KR101563173B1 - Monoolein cubic phase with pH sensible polymer and glucose oxidase and method for production thereof - Google Patents

Abstract

The present invention relates to a pH reactive monoolein cubic phase composition which has an excellent effect of controlling release of a target substance collected inside a cubic phase depending on glucose concentration.

Description

[0001] The present invention relates to a monoolein cubic phase in which a pH sensitive polymer and a glucose oxidase are immobilized, and a method for producing the same,

The present invention relates to a monoolein cubic phase in which a pH sensitive polymer and a glucose oxidase are immobilized, more specifically, a pH sensitive polymer is immobilized on a cubic phase water channel, and a glucose oxidase, GOD) relates to a monoolein cubic phase which is immobilized on the surface of cubic to change the pH according to the glucose concentration to control the release of the target substance.

The cubic phase has attracted much attention as a drug delivery vehicle due to its various advantages. The most commonly used material when making cubic phases is monoolein. Monoolein does not cause an immune response to the human body, can be eaten, and is often used as an emulsifier in food and pharmaceutical fields (A. Ganem-Quintanar, D. Quintanar-Guerrero, P. buri, Moolein: A review of the Pharmaceutical Applications, Drug Development and Industrial Pharmacy, 26 (2000) 809-820).

On the other hand, monoolefins spontaneously form cubic phases by entropy-driven processes upon contact with excess water (LW Hamley, Self-assembly of amphiphilic peptides, Soft Matter . 7 (2011) 4122-4138 ). The cubic phase thus formed passes through the water channels 10 to 20 nm in diameter crossing each other and the water channels are separated by the monoolein bilayer (H. Zhang, JC Kim, Preparation and photothermal induced release from cubic phase gold nanoparticle, Colloid and Surfaces A: Physiochemical and Engineering Aspects, 465 (2015) 59-66). These water channels are known to capture hydrophilic compounds and the monoolein bilayer can carry hydrophobic compounds.

By incorporating the stimulus-sensitive polymer into the aqueous phase of the monoolein cubic, the stimuli-sensitive cubic phase can be designed. For example, a cubic phase containing an alginate exhibits a release characteristic depending on the calcium ion concentration, which is followed by a process in which the alginate in the water channel becomes gel by the calcium ion and inhibits drug release from the cubic phase .

In addition, the cubic phase containing poly (N-isopropylacrylamide) (PNIPAM) exhibits temperature-dependent emission characteristics, which means that when the temperature increases above the lower critical solution temperature (LCST) Lt; RTI ID = 0.0 > cubic < / RTI >

However, up to now, there is no known technique for controlling the release of the contents in the cubic phase by changing the pH according to the glucose concentration.

Korean Patent No. 10-1055804 entitled "Monoolein cubic phase nanoparticles containing water-soluble extracts of Hodgkin's lacquer and method for producing the same"), there is disclosed a monoolein cubic bicarbonate nanoparticle containing a water- ≪ / RTI > monoolein cubic phase nanoparticles. However, the above document does not disclose a monoolein cubic phase in which the pH-sensitive polymer and glucose oxidase are immobilized on a cubic phase and the pH is changed according to the glucose concentration to control the release of the target substance. Korean Patent No. 10-0891545 entitled Polyisopropylacrylamide-immobilized monoolein cubic phase and its production method, hydrophobized polyisopropylacrylamide (PNIPAM) The present invention relates to a monoolein cubic phase prepared by immobilizing a water-soluble polymer in a water channel inside a cubic phase, and relatively less of the collected component is released below the phase transition temperature of the polyisopropylacrylamide, Can release a relatively large amount of the target substance, and the target substance can be released as needed.

The above documents do not disclose a monoolein cubic phase that fixes a pH sensitive polymer and a glucose oxidase on a cubic phase and controls the release of a target substance by changing the pH according to the glucose concentration.

Accordingly, it is an object of the present invention to develop a pH-responsive monoolein cubic phase composition capable of controlling the release of a target substance in a cubic phase by changing the pH according to the glucose concentration.

In order to accomplish the above object, the present invention provides a method for preparing a monoolein cubic phase, wherein the pH-sensitive polymer is immobilized on a water channel of a monoolein cubic phase and the glucose oxidase is immobilized on the surface of a monoolein cubic phase the present invention provides a pH-responsive monoolefin cubic phase composition characterized by being immobilized and controlled to release a target substance according to glucose concentration.

In the pH-responsive monoolein cubic phase composition of the present invention, the pH-sensitive polymer preferably has an ionizable functional group. For example, the pH-sensitive polymer may be selected from chitosan, basic proteolide, alginic acid, It can be one. In the present invention, the term "prostanoid" refers to a compound prepared by applying heat to an acidic or basic amino acid to attach a hydrophobic amino acid thereto.

In the pH-responsive monoolein cubic acid composition of the present invention, the target substance may be preferably a hypoglycemic agent, and may be, for example, one selected from among rutin, insulin and cinnamon.

In the meantime, the present invention provides a method for producing a protein comprising the steps of: (a) hydrophobizing a pH-sensitive polymer and a glucose oxidase; (B) preparing a mixed solution by mixing the hydrophobic pH-sensitive polymer, the hydrophobicized glucose oxidase, and the target material, after the step (a); And (c) after the step (b), adding the mixed solution to the monoolein melt to prepare a transparent gel; The present invention provides a method for preparing a pH-responsive monoolefin cubic phase composition.

Meanwhile, in the method for preparing the pH-responsive monoolefin cubic phase composition of the present invention, the hydrophobic method of the pH-sensitive polymer in step (a) is preferably performed by mixing the pH-sensitive polymer with a hydrophobic anchor The hydrophobic anchor may be, for example, monoalkylacrylate, dialkylacrylate, palmitic acid, stearylamine, and stearic acid. , And hydrophobic amino acids.

The ratio of the pH sensitive polymer to the hydrophobic anchor is preferably 20: 1 to 1000: 1 by weight. If the content of the hydrophobic anchor is lower than the above range, the pH-sensitive polymer is insufficiently hydrophobic and the pH-sensitive polymer may not be properly immobilized on the cubic phase water channel. On the other hand, if the content of the hydrophobic anchor is high, the properties of the pH sensitive polymer may be lost.

In the method of preparing the pH-responsive monoolein cubic acid composition of the present invention, the pH-sensitive polymer preferably has an ionizable functional group, and examples thereof include chitosan, basic proteolide, alginic acid, Can be selected. The basic proteolyte may be prepared by condensing a hydrophobic amino acid to lysine or arginine, and the acidic proteolide may be prepared by condensing a hydrophobic amino acid to glutamic acid or aspartic acid. For example, hydrophobic pH sensitive polymer can be prepared by condensation reaction of an acidic amino acid, aspartic acid, with a hydrophobic amino acid such as leucine.

Meanwhile, in the method for preparing the pH-responsive monoolein cubic acid composition of the present invention, the method of hydrophobizing the glucose oxidase of step (a) preferably comprises mixing the glucose oxidase in an aqueous buffer in which the dispersant is dissolved, (A); (B) adding a hydrophobic anchor dissolved in an organic solvent to the aqueous solution in which the glucose oxidase is dissolved; And (b) removing the dispersant through a filtration process and lyophilizing to obtain a hydrophobic glucose oxidase (c); . ≪ / RTI >

In the method of preparing the pH-responsive monoolein cubic phase composition of the present invention, the hydrophobic anchor of step (b) may be, for example, monoalkylacrylate, dialkylacrylate, Palmitic acid, stearylamine and stearic acid, and hydrophobic amino acids.

In addition, the ratio of the glucose oxidase in step (b) to the hydrophobic anchor is preferably 20: 1 to 1000: 1 by weight. If the content of the hydrophobic anchor is lower than the above range, hydrophobicization of the glucose oxidase may be insufficient and the glucose oxidase may not be immobilized properly. On the other hand, if the content of the hydrophobic anchor is high, the characteristics of the glucose oxidase may be lost.

In the method for preparing the pH-responsive monoolefin cubic phase composition of the present invention, the dispersant of the step (a) preferably has a hydropolilic liphophilic balance number (HLB no.) Of 4 to 15, , Preferably from 6 to 14, and most preferably from 7 to 13. When a surfactant higher than the above range is used, the glucose oxidase is denatured, and when it is lower than the range, the hydrophobic anchor can not be effectively dispersed.

In the method for preparing the pH-responsive monoolein cubic acid composition of the present invention, the surfactant may be, for example, sorbitan laurate, sorbitan palmitate, PEO (20) sorbitan tri Sorbitan trioleate, deoxycholate, and the like.

In the method for preparing the pH-responsive monoolefin cubic phase composition of the present invention, the concentration of the dispersant in the aqueous phase is preferably 0.1-5.0 wt%, more preferably 0.5-4.0 wt%, and most preferably 1.0-3.0 wt% Weight%. If the pH is outside the above range, the glucose oxidase may not be denatured or a hydrophobic anchor may not be effectively dispersed.

In the method for preparing the pH-responsive monoolein cubic acid composition of the present invention, the concentration of the glucose oxidase in the aqueous phase is preferably 0.1 to 1.0% by weight, more preferably 0.2 to 0.8% by weight, most preferably 0.3 To 0.6% by weight. If it is outside the above range, the reaction can not proceed.

As an example, when palmitic acid hydroxysuccinimide ester (PAHE) is used as a hydrophobic anchor, the concentration is preferably 0.3 to 3.0% by weight, more preferably 0.5 to 2.0% by weight, most preferably, Is preferably 0.7 to 1.5% by weight. If the concentration is lower than the above range, a large amount of the organic solvent may cause denaturation of the glucose oxidase. If the concentration is higher than the range, the palmitic acid hydroxysuccinimide ester (PHAE) may aggregate or be difficult to dissolve The reaction does not proceed.

Also, when palmitic acid hydroxysuccinimide ester (PAHE) is used as a hydrophobic anchor, in step (b), the molar amount of palmitic acid hydroxysuccinimide ester (PHAE) to glucose oxidase Ratio is preferably from 1: 200 to 1:10, more preferably from 1: 100 to 1:20, and most preferably from 1:50 to 1:30. When the ratio is lower than the above ratio, hydrophobic alkyl groups covalently bonded to the glucose oxidase are not so hydrophobic and hydrophobic effects are insignificant. Therefore, the hydrophobic glucose oxidase can not be immobilized on the surface of the liposome. When the ratio is higher than the above ratio, Excessively covalently bound to lower the enzyme activity of glucose oxidase.

Meanwhile, in the method for preparing the pH-responsive monoolefin cubic phase composition of the present invention, the temperature of the monoolein melt of step (c) is preferably 35 to 70 ° C.

In the method for preparing the pH-responsive monoolefin cubic phase composition of the present invention, the mixed solution of step (c) and the monoolefin melt are preferably mixed in a weight ratio of 3: 2 to 1: 3.

In the process for preparing the pH-responsive monoolein cubic acid composition of the present invention, the concentration of the hydrophobic pH-sensitive polymer and the hydrophobic glucose oxidase is preferably 1 to 15 wt% in the mixed solution.

In the method for preparing the pH-responsive monoolein cubic acid composition of the present invention, the target substance may be preferably a hypoglycemic agent, and may be, for example, one selected from among rutin, insulin and cinnamon.

In the present invention, glucose oxidase (GOD) immobilized on the surface of a monoolein cubic phase produces gluconic acid from glucose, whereby when the pH value of the medium is decreased, The shape of the pH responsive polymer immobilized on the surface of the water channel is changed, thereby releasing the target substance trapped in the water channel. That is, according to the present invention, an excellent effect of controlling the release behavior of the target substance trapped in the cubic phase can be exhibited according to the concentration of glucose.

1 is a ¹³ C NMR spectrum of a prostanoid composed of aspartic acid (Asp) and leucine (Leu).
FIG. 2 is a ¹ H NMR spectrum of a proteoid composed of aspartic acid (Asp) and leucine (Leu).
3 is an FT-IR spectrum of aspartic acid (Asp) (a), leucine (Leu) (b) and protonoid (c).
4 is a graph showing the change in the size of the protonoid particles according to the pH value of the solution.
FIG. 5 is a graph of pH change of the hydrophobic glucose oxidase / glucose mixed solution with time. DL (?), 50 mg / dL (?), 100 mg / dL (?), 200 mg /
FIG. 6 is a graph showing pH-dependent release over time of cubic phase on which hydrophobic glucose oxidase and proteinase are immobilized. FIG. pH 4.5 (●), pH 6.0 (○), pH 7.4 (▼)
FIG. 7 is a graph showing glucose concentration-dependent release over time of a cubic phase on which hydrophobic glucose oxidase and a proteolipid are immobilized. (), 100 mg / dL (?), 200 mg / dL (?), 400 mg /

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

< Manufacturing example  1: hydrophobized pH Sensitivity  As a polymer Of prostanoid  Synthesis>

In this example, asp acid (Asp), which is an acidic and pH sensitive polymer, is used, and pH-responsive polymer hydrophobicized using Leu, a hydrophobic amino acid, as a hydrophobic anchor To synthesize the protonoids.

10 g of aspartic acid (Asp) and 0.15 g of leucine (Leu) were mixed with 3 ml of glycerol in a three-necked flask (250 ml) to prepare a reaction product. Thereafter, the flask was filled with nitrogen gas, and the reaction product contained in the flask was heated in an oil bath (about 160 ° C) to melt the reaction product. This was continuously reacted at the same temperature for 12 hours to prepare a reaction mixture. After the reaction mixture was cooled to room temperature, 40 ml of an aqueous sodium bicarbonate solution (10%, w / v) was poured into the reaction mixture and dissolved. After the dissolution, the reaction mixture dissolved in the dialysis bag (MWCO 1,000, Spectrum (CA, USA)) was put in the dialysis bag and immersed in 1 L of distilled water for 10 hours while replacing the same amount of distilled water for 60 hours Lt; / RTI &gt;

The dialyzed and purified proteoids were lyophilized, sealed in glass vials and stored. In order to confirm that the amino acids were synthesized through the condensation reaction, the lyophilized proteolide was dissolved in deuterium oxide, and then ¹³ C NMR spectrum and ¹ H NMR spectroscopy on a Varian VXR-500S spectrometer The spectrum was obtained.

The carboxyl group of the aspartic acid (Asp) residue was observed at around 177 ppm, the amide bond was observed at around 172 ppm, and the methylene group of aspartic acid (Asp) and leucine residue was observed at about 38 ppm ). 1 is a ¹³ C NMR spectrum of a prostanoid composed of aspartic acid (Asp) and leucine (Leu).

On the other hand, the methine group of the aspartic acid residue (Asp) was observed at 4.6 ppm and the methyl group of leucine was observed at 0.8 ppm. From this, it was confirmed that a protode composed of aspartic acid (Asp) and leucine (Leu) was synthesized.

From the above results, it was clearly confirmed that a synthesized proteolide composed of aspartic acid (Asp) and leucine (Leu) was synthesized (Fig. 2). FIG. 2 is a ¹ H NMR spectrum of a proteoid composed of aspartic acid (Asp) and leucine (Leu).

In addition, each of aspartic acid (Asp), leucine (Leu), and prothonoids was prepared in the form of pellets together with potassium bromide (Kbr) and then analyzed by an infrared spectrophotometer (FT-IR spectrophotometer, PerkinElmer, Frontier) The spectrum was obtained.

As a result, the amine groups and carboxyl groups of aspartic acid (Asp) and leucine (Leu) were observed at about 3373 cm ^-1 and 1617 cm ^-1 , respectively. Amino groups were not observed in the spectra of the protonoids, and amide bonds formed by the condensation reaction of amine groups and carboxyl groups were observed at around 1738 cm ^-1 . In addition, the carboxyl group of aspartic acid (Asp) residues 1602 cm ^- was observed at about ^1.

Thus, it was once again confirmed that a protode composed of aspartic acid (Asp) and leucine (Leu) was synthesized (FIG. 3). Figure 3 is the FT-IR spectrum of aspartic acid (Asp), leucine (Leu), and prothonoids.

EXPERIMENTAL EXAMPLE 1 Observation of pH Dependency of Protonoid Particle Size [

 In this Experimental Example, the particle size according to the pH of the prostanoid prepared in Preparation Example 1 was observed.

Proteinoids composed of aspartic acid (Asp), a pH sensitive polymer, and leucine (Leu), a hydrophobic anchor, were dissolved in distilled water to make the concentration of proteoid 0.1%. The pH of the aqueous solution of prostanoid was adjusted using 1N aqueous NaOH solution and 1N HCl.

The size of the protonoid particles in the aqueous solution was measured using a light scattering apparatus (Brookhaven Inst. Co., Zetaplus), so that the light scattering intensity (count per second) of the aqueous solution of the protenoid was 50-200 CPS And diluted with distilled water to measure particle size.

As a result of the experiment, the formation of the prostanoid particles in the aqueous phase is due to the hydrophobic amino acid residues (leucine residues) of the prostanoids, resulting in intermolecular / intramolecular hydrophobic interactions, At acidic conditions such as pH 4, pH 5 and pH 6, the size was more than 1000 nm, and the size of the protonoid particles decreased as the pH value increased. As the pH of the water phase increased from pH 3 to pH 9, the particle size decreased from 1284.5 nm to 832.4 nm.

It was inferred that the increase in pH value was due to the ionization of the carboxyl group of the aspartic acid (Asp) residue of the proteolide resulting in increased hydrophilicity and solubility in aqueous phase, thereby reducing the degree of molecular association by hydrophobic attraction 4). Figure 4 is the size of the protonoid particle according to the pH value of the solution.

< Manufacturing example  2: hydrophobized Glucose  Oxidase production>

In this production example, the glucose oxidase was tried to be hydrophobized.

0.24 g of deoxycholate was dissolved in 12 ml of phosphate buffer (pH 8.8, 0.16 M), and 0.048 g of glucose oxidase was dissolved successively in an aqueous solution of deoxycholate.

After 0.0228 g of palmitic acid N-hydroxysuccinimide ester (PAHE) was dissolved in 2 ml of dioxane, 0.4 ml of this solution was added to the aqueous glucose oxidase solution prepared above The reaction was gradually added.

The molar ratio of 'palmitic acid ester: glucose oxidase' in the reaction mixture was 1:40. The reaction mixture was stirred at 30 DEG C for 12 hours to allow the reaction to proceed. In order to remove insoluble urea, filtration was performed using a filtration membrane (pore diameter 0.45 μm).

Then, the filtrate was put in a dialysis bag (MWCO 5000) to remove water-soluble impurities such as dioxycholate from the filtrate. The dialysis bag was immersed in 1 L of a phosphate buffer (pH 7.4, 0.15 M) and dialyzed while changing the buffer three times for 15 hours. The dialyzed solution was lyophilized to obtain a dried, hydrophobic glucose oxidase.

Experimental Example 2: Analysis of palmitic acid bound to hydrophobic glucose oxidase [

In this experiment, trinitrobenzenesulfonic acid (TNBS) method was used to determine the number of palmitic acid bound to one molecule of glucose oxidase.

First, 0.025 g of sodium lauryl sulfate was dissolved in 50 ml of phosphate buffer (pH 8.9, 0.01 M). Then, 0.5 ml of trinitrobenzenesulfonic acid solution was added to 83 ml of sodium lauryl sulfate aqueous solution prepared above to prepare a trinitrobenzenesulfonic acid (TNBS) solution (0.03%).

Then, 1 ml of a sodium lauryl sulfate aqueous solution, 1 ml of trinitrobenzenesulfonic acid (TNBS) solution, and 0.5 ml of a hydrophobic binding substance-bound aqueous glucose oxidase solution (3.55 mg / ml) , And reacted at 50 DEG C for 1 hour.

After the reaction, 1.9 ml of aqueous HCl solution (0.21 M) was added to the reaction mixture, allowed to stand at room temperature for 30 minutes, absorbance of the reactant was measured at 335 nm and the absorbance was compared to the standard curve of the amino acid , And the number of amine groups of the glucose oxidase to which the hydrophobic substance was bound was determined.

The number of amine groups was also determined by the same procedure as described above for the glucose oxidase without the hydrophobic binding substance. The amino acid standard curve was developed by the same reaction procedure as described above, and the absorbance at 335 nm was measured according to the concentration. As a result, the standard curve equation was Y = 0.572 X + 0.0062, where the unit of X is μm and Y is the absorbance at 330 nm.

As a result, the number of amine groups per molecule of hydrophobic glucose oxidase was 16.8, and the number of amine groups per one molecule of glucose oxidase without hydrophobicity was 23.1. Therefore, the number of palmitic acid bound per molecule of hydrophobized glucose oxidase was calculated to be 6.3.

< Experimental Example  3: hydrophobic Glucose  Oxidase Glucose  Due to oxidation pH  Change>

In this Experimental Example, the pH change according to the glucose concentration of the hydrophobic glucose oxidase prepared in Preparation Example 2 was observed.

2.5 mg of hydrophobic glucose oxidase was dissolved in 5 ml of glucose aqueous solution. The concentration of glucose in the aqueous glucose solution was 0 mg / dL, 50 mg / dL, 100 mg / dL, 200 mg / dL and 400 mg / dL. The pH change was measured for 36 hours while the mixed solution was left at room temperature.

As a result of the experiment, the pH of the solution containing no glucose was kept almost constant for 36 hours at around 7.3. However, when the glucose concentration was 50 mg / ml, the pH value decreased from 7.3 to 6.72 during the initial 4 hours and remained almost constant thereafter. The reason why the pH value is decreased is that the glucose is converted to gluconic acid by the hydrophobic glucose oxidase and the acidity of the solution is increased.

In addition, the decrease in pH value during the initial 4 hours means that the enzyme reaction was terminated within 4 hours. Even when the glucose concentration was 100 mg / dL, 200 mg / dL or 400 mg / dL, Time, and thereafter maintained a constant value.

The higher the glucose concentration, the higher the pH value of the solution decreased. For example, after 4 hours, the pH values of solutions having concentrations of 50 mg / dL, 100 mg / dL, 200 mg / dL and 400 mg / dL were 6.72, 6.63, 6.37 and 6.19, respectively.

It was judged that the larger the amount of glucose as a reactant, the more gluconate was produced (Fig. 5). FIG. 5 is a graph showing changes in pH of the 'hydrophobic glucose oxidase / glucose mixed solution' with time.

&Lt; Example 1: Preparation of cubic phase in which glucose oxidase and proteinase are immobilized >

In this Example, a cuboid phase in which the proanthoid of Production Example 1 and the hydrophobic glucose oxidase of Production Example 2 were immobilized was prepared by melt-hydration.

2 g of monoolein was placed in a 10 ml glass vial and melted in a 60 占 폚 bath. Hydrophobic glucose oxidase, protonoid, and carboxylic fluororecein (CF) were dissolved in distilled water to give a concentration of 1.1% (w / w), 2.2% (w / w), and 0.01% (w / w). 0.9 g of the mixed aqueous solution was heated to 60 DEG C and the heated solution was carefully added to the monoolein melt. Thereafter, the solution was allowed to stand at room temperature until the added aqueous solution was completely absorbed and a transparent semi-solid gel was obtained. Thus, a cubic phase in which a hydrophobic glucose oxidase and a hydrophobic pH sensitive polymer were immobilized was prepared.

EXPERIMENTAL EXAMPLE 4: pH-dependent Release Experiment [

In this experimental example, 5 ml of buffer solution (pH 4.5, pH 6 and pH 7.4) was added to the cubic phase prepared in Example 1 to observe the release of CF 3 as a target substance for 96 hours.

Glycine buffer (10 mM) was used as the release medium to observe the release behavior at pH 4.5. When the release behavior was observed at pH 6, MES buffer (10 mM) was observed as the released medium. When observing the release behavior, HEPES buffer (10 mM) was observed as a release medium.

At a given time, 0.1 ml of the released medium was taken and the fluorescence of the CF was measured with a fluorescence spectrometer (Hitachi, F2500). At this time, the excitation wavelength was 494 nm, and the emission wavelength measured for the fluorescent intensity was 518 nm.

As a result of the experiment, when the pH value of the release medium was 7.4, the release rate increased with saturation curve for 72 hours, and the release rate was about 85%. When the pH value of the release medium was 6, the release rate also increased with saturation curve, and the release rate was about 35% at 72 hours. When the pH value of the release medium was 4.5, the release rate also increased with saturation curve, and the release rate was about 17% at 72 hours. Overall, the lower the pH value of the released medium, the lower the% of release.

At pH 7.4, as shown in FIG. 4 of Experimental Example 1, the degree of ionization of the acidic proteolinide is relatively high, so that the proteolytic acid is hydrophilic and the degree of association of the proteolytic acid is small. Therefore, at pH 7.4, it was judged that the proteolyte could not effectively block the water channel of the cubic phase, and the channel was opened, resulting in a relatively high% release. Further, when the pH value of the medium is decreased, as shown in FIG. 4 of Experimental Example 1, the ionization degree of the acidic proteolide is decreased, the hydrophobicity of the prostanoid is increased, and the degree of association of the proteoid is increased , it was determined that at pH 6 and pH 4.5, the proteolyte efficiently blocked the water channel on the cubic, the channel was closed and the% of release was relatively low (Fig. 6). FIG. 6 is a graph showing pH-dependent release over time of cubic phase on which hydrophobic glucose oxidase and proteinase are immobilized. FIG.

As a result, it was confirmed that the pH-responsive monoolefin cubic phase composition of the present invention can control the release of the target substance according to the change of pH.

Experimental Example 5: Glucose-dependent release &gt;

In this Experimental Example, glucose solution (50 mg / dL, 100 mg / dL, 400 mg / dL) dissolved in 5 ml of distilled water or distilled water was added to the cubic phase prepared in Example 1, CF release behavior.

At a given time, 0.1 ml of the released medium was taken and CF fluorescence was measured with a fluorescence spectrometer (Hitachi, F2500). At this time, the excitation wavelength was 494 nm, and the emission wavelength measured for the fluorescent intensity was 518 nm.

As a result of the experiment, when the release medium was distilled water (when the glucose concentration was zero), the% of release increased with saturation curve and the release rate was about 90% over 72 hours. When the release medium was a glucose solution, the% of release increased with saturation curve, and the higher the glucose concentration, the lower the% release.

When the glucose concentration was 50 mg / dL, 100 mg / dL, 200 mg / dL and 400 mg / dL, the release rates at 72 hours were 90%, 84%, 63% and 54%, respectively. Thus, the higher the glucose concentration, the lower the release of gluconic acid was attributed to the greater amount of gluconic acid produced by the enzyme reaction, and the higher the glucose concentration, the more produced by the glucose oxidase immobilized on the cubic phase.

Thus, the higher the glucose concentration, the lower the pH value of the medium, the more the acidity of the protonoids decreases and the greater the hydrophobicity of the protonoids. In addition, it was judged that this increased the degree of association of the proteoid (see FIG. 4).

Thus, at high glucose concentrations, the proteolyte effectively blocks the aqueous channel on the cubic, the channel is closed and the percent release is relatively low (FIG. 7). FIG. 7 shows glucose concentration-dependent release results with time of cubic phase on which hydrophobic glucose oxidase and proteinase are immobilized.

As a result, it was confirmed that the monoolein cubic phase of the present invention immobilized with the glucose oxidase and the pH sensitive polymer controls the release of the content according to the glucose concentration.

Claims (15)

the pH-sensitive polymer is immobilized on the water channel of the monoolein cubic,
Wherein the glucose oxidase is immobilized on the surface of the monoolein cubic phase,
The release of the target substance located in the water channel is controlled according to the glucose concentration,
Wherein the pH-sensitive polymer is hydrophobized and is a prostanoid composed of aspartic acid and leucine.
delete The method according to claim 1,
The above-
Wherein the pH-responsive monoolefin cubic phase composition is a hypoglycemic agent.
(a) hydrophobizing the pH-sensitive polymer and the glucose oxidase, respectively;
(B) preparing a mixed solution by mixing the hydrophobic pH-sensitive polymer, the hydrophobicized glucose oxidase, and the target material, after the step (a); And
(C) adding the mixed solution to the monoolein melt after the step (b) to prepare a transparent gel; Lt; / RTI &gt;
Wherein the hydrophobic pH-sensitive polymer is a prostanoid composed of aspartic acid and leucine.
delete delete delete 5. The method of claim 4,
The method for hydrophobizing glucose oxidase of step (a)
(A) preparing an aqueous solution by mixing a glucose oxidase with an aqueous buffer in which a dispersant is dissolved;
(B) adding a hydrophobic anchor dissolved in an organic solvent to the aqueous solution in which the glucose oxidase is dissolved; And
(C) removing the dispersant through filtration and lyophilizing to obtain a hydrophobic glucose oxidase; &Lt; / RTI &gt; wherein the pH-responsive monoolein-cubic-phase composition is prepared by reacting a compound of formula
9. The method of claim 8,
The hydrophobic anchor may be a hydrophobic anchor,
Wherein the pH is at least one selected from the group consisting of monoalkylacrylate, dialkylacrylate, palmitic acid, stearylamine and stearic acid, and hydrophobic amino acids. A process for preparing a monoolefin cubic phase composition.
9. The method of claim 8,
The ratio of the glucose oxidase to the hydrophobic anchor in step (b)
Wherein the weight ratio of the polyolefin is 20: 1 to 1000: 1.
5. The method of claim 4,
The temperature of the monoolein melt of step (c)
Wherein the pH-responsive monoolefin cubic phase composition is in the range of 35 to 70 占 폚.
5. The method of claim 4,
The ratio of the mixed solution of the step (c) and the monoolein melt is,
Wherein the weight ratio of the monoolein to the cubic phase is from 3: 2 to 1: 3.
delete 5. The method of claim 4,
The concentration of the hydrophobic pH-sensitive polymer and the hydrophobized glucose oxidase may be,
And 1 to 15% by weight of the liquid mixture.
5. The method of claim 4,
The above-
Wherein the pH-responsive monoolefin cubic phase composition is a hypoglycemic agent.

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