 mg / mL, the CU value of DOX@CholPCL-NP:UC decreased by 17% and 50%, respectively, compared to when choline was 0 mg / mL, but DOX@PCL diOH- The CU value of NP:UC was not affected. In addition, DOX@CholPCL-NP:UC and DOX@PCL in control MRC-5 cells and Jurkat cells diOH- The CU value of NP:UC was not affected by choline. This result suggests that CholPCL-NP enters cancer cells via choline transporters, a pathway through which choline enters cells.

[0097]

[0098] <Example 8> Evaluation of intracellular choline concentration of CholPCL-NP

[0099] When CholPCL-NPs are introduced into cancer cells via choline transporters, the amount of choline introduced into the cells can change, and this was evaluated according to the Total choline detection assay protocol. Specifically, 500,000 HCT116 colon cancer cells were plated in 6-well plates and cultured for 24 hours, and the cancer cells were treated with 0 to 100 μg / mL of CholPCL-NP:UC prepared in Example 2 for 30 minutes or 4 hours. The cell culture medium was removed, and the cancer cells were washed twice with phosphate-buffered saline (DPBS), followed by addition of lysis buffer and separation with a scraper. The cell lysis suspension was collected and centrifuged at 13,000 rpm for 5 minutes to separate the supernatant. A choline fluorescent probe solution was added to the supernatant, and the fluorescence of the choline probe excited at 540 nm was detected at 590 nm. As shown in Fig. 9, as the concentration of CholPCL-NP:UC increased, the amount of choline introduced into the cells decreased, and in particular, when treated with 100 μg / mL, the amount of choline introduced into the cells decreased by approximately 37%. In addition, the fact that there was no significant difference between the change in the amount of choline introduced into the cells after 30 minutes of treatment with CholPCL-NP:UC and the change after 4 hours of treatment indicates that CholPCL-NP:UC rapidly introduced into the cells using choline transporters after cell treatment and prevented choline from entering the cells using choline transporters. This result implies that, unlike the pathway in which choline enters the cells using choline transporters, CholPCL-NP:UC binds to choline transporters and is endocytosed into the cells, preventing choline from using choline transporters.

[0100]

[0101] <Example 9> Evaluation of the effect of CholPCL-NP on mitochondrial membrane potential in cancer cells

[0102] It was confirmed that CholPCL-NP binds to a choline transporter, and in particular, it was confirmed in Example 8 that CholPCL-NP can reduce the influx of choline into cells when it binds to a choline transporter present on the cell membrane surface of cancer cells. In addition, since choline transporters are also present in the inner membrane of mitochondria, it was evaluated whether binding to this affects the mitochondrial inner membrane potential. Specifically, 200,000 HCT116 colon cancer cells were plated on a 6-well plate and cultured for 24 hours, and 50 μg / mL or 100 μg / mL of CholPCL-NP:UC prepared in Example 2 were treated to the cancer cells for 4 hours. According to the analysis method of the MitoProbe TM JC-1 assay kit, 2 μM of JC-1 reagent was treated to the cells 15 minutes before the end of the experiment, and the light emitted by the fluorescent factor excited at 488 nm was measured at 530 nm and 585 nm using a flow cytometer and a plate reader to analyze the polarization of the mitochondrial membrane potential. In general, JC-1 emits red light when the mitochondrial membrane potential is normal, and emits green light when the mitochondrial membrane potential is abnormal, causing depolarization and apoptosis. When the JC-1 Red / Green fluorescence ratio of cancer cells alone was set to 100%, it could be predicted that apoptosis occurred when the JC-1 Red / Green fluorescence ratio of the cells treated with nanoparticles decreased due to depolarization. As shown in Figures 10(a) and 10(b), the control nanoparticle, PCL diOH -Although the decrease in the JC-1 Red / Green fluorescence ratio induced by NP:UC was not significant, increasing its concentration slightly decreased the JC-1 Red / Green fluorescence ratio. Compared to the decrease in the JC-1 Red / Green fluorescence ratio (approximately 75%) induced by carbonylcyanide m-chlorophenylhydrazone (CCCP; 20.46 μg / mL), a control drug known to induce depolarization, the decrease in the JC-1 Red / Green fluorescence ratio induced by CholPCL-NP was smaller, but a decrease in the fluorescence ratio according to the nanoparticle concentration was observed. In particular, it was confirmed that approximately 30% of mitochondrial membrane potential was damaged when CholPCL-NP was present at a concentration of 100 μg / mL.

[0103]

[0104] <Example 10> Evaluation of the cancer cell death mechanism of CholPCL-NP

[0105] Mitochondrial membrane potential damage can induce apoptosis, so the cancer cell death mechanism of CholPCL-NP was analyzed. Specifically, 200,000 HCT116 colon cancer cells were plated in a 6-well plate and cultured for 24 hours, and 40 μg / mL of CholPCL-NP:UC prepared in Example 2 and 40 μg / mL of DOX@CholPCL-NP:UC prepared in Example 4 ([DOX] = 2 μg / mL) were treated to the cancer cells for 24 hours. The cell necrosis and apoptosis of cancer cells exposed to the nanoparticles were evaluated using a flow cytometer using double fluorescent staining with propidium iodide (PI) and Annexin V. As shown in Figures 11(a) and 11(b), 31.0% and 22.7% of cells treated with DOX or CholPCL-NP:UC were in apoptotic state, and 8.6% and 0.4% were in necrotic state, respectively. However, 56.5% and 21.2% of cells treated with DOX@CholPCL-NP:UC were in apoptotic and necrotic state, respectively. This result indicated that DOX@CholPCL-NP:UC induced apoptosis 1.82 times more than DOX in HCT116 colon cancer cells.

[0106]

[0107] <Example 11> Intravenous administration of CholPCL-NP and DOX@CholPCL-NP on cancer growth inhibition in a mouse model of cancer

[0108] To confirm the anticancer effect of nanoparticles in a cancer-induced mouse model, a mouse model induced with colon cancer cells (HCT116) was first prepared. All animal care and experimental protocols were performed in accordance with the guidelines of the Animal Ethics Committee of the Catholic University of Korea. The tumor xenograft model was implanted 5 × 10 into the thigh (flank) of female BALB / C nude mice (5 weeks old). 6It was created by subcutaneously injecting canine HCT116 cells (100 μL), and the tumor volume was approximately 50 mm 3 When the tumor volume reached , a tumor inhibition experiment was conducted by administering DOX·HCl, CholPCL-NP:UC, or DOX@CholPCL-NP:UC. Doxorubicin solution (DOX·HCl, 5 mg / kg; equivalent to 100 μg of drug), CholPCL-NP:UC (based on material amount, 100 mg / kg; equivalent to 2 mg of drug delivery system), and DOX@CholPCL-NP (based on DOX amount, 5 mg / kg; equivalent to 100 μg of drug) were administered twice via the tail vein at 2-day intervals to the prepared HCT116 cancer cell-induced mouse model. The tumor volume was approximately 1000 mm 3 Tumor volume was measured until it reached the size of . Tumor volume (mm 3 ) was calculated as long axis × short axis × short axis ÷ 2. As shown in Fig. 12(a), the tumor sizes of mice treated with DOX·HCl, CholPCL-NP:UC, or DOX@CholPCL-NP:UC were about 1.68 times, about 1.38 times, and about 3.52 times smaller than those of mice treated with saline. CholPCL-NP:UC showed 1.32 times lower anticancer effect than DOX·HCl, but DOX@CholPCL-NP:UC showed 2.10 times higher anticancer effect than DOX·HCl. In addition, as shown in Fig. 12(b), almost no change in body weight was observed in the CholPCL-NP:UC and DOX@CholPCL-NP:UC administration groups, indicating that CholPCL-NP:UC and DOX@CholPCL-NP:UC had no serious toxicity.

[0109]

[0110] The foregoing description of the present invention is for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0111] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Choline-Poly(ε-caprolactone) conjugate represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, n and m may be equal to or different from each other and are in the range of 1 to 100.

2. A choline-poly(ε-caprolactone) conjugate according to claim 1, characterized in that the molecular weight of the choline-poly(ε-caprolactone) conjugate is 500 to 10,000 Da.

3. Self-assembled nanoparticles comprising the choline-poly(ε-caprolactone) conjugate of claim 1.

4. Self-assembled nanoparticles according to claim 3, characterized in that the average particle size of the self-assembled nanoparticles is 10 to 300 nm.

5. A self-assembled nanoparticle according to claim 3, characterized in that the self-assembled nanoparticle further comprises a drug, and the drug is a chemical drug, a protein drug, or a genetic drug.

6. In claim 5, the chemical drug is doxorubicin, pheophorbide a, paclitaxel, dexamethasone, curcumin, epirubicin, gemcitabine, cilolimus, etoposide, vinblastine, vinca alkaloid, docetaxel, Gleevec, cyclophosphamide, teniposide, 5-fluorouracil, camptocecin, tamoxifen, anasterozole, floxuridine, leuprolide, flotamide, zoledronate, vincristine, streptozotocin, ifosfamide, topotecan, belotecan, irinotecan, vinorelbine, hydroxyurea, valrubicin, methotrexate, mechlorethamine, chlorambucil, busulfan, doxifluridine, prednisone, testosterone, mitoxantrone and Self-assembled nanoparticles comprising at least one selected from the group consisting of prednisolone.

7. In claim 5, the protein drug is a self-assembled nanoparticle comprising at least one selected from the group consisting of bovine serum albumin (BSA), immunoglobulin G, insulin, erythropoietin, interferon, interleukin, growth hormone, beta-glucocerebrosidase, alpha-glucosidase, lactase, lipase, adenosine deaminase, human serum albumin, oprelvekin, aldesleukin, alteplase, reteplase, tenecteplase, urokinase, teriparatide, exenatide, octreotide, glucagon, papain, and lepirudin.

8. In claim 5, the genetic drug is a self-assembling nanoparticle comprising at least one selected from the group consisting of plasmid DNA, mRNA, siRNA, miRNA, shRNA, antisense oligonucleotide (ASO), and oligonucleotide.

9. A drug delivery composition comprising the self-assembled nanoparticle of claim 3 as an active ingredient.

10. A drug delivery composition according to claim 9, characterized in that the drug delivery composition is absorbed in the gastrointestinal tract.

11. A pharmaceutical composition for treating or preventing cancer, comprising the self-assembled nanoparticle of claim 3 or claim 5 as an active ingredient.

12. A pharmaceutical composition according to claim 11, wherein the cancer disease is any one selected from the group consisting of colorectal cancer, breast cancer, triple-negative breast cancer (TNBC), cervical cancer, lung cancer, pancreatic cancer, colon cancer, bone cancer, skin cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

13. A health functional food composition for improving or preventing cancer, comprising the self-assembled nanoparticle of claim 3 or claim 5 as an active ingredient. 14.a) A step of dissolving the choline-poly(ε-caprolactone) conjugate of claim 1 in an organic solvent; and b) A method for manufacturing self-assembled nanoparticles, characterized by comprising a step of concentrating a solution dissolved in an organic solvent in step a) to form a thin film.

15. A method for manufacturing self-assembled nanoparticles, characterized in that, in claim 14, after step b), the method further includes step c), which comprises adding water to the formed thin film and then applying ultrasonic waves to break it up.

16. A method for producing self-assembled nanoparticles, characterized in that the organic solvent according to claim 14 is any one of ethanol, methanol, and chloroform.

17. A method for producing self-assembled nanoparticles, characterized in that in claim 14, a chemical agent is further added in step a).

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