KR20170124689A - Nanoparticle for improving intestinal permeability and thereof production method - Google Patents

Abstract

The present invention relates to intestinal permeable nanoparticles manufactured by mixing chitosan and dextran sulfate at a weight ratio of 1 : 0.2-1, and to a production method thereof. The chitosan/dextran sulfate nanoparticles manufactured by the present invention shows excellent enterocyte adsorption abilities and collects an object to be collected (target material) which has low solubility, is unstable and has low internal resorption rates such as coenzyme Q10, thereby significantly increasing absorption of the object to be collected to enterocyte and increasing internal resorption rates of the object to be collected.

Description

[0001] Nanoparticle for improving intestinal permeability and their production method [0002]

The present invention relates to intestinal permeable nanocapsules and a process for producing the same, and more particularly to a nanocapsule having enhanced permeability and produced by using chitosan and dextran sulfate, and a process for producing the same.

With increasing interest in functional foods, techniques for efficient use of functional ingredients are drawing attention. Functional substances such as natural extracts, drugs, vitamins, antioxidants, perfumes and colorants are susceptible to damage from external factors such as light, oxygen, moisture, and temperature, and their stability is lowered during processing and distribution, . In addition, when the functional substance is directly ingested, stability is lowered due to low permeability, acidic conditions in the stomach, enzymes, and other influencing factors, thereby limiting the physiological activity that they possess. Accordingly, most of functional materials are used in combination with delivery systems such as particles and capsules in order to protect from external environment and maintain stability.

The carrier provides a protective membrane function that helps maintain activity until ingestion of the functional material in vivo. Synthetic polymers and biopolymers are widely used as raw materials for the particles and capsules serving as carriers. For use in foods, it is suitable to make them naturally friendly and safe. Carbohydrate materials such as alginic acid, chitosan, and starch and protein materials such as albumin, whey protein, and soy protein are mainly used for manufacturing particles and capsules, which are biocompatibility and biodegradability characteristics. (Ko Sang-hoon, Manufacturing of nanoparticles and nanoparticles using food materials, Food Science and Industry, v.41 no.1, 2008, pp.25-32).

On the other hand, among the coating components of nanoparticles, chitosan is a substance processed from chitin contained in crustaceans such as crabs, lobster and shrimp shell, and is a polysaccharide composed of amino sugars, and N-acetyl-D-glucosamine N-acetyl-D-glucosamine) is obtained by deacetylation of chitin polymerized with beta-1-4 linkage. Chitosan is insoluble at neutral or basic pH, but when dissolved in an acidic solution such as acetic acid, the amino group becomes positively charged and becomes able to bind to negative charge substances such as TPP (tripolyphosphate). Since chitosan is not hydrolyzed by in vivo digestive enzymes and is hydrolyzed selectively by a specific enzyme such as lysozyme, chitosan is effective for selective release of drugs (Agnihotri et al., 2004; Jang & Lee , 2008; Sinha & Kumria, 2001). Since chitosan is non-toxic, biocompatible, and biodegradable, it is widely used as a coating material for encapsulating various drugs and physiologically active substances.

However, there have been a number of studies to improve the solubility and stability of functional materials using chitosan, but studies have not yet been conducted to effectively adsorb and adsorb to the mucosa during the absorption process for metabolism in the body.

Korean Registered Patent No. 10-1199251 (Registration Date: 2012.02.02) describes a method for producing chitosan by adding s-allyl cysteine (s-1) to dichloromethane to a solution of chitosan in a concentration of 1.0% (w / v) allylcysteine in a ratio of 10% (w / v) and homogenizing at 15,000 rpm for 2 minutes to form an oil in water (o / w) emulsion; And (ii) mixing the formed oil-in-water emulsion with TPP (tripolyphosphate) as a crosslinking agent at a ratio of 5: 3 (w / v), stirring the mixture at 3,000 rpm for 30 minutes, and removing dichloromethane The contents of the production method of chitosan-TPP nanoparticles are described. Korean Patent No. 10-1066197 (Registration Date: 2011.09.14) discloses a method for producing Coenzyme Q10, comprising the steps of: 1) dissolving coenzyme Q10 in a water-miscible organic solvent to prepare a mixed solution of coenzyme Q10 dissolved therein; 2) a mixed solution of step 1) is added to an aqueous solution containing one or two or more components selected from the group consisting of amino acids and proteins, and the mixture is stirred to prepare a coenzyme having one or more components selected from the group consisting of amino acids and proteins Generating nanoparticles of Q10; And 3) removing the water-miscible organic solvent. The present invention also relates to a method for producing solubilized coenzyme Q10 nanoparticles.

In the present invention, chitosan and dextran sulfate are selected as coating materials to develop nanoparticles having nanoparticles of a target substance (target substance) and enhanced permeability.

To achieve the above object, the present invention provides a first aspect of the present invention, wherein the intestinal permeable nanoparticles are prepared by mixing chitosan and dextran sulfate in a weight ratio of 1: 0.2 to 1: 1.

In the first aspect of the present invention, the nanoparticles preferably contain a substance to be trapped therein.

The target substance is preferably selected from the group consisting of Coenzyme Q10, catechin, resveratrol, anthocyanin, beta-carotene, it may be any one selected from the group consisting of vitamins, minerals, polyphenols, and ginsenosides.

The present invention also relates to a second aspect of the present invention, wherein a solution containing dextran sulfate is added to a solution containing chitosan and stirred, wherein the chitosan and dextran sulfate are mixed at a weight ratio of 1: 0.2 to 1: The present invention provides a method for producing nanoparticles for enhancing nanoparticles.

In the second aspect of the present invention, the solution containing the chitosan may preferably contain a substance to be trapped.

The target substance is preferably selected from the group consisting of Coenzyme Q10, catechin, resveratrol, anthocyanin, beta-carotene, it may be any one selected from the group consisting of vitamins, minerals, polyphenols, and ginsenosides.

Meanwhile, the third aspect of the present invention provides a method for enhancing intestinal permeability of a substance to be trapped, characterized in that the substance to be trapped is loaded on nanoparticles prepared by mixing chitosan and dextran sulfate in a weight ratio of 1: 0.2 to 1 .

In a third aspect of the present invention, the substance to be trapped is preferably Coenzyme Q10 (Coenzyme Q10), catechin, resveratrol, anthocyanin, beta-carotene, isoflavone isoflavones, gamma-orazanol, vitamins, minerals, polyphenols, and ginsenosides. The term " ginsenoside "

 Conventional nanocapsulization studies have been aimed at increasing solubility and enhancing absorption capacity in response to an increase in surface area due to nanoparticle formation. However, the present invention promotes intimal absorption by enhancing intestinal mucosal adhesion and penetration into intestinal cells It is an invention to be desired.

In the present invention, chitosan and dextran sulfate were selected as coating materials, and it is important that the mixing weight ratio of chitosan and dextran sulfate is 1: 0.2-1. In this range, the physical properties of the nanoparticles are the most excellent, and the mucoadhesive ability, the cell permeability, and the cell absorption ability are excellent.

The intestinal permeable nanoparticles prepared by mixing the chitosan of the present invention with dextran sulfate at a weight ratio of 1: 0.2 to 1 have a nanoparticle size of 200 to 300 nm, have excellent collection efficiency, have mucoadhesive ability and cell permeability great.

In the present invention, the polyphenols refer to all natural physiologically active materials containing polyphenols, such as jujube extract and grape extract, and the Jinsen-no-sae is a natural physiologically active material containing ginsenoside, such as red ginseng extract and ginseng extract Means all of the extracts.

In the present invention, Coenzyme Q10, which is low in solubility and low in absorption rate, is limited in utilization, is poor in storage and activity preservation with low heat and light stability, and has low absorption coefficient in the body. Thus, chitosan / Dextran sulfate nanoparticles. As a result, it was confirmed that coenzyme Q10 nano-encapsulated with chitosan / dextran sulfate exhibited increased solubility, increased stability to heat and light, increased permeability into colonic cell Caco-2 cells, Respectively. As can be seen from these results, the chitosan / dextran sulfate nanoparticles of the present invention have a low stability against solubility, heat and light, and nanocapsulate the target substance (target substance) having a low water absorption rate. Can be solved.

The nanoparticles produced by the present invention are functional materials and are applicable to various food formulations. Examples of formulations are cereals, caffeinated beverages, regular beverages, dairy products, chocolate, bread, snacks, confectionery, tea, pizza, jelly, noodles, gums, ice creams, alcoholic beverages, alcoholic drinks, vitamin complexes, But it is not necessarily limited thereto.

The chitosan / dextran sulfate nanoparticles produced by the present invention exert excellent intestinal cell adsorbing ability. By capturing a target (target substance) having low solubility, unstability and low absorption rate in the body, like Coenzyme Q10, The absorption into the intestinal cells can be remarkably improved and the absorption rate of these substances to be captured can be increased.

FIG. 1 shows the physical properties of nanoparticles according to the concentration of dextran sulfate (DS) solution of CS / DS nanoparticles. (A) shows the results of measuring the particle size and polydispersity index (PDI) (b) is the result of measurement of mucoadhesive ability.
Figure 2 is an image of CS / DS nanoparticles photographed using TEM.
FIG. 3 is a graph showing the results of the measurement of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3).
Fig. 4 shows the results of the collection efficiency of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP) and CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2 and CS / DS NP3).
FIG. 5 is a graph showing the results of the determination of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3). ≪ / RTI >
FIG. 6 is a graph showing the results of the determination of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3) was confirmed to be stable against heat.
FIG. 7 is a graph showing the distribution of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3) was confirmed to be stable to light.
Figure 8 is a graph showing the results of the determination of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3) in the colon cell Caco-2.
FIG. 9 shows the results of confirming the Caco-2 cell permeability of CS / DS nanoparticles using a confocal microscope. (a) is an unencapsulated coumarin 6, and (b) is an image of coumarin 6 captured in CS / DS capsules.
FIG. 10 is a graph showing the distribution of CoQ10-loaded CS / TPP nanoparticles (CS / DS NP1, CS / DS NP2, CS / DS) NP3) in the colonic cell Caco-2 cells.
Fig. 11 shows the antioxidative activity of coenzyme Q10 according to nano-encapsulation. Fig. 11 (a) shows DPPH radical scavenging activity and Fig. 11 (b) shows cell antioxidation results using HePg2 cells.

Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

<Preparation of materials>

Coenzyme Q10 (CoQ10) was purchased from Tokyo Chemical Industry (Tokyo, Japan) and chitosan (water soluble, 24 cps, 95% deacetylated) was purchased from Kittolife Co. (Seoul, Korea). Dextran sulfate (MW: 15,000), sodium triphosphate pentabasic (TPP), coumarin 6, C6, and mucin (extracted from porcine stomach, type III) Sigma-Aldrich Co. (St Louis, MO, USA).

In addition, Caco-2 (Colon carcinoma) and HepG2 (cell line human hepatocellular carcinoma) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and Korean Cell Line Bank . DMEM (Dulbecco's Modified Eagle's culture medium), NEAA (non-essential amino acid), FBS (fetal bovine serum), HBSS (Hank's balanced salt solution) and 0.25% trypsin-EDTA were purchased from Gibco Invitrogen Co. (Grand Island, NY, USA).

<Statistics Processing>

All results were expressed as mean ± standard deviation (SD) in triplicate and statistical analysis was performed using ANOVA. Statistical analysis was performed using SPSS Version 2.10 statistical package (SPSS Inc., Chicago, IL, USA).

[ Example  One: Coenzyme Q10  Manufacture of mounted nanoparticles]

In this example, CoQ10-loaded nanoparticles with coenzyme Q10 (CoQ10) were prepared.

The nanoparticles were prepared by using chitosan (CS), dextran sulfate (DS), sodium triphosphate pentabasic (TPP), and chitosan / dextran sulfate (CS / DS) nanoparticles and chitosan / sodium triphosphate penta-basic (CS / TPP) nanoparticles, respectively (Cho, Y., Shi, R., & Borgens, RB Chitosan nanoparticle-based neuronal membrane sealing and neuroprotection following acrolein -induced cell injury. Journal of Biological Engineering 4: 2 (2010)).

The concrete method was as follows. Chitosan (CS) was dissolved in distilled water to prepare a chitosan solution at a concentration of 0.5 mg / mL. CoQ10 was dissolved in ethanol to prepare a CoQ10 solution at a concentration of 2.0 mg / mL. Then, 0.75 mL of distilled water, 0.75 mL of CoQ10 solution and 3 mL of CS solution were mixed to prepare a CS / CoQ10 mixture.

The CS / DS nanoparticles were prepared by adding 3 mL of dextran sulfate solution (0.1, 0.3, 0.5 mg / mL) dissolved in distilled water to 4.5 mL of the CS / CoQ10 mixture and stirring for 10 minutes (1,000 rpm) Rod CS / DS nanoparticles were prepared.

The CS / TPP nanoparticles were prepared by adding 3 mL of TPP solution (0.05 mg / mL) dissolved in distilled water to 4.5 mL of the above CS / CoQ10 mixture and stirring and mixing at 1,000 rpm to prepare CoQ10-rod CS / TPP nanoparticles .

[ Experimental Example  1: chitosan / dextran Sulfate (CS / DS ) Confirmation of coating material concentration of nanoparticles]

In this experiment, DS solution concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.5, and 0.5) were measured when the concentration of CS solution was 0.5 mg / Nanoparticle size, polydispersity index (PDI), mucoadhesive ability (mucin adsorption capacity) and morphology of nanoparticles were determined according to the concentration of the nanoparticles.

The preparation of nanoparticles was the same as in Example 1 above. Specifically, 3 mL of dextran sulfate solution (0.1, 0.3, 0.5 mg / mL) was added to 4.5 mL of a mixture of 3 mL of 0.5 mg / mL CS solution and 1.5 mL of distilled water, and the mixture was stirred at 1,000 rpm for 10 minutes. To prepare CS / DS nanoparticles.

The particle size and polydispersity index (PDI) of the nanoparticles were measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK). Measurements were performed at 25 ± 1 ° C with 'multiple narrow modes'.

Measurement of mucosal adsorption (mucin adsorption) was performed by modifying the previously reported method (Shao, Y., Yang, L., & Han, HK TPGS-chitosome as an effective oral delivery system for improving bioavailability of coenzyme Q10, European Journal of Pharmaceutics and Biopharmaceutics 89: 339-346 (2015)).

Specifically, 0.6 mL of mucin solution (0.5 mg / mL) was mixed with 0.6 mL of nanoparticle suspension (nanoparticle dispersion) and cultured in a shaking water bath at 37 DEG C for 1 hour. Then, after centrifugation at 10,000 rpm for 5 minutes, the supernatant was separated and the amount of free mucin was measured through a 'Bradford protein assay'.

The supernatant was incubated with Bradford reagent for 5 minutes and absorbance was measured at 595 nm using a spectrophotometer (Biomate 3S, Thermo scientific, Waltham, Massachusetts, USA). The amount of mucin-absorbing nanoparticles was calculated as the difference between the amount of mucin added and the amount of mucin remaining in the supernatant, and the mucin concentration was calculated from the standard curve (Pengpong, T., Sangvanich, P., Sirilertmukul, K , & Muangsin, N. Design, Synthesis and in vitro evaluation of mucoadhesive p-coumarate-thiolated-chitosan as a hydrophobic drug carriers. European Journal of Pharmaceutics and Biopharmaceutics 86: 487-497 (2014)).

As a result of the particle size and PDI measurement of the nanoparticles, it was confirmed that the particle size and the PDI of the nanoparticles were both increased as the concentration of the DS solution increased. Particularly, when the DS solution concentration was 0.7 mg / mL or more, the particle size was increased to 500 nm or more. Also, it was confirmed that the PDI was 0.5 or more, and the particle distribution was not uniform (Fig. 1 (a)).

As a result of measurement of the mucoadhesion ability of the nanoparticles, the adsorbed mucin content increased in a concentration-dependent manner at a DS concentration of 0.5 mg / mL, but it was found that when the concentration exceeded 0.5 mg / mL, the mucosal adhesion was no longer increased 1 (b)).

On the other hand, the morphology of the nanoparticles was determined by TEM (transmission electron microscopy, JEM 2100F, JEOL, Tokyo, Japan) using a 0.5 mg / mL CS solution and a 0.3 mg / mL DS solution (Muthu, MS, Kulkarni, SA, Raju, A., & Feng, S.-S. Theranostic liposomes of TPGS coatings for targeted co- delivery of docetaxel and quantum dots. Biomaterials 33: 3494-3501 ). Samples to be analyzed by TEM were placed on a carbon-coated copper grid coated with 200-mesh carbon and dyed for 30 minutes using 2% phosphotungstic acid solution (Sigma-Aldrich Co.) . After dyeing, the samples were air-dried at 37 [deg.] C before being placed on the microscope.

As a result of the observation, it was confirmed that the particles have a spherical shape, a low PDI, and an average size of 200 to 300 nm (FIG. 2).

From the above results, it was confirmed that the concentration of dextran sulfate (DS) solution for preparing nanoparticles was suitably from 0.1 to 0.5 mg / mL.

FIG. 1 shows the physical properties of nanoparticles according to the concentration of dextran sulfate (DS) solution of CS / DS nanoparticles. (A) shows the results of measuring the particle size and polydispersity index (PDI) (b) is the result of measurement of mucoadhesive ability. Figure 2 is an image of CS / DS nanoparticles photographed using TEM.

[ Experimental Example  2: Coenzyme Q10  Mounted chitosan / dextran Sulfate (CS / DS ) Identification of physical properties of nanoparticles]

In order to examine the physical properties of the CoQ10-loaded CS / DS nanoparticles prepared according to the concentration of the dextran sulfate (DS) solution selected in Experimental Example 1, the particle size, the polydispersity index Polydispersity index, Derived count rate, and morphology of nanoparticles.

The particle size, polydispersity index, and dispersion ratio of the prepared CoQ10-rod nanoparticles were as shown in Table 1 below.

The particle size, polydispersity index, and dispersion were measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK).

Polymer Nanoparticle Chitosan solution
(mg / mL) Dextran sulfate solution
(mg / mL) Sodium triphosphate solution
(mg / mL) Particle Size (nm) Polydispersity index Dispersion ratio
(kcps) CS / DS 0.5 0.1 - 402.4 ± 38.0 ^ab 0.2 ± 0.0 ^b 169, 224 ± 31,495 ^b 0.3 329.1 ± 22.9 ^b 0.2 ± 0.0 ^b 229,567 ± 50,154 ^a 0.5 447.1 ± 68.3 ^a 0.3 ± 0.2 ^a 219,438 ± 22,513 ^ab CS / TPP - 0.05 358.4 ± 50.7 ^b 0.2 ± 0.0 ^b 188,267 ± 16,319 ^{ab ab} Means with different letters are significantly different at p <0.05.

As a result of the particle size measurement, the particle size of CoQ10-rod CS / DS nanoparticles and CoQ10-rod-CS / TPP nanoparticles was 329 to 447 nm, And it was not related to the concentration.

As a result of measuring the polydispersion index (PDI), the PDI of the CoQ10-rod nanoparticles was 0.2 to 0.3, and it was judged that the nanoparticles of the present invention had the homogeneity of the particle population (Gaumet, M., Vargas, A. Gurny, R., & Delie, F. Nanoparticles for drug delivery: The need for precision in reporting particle size parameters. European Journal of Pharmaceutics and Biopharmaceutics 69: 1-9 (2008)).

Dispersion ratio (DCR) represents the intensity of light scattering in the presence of particles, which can be affected by the number and size of particles. As the number or size of particles increases, DCR increases (Piest, M. & Engbersen, JF Role of boronic acid moieties in poly (amido amine) s for gene delivery. Journal of Controlled Release 155: 331-340 (2011)). Therefore, it was confirmed that when the DS solution concentration was 0.3 mg / mL, small-sized nanoparticles were produced.

[ Experimental Example  3: Coenzyme Q10  Mounted chitosan / dextran Sulfate (CS / DS ) Confirmation of mucoadhesion properties of nanoparticles]

In this Experimental Example, the mucoadhesion characteristics of the CoQ10-loaded CS / DS nanoparticles prepared according to the concentration of the dextran sulfate (DS) solution selected through Experimental Example 1 were examined.

CS / DS NP1 '(0.5 mg / mL of CS solution, 0.1 mg / mL of DS solution) prepared according to the concentration of dextran sulfate (DS) solution loaded with coenzyme Q10, CS / DS NP3 '(prepared from CS solution 0.5 mg / mL, DS solution 0.3 mg / mL) and CS / DS NP3 (CS solution 0.5 mg / mL, DS solution 0.5 mg / mL) Uncoated coenzyme Q10 was referred to as 'Free CoQ10' and CoQ10-loaded CS / TPP nanoparticles as 'CS / TPP NP' and then used as a sample.

The experimental method was the same as that described in Experimental Example 1 above.

As a result of the experiment, it was confirmed that the amount of mucin adsorbed on the nanoparticles increases with increasing concentration of the DS solution. In addition, the amount of mucin attached to CoQ10-loaded CS / DS nanoparticles prepared with DS solution 0.3 mg / mL and 0.5 mg / mL was about 0.24 mg / mL, about 0.51 mg / mL, It was confirmed that mucins were adsorbed about 24 times and 50 times more than the nanoparticles (about 0.01 mg / mL) (FIG. 3).

FIG. 3 is a graph showing the results of the measurement of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3).

From the above results, it was confirmed that the chitosan / dextran sulfate nanoparticles of the present invention had excellent mucoadhesive ability.

[ Experimental Example  4: Coenzyme Q10  Mounted chitosan / Dextran Sulfate (CS / DS ) Of nanoparticles Collection efficiency  Confirm]

In this Experimental Example, the entrapment efficiency (EE) of CoQ10-loaded CS / DS nanoparticles prepared according to the concentration of the dextran sulfate (DS) solution selected in Experimental Example 1 was examined.

CS / DS NP1 '(0.5 mg / mL of CS solution, 0.1 mg / mL of DS solution) prepared according to the concentration of dextran sulfate (DS) solution loaded with coenzyme Q10, CS / DS NP3 '(prepared from CS solution 0.5 mg / mL, DS solution 0.3 mg / mL) and CS / DS NP3 (CS solution 0.5 mg / mL, DS solution 0.5 mg / mL) CoQ10-loaded CS / TPP nanoparticles were referred to as 'CS / TPP NP' and were used as samples.

The collection efficiency of CoQ10-loaded CS / DS nanoparticles, CoQ10-loaded CS / TPP nanoparticles was measured using HPLC (validated high performance liquid chromatography) (Ankola, DD, Viswanad, B., Bhardwaj, V., Ramarao, P., & Kumar, MNVR Development of potent oral nanoparticulate formulation of coenzyme Q10 for treatment of hypertension: Can the simple nutritional supplements be used as first line therapeutic agents for prophylaxis / therapy? European Journal of Pharmaceutics and Biopharmaceutics 67: 361-369 2007).

 Specifically, CoQ10-rod nanoparticles were determined by analyzing the amount of free CoQ10 in the supernatant obtained by ultracentrifugation (Optima TL ultracentrifuge, Beckman, Fullerton, Calif., USA) at 30,000 × g for 30 minutes. The collection efficiency was calculated using the following equation (1).

To quantify CoQ10, reverse phase HPLC (HPLC, Waters 486 Tunable Absorbance Detector, Waters Corporation, Milford, MA, USA) was used. The HPLC system consisted of 'Waters 515 pump' and 'Waters 486 tunable absorbance detector'. 'A CAPCELL PAK C18 column (250 × 4.6 mm, 5 μm)' was used and the mobile phase consisted of ethanol and methanol %) was UV detected at 275 nm at a flow rate of 1.0 mL / min (Sakchareonkeat, P., Huang, TC, Suwannaporn, P., Hsuan Chiang, Y., Liang Hsu, Han Hong, Y. Encapsulation efficiency of coenzyme Q10- liposomes in alginate. Nutrition & Food Science 43: 150-160 (2013)).

As a result, the collection efficiency of CS / DS NP1 was about 65% and the collection efficiency of CS / DS NP2, 3 was about 95%. As the concentration of DS solution increased, the collection efficiency was increased. On the other hand, the CS / TPP NP showed a collection efficiency of about 59%, indicating a lower collection efficiency than the CS / DS NP2, 3 (FIG. 4).

Fig. 4 shows the results of the collection efficiency of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP) and CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2 and CS / DS NP3).

From the above results, it was found that the chitosan / dextran sulfate nanoparticles of the present invention are excellent in the collection efficiency, and thus are suitable for use as a carrier for carrying a functional substance containing coenzyme Q10.

[ Experimental Example  5: Coenzyme Q10  Mounted chitosan / Dextran Sulfate (CS / DS ) Determination of solubility of nanoparticles]

In this Example, the solubility of CoQ10-loaded CS / DS nanoparticles prepared according to the concentration of the dextran sulfate (DS) solution selected through Experimental Example 1 was examined.

CS / DS NP1 '(0.5 mg / mL of CS solution, 0.1 mg / mL of DS solution) prepared according to the concentration of dextran sulfate (DS) solution loaded with coenzyme Q10, CS / DS NP3 '(prepared from CS solution 0.5 mg / mL, DS solution 0.3 mg / mL) and CS / DS NP3 (CS solution 0.5 mg / mL, DS solution 0.5 mg / mL) Uncoated coenzyme Q10 was referred to as 'Free CoQ10' and CoQ10-loaded CS / TPP nanoparticles as 'CS / TPP NP' and then used as a sample.

The solubilities of Free CoQ10 and CoQ10-rod nanoparticles are described in Sun et al. (Wang, W., Wang, W., & Deng, Y. Effect of particle size on the particle size on solubility, dissolution rate, and oral bioavailability: Evaluation using coenzyme Q10 as naked nanocrystals. International Journal of Nanomedicine 7: 5733-5744 (2012)).

Specific experimental methods were as follows. To quantify dissolved CoQ10, extraction of free CoQ10 and CoQ10-rod nanoparticle suspensions was carried out. First, 200 μL of methanol and 600 μL of hexane were added to 100 μL of the sample, followed by vortexing for 3 minutes. Then, the mixture was centrifuged at 10,000 rpm for 10 minutes (Combi-408, Hanil Science Inc., Incheon, Korea) to remove 500 μL of supernatant. This extraction procedure was repeated twice and 600 μL of supernatant was removed. Total supernatant was collected and dried with 'speed vacuum concentrator (Ecospin 3180C, BioTron, Daejeon, Korea). The residue was reconstituted by pouring 300 μL of ethanol (10%) and the suspension was filtered through a 0.45 μm pore size polytetrafluoroethylene membrane filter (Xiboshil, Tianjin Fuji Tech Co., Tianjin, China) I filtered it. The CoQ10 present in the filtered solution was determined by HPLC.

As a result, it was confirmed that the solubility was increased according to the concentration of DS solution. Solubility of CS / DS NP was 140 ~ 164 ㎍ / mL, which was higher than solubility (about 128 ㎍ / mL) of Free CoQ10 and solubility (about 132 ㎍ / mL) of CS / TPP NP ) .

FIG. 5 is a graph showing the results of the determination of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3). &Lt; / RTI &gt;

From the above results, it was found that the chitosan / dextran sulfate nanoparticles of the present invention increase the solubility of the functional material by encapsulating the functional material containing coenzyme Q10.

[ Experimental Example  6: Coenzyme Q10  Mounted chitosan / dextran Sulfate (CS / DS ) Confirmation of stability of nanoparticles against heat and light]

In this experimental example, the effect of CS / DS nanocapsulation on the thermal and light stability of coenzyme Q10 was examined.

CS / DS NP1 '(0.5 mg / mL of CS solution, 0.1 mg / mL of DS solution) prepared according to the concentration of dextran sulfate (DS) solution loaded with coenzyme Q10, CS / DS NP3 '(prepared from CS solution 0.5 mg / mL, DS solution 0.3 mg / mL) and CS / DS NP3 (CS solution 0.5 mg / mL, DS solution 0.5 mg / mL) Uncoated coenzyme Q10 was referred to as 'Free CoQ10' and CoQ10-loaded CS / TPP nanoparticles as 'CS / TPP NP' and then used as a sample.

(1) Confirmation of thermal stability

CoQ10-rod CS / DS nanoparticles, CoQ10-rod CS / TPP nanoparticles, and free CoQ10 were prepared in a water bath at 60 DEG C to evaluate heat stability. The amount of coenzyme Q10 in each sample was evaluated by HPLC for 4 days at intervals of 24 hours after the same extraction procedure as in Experimental Example 5 above.

As a result, free CoQ10 showed high degradation (about 70%) after 3 days, and the remaining amount of coenzyme Q10 was about 38%, while that of nano-encapsulated coenzyme Q10 remained in the range of 67 to 81% And the thermal stability was remarkably improved (FIG. 6).

FIG. 6 is a graph showing the results of the determination of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3) was confirmed to be stable against heat.

From the above results, it was confirmed that the chitosan / dextran sulfate nanoparticles of the present invention can be used as an efficient carrier for stabilizing heat-labile functional materials including coenzyme Q10 from heat.

(2) Confirmation of stability against light

To confirm the stability to light, CoQ10-loaded CS / DS nanoparticles and CoQ10-loaded CS / TPP nanoparticles, free CoQ10, were incubated for 8 hours at 25 ° C using a UV lamp (Vilber Lourmat, Torcy, France) And light of 254 nm was irradiated. The distance between the sample and the UV light source was 15 cm, and the amount of coenzyme Q10 was measured using HPLC at 1 hour intervals after the same extraction process as described in Experimental Example 5 (Fir, MM, Milivojevic, L., Smidovnik, A., Milivojevic, L., Zmitek, J., Fir, MM, Smirnov, A., Prosek, M., & Smidovnik, A. Property studies of coenzyme Q10-cyclodextrins complexes. Acta Chimica Slovenica 56: 885-891 (2009a) & Prosek, M. Studies of CoQ10 and cyclodextrin complexes: Solubility, thermo-and photo-stability. Journal of Inclusion Phenomena and Macrocyclic Chemistry 64: 225-232 (2009b)).

As a result, it was confirmed that the remaining amount of coenzyme Q10 after nano-encapsulation was about 47% to 50% after 8 hours of light irradiation, while the remaining amount of coenzyme Q10 without nano-encapsulation was about 28% (FIG.

FIG. 7 is a graph showing the distribution of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3) was confirmed to be stable to light.

From the above results, it was confirmed that the chitosan / dextran sulfate of the present invention can be used as an efficient carrier for stabilizing a light-unstable functional substance from light, including coenzyme Q10.

[ Experimental Example  7: Coenzyme Q10  Mounted chitosan / dextran Sulfate (CS / DS ) Nanoparticles of Caco-2 cells Monolayer  group Permeability  Confirm]

In this Experimental Example, the permeability of the CoQ10-rod nanoparticles prepared according to the concentration of the dextran sulfate (DS) solution selected in Experimental Example 1 to the colonic Caco-2 monolayer tissue (intestinal cell layer constituting the surface of the intestinal membrane) Respectively.

CS / DS NP1 '(0.5 mg / mL of CS solution, 0.1 mg / mL of DS solution) prepared according to the concentration of dextran sulfate (DS) solution loaded with coenzyme Q10, CS / DS NP3 '(prepared from CS solution 0.5 mg / mL, DS solution 0.3 mg / mL) and CS / DS NP3 (CS solution 0.5 mg / mL, DS solution 0.5 mg / mL) Uncoated coenzyme Q10 was referred to as 'Free CoQ10' and CoQ10-loaded CS / TPP nanoparticles as 'CS / TPP NP' and then used as a sample.

The monolayer tissue was transferred to a 12-well transwell and the medium was replaced every two days before the transport experiment. TEER (Transepithelial electrical resistance) was measured to determine the monolayer cell applicable for the transmission experiment. When the cell monolayer of the cell was 400 Ω cm ^{2 or} more, it was considered to be applicable to the permeation experiment.

The HBSS-HEPES buffer was used for cell culture for cell permeation experiments. After removing the medium from the apical side and basolateral side of the monolayer, the cells were suspended in HBSS-HEPES buffer (12 1.5 mL of basolateral and 0.5 mL of apical) for 10 min at 37 ° C. After the cultured medium was removed, a medium containing coenzyme Q10 was added to the outside.

In the efflux experiment, the medium containing the nanoparticle dispersion (nanoparticle) was placed on the inside and a monolayer was incubated for 120 minutes. For permeation measurement, a small aliquot of the culture was released to the apical side of the cell at a designated time and the sample was collected again for immediate analysis. Papp (apparent permeability coefficient) was calculated using the following equation (2).

dQ / dt: linear appearance rate of receiver solution mass

A: area of cell monolayer (1.13 cm ² )

Co: initial substrate concentration

As a result, it was confirmed that the coenzyme Q10 captured by the nanoparticles decreased by about 50% from the basolateral to the apical side compared to the unencapsulated coenzyme Q10 (FIG. 8)

Figure 8 is a graph showing the results of the determination of CoQ10-loaded CS / TPP nanoparticles (CS / TPP NP), CoQ10-loaded CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, NP3) in the colon cell Caco-2.

From the above results, it was confirmed that the chitosan / dextran sulfate nanoparticles of the present invention inhibit the excretion of functional substances such as Coenzyme Q10 in vitro.

[ Experimental Example  9: chitosan / dextran Sulfate  Of nanoparticles Colonic cell  of mine Absorption capacity  Confirm]

In this Experimental Example, CS / DS nanoparticles prepared according to the concentration of the dextran sulfate (DS) solution selected in Experimental Example 1 were tested for their ability to absorb in the colon.

(1) Confocal laser scanning microscopy

To confirm the ability of CS / DS nanoparticles to absorb Caco-2 cells by confirming the ability of coumarin 6 to undergo nanocapsulization using CLSM (Confocal laser scanning microscopy).

Caco-2 cells (4 × 10 ⁵ cells / well) were injected into a glass-bottom dish and cultured at 37 ° C. until 50-70% of the cells were attached. Cells were then seeded with free Coumarin 6 and Coumarin 6-loaded CS / DS nanoparticles (CS / DS nanoparticles prepared with 0.5 mg / mL CS solution, 0.5 mg / mL DS solution) (Equivalent to 1 [mu] g / mL) for 2 hours. After incubation, the cell culture medium was aspirated and washed with cold PBS (phosphate buffered saline). The cells were then fixed with 70% ethanol in PBS (pH 7.4) for 15 minutes at room temperature. Then, the cells were washed twice with PBS, and the nuclei were stained with PI (propidium iodide) for 30 minutes. The cells were then carefully removed from the cover glasses and attached to slide glasses.

The cellular uptake of the nanoparticles was measured using confocal laser scanning microscopy (TCS SP5, Leica Co., Ltd., Wetzlar, Germany) (emission at 505 nm, excitation at 420 nm). The fluorescence intensity of the sample was analyzed by confocal images using Leica confocal software (Leica Microsystems, Heidelberg, Germany) and the ImageJ software system (NIH, Bethesda, MD, USA)

In the fluorescence (FITC) channel, the absorbance of Coumarin 6-rod CS / DS nanoparticles was shown to be green. In the PI channel, the nucleus of PI-stained intestinal cells was red have. When FITC and PI were combined, it was found that the CS / DS nanoparticles were introduced into the Caco-2 cells because green fluorescence surrounds the cytoplasm of nuclear periplasm (FIG. 9).

FIG. 9 shows the results of confirming Caco-2 cell permeability of CS / DS nanoparticles measured using a confocal microscope. (a) is an unencapsulated coumarin 6, and (b) is an image of coumarin 6 captured in CS / DS capsules.

On the other hand, the fluorescence intensity of coumarin 6 and encapsulated coumarin 6 was measured by ImageJ, and the average pixel intensities were 23.9 and 27.8.

From the above results, it was confirmed that the chitosan / dextran sulfate nanoparticle of the present invention was excellently absorbed into intestinal cells.

(2) Quantitative Experiment

(Russell-Jones, G., Arthur, L., & Walker, H.) Vitamin B12-mediated transport of nanoparticles (Russell-Jones et al., 1999; Saito et al., 2005) Cobata, Y., Okumura, H., Kobayashi, M., Hirano (1999), Saito, Y., Itagaki, S., Otsuka, Y., Kobayashi, Y. , T., & Iseki, K. Substrate specificity of the nateglinide / H + cotransport system for phenolic acids. Journal of Agricultural and Food Chemistry 53: 6100-6104 (2005)).

CS / DS NP1 '(0.5 mg / mL of CS solution, 0.1 mg / mL of DS solution) prepared according to the concentration of dextran sulfate (DS) solution loaded with coenzyme Q10, CS / DS NP3 '(prepared from CS solution 0.5 mg / mL, DS solution 0.3 mg / mL) and CS / DS NP3 (CS solution 0.5 mg / mL, DS solution 0.5 mg / mL) Uncoated coenzyme Q10 was referred to as 'Free CoQ10' and CoQ10-loaded CS / TPP nanoparticles as 'CS / TPP NP' and then used as a sample.

Specific experimental methods were as follows. The absorption of coenzyme Q10 measured the growth of monolayer cultures in a 24-well transwell. Cell monolayers were replaced every two days until transport experiments. At this time, it was judged that TEER was applicable to the experiment when the tissue was 350 Ω cm ^{2 or} more.

After removing the medium from apical side and basal chamber, 0.6 mL medium was placed on the basolateral side and preliminarily cultured at 37 ° C for 10 minutes. After removing the medium, 0.2 mL of coenzyme Q10 nanoparticles were injected into the cell membrane and cultured at 37 ° C for 120 minutes. The cell monolayer was then washed with 1 mL of the rapidly cooled culture medium. For extraction, the cells were dispersed in 1 mL of methanol and subjected to sonication to prevent cell integrity. The extraction solution was centrifuged at 10,000 rpm for 5 minutes to measure the absorption rate of CoQ10.

As a result, it was confirmed that CS / DS NP3 increased more than 3-fold in Caco-2 uptake ability than free coenzyme Q10. CS / TPP nanoparticles showed similar Caco-2 cell uptake as free coenzyme Q10 (FIG. 10).

FIG. 10 is a graph showing the results of the measurement of CoQ10-load CS / TPP nanocapsules (CS / TPP NP), CoQ10-load CS / DS nanoparticles (CS / DS NP1, CS / DS NP2, CS / DS) NP3) in the colonic cell Caco-2 cells.

[ Experimental Example  10: chitosan / dextran Sulfate  Identification of Antioxidant Activity of Nanoparticles]

In this experiment, the antioxidant activity of Coenzyme Q10 was examined by chitosan / dextran sulfate (CS / DS) nanocapsulation.

CS / DS NP1 '(0.5 mg / mL of CS solution, 0.1 mg / mL of DS solution) prepared according to the concentration of dextran sulfate (DS) solution loaded with coenzyme Q10, CS / DS NP3 '(prepared from CS solution 0.5 mg / mL, DS solution 0.3 mg / mL) and CS / DS NP3 (CS solution 0.5 mg / mL, DS solution 0.5 mg / mL) Uncoated coenzyme Q10 was referred to as 'Free CoQ10' and CoQ10-loaded CS / TPP nanoparticles as 'CS / TPP NP' and then used as a sample.

(One) DPPH  Radical Scatters Assay ( Diphenyl -One- picrylhydrazyl  ( DPPH ) radical scavenging assay)

The DPPH radical scavenging activity was tested by modifying the Brand-Wiliams method (Brand-Williams, W., Cuvelier, ME, & Berset, C. Use of a free radical method to evaluate antioxidant activity. and Technology 28: 25-30 (1995)).

Specific experimental methods are as follows. The DPPH solution was dissolved in anhydrous ethanol (100 μL, 0.5 mM) and reacted with 100 μL of the sample. The reactants were shaded, reacted at 25 ° C for 30 minutes, and the absorbance was measured at 517 nm using Synergy HT Multi-microplate reader (BioTek Instruments, Winooski, VT, USA). The radical scavenging effect was calculated using the following equation (3).

Ac: absorbance of control (control)

As: the absorbance of the sample

Ab: absorbance of blank

As a result of the experiment, it was confirmed that there is no significant difference between the samples in the DPPH experiment (Fig. 11 (a)). These results indicate that the nanocapsule does not affect the antioxidant activity measurement of coenzyme Q10.

11 (a) shows the result of confirming the antioxidant activity of Coenzyme Q10 according to nano-encapsulation using the DPPH radical scavenging ability.

(2) Cell Antioxidant Activity CAA )

HepG2 cells were seeded in a 96-well plate at a density of 6 × 10 ⁴ , cultured for 24 hours, and then the culture medium was removed and washed with PBS. To the triplicate wells were added 25 μM DCFH-DA (dichloro-dihydro-fluorescein diacetate) dissolved in 100 μL of the coenzyme Q10 solution or coenzyme Q10 nanoparticles for 1 hour. After incubation for 1 hour, the wells were washed with 100 μL PBS and 100 μL of HBSS was treated with 600 μM ABAP (2,2'-Azobis dihydrochloride). Using a Synergy HT Multi-microplate reader (BioTek Instruments, Winooski, VT, USA), 96-well microplates were excited at 538 nm for 5 minutes at 485 nm Respectively. Each plate contained a triplicate control (containing cells treated with DCFH-DA and HBSS with ABAP) and a blank (containing cells treated with DCFH-DA and HBSS without ABAP) . The CAA values were calculated using the following equation (4): Hu, B., Ting, Y., Zeng, X., & Huang, Q. Bioactive peptides / chitosan nanoparticles enhanced cellular antioxidant activity of (-) - epigallocatechin- -gallate. Journal of Agricultural and Food Chemistry 61: 875-881 (2013)).

&Quot; (4) &quot;

CAA value = 100 - (?? S A) / (?? C A) × 100

∫ ▒ ▒ SA: Integral area of fluorescence versus time curve of sample

∫▒CA: Integral area of control (control)

As a result, it was confirmed that the CAA value of CoQ10-loaded CS / DS nanoparticles was higher than that of CoQ10-loaded CS / TPP nanoparticles and free CoQ10 (FIG. 11 (b)).

11 (b) shows the result of confirming the cell antioxidant activity of Coenzyme Q10 according to nano-encapsulation using HePg2 cells.

From the above results, it was found that the chitosan / dextran sulfate nanoparticle of the present invention is a drug delivery system capable of effectively delivering a functional substance such as Coenzyme Q10.

Claims (8)

Chitosan and dextran sulfate in a weight ratio of 1: 0.2 to 1: 1.
The method according to claim 1,
The nano-
Permeable nanoparticle characterized by containing a substance to be trapped therein.
3. The method of claim 2,
The above-
Coenzyme Q10, catechin, resveratrol, anthocyanin, beta-carotene, isoflavones, gamma-orazanol, vitamins, Wherein the nanoparticles are any one selected from the group consisting of minerals, polyphenols, and ginsenosides.
A solution containing dextran sulfate is added to a solution containing chitosan and stirred,
Wherein the chitosan and dextran sulfate are mixed at a weight ratio of 1: 0.2 to 1: 1.
5. The method of claim 4,
Wherein the solution containing the chitosan contains a substance to be trapped.
6. The method of claim 5,
The above-
Coenzyme Q10, catechin, resveratrol, anthocyanin, beta-carotene, isoflavones, gamma-orazanol, vitamins, Wherein the nanoparticles are any one selected from the group consisting of minerals, polyphenols, and ginsenosides.
Wherein the nanoparticles are prepared by mixing chitosan and dextran sulfate at a weight ratio of 1: 0.2 to 1: 1.
8. The method of claim 7,
The above-
Coenzyme Q10, catechin, resveratrol, anthocyanin, beta-carotene, isoflavones, gamma-orazanol, vitamins, Wherein the target substance is any one selected from the group consisting of minerals, polyphenols, and ginsenosides.

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