CN111603437A - A kind of delivery complex containing medicine or live bacteria and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of composite gel carriers, in particular to a delivery compound encapsulating a drug or live bacteria and a preparation method thereof. The method comprises the steps of: mixing a sodium alginate solution with a target drug solution or a target live bacteria mixed solution of sodium alginate, and then drop it into the CaCl ₂ solution to obtain calcium alginate gel microspheres. Absorb the surface moisture, immerse the microspheres in the glyceride of unsaturated fatty acids, take out the microspheres and mix with the uncoagulated gelatin gel, add it to the mold, cool down until the gelatin gel is completely solidified, and after standing, take off the mold, that is, delivery complex. The present invention is suitable for drugs such as proteins and polypeptides that are easily affected by the gastrointestinal environment, as well as probiotic live bacteria preparations, and can effectively protect the loaded drugs or live bacteria from the impact of the gastric environment, while the drug or live bacteria can be protected from the effects of the gastric environment. Reach the target site in the intestine for release.

Description

A kind of delivery complex containing medicine or live bacteria and preparation method thereof

technical field

The invention relates to the technical field of composite gel carriers, in particular to a delivery compound containing drugs or live bacteria and a preparation method thereof.

Background technique

Oral administration is the most convenient, safe and comfortable route of administration, and it is also the preferred route for many drug design and development. However, due to the complexity of the digestive tract environment, protein and polypeptide drugs are easily destroyed by gastric acid and protease in the digestive tract, resulting in low drug absorption and availability. In the case of oral administration, due to the strong acid environment in the stomach and the acid-base changes in the gastrointestinal environment, the number of live probiotics taken is greatly reduced.

Gel is a special semi-solid state between solid and liquid. Many polymer materials can be prepared into gel carriers. These carriers have a certain protective effect on drugs. At the same time, they can change their state according to the changes of pH, ionic strength, temperature, redox conditions, etc. Site-specific controlled loading and release, while the aqueous core of the gel enhances drug stability and bioactivity.

Sodium alginate is a water-soluble natural linear unbranched polysaccharide that can form hydrogels with divalent metal ions such as calcium ions. Due to its good biocompatibility, mucosal adsorption, biodegradability, and mild gelation conditions, it is often used as a safe biomaterial in the fields of food and medicine. Gelatin is a natural macromolecular polypeptide compound obtained by degrading fibrous collagen. It is odorless, translucent, slightly glossy, and can be stored for a long time under dry conditions. It has a wide range of uses in food, medicine, industry and other fields. In recent years, due to its excellent performance, it has been widely used in drug controlled release materials in the field of medicine.

To sum up, to develop an oral administration carrier suitable for protein, polypeptide drugs and live probiotics preparations, by encapsulating proteins, polypeptide drugs and live probiotics preparations, improving its oral bioavailability is still an oral drug. Key issues in drug delivery formulation development.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a drug-loaded or live bacteria-encapsulating delivery compound and a preparation method thereof. To achieve the above object, the present invention adopts the following technical solutions:

The first object of the present invention is to provide a preparation method of a delivery compound containing a drug or live bacteria, comprising the steps of:

S1, mix the sodium alginate solution with an equal volume of the target drug solution or the target viable bacteria solution, and configure it into a sodium alginate mixed solution with a sodium alginate mass fraction of 1-3%;

S2, drop the _sodium alginate mixed solution into a CaCl solution with a concentration of 0.1 mol/L to form calcium alginate gel microspheres, and let stand for 10-15min to obtain morphologically stable calcium alginate gel microspheres ball;

S3, after the described calcium alginate gel microspheres are washed, the surface moisture is absorbed, and then the calcium alginate gel microspheres are immersed in the glyceride of unsaturated fatty acids;

S4. Mix the glyceride-treated calcium alginate gel microspheres with the unsolidified gelatin gel and add it to the mold, cool down until the gelatin gel is completely solidified, and after standing for 20-30min, remove the mold to obtain composite gel.

Further, the target drug is a protein or a polypeptide, and the target live bacteria are probiotics.

Further, in S4 of the above preparation method, the gelatin gel is prepared by the following steps:

(1) mix the gelatin particles and water according to the ratio of 10g: 15mL, heat and stir until the gelatin particles are completely dissolved to obtain a gelatin solution;

(2) Weigh 1.2 g of pectin, 5.9 g of glucose, 1 g of citric acid, 0.8 g of sodium citrate, and 27 g of maltose, dissolve in 15 mL of water, mix with the gelatin solution of step (1), add 10 mL of water, and remove air bubbles , to obtain a gelatin gel.

Further, in S4 of the above-mentioned preparation method, the gelatin solution is heated to 70-80° C., and then the temperature is lowered to 35° C., and the temperature is maintained at 35° C. to obtain an uncoagulated gelatin gel. The microspheres treated with glycerolipid are added, the two are mixed evenly, added to the mold, cooled until the gelatin gel is completely solidified, and after standing for 30-40 minutes, the mold is removed to obtain the delivery compound.

Further, the shape of the mold is a cube.

The second object of the present invention is to provide a drug-loaded or live bacteria-encapsulating delivery complex prepared by the above method.

Compared with the prior art, the present invention has the following beneficial effects:

1. The carrier formed by a single gel often has obvious shortcomings. The network structure in the calcium alginate gel microspheres is very loose, which leads to the permeation of the loaded material during the preparation of the calcium alginate gel microspheres. leakage losses and rapid release, which limit its application. Gelatin is brittle, has poor thermal stability, is easily degraded in water environment, and has poor solubility in cold water, so it is difficult to achieve the effect of sustained release. The drug-loaded or live-bacteria-encapsulated delivery compound developed in the present invention can make up for the defects of a single gel and take advantage of both to effectively protect the loaded drug or live bacteria so that it can be released after reaching the intestinal tract.

2. The drug-loaded or live bacteria-encapsulating delivery complex of the present invention combines the advantages of two gels, and the protein, polypeptide drugs and live probiotic bacteria preparations are loaded into the inner gel calcium alginate microspheres, which The higher water content can play an effective protective role. The glycerides of unsaturated fatty acids in the outer layer of the microspheres can prevent the loss of water. Its nature makes it gradually depolymerize in an alkaline environment to achieve a slow release in the intestine. the goal of. The outer layer of gelatin gel can protect the calcium alginate gel microspheres on the inner side, maintain the structure of the gel, slow down the impact of gastric digestion, and avoid the loss and early release of internal proteins and polypeptides.

Description of drawings

Figure 1 is a schematic diagram of the construction process of the delivery complex of the present invention.

FIG. 2 is a physical diagram of the calcium alginate gel microspheres prepared in Example 1. FIG.

FIG. 3 shows the BSA-loaded delivery complex prepared in Example 1. FIG.

FIG. 4 is the result of the in vitro release experiment of Example 1. FIG.

Figure 5 shows the results of the in vitro release experiment of Example 2, in which A is the experimental group and B is the control group.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

A kind of preparation method of drug-loaded delivery complex

The model drug in this embodiment is Bovine Serum Albumin (BSA) (concentration is 1 mg/mL), which specifically includes the following steps:

S1. Dissolve 4 g of sodium alginate powder in 100 mL of sterile water to prepare a sodium alginate solution with a mass fraction of 4%, measure a certain volume of sodium alginate solution, add an equal volume of BSA solution to mix, and configure it into a mass fraction It is 2% sodium alginate mixed solution;

S2. Absorb 2% sodium alginate mixed solution, and drop the droplets into the CaCl ₂ solution with a concentration of 0.1mol/L using a constant current pump. The droplet position is 30cm away from the liquid surface. The magnetic stirrer was stirred at 250 rpm, and the droplets entered the CaCl ₂ solution to form calcium alginate gel microspheres immediately, as shown in Figure 2, and let stand for 10 min, so that calcium ions and sodium alginate formed a polymerized structure to obtain morphologically stable microspheres. Calcium alginate gel microspheres;

S3, the above-mentioned calcium alginate gel microspheres are washed once with sterile water, and the surface moisture is absorbed by filter paper, and then the microspheres are immersed in the glyceride of unsaturated fatty acids, so that a layer of grease is adsorbed on the surface of the microspheres;

S4. Preparation of gelatin gel: Weigh 10 g of food-grade gelatin granules, add 15 mL of sterile water, and soak for 15 min at room temperature to make it completely swollen and soft. Heat in a water bath at 80°C with constant stirring until the gelatin particles are completely dissolved. Weigh 1.2 g of pectin, 5.9 g of glucose, 1 g of citric acid, 0.8 g of sodium citrate, and 27 g of maltose, respectively, dissolve them in 15 mL of sterile water, and stir to dissolve the components completely. Then, it was added to the dissolved gelatin solution, and then 10 mL of sterile water was added, the gelatin solution was mixed uniformly, and the bubbles in the solution were removed by ultrasonic wave method to obtain gelatin gel;

The gelatin gel was heated to 70°C, then cooled to 35°C, and the temperature was maintained at 35°C to obtain an uncoagulated gelatin gel, and glyceride-treated microspheres were added to the uncoagulated gelatin gel, and the two Mix evenly, add it to the cube mold, cool down until the gelatin gel is completely solidified, and after standing for 30 minutes, remove the mold to obtain a drug-loaded delivery compound, as shown in Figure 3.

The drug-encapsulated delivery complex prepared in this example was subjected to an in vitro release experiment, and the method was as follows:

The drug-loaded delivery complex was placed in 20 mL of simulated gastric juice (sodium chloride 2 g, 7 mL of concentrated hydrochloric acid, and ultrapure water was added to dilute to 1 L, the pH of the solution was 1.2), incubated at a constant temperature of 100 rpm in a water bath at 37 °C, and sampled every 30 min. For detection, continuous sampling was performed 6 times until incubation for 3h. Then remove the simulated gastric juice, add 20 mL of simulated intestinal juice (6.8g potassium dihydrogen phosphate, add ultrapure water to make up to 1L, adjust the pH of the solution to 7.4,), continue to incubate in a 37°C water bath at 100rpm, take samples every 30min for detection, continuously Sampling 10 times and incubating for 5h. The protein concentration in the sample was determined using the Bradford method, and the protein release curve was drawn.

The results are shown in Figure 4. After the drug-loaded delivery complex was treated in simulated gastric fluid for 3 h, the protein was released by about 10%. When the microspheres were moved into the simulated intestinal fluid, with the increase of pH, the gel in the outer layer gradually absorbed and swelled, the pores became larger, the electrostatic interaction between the inner seaweed calcium microspheres and the amino groups weakened, and the aggregated structure was also dissolved. polymerized, the protein diffused to the outside of the microspheres along with the leaking moisture. The protein in the composite gel was basically released completely within 4 hours.

Example 2

A kind of preparation method of delivery complex encapsulating live bacteria

In this embodiment, other preparation methods are the same as those in embodiment 1, except that the objective viable bacteria are lactic acid bacteria model strains (concentration is 1×10 ¹⁰ cfu/mL).

The in vitro release experiment was carried out on the delivery complex containing live bacteria prepared in this example, and the method was as follows:

In the experimental group, the delivery complex encapsulated with live bacteria was placed in 20 mL of simulated gastric juice (2 g of sodium chloride, 7 mL of concentrated hydrochloric acid, and ultrapure water was added to make the volume to 1 L, and the pH of the solution was about 1.2), and incubated at a constant temperature of 100 rpm in a 37 °C water bath for 3 h. Then remove the simulated gastric juice, add 20 mL of simulated intestinal juice (6.8 g potassium dihydrogen phosphate, add ultrapure water to make the volume to 1 L, adjust the pH of the solution to 7.4), and continue to incubate in a 37°C water bath at 100 rpm for 1 h. The delivery complex was then taken out, completely lysed under mild conditions, centrifuged at 1000 rpm to remove undissolved gel particles, diluted with the supernatant, coated on a plate, incubated at 37°C for 24 hours, and the number of single clones on the plate was detected. Calculate the number of viable bacteria. In the control group, the bacterial solution of the same concentration was put into a 3KD dialysis bag and treated in the same way.

The results are shown in Figure 5. The results of plate coating show that the delivery complex has an effective protective effect on the loaded lactic acid bacteria. After the treatment of simulated gastric juice and intestinal juice, the number of viable bacteria of the delivery complex can reach about 1.2×10 ⁹ cfu/mL, while the number of viable bacteria in the control group without carrier protection was only 3×10 ⁷ cfu/mL.

It should be noted that when a numerical range is involved in the claims of the present invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. In order to prevent repetition, the present invention describes the preferred Example.

Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be construed to include the preferred embodiment and all changes and modifications that fall within the scope of the present invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, provided that these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (6)

1. a preparation method of the delivery compound of a package drug or viable bacteria, is characterized in that, comprises the steps: S1, mix the sodium alginate solution with an equal volume of the target drug solution or the target viable bacteria solution, and configure it into a sodium alginate mixed solution with a sodium alginate mass fraction of 1-3%; S2, drop the _sodium alginate mixed solution into a CaCl solution with a concentration of 0.1 mol/L to form calcium alginate gel microspheres, and let stand for 10-15min to obtain morphologically stable calcium alginate gel microspheres ball; S3, after the described calcium alginate gel microspheres are washed, the surface moisture is absorbed, and then the calcium alginate gel microspheres are immersed in the glyceride of unsaturated fatty acids; S4. Mix the glyceride-treated calcium alginate gel microspheres with the unsolidified gelatin gel and add it to the mold, cool down until the gelatin gel is completely solidified, and after standing for 20-30min, remove the mold to obtain Drug-loaded or live bacteria-encapsulated delivery complexes. 2 . The preparation method of a drug-loaded or live bacteria-encapsulating delivery complex according to claim 1 , wherein, in S1 , the target drug is a protein or a polypeptide, and the target live bacteria are probiotics. 3 . 3. the preparation method of the delivery compound of a kind of drug-loaded or viable bacteria according to claim 1, is characterized in that, in S4, gelatin gel is prepared from the following steps: (1) mix the gelatin particles and water according to the ratio of 10g: 15mL, heat and stir until the gelatin particles are completely dissolved to obtain a gelatin solution; (2) Weigh 1.2 g of pectin, 5.9 g of glucose, 1 g of citric acid, 0.8 g of sodium citrate, and 27 g of maltose, dissolve in 15 mL of water, mix with the gelatin solution of step (1), add 10 mL of water, and remove air bubbles , to obtain a gelatin gel. 4. the preparation method of the delivery compound of a kind of encapsulated drug or viable bacteria according to claim 3, is characterized in that, in S4, gelatin gel is heated to 70-80 ℃, and then the temperature is lowered to 35 ℃, and Maintain the temperature at 35°C to obtain an uncoagulated gelatin gel, add glyceride-treated calcium alginate gel microspheres to the uncoagulated gelatin gel, mix the two, add them into the mold, and cool down to the gelatin gel It is completely solidified, and after standing for 30-40 minutes, the mold is removed, and the delivery compound is obtained. 5. the preparation method of a kind of drug-loaded gelatin-calcium alginate composite gel according to claim 4, is characterized in that, described mould shape is cube. 6. The drug-loaded or live bacteria-encapsulating delivery complex prepared by the method of any one of claims 1-5.

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