LU502172B1 - Preparation method of enteric hybrid hydrogel based on tyrosine - Google Patents

Abstract

A preparation method of enteric hybrid hydrogel based on tyrosine: mixing the caustic soda solution of tyrosine with gelatin, uniformly stirring at 50-57 degrees celsius at a speed of 40-80 rpm until the gelatin solid is completely dissolved, performing ultrasonic treatment for 10-50 s, then adjusting the pH to below 6.5, oscillating at a speed of 1000-1200 rpm for 10-30 s, and performing ultrasonic treatment for 10-30 s. The hybrid hydrogel prepared by the invention has pH responsiveness, can be gelled in acid at above 33 degrees celsius, and can be sol in neutral or alkaline environment, meeting the requirements of enteric intelligent materials. The preparation method of that invention has simple step and low cost, can be gelled only by mix and stirring, is not easy to deteriorate, is environment-friendly, and can keep the colloidal state in the simulated gastric juice environment and dissolve in the simulated intestinal environment.

Description

DESCRIPTION LU502172

PREPARATION METHOD OF ENTERIC HYBRID HYDROGEL BASED ON TYROSINE

TECHNICAL FIELD The invention relates to a preparation method of enteric hybrid hydrogel based on tyrosine.

BACKGROUND At present, some substances (such as drugs, enzyme preparations, lactic acid bacteria, etc.) that play an active role in the intestinal tract will be destroyed by gastric juice when taken orally, and cannot reach the intestinal tract to play a role (the PH value of human stomach is about 2.0, while that of small intestine is about 7.4-7.6). To solve this problem, at present, the design of embedded materials for oral intestinal release is a hot and difficult point at home and abroad. At present, the embedding materials for intestinal release are mostly cellulose, poly (vinyl ether)-maleic acid copolymer, formaldehyde gelatin, acrylic acid-methacrylic acid copolymer, shellac, etc. However, there are some problems in these materials: cellulose materials, poly (methyl vinyl ether-maleic acid) copolymer, formaldehyde gelatin, etc. need to be modified on the basic structure, so the operation is complicated and there are many processes; acrylic acid-methacrylic acid copolymer materials are brittle, so it is necessary to add plasticizer to gel. The disintegration of shellac in intestinal tract is unstable.

Hydrogel is a semi-solid colloidal substance prepared from gel factor and water. In recent years, supramolecular hydrogels with peptides or amino acids as gel factors have attracted much attention. Because the preparation process of polypeptide hydrogel is generally in an aqueous environment, hydrogel factors self-assemble to form a three-dimensional network structure through intermolecular non-chemical bond forces (hydrogen bond, ionic bond, hydrophobic interaction, -x stacking, Van der Waals force, etc.), thereby binding water molecules to form a semi-solid jelly-like gel. Therefore, polypeptide hydrogel has better biocompatibility and biodegradability than other supramolecular hydrogels and polymer gels, and it can self-assemble into colloid, with low manufacturing cost and simple operation.

Commercial amino acids include commercial amino acids with protection, generally tert-butoxycarbonyl (Boc) protected amino acids and N-fluorene methoxycarbonyl (Fmoc) protected amino acids. These protected amino acids have been industrialized and are mainly used in the synthesis of peptides or protein. Since R. Bruce Merrifield, the Nobel Prize winner 14502172 chemistry in 1984, developed the solid-phase synthesis method of peptides, the artificial synthesis of peptides has been industrialized rapidly. Up to now, most commercial customized polypeptide chains have been obtained by peptide solid-phase synthesis technology using Fmoc-protected amino acids. The commercialization of Fmoc amino acids has greatly promoted the research and application of peptides and protein.

As a member of polypeptide hydrogel factor, amino acid or short peptide hydrogel of N- fluorene methoxycarbonyl (Fmoc) has many excellent properties, such as good biocompatibility, biodegradability, chemical modification, low cost and easy preparation, and has been widely used in cell culture, tissue engineering, drug sustained release, biomedicine and other fields. Fmoc-Y(Fmoc-Tyrosine, N-fluorene methoxycarbonyl-tyrosine), as a member of N-fluorene methoxycarbonyl amino acid, is aromatic and can self-assemble under the action of x-x stacking and hydrogen bonding. The gel formed by commercial tyrosine self-assembly has special correspondence to pH: it can gel in acidic conditions and sol in neutral alkaline environment (Aufderhorst-Roberts, et al, Soft Matter, 2012, 8, 5940-5946). However, commercial tyrosine hydrogels have low mechanical properties and large pore size, which makes them unable to resist the damage of acid molecules in gastric acid to embedded substances (such as enzymes). Therefore, the hydrogel formed by commercial tyrosine alone can't achieve the effect of protecting the embedding substance in gastric juice and releasing the embedding substance in intestinal tract.

Gelatin is a macromolecular protein obtained by partial acid hydrolysis (type A gelatin), alkali hydrolysis (type B gelatin) and enzymatic hydrolysis and purification of collagen. Gelatin has a very stable molecular structure, can absorb water and swell to form hydrogel, and has good biocompatibility and taste, and is widely used as a food functional substance and nutritional ingredient. At the same time, the unique macromolecular protein structure of gelatin also makes it have the ability to resist gastric acid. However, its thermal stability is poor, it has melted at 37°C of normal body temperature, and it has no environmental responsiveness, which limits its application as an embedding material for oral intestinal release alone.

SUMMARY

The technical problem to be solved by the invention is to overcome the above-mentionéd/5021 72 defects of the existing embedding materials and provide a preparation method of tyrosine-based enteric hybrid hydrogel. The obtained enteric hybrid hydrogel is gelled at above 33°C under acidic conditions and sold under neutral or alkaline conditions.

A further technical problem to be solved by the present invention is to overcome the above defects of the existing embedding materials and provide a preparation method of tyrosine-based enteric hybrid hydrogel with good biocompatibility, simple preparation method and low cost.

The technical scheme adopted by the invention to solve the technical problems is as follows: A preparation method of enteric hybrid hydrogel based on tyrosine is prepared by stirring and mixing tyrosine solution and gelatin.

The mass ratio of tyrosine to gelatin in the tyrosine solution is 1: 1-18 (preferably 1: 12-16).

Further, the tyrosine is preferably N- fluorene methoxycarbonyl-tyrosine (fmoc- tyrosine, Fmoc-Tyr-OH for short).

The gelatin can be obtained through commercial channels.

Furthermore, the tyrosine solution contains edible caustic soda, which is a caustic soda solution of tyrosine.

Further, the invention comprises the following steps: mixing the caustic soda solution of tyrosine with gelatin, uniformly stirring at 50-57°C at a speed of 40-80 rpm until the gelatin solid is completely dissolved, performing ultrasonic treatment for 10-50 s, adjusting the pH to below

6.5 (preferably 1.5-6.4), and oscillating at a speed of 1000-1200 rpm for 10-30 seconds, standing at 0-50°C for more than 10 minutes, and gelling.

Furthermore, the concentration of tyrosine in the caustic soda solution of tyrosine is

1.05-147.5 mg/mL. If the concentration of tyrosine is lower than 1.05 mg/mL, it is not conducive to the gelation of the later hybrid hydrogel, and affects its pH response. However, if the concentration of tyrosine is higher than 147.5 mg/mL, the concentration of tyrosine in the solution will be inaccurate because of its low solubility, and it will be mixed unevenly with gelatin, which will eventually lead to uneven hybrid hydrogel and unstable properties.

Furthermore, edible caustic soda is added into the tyrosine solution to adjust the pH, and the added amount of edible caustic soda is 8-12% of the tyrosine contained in the tyrosine solution.

When the concentration of edible caustic soda is less than 8% of tyrosine, the solubility 61502172 tyrosine will be reduced; when the concentration of edible caustic soda is more than 12% of tyrosine, the pH of the solution will be affected, and finally the formation of hybrid hydrogel will be affected.

Furthermore, the mass ratio of tyrosine to gelatin in the tyrosine solution is 1: 1-18, and when the proportion of gelatin is lower than this ratio, the acid resistance of hybrid hydrogel is not high; when the proportion of gelatin is higher than this ratio, the temperature tolerance of hybrid hydrogel decreases, that is, it will gel at 37°C.

Further, that gel temperature is between 0 and 50°C; when the temperature is lower than 0°C or higher than 50°C, the hybrid adhesive is difficult to form.

The principle of the invention is that under acidic conditions, tyrosine and gelatin self-assemble to form a three-dimensional network structure through the synergistic action of aromatic accumulation and hydrogen bonds, so as to bind water molecules, enzymes or drug molecules. However, under neutral or alkaline conditions, the charge carried by commercial tyrosine changes, the intermolecular repulsion force increases, and the repulsion force is greater than the synergistic force of aromatic accumulation and hydrogen bond, which leads to the collapse of the cross-linked structure related to tyrosine, thus leading to the dissolution of colloid.

The enteric hybrid hydrogel based on tyrosine prepared by the invention has the following characteristics: above 33°C, the colloid can form a colloid in an acidic environment, and is not affected by acidic solution or simulated gastric juice; when the hybrid hydrogel is put into a neutral or alkaline solution, or a simulated intestinal solution, the gel will automatically dissolve. Therefore, the hybrid hydrogel prepared by the invention can coat dyes, enzymes and drug molecules such as direct red 80, and its gelling property is not affected by these dyes, enzymes or drug molecules. Therefore, the invention is expected to be applied to the intestinal tract controlled release of active substances such as drugs or enzymes which are easily damaged by gastric acid and need to reach the intestinal tract for controlled release.

The invention has the following beneficial effects: (1) The hybrid hydrogel prepared by the invention has pH responsiveness, can gel in acid at above 33°C (the temperature at which gelatin can't gel), and can be sol in neutral or alkaline environment, meeting the requirements of enteric intelligent materials. The preparation methdd/5021 72 of that invention has simple step and low cost, can be gelled only by mix and stirring, is not easy to deteriorate, is environment-friendly, and can keep the colloidal state in the simulated gastric juice environment and dissolve in the simulated intestinal environment.

(2) The hybrid hydrogel prepared by the invention can wrap dyes, enzymes or drug molecules such as direct red 80, and its gel property is not affected by these dyes, enzymes or drug molecules. In addition, in view of the good biocompatibility of tyrosine and gelatin, the hybrid hydrogel prepared by the invention is expected to be applied to the field of intestinal controlled release of active substances such as drugs (such as enzymes, acid-intolerant lactic acid bacteria, etc.).

(3) The hybrid hydrogel prepared by the invention can still exist in high concentration salt solution, that is, the ionic strength has little effect on its gelation. Therefore, the hydrogel is a salt-tolerant hydrogel.

(4) The preparation method of the invention is simple in steps, low in cost, difficult to deteriorate and environment-friendly.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a digital photo of tyrosine/gelatin in different proportions. A-E are hybrid hydrogels formed when the concentration of tyrosine is 10 mg/mL, the mass concentrations of gelatin are 10, 50, 100, 140, 180 mg/ml, and the pH is 5.5. F is the photo of gel corresponding to A after adding buffer solution with pH 7.5, and so on, and G-J is the digital photo of hybrid hydrogel corresponding to B-E after adding buffer solution with pH 7.5.

Fig. 2 is a scanning electron microscope (SEM) image of the hybrid hydrogel prepared in Example 1 (the mass ratio of tyrosine to gelatin is 1:8). (As can be seen from the figure, there are 10-50 um pores in the microstructure of tyrosine/gelatin hybrid hydrogel, which can be used to embed dyes, enzymes or drugs).

Fig. 3 shows the release curves of direct red 80 dye embedded in the hybrid hydrogel of Example 3 at different pH values. (from top to bottom, the pH values are 9.0, 7.5, 7.0, 6.0 and

1.7 in turn).

Fig. 4 is the activity of lactase treated by different treatment methods measured in Example 4 of the present invention;

Unembedded lactase and gelatin-embedded lactase are the retention rates of enzyme activit#J5021 72 in the simulated gastric juice after adding the simulated gastric juice. Tyrosine-embedded lactase and hybrid hydrogel-embedded lactase can simulate the retention rate of lactase activity in intestinal juice by simulating gastric juice and adding simulated intestinal juice.

DESCRIPTION OF THE INVENTION The invention will be further explained with reference to the following examples and drawings.

The tyrosine used in the embodiment of the invention is N- fluorene methoxycarbonyl-tyrosine (fmoc- tyrosine, Fmoc-Tyr-OH for short), which is purchased from Jill Biochemical (Shanghai) Co., Ltd.; other chemical reagents used, unless otherwise specified, are obtained through conventional commercial channels.

Example 1 The preparation method of enteric hybrid hydrogel based on tyrosine in this embodiment includes the following steps: weighing 4 mg of edible caustic soda, adding ImL of deionized water, and fully dissolving to obtain caustic soda solution, taking 40 mg of fmoc- tyrosine powder, adding it into caustic soda solution, dissolving it by ultrasonic, adding 320 mg gelatin, putting it in a magnetic stirrer at 50°C and stirring it evenly at a speed of 60 rpm until the gelatin solid is completely dissolved, performing ultrasonic treatment for 10 s, adjusting the pH to 5.5 with hydrochloric acid solution, oscillating with a vortex shaker at 1000 rpm for 10 s, and standing in a water bath at 37°C for 0.5 h.

Example 2 The preparation method of enteric hybrid hydrogel based on tyrosine in this embodiment includes the following steps: weighing 1 mg of edible caustic soda, adding ImL of deionized water, fully dissolving, taking 10 mg of fmoc- tyrosine powder, adding into caustic soda solution, dissolving by ultrasonic, adding 140 mg of gelatin, putting it /min a magnetic stirrer at 50°C for 60 rpm, stirring it evenly until the gelatin solid is completely dissolved, ultrasonic for 20 s, adjusting the pH value to 5.5 with hydrochloric acid solution, shaking it with a vortex shaker at 1000rpm for 10 s, standing at room temperature for 70 minutes to obtain the enteric hybrid hydrogel based on tyrosine (see Figure 1, D), adding a phosphate buffer solution with a pH of 7.5 to the hybrid hydrogel, and colloidal sol after 1h (see Figure 1, L).

Example 3 LU502172 The preparation method of tyrosine-based enteric hybrid hydrogel in this embodiment includes the following steps (tyrosine/gelatin hybrid hydrogel embeds direct red 80 dye, and studies the release effect of hybrid gel on direct red 80 dye at different pH): weighing 2 mg of edible caustic soda, adding 1mL of deionized water, and after fully dissolving, taking 20 mg of fmoc- tyrosine powder, adding into caustic soda solution, dissolving by ultrasonic, adding 100 mg gelatin, uniformly stirring in a magnetic stirrer at 50°C at a speed of 60 rpm until the gelatin solid is completely dissolved, performing ultrasonic treatment for 20 s, adjusting the pH to 5.5 with hydrochloric acid solution, and quickly adding 0.7 mmol/L of direct red 80 dye, after shaking for 10 s with a vortex shaker at 1000rpm and standing at room temperature for 100 minutes, the enteric hybrid hydrogel with direct red 80 was formed. The enteric hybrid hydrogel coated with direct red 80 dye was placed in buffer solutions with pH values of 1.7, 6, 7, 7.5 and 9, respectively, and the absorbance of the solutions was detected by ultraviolet spectrophotometer at 525nm every 30 minutes. Compared with the absorbance of the same concentration of direct red 80 dye without embedding at 525nm, the release effect of hybrid glue on embedded direct red 80 dye in buffer solutions with different pH values was detected (see Figure 3).

Example 4 The preparation method of enteric hybrid hydrogel based on tyrosine in this embodiment includes the following steps (embedding lactase in hybrid hydrogel, and studying the release effect of hybrid gel on lactase at different pH): weighing 6 mg of edible caustic soda, adding ImL of deionized water, fully dissolving, taking 60mg of fmoc- tyrosine powder, adding into caustic soda solution, ultrasonic dissolving, adding 300 mg of gelatin, putting it /min a magnetic stirrer at 50°C and stirring at a speed of 60 rpm until the gelatin solid is completely dissolved, after ultrasonic treatment for 20 s, adjusting the pH value to 6 with hydrochloric acid solution, quickly adding 0.5 mL of 10 mg/mL lactase aqueous solution, oscillating for 10s with a vortex shaker at 1000 rpm, and standing at 37°C for 120 minutes to obtain the hybrid hydrogel embedded with lactase.

Taking 0.5 g of hybrid hydrogel embedding lactase, 0.5 g of tyrosine hydrogel embedding the same amount of lactase (only tyrosine embedding), 0.5 g of gelatin block embedding the same amount of lactase (only gelatin embedding) and 0.5 g of single lactase embedding the same amount (the amount of lactase added in each test tube is the same) in this example, putting it inl4)502172 test tube containing 5 mL of simulated gastric juice (corresponding to test tubes No.1, No.2, No.3 and No.4, respectively), and putting it in a water bath shaker at 37°C and 100rpm/min for 2 h.

The results showed that the gelatin block embedded with the same amount of lactase had dissolved at 37°C. The enzymatic activity of lactase in the simulated gastric juice (No.4 test tube) added with an unencapsulated single lactase and the simulated gastric juice (No.3 test tube) added with gelatin blocks embedded with the same amount of lactase was directly determined by o-nitrobenzene -B-D- galactoside method.

After the same simulated gastric juice treatment, the lactase gel block embedded with tyrosine hydrogel (No.2 test tube) and the lactase gel block embedded with hybrid hydrogel (No.1 test tube) did not dissolve. Therefore, take out the gel block in simulated gastric juice and put it in Sml of simulated intestinal juice with pH 7.5 (corresponding to test tubes 5 and 6, respectively). After the gel block is completely dissolved at 37°C, the enzyme activity in test tube solutions 5 and 6 is determined by o-nitrobenzene -B-D- galactoside method.

The enzyme activity of the same amount of lactase without simulated gastric juice treatment is 100% in triplicate, and the relative enzyme activity is calculated, which is the enzyme activity retention rate (see Figure 4 for the results).

Claims (4)

CLAIMS LU5021 72

1. A preparation method of enteric hybrid hydrogel based on tyrosine, characterized by stirring and mixing tyrosine solution and gelatin, wherein the mass ratio of tyrosine to gelatin in the tyrosine solution is 1:1-18, and the tyrosine solution contains edible caustic soda, which is the caustic soda solution of tyrosine; the preparation method comprises the following steps: mixing the caustic soda solution of tyrosine with gelatin, uniformly stirring at 50-57°C at a speed of 40-80 revolutions per minute until the gelatin solid is completely dissolved, performing ultrasonic treatment for 10-50 seconds, then adjusting the pH to below 6.5, oscillating at a speed of 1000-1200 revolutions per minute for 10-30 seconds, and standing at 0-50°C, wherein in the caustic soda solution, the concentration of tyrosine is 1.05-147.5 mg/ml, and the added amount of edible caustic soda in the tyrosine solution is 8-12% of the tyrosine contained in the tyrosine solution.

2. The preparation method of enteric hybrid hydrogel based on tyrosine according to claim 1, characterized in that the mass ratio of tyrosine to gelatin in the tyrosine solution is 1:12-16.

3. The preparation method of enteric hybrid hydrogel based on tyrosine according to claim 1 or 2, characterized in that the tyrosine is N-fluorene methoxycarbonyl-tyrosine.

4. The preparation method of tyrosine-based enteric hybrid hydrogel according to claim 1 or 2, characterized in that the pH is adjusted to 1.5-6.4.