KR102292747B1 - Thermosponge nanoparticle platform coated with chitosan for delivery bioactive materials and its use - Google Patents

Abstract

The present invention relates to a chitosan coated thermosponge nanoparticle (CTNP) platform for drug delivery and uses thereof. The temperature-sensitive nanosponge platform coated with chitosan for delivery of physiologically active substances according to the present invention has improved cellular uptake, so that more drugs, for example, anticancer drugs, can be delivered into cells, and p-gp inhibition is weakly performed by Pluronic. It can effectively treat drug-resistant tumor cells without a separate p-gp inhibitor.

Description

Thermosponge nanoparticle platform coated with chitosan for delivery bioactive materials and its use

The present invention relates to a chitosan-coated thermosponge nanoparticle (CTNP) platform for delivery of bioactive substances and uses thereof. The temperature-sensitive nanosponge platform coated with chitosan for delivery of physiologically active substances according to the present invention has improved cellular uptake, so that more drugs, for example, anticancer drugs, can be delivered into cells, and p-gp inhibition is weakly performed by Pluronic. It can effectively treat drug-resistant tumor cells without a separate p-gp inhibitor.

Cancer cells refer to cells that do not die and continue to live as normal cells lose the limit of the number of divisions due to genetic mutations. They continue to proliferate even when attached to other cells, and even expand their territory by pushing out normal cells, eventually spreading throughout the body, leading to death. According to estimates by the International Agency for Research on Cancer (IARC), cancer is the second most common cause of death in both economically developing and developed countries, with an estimated 13% of global cancer cases occurring in southwestern Asia. . In particular, the severity of the disease is high as it has been the number one cause of death in the Republic of Korea for over 10 years.

The overexpressed p-glycoprotein (p-gp) on the surface of cancer cells pumps out the drug to prevent the drug from accumulating in the cell, so the anticancer drug goes out of the cell before it even reaches the target. This is defined as multi-drug resistance (MDR), which increases the resistance of cells to the effects of anticancer drugs during chemotherapy, which is a major cancer treatment method, and has been regarded as a major obstacle in clinical cancer treatment. In order to overcome this limitation, a treatment method using nanoparticles (NP) has been developed.

Nanoparticles are characterized by being able to target specific sites by surface modification or to increase cellular uptake by coating. In particular, coating with a positively charged biocompatible chitosan material increases affinity for the cell membrane and increases cellular uptake. In addition, by simultaneously delivering two or more different drugs to the nanoparticles, it is possible to overcome the problems of side effects due to drug resistance or high toxicity.

On the other hand, chitosan is a pyranose unit of glucosamine, which is β-1,4 bonded, and has a molecular weight of 1 million or more in which 5,000 or more glucosamine residues are bonded and a polysaccharide having a polyvalent cation. It is a series of biopolymer materials that can be extracted from aquatic systems including crustaceans such as crab shells and shrimps and squid. In terms of its molecular structure, it has a structure similar to cellulose, a type of polysaccharide, and has excellent biocompatibility, so that it can be used during immune response. Since rejection does not occur, it has been applied to the pharmaceutical industry, and after being recently certified as a food by the US FDA, chitosan is being applied as an important biological industry and biomedical material in the 21st century. In particular, chitosan with a specific molecular weight within 20,000 ~ 100,000 is known to have strong physiological activity, so it is expected to have applications in the health food field, food and beverage field, cosmetics field, health hygiene field, and pharmaceutical field. can

prior literature

[Patent Document 1] Republic of Korea Patent Publication No. 2009-0088614 (2009. 08. 20)

As a result of the present inventors' earnest efforts to develop a bioactive substance delivery system capable of cellular uptake and tumor targeting, the present inventors' conventionally developed temperature-sensitive nanosponge (thermosponge nanoparticle, TNP) is coated with chitosan to increase affinity to the cell membrane. cellular uptake is improved, allowing more physiologically active substances to be delivered into cells than TNP. Also, since Pluronic has weak p-gp inhibition, it can effectively treat drug-resistant tumor cells without a separate p-gp inhibitor. It can be confirmed that the present invention has been completed.

An object of the present invention is to provide a temperature-sensitive nano-sponge platform (Chitosan coated thermosponge nanoparticle (CTNP) platform) coated with chitosan for delivery of a physiologically active substance.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of cancer comprising the temperature-sensitive nano-sponge platform coated with the chitosan.

The present invention also aims to provide a cosmetic composition comprising the temperature-sensitive nano-sponge platform coated with the chitosan.

The present invention also aims to provide a food composition comprising a temperature-sensitive nano-sponge platform coated with the chitosan.

As used herein, the term "biodegradable" means that a polymer can be chemically degraded in the body to form non-toxic compounds, the rate of degradation being equal to or different from the rate of drug release.

As used herein, the term “biocompatibility” refers to a substance that interacts with the human body without undesirable subsequent effects.

As used herein, the term “bioactive material” refers to, after being administered to a recipient (animal subject) that is positive or positive for physical, physiological, mental, biochemical, biological, or other bodily function. It is meant to include any drug that can affect in a negative way, for example, the 'bioactive substance' is a drug, protein, peptide, miRNA, siRNA, DNA, plasmid DNA, small molecule ( small molecules), antibodies, viruses, microorganisms, somatic cell nuclei, organelles, mitochondria, enzymes, conutrients, vitamins, natural products, extracts, or other active agents, but are not limited thereto. Silver proteins, peptides, nucleosides, nucleotides, endogenous ligands, neurotransmitters, hormones, autacoids, cytokines, antiviral agents, anticancer agents, antibiotics, oxygen-enhancing agents, oxygen-containing agents, antibiotics selected from the group consisting of anticonvulsants and anti-inflammatory drugs.

As used herein, the term "therapeutic agent" means any agent that, when administered to an organism (human or non-human), induces a desired pharmacological, immunogenic and/or physiological effect by local and/or systemic action. compound or composition. Thus, the term includes compounds or chemicals traditionally considered drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, genetic constructs, and the like. "Therapeutic agent" includes compounds or compositions used in all major therapeutic fields, including but not limited to: anti-infective agents such as antibiotics and antiviral agents; analgesic and analgesic combinations; local and general anesthetics; anorectics; anti-arthritis; anti-asthma; anticonvulsant; antidepressants; antihistamines; anti-inflammatory; antiemetic; antimigraine; anti-neoplastic agents; anti-itch drugs; antipsychotics; fever remedy; anti-smoke festival; cardiovascular preparations (including calcium channel blockers, β-blockers, β-agonists and antiarrhythmic agents); antihypertensives; chemotherapeutic agents; diuretic; vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormone; osteogenic stimulants and bone resorption inhibitors; immunosuppressants; muscle relaxants; psychostimulants; sedative; tranquilizer; proteins, peptides and fragments thereof (naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (ribonucleotides (RNA) or deoxyribonucleotides (DNA), gene constructs, expression vectors, plasmids, including polymeric forms of two or more nucleotides, double- and single-stranded molecules and supercoiled or condensed molecules; antisense molecules, etc.).

As used herein, the terms "peptide", "polypeptide", "oligopeptide" and "protein" may be used interchangeably to refer to a peptide or protein drug and are limited to a particular molecular weight, peptide sequence or length, bioactivity or therapeutic field. doesn't happen

As used herein, the term "therapeutic effect" refers to any improvement in the disease of a subject, human or animal treated according to the method, and has a preventive or preventive effect, or a physical investigation, laboratory or mechanical method. any amelioration in the severity of the signs and symptoms of a disease, condition or condition that can be detected by Unless defined otherwise with respect to a particular disease or condition, as used herein, the term "treatment" or "treating" means (i) an animal susceptible to but not yet diagnosed with a disease, disorder and/or condition, or preventing the development of a disease, disorder or condition in a person; (ii) inhibiting the disease, disorder or condition, ie inhibiting its progression; and/or (iii) alleviating the disease, condition or condition, ie causing regression of the disease, condition and/or condition.

According to the first embodiment,

The present invention is a physiologically active material comprising a core portion containing aliphatic polyester, and a shell portion containing a polymer surrounding the core portion, and coated with chitosan. To provide a temperature-sensitive nano-sponge platform coated with chitosan for delivery. A schematic diagram of a temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active substance according to the present invention is shown in FIG. 1 .

In the temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active material according to the present invention, the weight ratio of the aliphatic polyester in the core part and the polymer in the shell part is 1:10 to 1:20.

In the temperature-sensitive nanosponge platform coated with chitosan for delivery of physiologically active substances according to the present invention, the chitosan is characterized in that it has a deacetylation degree of 50 to 90 and a molecular weight of 10 to 100 kDa.

In the temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active material according to the present invention, the weight ratio of the polymer and chitosan in the shell part is 1:1 to 1:3, preferably 1:1.

In the temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active material according to the present invention, the aliphatic polyester is polylactic acid (PLA), polyglycolic acid (PGA) or polylactic acid-glycol It is characterized in that it is an acid copolymer (Polylactide-co-Glycolide, PLGA). The polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (Polylactide-co-Glycolide, PLGA) is approved for clinical use by the US Food and Drug Administration (FDA) As a synthetic polymer, it is decomposed in a living body and has the advantage of having excellent biocompatibility.

In the temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active substance according to the present invention, the polymer in the shell part is characterized in that it is a pluronic series. Pluronic copolymer is a linear triblock copolymer composed of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) as a temperature-sensitive polymer showing a sol/gel transition in an aqueous solution phase. For example, the polymer in the shell part may be Pluronic F127 (Poloxamer 407).

In the temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active substance according to the present invention, the nano-sponge has an average particle diameter of 50 to 500 nm. While minimizing biological stimulation within the above range, it is possible to efficiently deliver nanoparticles to a target site.

In the temperature-sensitive nanosponge platform coated with chitosan for delivery of a physiologically active substance according to the present invention, the drug is nucleic acid, protein, polypeptide, carbohydrate, inorganic substance, antibiotic, anticancer agent, antibacterial agent, steroid, anti-inflammatory analgesic, sex hormone, Immunosuppressant, antiviral, anesthetic, antiemetic, antihistamine, local anesthetic, antiangiogenic, vasoactive agent, anticoagulant, immunomodulatory agent, cytotoxic agent, antibody, neurotransmitter, psychoactive agent, anti-inflammatory agent, anti-wrinkle agent, hair loss agent , hair growth agent, blood circulation improving agent, anti-aging agent, diet improving agent, oligonucleotide, lipid, cell, tissue, characterized in that at least one substance selected from the group consisting of cancer chemotherapeutic agents and vaccines.

In the temperature-sensitive nano-sponge platform coated with chitosan for delivery of physiologically active substances according to the present invention, the nano-sponge platform is used as a molding material, a cosmetic material, or a support for tissue regeneration.

The temperature-sensitive nanosponge platform coated with chitosan for delivery of physiologically active substances according to the present invention has improved cellular uptake, so that more drugs, for example, anticancer drugs, can be delivered into cells, and p-gp inhibition is weakly performed by Pluronic. It can effectively treat drug-resistant tumor cells without a separate p-gp inhibitor.

According to the second embodiment,

The present invention is to provide a pharmaceutical composition for the treatment of cancer comprising the temperature-sensitive nano-sponge platform coated with the chitosan. The present invention is shown in Figure 2 a schematic diagram of the cancer treatment process using the temperature-sensitive nano-sponge platform coated with chitosan for delivery of the physiologically active substance.

In the pharmaceutical composition for treating cancer according to the present invention, the cancer is pancreatic cancer, liver cancer, breast cancer, lung cancer, stomach cancer, rectal cancer, gallbladder cancer, ovarian cancer, bladder cancer, colorectal cancer, lymphoma, brain cancer, uterine cancer, prostate cancer and malignant melanoma Or it is characterized in that it is biliary tract cancer.

In the pharmaceutical composition for treating cancer according to the present invention, the drug is a hydrophobic drug, a hydrophilic drug, or a hydrophobic drug and a hydrophilic drug. The hydrophobic drug is vinblastine, etoposide, Alkeran, Cytoxan, Daunorubicin, Hydrea, Mithramycin, Mitomycin ), Mitoxantrone, Nitrogen Mustard, Velban, Vincristine, Carboplatinum, Idarubicin, Irinotecan, Leustatin ( Leustatin), Navelbine (Navelbine), taxotere (Taxotere), topotecan (Topotecan) may be at least one selected from the group consisting of adriamycin, daunomycin and paclitaxel. The hydrophilic drug is Busulfan, Chlorambucil, Cyclophosphamide, Melphalan, Cisplatin, Ifosfamide, Cytarabine, 5 -Fluorouracil (5-FU), methotrexate (Methotrexate, MTX), actinomycin-D (Actinomycin-D), it may be one or more selected from the group consisting of bleomycin and gemcitabine (gemcitabine).

In the pharmaceutical composition for the treatment of cancer according to the present invention, the pharmaceutical composition is characterized in that it further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes carriers and vehicles commonly used in the pharmaceutical field, and specifically, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances ( For example, various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salt), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic matrix, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool paper and the like.

According to the third embodiment,

The present invention is to provide a cosmetic composition comprising a temperature-sensitive nano-sponge platform coated with the chitosan.

In the cosmetic composition according to the present invention, the cosmetic composition is characterized in that it comprises a wrinkle improving agent, a skin aging inhibitor or a skin whitening agent.

In the cosmetic composition according to the present invention, the temperature-sensitive nano-sponge platform coated with chitosan is characterized in that it comprises a hydrophilic material, a hydrophobic material or a hydrophilic material and a hydrophilic material. The hydrophilic material may include arbutin, adenosine, vitamin C, vitamin B3, vitamin B5, vitamin H, acetylglucosamine, madecassoside, or selenium aspartate. The hydrophobic material is eugenol, retinol, astaxanthin, caffeine, carnosinic acid, catechin, coenzyme-Q10, curcumin, ellagic acid, ferric acid, idebenone, isoflavone, linoleic acid, lipoic acid, lycopene, olenoic acid, may contain phloretin, quercetin, resveratrol, squalane, squalene, tannic acid, vitamin A, vitamin B, vitamin E, vitamin F, cholesterol, phytosphingosine, glycosphingolipid, beta-sitosterol, lauric acid or lecithin have.

In the cosmetic composition according to the present invention, the cosmetic composition is prepared in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder. characterized.

According to the fourth embodiment,

The present invention is to provide a food composition comprising a temperature-sensitive nano-sponge platform coated with the chitosan.

In the food composition according to the present invention, the food composition includes all types of food such as health functional food, nutritional supplement, nutritional supplement, pharmaceutical food, health food, nutraceutical, designer food, or food additive. may include. For example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc. may include

In the food composition according to the present invention, the food composition includes components commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

In the food composition according to the present invention, the food composition includes various nutrients, vitamins, minerals (electrolytes), dietary components, 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 colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, or carbonation agents used in carbonated beverages, and the like. For example, in one embodiment of the present application, when the food composition is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, or various plant extracts may be additionally included in addition to the active ingredient of the present application. have.

The temperature-sensitive nano-sponge platform coated with chitosan for delivery of physiologically active substances according to the present invention has the advantage of loading various drugs as well as anti-cancer drugs, so it can be applied to the field of cancer treatment and can treat various diseases. The scope of application in the field of treatment is wide. In addition, since chitosan has a skin penetrating function, it is used in functional cosmetics fields (atopy, acne, psoriasis, wrinkle improvement, hair loss improvement, etc.), toothpaste, shampoo, soap and medical devices (drug skin patch, catheter coating material, drug release system) etc.) is expected to be used in the field.

1 shows a schematic diagram of a temperature-sensitive nano-sponge platform (Chitosan coated thermosponge nanoparticle (CTNP) platform) coated with chitosan for delivery of a physiologically active substance according to the present invention.
2 shows a schematic diagram of an effective cancer treatment process of drug-loaded CTNP.
3 shows the size of the temperature-sensitive nano-sponge platform (TNP platform) and the temperature-sensitive nano-sponge platform (CTNP platform) coated with chitosan.
Figure 4 shows the dispersion of the temperature-sensitive nano-sponge platform (TNP platform) and chitosan-coated temperature-sensitive nano-sponge platform (CTNP platform).
5 shows the surface charge of the temperature-sensitive nano-sponge platform (TNP platform) and the temperature-sensitive nano-sponge platform (CTNP platform) coated with chitosan.
Figure 6 shows the size of the temperature-sensitive nano-sponge platform (TNP platform) and chitosan-coated temperature-sensitive nano-sponge platform (CTNP platform) before and after drug loading.
7 shows the dispersion of the temperature-sensitive nano-sponge platform (TNP platform) and the chitosan-coated temperature-sensitive nano-sponge platform (CTNP platform) before and after loading the hydrophobic drug.
Figure 8 shows the surface charge of the temperature-sensitive nano-sponge platform (TNP platform) and the chitosan-coated temperature-sensitive nano-sponge platform (CTNP platform) before and after loading the hydrophobic drug.
9 shows the cytotoxicity of each concentration of the TNP platform itself and the chitosan coated TNP (CTNP) platform.
10 shows the anticancer effect according to the type of inhibitor.
11 shows the anticancer effect of PTX-loaded TNP and CTNP (PTX@TNP, PTX@CTNP).

Hereinafter, various examples are presented to help the understanding of the invention. The following examples are provided for easier understanding of the invention, and the protection scope of the invention is not limited to the following examples.

<Example>

Example 1. Preparation of chitosan-coated temperature-sensitive nano-sponge (CTNP) platform

The temperature-sensitive nano sponge coated with chitosan was prepared by nanoprecipitation method (poly-D, L-lactide, ~18 kDa, PLA-COOH) and Pluronic F127 (Pluronic F127, 12.6 kDa) in a weight ratio of 1:20 After preparation through nanoprecipitation), it was completed by coating with chitosan (DAC90, 10kDa). PLA (10mg) was dissolved in acetone (99%, sigma aldrich) 1mL, and after dissolving Pluronic F127 in the reaction solution, the reaction was maintained at room temperature for 2 hours. The reaction solution was slowly dropwise in 5 mL of deionized water (DIW) stirred at 530 rpm, and then acetone was removed in a fume hood for 6 hours or more. Then, chitosan was added and stirred at 530 rpm for 2 hours, and finally ultrafiltration (Amicon Ultra-15 filter) was performed to increase the purity of the nanoparticles.

Example 2. Analysis of size, dispersion and surface charge of CTNP platform

The size, dispersion, and surface charge of the CTNP platform prepared according to Example 1 and the TNP platform as a control were analyzed using Zetasizer (Nano-ZS, Malvern) equipment, and the results are shown in FIGS. 3 to 5, respectively. .

As a result, the TNP platform that was not coated with chitosan appeared to have a size of about 70 nm at 37 °C, and CTNP (Pluronic: Chitosan = 1:3) coated with 600 mg or less of chitosan had a size of about 100 nm or less. It was confirmed that the coating did not significantly affect the physicochemical properties of nanoparticles. However, in the case of CTNP (Pluronic: Chitosan = 1:3.5) using 700 mg or more of chitosan, it was prepared as an unstable formulation having a size of about 150 nm (FIG. 3). In addition, it was confirmed that the TNP and all CTNP platforms had a dispersion (PDI) of 0.3 or less and were monodisperse (Fig. 4), and all of the CTNP platforms coated with chitosan showed a positive charge value of about 20 mV, so that chitosan was deposited on the surface. It was confirmed that a stable coating was formed and existed (FIG. 5).

Example 3. Preparation and characterization of drug-loaded CTNP platform

As a hydrophobic anticancer agent, Paclitaxel (200μg) was dissolved in 1mL of acetone (99%, sigma aldrich) and then added to PLA (10mg) and reacted at room temperature for 1 hour. Then, after dissolving Pluronic F127 (200 mg) in the reaction solution and reacting for 2 hours, slowly dropwise into 5 mL of deionized water while stirring the reaction solution at 530 rpm, followed by a fume hood for at least 6 hours Acetone was removed from the PLA:Pluronic:Chitosan = 1:20:20, which is the optimal formulation condition for drug loading evaluation, was prepared. The size, dispersion, and surface charge of the TNP and CTNP platforms before and after drug loading were analyzed using an electrophoretic light scattering spectrophotometer (ELS-Z2, Otsuka) equipment.

As a result, when 5 wt% of Palitaxel (PTX) was loaded into TNP and CTNP, the size was about 70 nm, and it was confirmed that the physicochemical properties of nanoparticles according to drug loading were not significantly affected ( FIG. 6 ). Even in the case of the dispersion value (PDI), it was confirmed that drug loading was stably achieved by showing a result of 0.3 or less both before and after loading of the drug ( FIG. 7 ). In addition, in the case of surface charge, when drugs were loaded onto TNP and CTNP, respectively, TNP and PTX@TNP showed a difference of about -20 mV and CTNP and PTX@CTNP of about +20 mV, so that the hydrophobic drug was stable to both TNP and CTNP. It was confirmed that it can be loaded with (FIG. 8).

Example 4. Evaluation of intracellular safety of CTNP

For intracellular safety evaluation, fibroblast cells (NIH3T3, cell line bank) ^{were dispensed in a 96-well plate at a cell count of 1x10 4} per well and grown at 37°C and 5% CO2 for 12 hours. Cells were treated in DMEM containing 10% FBS and 1% AA at concentrations of TNP 100μg/ml, 200μg/ml, and CTNP 3.125μg/ml ~ 100μg/ml, and incubated for 24 hours. After mixing cck-8 reagent:medium in a ratio of 1:9, 110 μl of each cell was treated and incubated for 1 hour while blocking light. Then, absorbance (450 nm) was measured.

As a result, it was confirmed that the TNP platform had little difference in cell viability from the control group even when the concentration was high (FIG. 9a). In addition, even in the case of CTNP, even when the concentration of chitosan was increased, the cell viability was hardly different from that of the control group, so it was confirmed that there was no cytotoxicity (FIG. 9b). Therefore, it has been proven that the CTNP platform has excellent biocompatibility and can be used in future pharmaceuticals and medical devices.

Example 5. Evaluation of p-gp inhibition efficacy

Colorectal cancer cells (HCT15, ATCC) were seeded in a 96-well plate at 1x10 ⁴ cells per well and grown at 37°C and 5% CO2 for 12 hours. Paclitaxel in various concentrations including drug itself, tetrandrine (TET), pluronic F127 (PLU), thermosponge nanoparticle (TNP) in RPMI-1640 containing 10% FBS and 1% AA at a concentration of 0.1 μg/ml The cells were treated and incubated for 48 hours. After mixing cck-8:medium in a ratio of 1:9, each cell was treated with 110 μl and incubated for 1 hour while blocking light. Then, absorbance (450 nm) was measured.

As a result, HCT15 cells are resistant to paclitaxel (PTX), and when treated with anticancer drugs alone, they exhibited about 20% anticancer effect, but when treated with tetrandrine (TET), a 4th generation p-gp inhibitor, It was confirmed that the anticancer effect was about 40~70% depending on the concentration, and the efflux degree of PTX decreased due to the p-gp inhibition effect of TET, indicating a more effective anticancer effect. In addition, when an anticancer agent was treated with pluronic, known as a p-gp inhibitor, it showed an anticancer effect of about 40-50% compared to an anticancer agent that showed about 20% of an anticancer effect, and TNP containing pluronic was also treated with an anticancer agent alone. It showed a more effective anticancer effect (FIG. 10). Based on these results, it was confirmed that TNP itself can act as a p-gp inhibitor, although weakly.

Example 6. Evaluation of anticancer efficacy of drug-loaded CTNP

Colorectal cancer cells (HCT15, ATCC) were seeded in a 96-well plate at 1x10 ⁴ cells per well and grown at 37°C and 5% CO2 for 12 hours. Cells were treated with PTX, PTX@TNP, and PTX@CTNP in RPMI-1640 containing 10% FBS and 1% AA so that 0.1 μg/ml of PTX was contained and incubated for 48 hours. After mixing cck-8:medium in a ratio of 1:9, each cell was treated with 110 μl and incubated for 1 hour while blocking light. Then, absorbance (450 nm) was measured.

As a result, paclitaxel (PTX) itself showed an anticancer effect of about 38% due to drug resistance, and PTX-loaded TNP showed an anticancer effect of about 40%. On the other hand, when the CTNP according to the present invention is used, the intracellular drug delivery rate is increased and the p-gp inhibition effect can be obtained at least weakly by the component Pluronic, so it was confirmed that it exhibits an anticancer effect that is about 1.5 times more than that of TNP (Fig. 11). ). Therefore, it has been proven that CTNP is an excellent platform that can effectively treat drug-resistant tumors without a separate p-gp inhibitor. In addition, since CTNP can improve skin permeability and serous permeability, it can be used as a platform for treatment of various diseases. The CTNP platform technology according to the present invention is not limited to pharmaceuticals, but is expected to be widely used in cosmetics and medical devices, daily necessities, and dry food.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

Consists of a core portion (core portion) containing polylactic acid (Poly Lactic Acid, PLA) and a shell portion (shell portion) containing a pluronic copolymer surrounding the core portion,
Temperature-sensitive nano-sponge platform coated with chitosan for delivery of physiologically active substances, characterized in that it is coated with chitosan.
According to claim 1,
The weight ratio of polylactic acid (PLA) in the core part and the pluronic copolymer in the shell part is 1:10 to 1:20, which is characterized in that it is temperature sensitive coated with chitosan for delivery of physiologically active substances Nanosponge platform.
According to claim 1,
The chitosan is characterized in that it has a deacetylation degree of 50 to 90 and a molecular weight of 10 to 100 kDa, a temperature-sensitive nano-sponge platform coated with chitosan for delivering physiologically active substances.
According to claim 1,
The weight ratio of the pluronic copolymer and chitosan in the shell part is 1:1 to 1:3, which is a temperature-sensitive nano-sponge platform coated with chitosan for delivering physiologically active substances.
delete delete According to claim 1,
The physiologically active substances include nucleic acids, proteins, polypeptides, carbohydrates, inorganic substances, antibiotics, anticancer agents, antibacterial agents, steroids, anti-inflammatory analgesics, sex hormones, immunosuppressants, antivirals, anesthetics, antiemetics, antihistamines, local anesthetics, antivascular Forming agent, vasoactive agent, anticoagulant, immunomodulatory agent, cytotoxic agent, antibody, neurotransmitter, psychoactive agent, anti-inflammatory agent, wrinkle improving agent, hair loss improving agent, hair growth agent, blood circulation improving agent, anti-aging agent, diet improving agent, oligonucleotide, lipid, cell, A temperature-sensitive nanosponge platform coated with chitosan for delivery of physiologically active substances, characterized in that it is at least one material selected from the group consisting of tissue, cancer chemotherapeutic agent and vaccine.
According to claim 1,
The temperature-sensitive nano-sponge platform is a temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active material, characterized in that it is used as a support for molding material, cosmetic material or tissue regeneration.
A pharmaceutical composition for the treatment of cancer comprising a temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active substance according to any one of claims 1 to 4 and 7.
10. The method of claim 9,
The cancer is pancreatic cancer, liver cancer, breast cancer, lung cancer, stomach cancer, rectal cancer, gallbladder cancer, ovarian cancer, bladder cancer, colorectal cancer, lymphoma, brain cancer, uterine cancer, prostate cancer and malignant melanoma or biliary tract cancer, characterized in that, cancer treatment pharmaceutical composition for use.
A cosmetic composition comprising a temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active material according to any one of claims 1 to 4 and 7.
12. The method of claim 11,
The cosmetic composition is characterized in that it comprises a wrinkle improving agent, a skin aging inhibitor or a skin whitening agent, the cosmetic composition.
12. The method of claim 11,
The cosmetic composition is a cosmetic composition, characterized in that it is prepared in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.
A food composition comprising a temperature-sensitive nano-sponge platform coated with chitosan for delivery of a physiologically active substance according to any one of claims 1 to 4, and 7.
15. The method of claim 14,
The food composition is a health functional food, nutritional supplement, nutritional supplement, pharmaceutical food (pharmafood), health food, nutraceutical (nutraceutical), designer food, or food composition, characterized in that it comprises a food additive.

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