KR20120124191A - Microcapsule for oral administration and preparation method thereof - Google Patents

Abstract

PURPOSE: A microcapsule for oral administration and a method for manufacturing the same are provided to improve mucosal adhesion and durability. CONSTITUTION: A microcapsule for oral administration contains 40-85 wt% of alginate, 5-20 wt% of poly-L-lysine, 5-20 wt% of alginate with a thiol group, and 5-20 wt% of physiologically active substance. The alginate is denoted by chemical formula 1. A method for preparing the alginate with the thiol group comprises: a step of adding L-cysteine salt to a reaction mixture; a step of adjusting pH of reacted mixture; and a step of performing dialysis of the reaction mixture. The average particle diameter of the microcapsule is 5-5000 um. A method for preparing the microcapsule comprises: a step of suspending the physiologically active substance in saline solution and adding alginate to the suspension; a step of reacting the reaction mixture in a calcium salt solution; a step of reacting the reaction mixture in a poly-L-lysine solution; and a step of coating the reaction mixture with the alginate solution containing thiol groups.

Description

Microcapsules for oral administration and preparation method thereof {microcapsule for oral administration and preparation method}

The present invention relates to alginate, poly-L-lysine, alginate having a thiol group and a bioactive substance, and relates to oral microcapsules useful for oral administration of a bioactive substance, such as lactic acid bacteria or colonic target drugs, and a preparation method thereof. .

Probiotics have been extensively tried commercially and scientifically because of their significant health benefits in the gastrointestinal tract. Probiotics are recognized as beneficial bacteria that can reduce harmful bacteria from the small intestine. Lactobacillus or lactic acid bacteria are known as the most beneficial bacteria in the digestive tract. Thus, these bacteria compete with harmful bacteria such as E. Coli to maintain microbial balance in the host. In addition, Lactobacillus promotes beneficial effects such as immunomodulation, anticancer and antimicrobial activity.

Lactic acid bacteria must be delivered live to the small intestine through oral administration, but there are limitations such as gastric acid conditions, degradation of bacteria by bile acids and degrading enzymes. These barriers limit the survival and stability of living bacterial cells that reach the small intestine. Therefore, oral administration of Lactobacillus is extremely difficult.

Microencapsulation techniques facilitate the proper delivery system for this purpose and have been successfully used to capture probiotic bacteria or other therapeutic live bacteria. In comparison to other systems, alginate microcapsule systems have been widely used to encapsulate live bacteria.

Alginates have been successfully used for microencapsulation of living microorganisms because of their biocompatibility, nontoxicity and low immunogenicity, and have the activity of forming physiological hydrogels in the presence of divalent cations such as calcium or barium. However, it has been reported that microencapsulation of bacteria in alginates cannot effectively protect encapsulated organisms from high acidity. Thus, various cationic polymers, such as coatings, were used to coat the alginate microparticles to enhance the stability of the alginate microparticles and to prevent loss of encapsulated organism content.

Oral administration is the most desirable and attractive route of the drug delivery system, and although Lactobacillus delivery is the only way to improve the therapeutic effect, there are limitations to oral administration due to drug delivery to the target site and rapid drug clearance. Because of this, the delivered drugs do not exhibit adequate therapeutic activity.

In order to overcome the limitation of oral administration of bioactive substances such as lactic acid bacteria or colonic target drugs, for example, LS29, the present inventors have described alginate / PLL / thiol group-containing alginate (APTA) microcapsules using alginate having a thiol group. As a result of loading LS29 in the microcapsules, the microcapsules were found to be useful for in vitro and in vivo drug delivery of living bacterial cells such as LS29, thereby completing the present invention.

An object of the present invention and microcapsules useful for oral drug delivery that can effectively exhibit the activity of the delivered bioactive material by properly delivered to the target site during oral administration of a bioactive material such as lactic acid bacteria or colon target drug and its It is to provide a manufacturing method.

In order to achieve the above object, the present invention comprises 40 to 85% by weight of alginate, 5 to 20% by weight of poly-L-lysine, 5 to 20% by weight of alginate having a thiol group and 5 to 20% by weight of a bioactive material. Oral administration microcapsules are provided, and the oral administration microcapsules exhibit a three-layer coating structure.

The alginate is used for microencapsulation of bacteria, and beyond the content range may cause precipitation problems.

The poly-L-lysine plays a role of improving the stability of the alginate microcapsules, and if it is out of the content range, it may cause nutrient supply problems into the alginate microcapsules.

The alginate having the thiol group serves to improve the stability and colonic mucoadhesiveness of the microcapsules, and if it is outside the content range, a problem of nutrient supply into the alginate microcapsules may be caused.

In particular, the alginate having a thiol group is preferably a compound represented by the following general formula (1):

[Formula 1]

In the above formula, R ₁ and R ₂ are different from each other, and may be any one of hydrogen or a carboxyl group.

Alginate having the thiol group in the N (i) alginate (3-dimethylaminopropyl) -N- ethylcarbodiimide hydrochloride (EDAC) and N-phase of the reaction by the addition of hydroxyl-succinimide (NHS) (No. Stage 1); (ii) adding L-cysteine salt to the reaction mixture obtained in the first step (second step); (iii) adjusting the pH of the reaction mixture obtained in the second step (third step); And dialysis of the reaction mixture obtained in the third step.

More specifically, the first step is to react with (i) adding EDAC to the alginate to activate the carboxyl group of the alginate, and (ii) adding NHS to the reaction mixture to enhance the efficiency of the EDAC-mediated coupling reaction. It can be added and reacted. In the second step, the L-cysteine salt may be added at a ratio of 5 to 50 mol% based on 1 molar ratio of alginate. At this time, the addition of L-cysteine outside the content range may cause a problem in the stability of the microcapsules. The third step adjusts the pH of the reaction mixture to 6-7, and the fourth step purifies the reaction mixture through dialysis. These total reactions can be carried out under a nitrogen atmosphere to prevent oxidation of the thiol group.

The bioactive substance may be selected from a lactic acid bacterium or a large intestine targeting drug that exhibits efficacy in the large intestine, and the lactic acid bacteria may be Lactobacillus salivarius LS29 and the like, but is not limited thereto. May be, but is not limited to, peptides, proteins, and the like.

The average particle diameter of the microcapsules according to the present invention is preferably 5 to 5000 ㎛. If the average particle diameter is out of the above range, a problem may occur in the stability of the microcapsules.

In addition, the present invention comprises the steps of suspending the bioactive material in physiological saline, and adding alginate to the suspension (first step); Reacting the reaction mixture obtained in the first step in a calcium salt solution (second step); Reacting the reaction mixture obtained in the second step in a poly-L-lysine solution (third step); And (4) coating the reaction mixture obtained in the third step with an alginate solution having a thiol group (fourth step).

At this time, the final concentration of the alginate is 0.5 to 3% by weight, it is preferable to contain 8x10 ¹⁰ to 10x10 ¹⁰ CFU / mL when the bioactive material is lactic acid bacteria.

The microcapsules according to the present invention are microcapsules having a three-layer coating structure including alginate, poly-L-lysine, and alginate having a thiol group. Bioactive substances such as colonic target drugs can be usefully used for effective oral administration.

1 is a schematic of the synthesis of alginate having a thiol group,
Figure 2 is a schematic diagram showing the manufacturing process of the microcapsules according to an embodiment of the present invention,
3A and 3B are micrographs of microcapsules according to one embodiment of the present invention (a: microcapsules not loaded with LS29, b: microcapsules loaded with LS29),
Figure 4a is a survival analysis of LS29 loaded in microcapsules according to an embodiment of the present invention in artificial gastric juice (SGF),
Figure 4b shows the degree of release of LS29 from microcapsules according to an embodiment of the present invention in artificial intestinal fluid (SIF),
5 is an evaluation of adhesion in vitro adhesion of the microcapsules according to an embodiment of the present invention,
6 is to evaluate the in vivo adhesive adhesion of the microcapsules according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 Bacteria Culture Conditions and Cell Suspension Preparation

Lactobacillus salivarius 29 (LS29) was used by Professor Yoon-jae Choi of Seoul National University. 5 μL of LS29 was inoculated in 5 mL of MRS medium and incubated for 15 hours. Thereafter, 0.5 mL of cultured LS29 was suspended in 50 mL of fresh MRS medium and incubated for 12 hours. The culture was maintained at 170 rpm with shaking at 170 rpm. The culture was then centrifuged at 3,000 rpm for 10 minutes to recover bacteria and washed twice with sterile physiological saline (0.85%). The bacteria were then resuspended in 0.85% saline and either the cells themselves or microencapsulated cells were used.

Example 2 Preparation and Properties of Alginate (TA) Having Thiol Group

1 g of sodium alginate (viscosity: 250 cps) was dissolved in 100 mL of secondary distilled water. EDAC [N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride] was added to the polymer to a final concentration of 100 mM and reacted for 45 minutes to activate the carboxyl group of the polymer. Thereafter, NHS (N-hydroxyl succinimide) at the same concentration as EDAC was added to the reaction and reacted for 45 minutes to enhance the efficiency of the EDAC-mediated coupling reaction. The polymer and L-cysteine monohydrate hydrochloride were added at a weight ratio of 1: 4 (polymer: L-cysteine monohydrate hydrochloride), and the pH of the reaction mixture was adjusted to 6.0 using 1M NaOH solution. In order to prevent oxidation of the thiol group, the entire reaction was carried out under a nitrogen atmosphere. The reaction mixture was reacted for 10-12 hours with stirring at room temperature. The obtained alginate cysteine conjugate was dialyzed for 48 hours on an aqueous 1 mM HCl solution at room temperature, followed by dialysis on the same medium containing 1% NaCl for 48 hours. Thereafter, the polymer conjugate was lyophilized and stored in a sealed container at 4 ° C. until use. The schematic diagram of the reaction regarding the synthesis of alginate (TA) having a thiol group is shown in FIG. 1.

1) Thiol content analysis using Elman's reagent reaction

The alginate polymer having 1 mg thiol group was dissolved in 1 mL of 0.5% phosphate buffer (pH 8.0). Then, 1 mL of 0.03% (w / v) DTNB (5, 5-dithio-bis-2 nitrobenzoic acid) dissolved in 0.5% phosphate buffer (pH 8.0) was added to the alginate polymer solution. The resulting solution was reacted at room temperature for 1 hour. The absorbance was then measured in visible light at 405 nm. The titration curve obtained using L-cysteine was prepared and the concentration of thiol content in TA was evaluated using Elman reagent.

As a result, about 759 ± 32.4 μM of cysteine was introduced per gram of alginate.

2) Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

FTIR analysis of alginate (TA) with the prepared thiol group at a wavelength of 650 to 4000 cm ⁻¹ was performed using a Thermo-Nicolet 6700 Infrared Spectrometer (FTIR) (Midac, Atlanta, GA, USA).

The FTIR measurement of the TA showed several new bands located in the range of 1173-1728 cm ⁻¹ . That is, compared to the structural analysis of alginate itself, three major absorption bands were obtained at 1728 cm ⁻¹ (C═O of carboxyl group), 1641 cm ⁻¹ (C═O of amide), and 1546 cm ⁻¹ (NH of amide). Indicated. All of these absorption bands are due to the functional group of cysteine attached to the polymer conjugate, and it was found that cysteine was successfully bound to the alginate skeleton. Bands appearing in the range of 2550-2600 cm ⁻¹ belong to sulfide functional groups.

Example 3 Survival Assessment of LS29 in Artificial Gastric Fluid (SGF) and Intestinal Fluid (SIF)

1 mL of LS29 culture was reacted with 20 mL of SGF or SIF for 0, 5, 10, 20,30, 60, 90 and 120 minutes respectively. Thereafter, 1 mL of liquid was collected from each medium, and the colony forming unit (CFU) of LS29 was analyzed using test materials diluted in multiple stages on an MRS agar plate. The plates were incubated at 37 ° C. for 36-48 hours. CFU was measured to assess the survival of LS29.

As a result, when LS29 was exposed to SGF, no bacteria survived after 20 minutes. However, when the LS29 was exposed to SIF, the survival of the LS29 was maintained until 120 minutes of evaluation time. Therefore, LS29 was found to be very sensitive to acidic conditions and thus could not survive when administered orally due to the acidic environment of the stomach.

Example 4 Preparation of LS29-Loaded APTA Microcapsules

APTA microcapsules loaded with LS29 were prepared by Martoni et al. The method of ( J. Biomed. Biotechnol. 2007, 13684, 2007) was prepared with a slight modification. After LS29 was suspended in sterile saline solution (0.85%), sterile sodium alginate was added to bring the final alginate concentration to 1.5% (w / v). Cell density was maintained at -10 ¹⁰ CFU / mL for each microencapsulation. The alginate-LS29 mixture was extruded in a lightly stirred sterile 0.1 M CaCl ₂ solution while maintaining a sterile environment. The calcium alginate microcapsules formed were hardened in CaCl ₂ solution for 30 minutes and immersed in poly-L-lysine (PLL) HBr solution (molecular weight 30,000-70,000) for 10 minutes. The microcapsules were then coated with alginate (TA) with 0.1% (w / v) thiol group for 10 minutes to form a surface membrane. At this time, the microcapsules were washed twice with sterile saline solution after each process. Finally, the microcapsules were suspended in sterile saline and stored at 4 ° C.

Example 5 Shape and Size of Microcapsules

The shape of APTA and LS29-loaded APTA microcapsules was analyzed using an optical microscope (Carl Zeiss, Axiovert 40 CFL, Germany). Photomicrographs of microcapsules were taken and capsule size was measured using a hemocytometer scale bar.

3A and 3B show the shapes of APTA microcapsules (a) and LS29-loaded APTA microcapsules (b), respectively, and both APTA and LS29-loaded APTA microcapsules are spherical with smooth surfaces, which are useful for oral drug delivery. It is a useful shape. The average size of the APTA and LS-29 loaded APTA microcapsules was 1.11 ± 0.06 mm and 1.12 ± 0.05 mm, respectively, with almost no difference between the microcapsules.

Example 6 Loading Efficiency (%)

Supernatants were collected after microencapsulation and each coating time during microencapsulation and LS29 unloaded after step dilution on MRS agar plates. CFU was measured according to previously known methods. Unloaded LS29 (CFU / mL) was subtracted from the original bacterial load (CFU / mL) to yield the actual loading content and efficiency (%) of LS29-loaded APTA microcapsules.

The results showed higher loading content (? 10 ¹⁰ CFU / mL) and efficiency (99.7 ± 0.36%) of LS29 through APTA microcapsules.

Example 7 LS29 Survival Analysis in LS29-loaded APTA Microcapsules in SGF

LS29-loaded APTA microcapsules were incubated in 2 mL of SGF in a separation tube at 37 ° C. for various times, ie 0, 5, 10, 20, 30, 60 and 120 minutes, at which time they were stirred at 100 rpm. At each time point gastric juice medium was removed and washed with sterile physiological saline (0.85%) to remove trace amounts of acidic medium. Thereafter, the microcapsules were mechanically ruptured using a sterile homogenizer (T25 basic, IKA), and cells were separated using a 70 μm filter. Survival of microencapsulated live LS29 was step diluted in isolated test material and CFU was measured in MRS agar plates as previously described.

As a result, as shown in FIG. 4A, about 40% of the encapsulated LS29 survived for 60 minutes in SGF in the APTA microcapsules, whereas the LS29 not encapsulated in the APTA microcapsules survived within 20 minutes. Thus, LS29 significantly increased survival rate through APTA microencapsulation.

Example 8 Glass of LS29 from LS29-loaded APTA Microcapsules in SIF

LS29-loaded APTA microcapsules were incubated with 10 mL of SIF at 37 ° C. with 100 rpm shaking. 0.5 mL liquids were collected at time intervals of 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hours and the same amount of fresh medium was added. Samples were step diluted on MRS agar plates to analyze the glass of LS29 at different time intervals.

As a result, as shown in Figure 4b LS29 is released from the APTA microcapsules in SIF in a time-dependent manner, and maintained survival over the experimental period. About 76% of the encapsulated LS29 was released after 24 hours from the microcapsules and this glass continued thereafter. Therefore, the APTA microcapsules of this example were found to be useful for controlling the release of LS29.

≪ Example 9 > In vitro  Mucoadhesive evaluation

To investigate the mucoadhesive properties of APTA microcapsules in vitro, fluorescein isothiocyanate (FITC) -loaded APTA microcapsules were prepared as described above without LS29 addition. FITC-loaded APA microcapsules were prepared as controls. In vitro mucoadhesion was evaluated according to a previously known method ( Int. J. Pharm. 359, 205, 2008).

That is, freshly excised pig small intestine sections (5.5 cm x 2.5 cm) were attached on the stage and washed with 0.85% saline. The FITC-loaded APA and APTA microcapsules suspended in physiological saline were then transferred to the small intestine. The slide was then placed vertically in a plastic cylinder containing 40 mL of 0.85% saline. Incubated at 37 ° C. with 100 rpm shaking. At defined time intervals, the microcapsules remaining on the mucosa were scraped and mechanically ruptured. The supernatants were then collected by centrifugation at 13,000 rpm for 10 minutes. Unbound microcapsules precipitated at the bottom of the cylinder were scraped and ruptured, and the supernatant was collected by centrifugation in the same manner as before. Fluorescence was measured at 490 nm. The amount of FITC from bound or unbound capsules was considered as the total amount of FITC. The amount of FITC remaining on the excised pig small intestinal mucosa for APA and APTA microcapsules was calculated as a percentage of the total amount.

As a result, as shown in Figure 5 APA microcapsules were incubated in physiological saline (0.85%) for 1 hour at 37 ℃ after a few adhered to the small intestinal mucosa surface, after 2 hours hardly attached Most of the APTA microcapsules were well adhered on the mucosa under similar conditions throughout the experimental period.

The SH group of the alginate having a thiol group used as the surface film of the APTA microcapsules formed a disulfide bond with the mucin glycoprotein of the mucosa, thereby providing strong mucoadhesion compared with the APA microcapsules.

≪ Example 10 > In vivo  Mucoadhesive evaluation

APA and APTA microcapsules without LS29 were prepared according to the methods described above and then labeled with ^99m TcO4 according to known methods (J. Nucl. Med. 46, 141, 2005).

All animal experiments were performed with the approval of the Experimental Animal Steering Committee for animal treatment at Chonbuk National University. Female SD rats (8 weeks old, purchased from Orient-Bio) received a standard diet and water. Rats were anesthetized with 2-2.5% isoflurane and then in vivo images were obtained. Thereafter, the anesthetized rat was fixed on a plate with an attachment strip and mounted on a single head gamma camera (X-SPECT / CT, GE Healthcare, Uppsala, Sweden) with flat collimates, a window setting of 140 keV and a width of 20%. Placed in a lying position. Five-minute planar images were obtained at 30, 60, 120, 180 and 210 minutes to confirm the flow of radioactivity to the distal small intestine.

As a result, as shown in Figure 6, the distribution of the two microcapsules were found in the stomach of the rat 30 and 60 minutes after oral administration, and then moved to the small intestine after 120 minutes. After 120 minutes, two radioactive microcapsules were found in the small intestine, but the intensity of the radioactive color of ^99m Tc-APA was weaker than ^99m Tc-APTA. The radioactivity of the ^99m Tc-APA microcapsules was clearly weakened over time at 180 and 210 minutes, while the ^99m Tc-APTA microcapsules were still attached to the small intestine mucosa and showed stronger mucoadhesion than the APA microcapsules. . This is because they have alginates with thiol groups on the surface membrane that can form strong disulfide bonds with mucin glycoproteins of the small intestinal mucosa.

Therefore, APTA microcapsules can be usefully used as a mucosal delivery system for bioactive substances such as LS29.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (6)

Microcapsules for oral administration comprising 40 to 85% by weight of alginate, 5 to 20% by weight of poly-L-lysine, 5 to 20% by weight of alginate having a thiol group, and 5 to 20% by weight of a bioactive substance. The microcapsule for oral administration according to claim 1, wherein the alginate having a thiol group is represented by the following Chemical Formula 1.
[Formula 1]

In the above formula, R ₁ and R ₂ are each different, either hydrogen or carboxyl group. The alginate having a thiol group comprises: (i) adding EDAC and NHS to the alginate and reacting (first step); (ii) adding L-cysteine salt to the reaction mixture obtained in the first step (second step); (iii) adjusting the pH of the reaction mixture obtained in the second step (third step); And microcapsules for oral administration characterized in that it comprises a step of dialysis of the reaction mixture obtained in the third step. The microcapsule for oral administration according to claim 1, wherein the physiologically active substance is any one of a target drug for lactic acid bacteria or colon which is effective in the large intestine. The microcapsules for oral administration according to claim 1, wherein the microcapsules have an average particle diameter of 5 to 5000 µm. Suspending the bioactive substance in physiological saline and adding alginate to the suspension (first step);
Reacting the reaction mixture obtained in the first step in a calcium salt solution (second step);
Reacting the reaction mixture obtained in the second step in a poly-L-lysine solution (third step); And
Coating the reaction mixture obtained in the third step with an alginate solution having a thiol group (fourth step)
Method for producing oral microcapsules comprising a.

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