KR20230124430A - Composition for delivering poorly soluble drug comprising water-dispersed milk protein complex carrying poorly soluble drug complex and method for preparing the same - Google Patents

Abstract

It relates to a composition for delivery of a poorly soluble drug, including a poorly soluble drug, beta-lactoglobulin and tannic acid-metal ion, and a method for preparing the same, which has high stability and can adjust the drug content according to the number of repetitions of coating.

Description

Composition for delivering poorly soluble drug comprising water-dispersed milk protein complex carrying poorly soluble drug and method for preparing the same

The present specification relates to a composition for delivery of a poorly soluble drug, including a poorly soluble drug, beta-lactoglobulin and tannic acid-metal ion, and a method for preparing the same.

Poorly soluble drugs are drugs that exhibit low solubility in water, and include acetaminophen, ketoprofen, and acyclovir. Poorly soluble drugs have low solubility in water, resulting in low absorption rates and reduced physiological activity.

Beta-lactoglobulin (β-lactoglobulin) is a major component of whey protein and is a member of the lipocalin family of proteins. The characteristic affinity for hydrophobic compounds, including poorly soluble drugs, has promoted the development of hydrophobic drug delivery systems through microencapsulation technology. In this regard, research on improving the water solubility of resveratrol by generating a complex with resveratrol using beta-lactoglobulin has been conducted.

However, although the above studies improved the water solubility of resveratrol through complex formation, the protein exhibited limited applicability, including the problem of being sensitive to surrounding stimuli and being easily deformed, and the fact that it could not be controlled because it existed in a solution state.

Nanocoating is a method of modifying the surface properties by forming a thin film of 100 nm or less on the surface, and can combine various functions in a controlled manner. As a coating material, tannic acid is a polyphenolic material containing a galloyl group (1,2,3-trihydroxyphenyl group), and metal cations (Fe(III), Fe(II), Al(III), Zr(IV), Ru(III), Rh(III), Zn(II), V(III), Cr(III), Mn(II), Co(II), Ni(II), Cu(II), Mo(II), Cd(II), Eu(III), Gd(III), Tb(III), and Ce(III)) quickly form complexes to provide nano-coatings that can be adjusted in thickness regardless of material surface properties. carry out In addition, the galloyl group performs the function of a typical adhesive motif through various non-covalent interactions such as hydrogen bonds and π-π interactions, and thus has easy adhesion with other materials.

The process of immobilizing the poorly soluble drug-protein complex using the nanocoating was studied to compensate for the above-mentioned uncontrollable disadvantages, and it is expected that integrating drugs into multilayer nanofilms can be used to develop innovative drug delivery systems. Expect.

The present inventors discovered a poorly soluble drug-beta-lactoglobulin complex nanocoating method based on tannic acid-metal ion nanocoating and various intermolecular interactions, and developed a poorly soluble drug delivery composition having stability against various enzymes under various pH conditions. developed.

Depending on the number of repetitions of coating, the thickness can be precisely controlled at the nanometer level, so it can be applied as a drug delivery system that controls the content of poorly soluble drugs. By grafting onto the tannic acid-metal ion nanocoating, which is not limited to the shape and surface properties of the coating material, it was possible to easily introduce a poorly soluble drug-β-lactoglobulin complex to the surface of the desired material, and furthermore, it could be applied to a large area regardless of the size or shape of the material. possible. Since the manufacturing process is carried out under mild conditions (room temperature, neutral pH, aqueous phase), it is possible to form a poorly soluble drug-beta-lactoglobulin complex nanofilm on the surface of materials sensitive to acidity and temperature, and the materials used are inexpensive in the surroundings. It is an easily obtained material and is cost-effective because it does not require special equipment in the manufacturing process. In addition, it has stability against various pH ranges and digestive enzymes in the body.

The composite nanocoating can be performed on various substrates regardless of the structural dimension, and can be used as a drug delivery system by performing coating on a drug carrier in the form of particles. The designed sequential layered structure can contain fluorescent/radioactive marker proteins or target marker proteins for specific diseases and can function as intelligent theranostic agents, and the poorly soluble drug-protein nanocoating method and design can be used for next-generation drugs It can be applied to the development of delivery platforms.

One example of the present specification provides a composition for delivery of a poorly soluble drug, including a poorly soluble drug, beta-lactoglobulin and tannic acid-metal ion.

Other examples include substrate; and

Including a coating layer coating the surface of the substrate,

In the coating layer, (a) a nanofilm containing a poorly soluble drug and a beta-lactoglobulin complex and (b) a nanofilm containing tannic acid-metal ions are stacked in any order,

A composition for delivery of a poorly soluble drug is provided.

Another example provides a method for delivering the poorly soluble drug.

Another example is (1) adding a metal ion solution and a tannic acid solution to a substrate to form a tannic acid-metal ion nanofilm coating layer on the surface of the substrate; and

(2) The substrate obtained in step (1) is added to a solution of the poorly soluble drug and beta-lactoglobulin complex to form a poorly soluble drug and beta-lactoglobulin complex nanofilm coating layer on the tannic acid-metal ion nanofilm coating layer on the surface of the substrate. forming step

It provides a method for producing a composition for delivery of a poorly soluble drug comprising a.

One example of the present specification provides a composition for delivery of a poorly soluble drug, including a poorly soluble drug, beta-lactoglobulin and tannic acid-metal ion.

Another embodiment provides a method for delivering a poorly soluble drug comprising the step of administering a composition for delivery of a poorly soluble drug containing a poorly soluble drug, beta-lactoglobulin and tannic acid-metal ion to an administration subject.

The poorly soluble drug, beta-lactoglobulin and tannic acid-metal ion

(a) a nanofilm containing a poorly soluble drug and beta-lactoglobulin complex; and

(b) nanofilm containing tannic acid-metal ions

It may be included in the form of, but is not limited thereto.

The (a) nanofilm and (b) nanofilm may be included in an alternating stacked form regardless of the order, but is not limited thereto.

The (a) nanofilms and (b) nanofilms are alternately 1 to 100, 1 to 50, 1 to 20, 1 to 17, 1 to 15, respectively, regardless of order. 1 to 12, 1 to 10, 1 to 8, 1 to 6, 3 to 100, 3 to 50, 3 to 20, 3 to 17, 3 to 15, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 5 to 100, 5 to 50, 5 to 20, 5 to 17 , 5 to 15, 5 to 12, 5 to 10, 5 to 8, or 5 to 6 layers, such as 5 layers, but is not limited thereto.

In another example, (a) the content of the poorly soluble drug can be controlled by adjusting the number of layers of the nanofilm containing the poorly soluble drug and beta-lactoglobulin complex (e.g., as the number of layers increases, the content of the poorly soluble drug increases) (b) controlling the number of layers of the nanofilm containing tannic acid-metal ions can control the drug release time (e.g., as the number of layers increases, the release time of the drug is delayed, or as the number of layers increases, the release time of the drug decreases). becomes longer).

The thickness of the composition may be adjusted according to the number of layers of the (a) nanofilm and (b) nanofilm, but is not limited thereto.

Other examples include substrate; and

Including a coating layer coating the surface of the substrate,

In the coating layer, (a) a nanofilm containing a poorly soluble drug and a beta-lactoglobulin complex and (b) a nanofilm containing tannic acid-metal ions are stacked in any order,

A composition for delivery of a poorly soluble drug is provided.

The (a) nanofilm has a thickness of 0.1 to 10 nm, 0.1 to 7 nm, 0.1 to 4 nm, 0.1 to 3 nm, 0.1 to 2 nm, 0.1 to 1.8 nm, 0.5 to 10 nm, 0.5 to 7 nm, 0.5 to 4 nm, 0.5 to 3 nm, and 0.5 to 0.5 nm. 2 nm, 0.5 to 1.8 nm, 1 to 10 nm, 1 to 7 nm, 1 to 4 nm, 1 to 3 nm, 1 to 2 nm, 1 to 1.8 nm, 1.5 to 10 nm, 1.5 to 7 nm, 1.5 to 4 nm, 1.5 to 3 nm, 1.5 to 1.5 nm 2 nm, 1.5 to 1.8 nm, 1.7 to 10 nm, 1.7 to 7 nm, 1.7 to 4 nm, 1.7 to 3 nm, 1.7 to 2 nm, or 1.7 to 1.8 nm, for example, may have a thickness of 1.75 nm, It is not limited thereto.

The (b) nanofilm has a thickness of 0.1 to 10 nm, 0.1 to 8 nm, 0.1 to 6 nm, 0.1 to 4 nm, 0.1 to 3 nm, 0.1 to 2.8 nm, 1 to 10 nm, 1 to 8 nm, 1 to 6 nm, 1 to 4 nm, 1 to 4 nm. 3 nm, 1 to 2.8 nm, 2 to 10 nm, 2 to 8 nm, 2 to 6 nm, 2 to 4 nm, 2 to 3 nm, 2 to 2.8 nm, 2.5 to 10 nm, 2.5 to 8 nm, 2.5 to 6 nm, 2.5 to 4 nm, 2.5 to 2.5 nm It may have a thickness of 3 nm, 2.5 to 2.8 nm, 2.7 to 10 nm, 2.7 to 8 nm, 2.7 to 6 nm, 2.7 to 4 nm, 2.7 to 3 nm, or 2.7 to 2.8 nm, for example, may have a thickness of 2.78 nm, It is not limited thereto.

Other examples include substrate; and

Including a coating layer coating the surface of the substrate,

The coating layer is composed of (a) a nanofilm containing a poorly soluble drug and beta-lactoglobulin complex and (b) a nanofilm containing tannic acid-metal ions stacked in any order to give a composition for delivery of a poorly soluble drug to an administration subject. It provides a method for delivering a poorly soluble drug comprising the step of administering.

The coating layer may be formed at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, 100 or less, 50 It may be formed below, but is not limited thereto.

Another example is

(1) adding a metal ion solution and a tannic acid solution to a substrate to form a tannic acid-metal ion nanofilm coating layer on the surface of the substrate; and

(2) The substrate obtained in step (1) is added to a solution of the poorly soluble drug and beta-lactoglobulin complex to form a poorly soluble drug and beta-lactoglobulin complex nanofilm coating layer on the tannic acid-metal ion nanofilm coating layer on the surface of the substrate. forming step

A method for producing a composition for delivery of a poorly soluble drug comprising a.

Steps (1) to (3) are repeated 1 to 10 times, 1 to 8 times, 1 to 6 times, 3 to 10 times, 3 to 8 times, 3 to 6 times, 5 to 10 times times, 5 to 8 times, or 5 to 6 times, for example, it may be repeated 5 times, but is not limited thereto.

The thickness of the composition may be adjusted according to the number of repetitions of the steps (1) to (3), but is not limited thereto.

The poorly soluble drug and beta-lactoglobulin complex is prepared by mixing the poorly soluble drug and beta-lactoglobulin at 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4 , 1:6, 1:8, or may be included in a molar concentration ratio of 1:10, 10:1 to 1:10, 10:1 to 1:8, 10:1 to 1:6, 10:1 to 1:4, 10:1 to 1:2, 8:1 to 1:10, 8:1 to 1:8, 8:1 to 1:6, 8:1 to 1:4, 8:1 to 1 :2, 6:1 to 1:10, 6:1 to 1:8, 6:1 to 1:6, 6:1 to 1:4, 6:1 to 1:2, 4:1 to 1:10 , 4:1 to 1:8, 4:1 to 1:6, 4:1 to 1:4, 4:1 to 1:2, 2:1 to 1:10, 2:1 to 1:8, 2 :1 to 1:6, 2:1 to 1:4, or 2:1 to 1:2 may be included in a molar concentration ratio, but is not limited thereto.

The poorly soluble drug and beta-lactoglobulin complex may be characterized in that water solubility is improved compared to poorly soluble drugs, but is not limited thereto.

The substrate may be a gold plate, polystyrene, metal, metal oxide, ceramic, mineral, polymer, biocompatible polymer (e.g., polyacrylic acid, polyvinyl pyrrolidone), hydroxyalkyl cellulose, Carbomer, Polyvinyl alcohol, Chitosan, Alginate, Carboxymethyl Cellulose, Poly(hexamethylenebiguanide), Carrageenan , Polyethylene oxide, polysaccharide, Collagen, Gelatin, Hyaluronic acid, Xanthan gum, Acacia gum, Alginic acid acid, agar, gum arabic, tragacanth, karaya gum, pectin, mannitol, poloxamer, guar gum), polyethylene glycol (PEG) (eg, polyethylene glycol 6000), dextran, hydroxypropyl cellulose or hydroxypropyl methylcellulose), glass, and/or It may be one or more selected from the group consisting of wood, etc. (inanimate group), and the substrate may be one or more selected from the group consisting of fungi (eg, bacteria, fungi, etc.), plants, animals, etc. (biological group ), it may be a combination of the inanimate group and living organisms. The bacteria may be probiotics, and the probiotics are Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus It may be at least one selected from the group consisting of Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus fermentum, and Lactobacillus salivarius, but is not limited thereto. When the substrate is a plant or animal, it may be the plant or animal itself, or an organ or cell separated therefrom, but is not limited thereto.

The poorly soluble drug may be at least one selected from the group consisting of resveratrol, hydrophobic vitamins, polyphenols, and/or fatty acids, and specifically, vitamin D, quercetin, retinol, folic acid, omega 3-fatty acid, and docosa It may be at least one selected from the group consisting of docosahexaenoic acid (DHA), acetaminophen, ketoprofen, acyclovir and/or coenzyme Q10, but is not limited thereto.

The metal ion is iron ion, aluminum ion (Al(III)), zirconium ion (Zr(IV)), ruthenium ion (Ru(III)), rhodium ion (Rh(III)), zinc ion (Zn(II) ), vanadium ion (V(III)), chromium ion (Cr(III)), manganese ion (Mn (II)), cobalt ion (Co(II)), nickel ion (Ni(II)), copper ion (Cu(II)), molybdenum ion (Mo(II)), cadmium ion (Cd(II)), europium ion (Eu(III)), gadolinium ion (Gd(III)), terbium ion (Tb(III)) )) and at least one selected from the group consisting of cerium ions (Ce(III)), but is not limited thereto.

The composition for delivery of the poorly soluble drug may be administered by oral administration and/or transdermal administration, but is not limited thereto.

The administration subject may be one or more selected from the group consisting of human, cow, horse, sheep, pig, cat, dog, mouse, rat, rabbit, guinea pig, monkey, etc., but is not limited thereto.

Hereinafter, the present invention will be described in detail.

In the present specification, "poorly soluble drug" means a drug that is poorly soluble in water or other solvents.

The above “poor solubility” means that the water solubility is less than 20 mg/mL, less than 18 mg/mL, less than 16 mg/mL, less than 14 mg/mL, less than 12 mg/mL, less than 10 mg/mL, or less than 8 mg/mL. , It may mean less than 6 mg/mL, or less than 4 mg/mL, but is not limited thereto.

“Resveratrol” in this specification may have a water solubility of 0.3 g/L according to the solubility classification table listed in the US Pharmacopoeia, and may belong to “Very slightly soluble”.

In the present specification, the “composition for delivery of a poorly soluble drug” includes a poorly soluble drug contained in a coating layer coating a specific substrate to deliver the poorly soluble drug, and the coating layer is configured in a form in which one or more layers are stacked. may be, but is not limited thereto.

In the present specification, "beta-lactoglobulin (β-lactoglobulin)" is a globulin that accounts for the largest proportion among components of general milk, and is the main component of whey protein contained in cow's or sheep's milk.

In the present specification, "tannic acid" is a specific form of tannin, a type of polyphenol, and is composed of gallotannins obtained by solvent extraction of gall nut or nutmeg products, and is used as a clarifier, flavoring agent, and flavor adjuster in food. used, etc.

In the present specification, “resveratrol” is a type of polyphenol and is found in many plants, including berries such as mulberries, peanuts, grapes, raspberries, and cranberries. It has anti-cancer and strong antioxidant action and plays a role in lowering serum cholesterol.

In the present specification, “substrate” may be a material to be coated during coating in the process of preparing the composition of the present specification, and may be used in the meaning commonly used in the art, but is not limited thereto. In addition, it may refer to an object or object to be targeted for nanofilm or nanocoating, and any object having mass and volume may be used regardless of type. For example, the coating target may be at least one selected from inanimate objects such as metal, metal oxide, ceramic, mineral, polymer, glass, and/or wood. Specifically, the coating target may be one or more species selected from organisms such as fungi (eg, bacteria, fungi, etc.), plants, and animals, and may be an object in which an inanimate object and an organism are combined. When the coating target is a plant or animal, it may be the plant or animal itself, or an organ or cell separated therefrom.

In the present specification, "composite solution" means that the form of powder (powder), solution, etc. of each component of the complex is mixed and exists in the form of a solution, and the form of each component is not limited.

In the composition for delivery of a poorly soluble drug, the thickness of the coating layer may linearly increase according to the number of coatings, and the thickness may be finely adjusted to the nanometer level according to the number of coatings.

In the present specification, the composition may be a pharmaceutical composition, cosmetic composition, skin external application composition, health functional food, or quasi-drug composition, which is a solution, ointment, external ointment, cream, essence, foam, lotion, nutrient lotion, softening water, softening lotion , pack, emulsion, makeup base, soap, cleanser, bath additive, sun cream, sun oil, suspension, emulsion, paste, gel, lotion, powder, soap, cleansing with surfactant, oil, foundation, powder foundation, emulsion foundation , may have a formulation selected from the group consisting of a wax foundation, a patch, and a spray.

The composition may be a formulation for oral administration or parenteral administration, and if it is for oral administration (e.g., pharmaceutical composition (oral) or food composition), the composition may be a powder, granule, tablet, capsule, or suspension. , It may be formulated as an emulsion, syrup, aerosol, and the like.

The composition is suitable for humans, primates such as monkeys, rodents such as mice, rats, rabbits, other mammals including dogs, cats, cows, pigs, sheep, horses, goats, chickens, ducks, geese, etc. Reptiles including birds, snakes, lizards, turtles, crocodiles, and the like, amphibians, and vertebrates including fish may be administered to subjects selected from various routes.

The administration method of the composition may be any method commonly used, for example, oral administration, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal (intraperitoneal) administration, local administration at the lesion site, transdermal administration, It may be administered by a parenteral route such as rectal administration or intraperitoneal injection. All modes of administration can be envisaged, for example, by oral or parenteral administration (percutaneous administration, intraperitoneal, rectal, intravenous, intramuscular or subcutaneous injection). At this time, the parenteral route is preferably transdermal administration, and among them, topical application may be the most preferable. A preferred dosage of the pharmaceutical composition for whitening according to the present invention varies depending on the condition and weight of the patient, the severity of the disease, the type of drug, the route and period of administration, but can be appropriately selected by those skilled in the art. However, for desirable effects, the pharmaceutical composition for whitening according to the present invention may be administered in an amount of 0.1 to 100 mg/kg once or several times a day, but is not limited thereto.

The composition can be administered in a pharmaceutically effective amount. The dosage of the composition may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration interval, administration route, excretion rate and reaction sensitivity. there is. The dosage may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease, and may be divided into once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist.

For example, the once or daily dose of the composition is 0.001 to 10000 mg/kg, specifically, 0.01 to 10000 mg/kg, 0.01 to 5000 mg/kg, 0.01 to 5000 mg/kg, based on the solid weight of the active ingredient (poorly soluble drug). to 3000 mg/kg, 0.01 to 2500 mg/kg, 0.01 to 2000 mg/kg, 0.01 to 1800 mg/kg, 0.1 to 10000 mg/kg, 0.1 to 5000 mg/kg, 0.1 to 3000 mg/kg, 0.1 to 3000 mg/kg 2500 mg/kg, 0.1 to 2000 mg/kg, 0.1 to 1800 mg/kg, 1 to 10000 mg/kg, 1 to 5000 mg/kg, 1 to 3000 mg/kg, 1 to 2500 mg/kg, 1 to 2000 1 to 1800 mg/kg, 10 to 10000 mg/kg, 10 to 5000 mg/kg, 10 to 3000 mg/kg, 10 to 2500 mg/kg, 10 to 2000 mg/kg, 10 to 1800 mg /kg, 100 to 10000 mg/kg, 100 to 5000 mg/kg, 100 to 3000 mg/kg, 100 to 2500 mg/kg, 100 to 2000 mg/kg, or 100 to 1800 mg/kg, but It is not limited. The one-time or daily dose may be formulated as a single formulation in unit dose form, formulated in appropriate portions, or prepared by placing it in a multi-dose container. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.

Each of the above compositions may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or parenteral formulations in the form of sterile injectable solutions according to conventional methods and used.

In addition to the active ingredients described above, the composition is generally selected from the group consisting of pharmaceutically and / or physiologically acceptable carriers, excipients, diluents, etc. selected in consideration of the method of administration and standard pharmaceutical practice. It can be administered in admixture with one or more kinds of adjuvants. For example, the pharmaceutically and/or physiologically acceptable carrier may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, It may contain at least one selected from the group consisting of cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. However, it is not limited thereto, and may be any carrier commonly used in the pharmaceutical field.

The pharmaceutically and / or physiologically acceptable diluent and / or excipient is selected from the group consisting of all fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, etc. commonly used in the proper formulation of pharmaceutical compositions It may be one or more. For example, in the case of solid preparations for oral administration such as tablets, pills, powders, granules, or capsules, the excipient is at least one material for formulating such solid preparations, such as starch, calcium carbonate , sucrose, lactose, and may be one or more selected from the group consisting of gelatin. In addition, lubricants such as magnesium styrate, talc, and the like may also be used. In addition, in the case of formulation of liquid preparations for oral administration such as suspensions, internal solutions, emulsions, syrups, etc., at least one selected from the group consisting of commonly used simple diluents such as water, liquid paraffin, etc. may be used. , Various excipients commonly used, for example, wetting agents, sweeteners, aromatics, and / or preservatives may be included, but are not limited thereto.

For example, the composition may be administered orally, orally or in the form of tablets containing starch or lactose, or in capsules containing suitable excipients, or in the form of elixirs or suspensions containing flavoring or coloring chemicals. It may be administered sublingually. Such liquid formulations may be prepared by suspending agents (e.g. methylcellulose, semi-synthetic glycerides such as Witepsol, or mixtures of Apricot kernel oil and PEG-6 esters or PEG-8 and caprylic/capric acid). It may be formulated with pharmaceutically acceptable excipients such as mixtures of glycerides, but not limited thereto.

The present invention relates to a composition for delivery of a poorly soluble drug, including a poorly soluble drug, beta-lactoglobulin, and tannic acid-metal ion, and a method for preparing the same, wherein the thickness can be precisely adjusted to the nanometer level according to the number of repetitions of coating. It can be applied as a drug delivery system that controls the content of poorly soluble drugs. Since the manufacturing process is performed under mild conditions (room temperature, neutral pH, aqueous solution), it is possible to form nanofilms on the surface of materials sensitive to acidity and temperature. In addition, it has stability against various pH ranges and digestive enzymes in the body.

1a and 1b are photographs showing whey protein isolate used for beta-lactoglobulin extraction and beta-lactoglobulin extracted from the protein, respectively.
2 is a graph showing the results of MALDI-TOF MS analysis performed to measure the molecular weight of beta-lactoglobulin extracted from whey protein isolate.
3 is a photograph showing the prepared resveratrol-β-lactoglobulin complex solution.
4 is a diagram showing a schematic diagram of a nanocoating process of a tannic acid-ferric ion nanofilm and a resveratrol-β-lactoglobulin complex nanofilm.
5 is a graph showing the results of measuring the thickness according to the number of nano-coating layers generated according to the number of nano-coatings.
6 is a graph showing the analysis results of X-ray photoelectron spectroscopy performed for surface analysis (component analysis) of the prepared Fe(III)-TA nanofilm and Fe(III)-TA/R-BLG nanofilm. .
7 is a photograph showing the surface of the prepared Fe(III)-TA nanofilm and the Fe(III)-TA/R-BLG nanofilm produced by dropping water droplets to measure the water contact angle.
8 is a photograph showing an Fe(III)-TA/R-BLG nanocoating layer generated on polystyrene particles.
9 is a graph showing the results of measuring the surface charge of the Fe(III)-TA/R-BLG nanocoating nanofilm.
Figure 10 is a graph showing the results of measuring the thickness change to confirm the stability of the Fe (III) -TA / R-BLG nanocoating nanofilm according to pH.
Figure 11 is a graph showing the results of measuring the thickness change to confirm the stability according to the enzyme type of the Fe (III) -TA / R-BLG nanocoating nanofilm of Figure 10.

Hereinafter, the present invention will be described in detail through the following examples. The following examples are intended to illustrate the present invention, but the present invention is not limited by the following examples.

Example 1. Preparation of resveratrol and beta-lactoglobulin complex

Example 1-1. Beta-lactoglobulin extraction

In order to obtain beta-lactoglobulin (β-lactoglobulin), whey protein was isolated and extracted through a purification step, and the whey protein wasolate was purchased commercially.

Specifically, 40 mL of distilled water was added to 2.5 g of the whey protein isolate and allowed to stand in a refrigerator for 12 hours or more to swell. Thereafter, the whey protein solution was adjusted to pH 4.6 using 1M HCl, filled with distilled water to a total volume of 50 mL, and allowed to stand at room temperature for 1 hour. The settled solution was centrifuged at 8000 g for 30 minutes to remove settled casein, and only the supernatant was obtained.

After adding NaCl corresponding to 7% (w/v) of the total volume of the supernatant obtained above, the mixture was stirred until dissolved. After adjusting the pH to 2 using 1M HCl, the mixture was allowed to stand for 20 minutes. The settled solution was centrifuged at 8000g for 30 minutes to remove α-lactalbumin, immunoglobulins, and bovine serum albumin, and only the supernatant was obtained.

Thereafter, filtration was performed using a syringe filter having a pore size of 0.45 μm in order to remove microorganisms. Salts were removed from the filtered solution through ultrafiltration (10,000 MWCO), and beta-lactoglobulin was concentrated. The concentrated beta-lactoglobulin was lyophilized for 3 days to finally obtain beta-lactoglobulin in powder form. The whey protein isolate is shown in FIG. 1a and the beta-lactoglobulin obtained through the above process is shown in FIG. 1b.

Example 1-2. Molecular weight analysis of beta-lactoglobulin

MALDI-TOF MS analysis was performed to measure the molecular weight of beta-lactoglobulin extracted from whey protein isolate, and the results are shown in FIG. 2 . As a result of MALDI-TOF MS analysis, a unique m/z peak appeared at 18348.6, which was a value corresponding to the molecular weight of beta-lactoglobulin in monomeric form, confirming that the extracted material was beta-lactoglobulin.

Example 1-3. Preparation of resveratrol and beta-lactoglobulin complex

Resveratrol (Tokyo Chemistry Industry (TCI), Japan, purity: > 99.0%) was weighed to a concentration of 4 mM and dissolved in 75% ethanol. In order not to affect the protein structure, resveratrol was diluted to a concentration of 400 μM using 10 mM pH 7.4 phosphate buffer so that the final ethanol concentration was not higher than 7%.

In addition, the beta-lactoglobulin powder obtained in Example 1-1 was prepared using a 10 mM pH 7.4 phosphate buffer solution so that the beta-lactoglobulin solution concentration was 400 μM.

The prepared resveratrol solution and beta-lactoglobulin solution were mixed at a molar concentration of 1:1, respectively, and allowed to stand for 2 hours to prepare a resveratrol-β-lactoglobulin complex (R-BLG). The prepared complex solution is shown in FIG. was

Example 2. Nano coating progress

Example 2-1. Formation of tannic acid-ferric ion nanocoating

To manufacture the tannic acid-iron ion nanocoating, gold plate (manufactured in the process at the Institute of Nano Technology, produced by sequentially depositing 5 nm of titanium (Ti) and 100 nm of gold (Au) on a 4-inch silicon wafer), tannic acid (Sigma-Aldrich, USA) and iron ions (Sigma-Aldrich, USA) were prepared. Each solution was prepared using distilled water so that the concentration of the prepared tannic acid was 40 mg/mL and the concentration of the prepared iron ion (FeCl₃·6H2O) was 10 mg/mL. In addition, the prepared gold plate is washed, the washed gold plate is placed in a Petri dish, and 4900 μL of distilled water is added thereto.

50 μL of iron ion solution was added to the gold plate, and after 10 seconds, 50 μL of tannic acid solution was added and shaken using an orbital shaker for 1 minute.

Thereafter, the gold plate to which the above solution was added was placed in 5mL of 20mM pH 7.4 Tris-HCl buffer solution for 3 minutes, followed by washing with distilled water three times and blowing with nitrogen gas (N ₂ ) through an air blow gun. Distilled water was removed. The coating step was repeated as many times as the desired number of coating layers.

Example 2-2. Resveratrol-β-lactoglobulin complex nano-coating formation

The gold plate subjected to tannic acid-iron ion nanocoating in Example 2-1 was placed in a petri dish, and 5 mL of the resveratrol-beta-lactoglobulin complex solution prepared in Example 1-3 was added to orbital shaker for 15 minutes. shaken using a shaker).

After shaking using the orbital shaker, the washing process with distilled water was repeated three times, and then the remaining distilled water was removed by blowing with nitrogen gas (N ₂ ) through an air blow gun. The coating step was repeated as many times as the desired number of coating layers, and a schematic diagram of the nanocoating process is shown in FIG. 4 .

The tannic acid-iron ion nanofilm formed through the above process is Fe(III)-TA, and the resveratrol-beta-lactoglobulin complex nanofilm is R-BLG (Resveratrol) -beta -lactoglobulin ( B eta- L acto g lobulin)), and the tannic acid-ferric ion/resveratrol-β-lactoglobulin complex nanofilm is referred to as Fe(III)-TA/R-BLG.

Example 3. Tannic acid-ferric ion/resveratrol-beta-lactoglobulin complex (Fe(III)-TA/R-BLG) nanocoating analysis

Example 3-1. Thickness measurement of nanocoating nanofilm

In order to measure the thickness of the nanocoating Fe (III) -TA / R-BLG having 1 to 10 coating layers by repeating the coating step in Example 2 1 to 10 times, a spectroscopic ellipsometer , J. A. Woolam Co., USA) was measured with the Cauchy model, and the results are shown in Table 1 and FIG.

number of coating layers thickness (nm) One 4.04±0.05 2 9.96±0.23 3 14.95±0.39 4 21.87±0.12 5 25.86±0.25 6 31.3±0.26 7 34.51±0.25 8 38.43±0.28 9 41.19±0.25 10 45.1±0.29

As a result, an Fe(III)-TA/R-BLG nanofilm of about 4.31 nm, an Fe(III)-TA nanofilm of each layer of about 2.78 nm, and an R-BLG nanofilm of about 1.75 nm were formed. The thickness of (III)-TA/R-BLG increased linearly. Through this, the Fe(III)-TA/R-BLG nanofilm can be prepared by precisely controlling the thickness at the nanometer level according to the number of coatings.

Example 3-2. Nanocoating nanofilm surface analysis

X-ray photoelectron spectroscopy (XPS) was performed to analyze the surface of the Fe(III)-TA nanofilm and Fe(III)-TA/R-BLG nanofilm prepared in Example 2, and the analysis results are shown in FIG. 6 And shown in Table 2, Table 2 below is a result showing the ratio of surface elements.

C (carbon) 1s O (oxygen) 1s N (nitrogen) 1s Fe (iron) 2p [Fe(III)-TA] ₁ 61.56% 34.04% 2.35% 2.15% [Fe(III)-TA/R-BLG] ₁ 56.47% 27.26% 14.06% 2.20%

As a result, when comparing the nitrogen (N) ratio, the Fe(III)-TA nanofilm was 2.35% and the Fe(III)-TA/R-BLG nanofilm was 14.06%, increasing the nitrogen content by R-BLG. did Through this, it was confirmed that the R-BLG coating was formed by increasing nitrogen present in various amino acids and peptide bonds of R-BLG.

Example 3-2. Measurement of water contact angle of nanocoated nanofilm

The water contact angle of the Fe(III)-TA nanofilm and the Fe(III)-TA/R-BLG nanofilm prepared in Example 2 was measured by SmartDrop. It was obtained by measuring using a protractor manufactured by Plus (FEMTOBIOMED Inc., Republic of Korea), and the analysis results are shown in FIG. 7 and Table 3.

Water contact angle (°) [Fe(III)-TA] ₁ 25.0±0.2 [Fe(III)-TA/R-BLG] ₁ 48.8±0.4

As a result, it was measured as 25±0.2° for the Fe(III)-TA nanofilm and 49±0.4° for the Fe(III)-TA/R-BLG nanofilm. Through this, it was found that the Fe(III)-TA/R-BLG nanofilm has less hydrophilicity than the Fe(III)-TA nanofilm, so even if the R-BLG layer is formed at the nanometer level, the properties of the nanofilm surface change rapidly. Confirmed.

Example 4. Analysis after Fe(III)-TA/R-BLG nanocoating on polystyrene particles

Example 4-1. Fe(III)-TA/R-BLG nanocoating on polystyrene particles

To confirm the application of the Fe(III)-TA/R-BLG nanocoating, polystyrene particles (Sigma-Aldrich, USA) using particles having a diameter of 3 μm were prepared. The polystyrene particles prepared above were added to a 2mL microtube so as to be 1 (v/v)% of the total coating solution. Specifically, 50 μL of a 10 (v/v)% polystyrene particle dispersion and 440 μL of distilled water were added to make 1 (v/v)% of 500 μL of the total coating solution.

5 μL of 40 mg/mL tannic acid and 10 mg/mL iron ion prepared in Example 2-1 were added every 10 seconds, respectively. After shaking for 1 minute using an orbital shaker, 500 μL of 20 mM pH 7 MOPS buffer was added and mixed for 3 minutes. A washing step was performed in which the upper coating residue was removed by separating at 6000 rpm for 3 minutes using a centrifuge, dispersed by adding 1 mL of distilled water, and then centrifuged again. The Fe(III)-TA-coated polystyrene particles were dispersed in 1 mL of the 200 μM R-BLG complex solution prepared in Examples 1-3 and then shaken on an orbital shaker for 15 minutes.

Thereafter, the same centrifugation and washing steps were performed to obtain Fe(III)-TA/R-BLG nanocoated polystyrene particles. The above process was repeated to adjust the Fe(III)-TA/R-BLG nanocoating layer.

Example 4-2. Confirmation of Fe(III)-TA/R-BLG nano-coating on polystyrene particles

In order to check whether the nanocoating performed in Example 4-1 was coated or not, Fe(III)-TA/R-BLG nanocoated particles, which were subjected to the process of Example 4-1 5 times, were prepared and injected. It was confirmed with an electron microscope (Scanning Electron Microscope; SEM), and the results are shown in FIG. 8. Specifically, it was observed before and after nanocoating, and measured at 10,000 magnification and 50,000 magnification, respectively.

As a result, while showing a smooth surface before coating, it was confirmed that the surface was covered with small particles after coating. Through this, it was confirmed that the nanofilm was well formed not only on the plane but also on the particle.

Example 4-3. Surface charge measurement of Fe(III)-TA/R-BLG nanocoated nanofilms

The surface charge of the nanocoated nanofilm of Example 4-1 was measured. Specifically, the nanocoated polystyrene particles of Example 4-1 were dispersed in water to a concentration of 1 mg/mL, put into a measurement cell, and a voltage was applied to the cell to determine the scattering state and surface charge of the particles by Zetasizer Nano ZS (Malvern, UK). ), and the results are shown in FIG. 9 and Table 4.

Bare PS Fe(III)-TA R-BLG Fe(III)-TA R-BLG Zeta potential (mV) -17.5±0.5 -71.9±2.0 -63.8±1.1 -66.4±1.0 -55.4±1.0

As a result, it was confirmed that the nanofilms of each layer were negatively charged and were maintained negatively during the coating formation process, so that the polystyrene particles did not aggregate with each other and were well dispersed in the aqueous solution.

Example 5. Fe (III) -TA / R-BLG nano coating stability confirmation

Example 5-1. Check pH stability

For the pH stability test of the Fe(III)-TA/R-BLG nanocoating nanofilm, 50 mM pH 4.0 sodium acetate buffer, 10 mM pH 7.4 Tris-HCl buffer, and 100 mM pH 9.5 carbonate/bicarbonate /bicarbonate) buffer solution was prepared.

The coating step of Example 2 on a gold substrate (manufactured in the process at the Institute of Advanced Nanotechnology, in detail, produced by sequentially depositing 5 nm of titanium (Ti) and 100 nm of gold (Au) on a 4-inch silicon wafer) A gold substrate coated with the Fe(III)-TA/R-BLG nanofilm having 5 coating layers was prepared by proceeding 5 times. In order to test the stability in various pH ranges, each of the prepared 50mM pH 4.0 sodium acetate buffer solution, 10mM pH 7.4 Tris-HCl buffer solution and 100mM pH 9.5 carbonate/bicarbonate buffer solution After a certain period of time has elapsed since the prepared gold substrate was inserted, the substrate was taken out, washed with distilled water, and dried by blowing with nitrogen gas (N ₂ ) through an air blow gun. Stability was determined by using a spectroscopic ellipsometer (JA Woolam Co., USA) to detect and reflect light in the visible ray region (300-700 nm) on the surface of the nanofilm, and then using the Cauchy equation. Through the method applied to the model, the residual rate was analyzed by measuring the thickness change of the nanofilm according to the reaction time (1 hour, 6 hours, 12 hours, 1 day, 3 days, 7 days), and the results are shown in FIGS. Table 5 shows. The residual rate was calculated by Equation 1 below. The unit of Table 5 below is %.

[Equation 1]

Residual rate (%) = 100 X (Nanofilm thickness after reaction / Nanofilm thickness before reaction)

1 hours 6 hours 12 hours 1 day 3 days 7 days pH 4 95.96±2.82 98.89±0.61 99.80±3.03 99.35±1.68 100.82±2.77 99.78±1.25 pH 7.4 99.60±4.96 99.19±0.77 99.76±0.79 99.52±1.14 97.63±1.14 99.89±2.69 pH 9.5 98.20±5.64 96.55±4.43 100.23±2.39 100.16±1.83 96.30±6.26 95.49±6.55

As a result, it was confirmed that there was little change in thickness over time and that it was stable in various pH ranges.

Example 5-2. Confirmation of stability for digestive enzymes

To test the stability of the Fe(III)-TA/R-BLG nanocoating nanofilm to digestive enzymes, the concentration of pepsin in a NaCl solution corresponding to 0.2% (w/v) of the total volume of 10 mM HCl was dissolved. A 1 mg/mL pH 2 pepsin solution was prepared. A solution having a trypsin concentration of 0.1 mg/mL in 10 mM pH 7.4 phosphate buffer solution is prepared such that the concentration of alpha-amylase is 0.5 mg/mL in 10 mM phosphate buffer solution pH 5.5.

Coating step of Example 2 on a gold substrate (processed and manufactured at the Institute of Advanced Nanotechnology, in detail, manufactured by sequentially depositing 5 nm of titanium (Ti) and 100 nm of gold (Au) on a 4-inch silicon wafer) 5 times to prepare a gold substrate coated with Fe(III)-TA/R-BLG nanofilm having 5 coating layers. For the stability test in various enzymes, after putting the prepared gold substrate in each of the above-prepared pepsin solution, alpha-amylase solution, and trypsin solution, take out the substrate after a certain time, wash with distilled water, and dry with nitrogen gas (N ₂ ) made it Stability was analyzed by measuring the thickness change of the nanofilm according to the reaction time (1 hour, 6 hours, 12 hours, 1 day, 3 days, 7 days) in substantially the same way as in Example 5-1, and the residual rate was analyzed. , The results are shown in FIG. 11 and Table 6. The residual rate was calculated by Equation 1 above. The unit of Table 6 below is %.

1 hours 6 hours 12 hours 1 day 3 days 7 days Pepsin 104.64±2.10 103.51±0.60 99.74±1.06 100.3±0.50 101.76±0.42 98.85±1.29 α-amylase 99.30±0.50 100.15±0.99 94.27±4.06 97.76±1.49 101.89±3.59 106.11±4.73 trypsin 103.19±0.30 97.94±1.21 96.65±1.01 98.96±1.57 102.43±0.51 100.51±0.41

As a result, it was confirmed that there was little change in thickness over time and that it was stable against various digestive enzymes.

Claims (17)

A composition for delivery of a poorly soluble drug, comprising a poorly soluble drug, beta-lactoglobulin and tannic acid-metal ion. The method of claim 1, wherein the poorly soluble drug consists of resveratrol, vitamin D, quercetin, retinol, folic acid, omega 3-fatty acid, docosahexanoic acid (DHA), acetaminophen, ketoprofen, acyclovir and coenzyme Q10. At least one selected from the group, a composition for delivery of poorly soluble drugs. The method of claim 1, wherein the metal ion is iron ion, aluminum ion (Al (III)), zirconium ion (Zr (IV)), ruthenium ion (Ru (III)), rhodium ion (Rh (III)), zinc ion (Zn(II)), vanadium ion (V(III)), chromium ion (Cr(III)), manganese ion (Mn (II)), cobalt ion (Co(II)), nickel ion (Ni( II)), copper ion (Cu(II)), molybdenum ion (Mo(II)), cadmium ion (Cd(II)), europium ion (Eu(III)), gadolinium ion (Gd(III)), ether A composition for delivery of poorly soluble drugs, which is at least one member selected from the group consisting of empty ions (Tb(III)) and cerium ions (Ce(III)). The method of claim 1, wherein the poorly soluble drug, beta-lactoglobulin and tannic acid-metal ion
(a) a nanofilm containing a poorly soluble drug and beta-lactoglobulin complex; and
(b) nanofilm containing tannic acid-metal ions
Which is contained in the form of, a composition for delivery of poorly soluble drugs. [Claim 5] The composition for delivery of poorly soluble drugs according to claim 4, wherein the (a) nanofilm and (b) nanofilm are alternately stacked regardless of order. The composition for delivery of poorly soluble drugs according to claim 4, wherein the (a) nanofilm and (b) nanofilm are each alternately composed of 1 to 100 layers regardless of order. The composition for delivery of poorly soluble drugs according to claim 4, wherein the thickness of the composition is adjusted according to the number of layers of the (a) nanofilm and (b) nanofilm. substrate; and
Including a coating layer coating the surface of the substrate,
In the coating layer, (a) a nanofilm containing a poorly soluble drug and a beta-lactoglobulin complex and (b) a nanofilm containing tannic acid-metal ions are stacked in any order,
A composition for delivery of poorly soluble drugs. The composition for delivery of poorly soluble drugs according to claim 8, wherein two or more coating layers are formed. The composition for delivery of a poorly soluble drug according to any one of claims 1 to 9, which is administered by oral administration or transdermal administration. (1) adding a metal ion solution and a tannic acid solution to a substrate to form a tannic acid-metal ion nanofilm coating layer on the surface of the substrate; and
(2) The substrate obtained in step (1) is added to a solution of the poorly soluble drug and beta-lactoglobulin complex to form a poorly soluble drug and beta-lactoglobulin complex nanofilm coating layer on the tannic acid-metal ion nanofilm coating layer on the surface of the substrate. forming step
A method for producing a composition for delivery of a poorly soluble drug comprising a. The method of claim 11, wherein the poorly soluble drug is resveratrol, vitamin D, quercetin, retinol, folic acid, omega 3-fatty acid, docosahexanoic acid (DHA), acetaminophen, ketoprofen, acyclovir and coenzyme Q10. Method for producing a composition for delivery of at least one selected from the group consisting of poorly soluble drugs. The method of claim 11, wherein the metal ion is iron ion, aluminum ion (Al (III)), zirconium ion (Zr (IV)), ruthenium ion (Ru (III)), rhodium ion (Rh (III)), zinc ion (Zn(II)), vanadium ion (V(III)), chromium ion (Cr(III)), manganese ion (Mn (II)), cobalt ion (Co(II)), nickel ion (Ni( II)), copper ion (Cu(II)), molybdenum ion (Mo(II)), cadmium ion (Cd(II)), europium ion (Eu(III)), gadolinium ion (Gd(III)), ether A method for preparing a composition for delivery of poorly soluble drugs, which is at least one selected from the group consisting of empty ions (Tb(III)) and cerium ions (Ce(III)). The method of claim 11, wherein the substrate is at least one selected from the group consisting of gold plate, glass, polystyrene, bacteria and fungi. The method of claim 11, wherein steps (1) to (3) are repeated 1 to 10 times. The method of claim 15, wherein the thickness of the composition is adjusted according to the number of repetitions of steps (1) to (3). The method for preparing a composition for delivery of a poorly soluble drug according to any one of claims 11 to 16, wherein the composition is for oral administration or transdermal administration.