A composite modified hydrogel preparation and preparation method thereof

Abstract

The present invention belongs to the field of biomedical materials, and more particularly relates to a composite modified hydrogel formulation and its preparation method. This composite modified hydrogel formulation comprises the following raw materials, calculated by weight: 0.8-1.5% bovine milk lipid microspheres grafted with hyaluronic acid, 0.1-0.3% modified magnesium-aluminum layered double hydroxide, and the balance deionized water. This composite modified hydrogel formulation exhibits high drug loading and excellent sustained-release properties.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a composite modified hydrogel preparation and a preparation method thereof. Background Art

[0002] Local injection is a commonly used clinical treatment strategy that can increase drug concentration at the lesion site and reduce systemic adverse reactions. However, this method has many limitations, such as low drug delivery efficiency, short tissue retention time, rapid metabolic rate, and insufficient bioavailability, necessitating frequent and repeated administration. This not only affects the therapeutic effect but also may increase the risk of complications such as bleeding and infection. Therefore, the development of efficient drug delivery systems is of great significance.

[0003] Hydrogels are water-insoluble polymers with a three-dimensional network structure. Their network contains numerous hydrophilic groups, allowing them to absorb a significant proportion of water in an aqueous environment while maintaining stable water retention. Their excellent biocompatibility also lends them to a wide range of applications in tissue engineering, drug delivery, and wound healing.

[0004] Natural polysaccharide hydrogels (such as chitosan, hyaluronic acid, and sodium alginate) are ideal drug delivery carrier materials due to their wide availability, low cost, and ease of functional modification. Notably, hyaluronic acid, as one of the few natural polysaccharides with inherent cell-targeting properties, can enhance drug delivery efficiency through receptor-mediated endocytosis, a property that makes it more flexible in drug delivery systems. However, natural hyaluronic acid hydrogels suffer from problems such as loose structure, low drug loading, and significant drug burst release. Therefore, it is necessary to modify hyaluronic acid hydrogels to address these issues. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a composite modified hydrogel preparation having a high drug loading capacity and excellent sustained-release performance.

[0006] A second object of the present invention is to provide a method for preparing a composite modified hydrogel preparation with a simple process.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A composite modified hydrogel preparation, composed of the following raw materials, calculated by mass percentage: 0.8-1.5% of bovine milk lipid microspheres grafted with hyaluronic acid, 0.1-0.3% of modified magnesium aluminum layered double hydroxide, and the balance being deionized water;

[0009] The preparation method of the milk lipid microspheres grafted with hyaluronic acid comprises the following steps:

[0010]

[0011] (1) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to an aqueous solution of hyaluronic acid and adjusting the pH to 5-6, then adding α-amino acid ethyl ester containing a side chain terminal amino group to react, adjusting the pH to 7-7.5 to terminate the reaction, and after the reaction is completed, dialyzing and freeze-drying the reaction solution to obtain intermediate 1;

[0012] The structural formula of the α-amino acid ethyl ester containing a side chain terminal amino group is as follows, wherein m=1 or 2 or 3:

[0013] ;

[0014] (2) adding the intermediate 1 of step (1) to an aqueous solution of methanol, and then adding lithium hydroxide to react. After the reaction is completed, adding acetone to the reaction solution to obtain a precipitate, adding water to the precipitate to redissolve it, adjusting the pH to 4-5, dialyzing, and freeze-drying to obtain the intermediate 2;

[0015] (3) Irradiating the bovine milk exosomes to obtain bovine milk lipid microspheres; adding the intermediate 2 of step (2) to the PBS solution of the bovine milk lipid microspheres, and then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to react. After the reaction is completed, centrifuging and resuspending the precipitate to obtain the bovine milk lipid microspheres grafted with hyaluronic acid.

[0016] Preferably, the molar ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and α-amino acid ethyl ester containing a side chain terminal amino group in step (1) is 1:(50-55):(50-55):(50-55).

[0017] Preferably, the concentration of hyaluronic acid in the aqueous solution of hyaluronic acid in step (1) is 3-5 mg / mL; the molecular weight of the hyaluronic acid is 100-150 kDa; and the reaction time is 40-48 h.

[0018] Preferably, the molar ratio of the intermediate 1 to lithium hydroxide in step (2) is 1:(1-3); the concentration of the intermediate 1 in the methanol aqueous solution is 8-10 mg / mL; the methanol aqueous solution is composed of methanol and deionized water in a volume ratio of 2:1; and the reaction time is 3-5 days.

[0019] Preferably, in step (3), the dosage ratio of the intermediate 2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and the PBS solution of bovine milk lipid microspheres is 1 mg: (2.5-2.8) mg: (1.5-1.7) mg: (0.5-1) mL; the concentration of the PBS solution of bovine milk lipid microspheres is 1 mg / mL; and the reaction time is 12-15 h.

[0020] Preferably, the preparation method of the modified magnesium-aluminum layered double hydroxide comprises the following steps:

[0021] (a) adding magnesium nitrate and aluminum nitrate to deionized water to obtain a mixed solution; adjusting the pH of the mixed solution to 8.5-9.5 with a sodium hydroxide solution, stirring the solution for reaction; after completion of the reaction, centrifuging, washing, and vacuum drying the reaction solution to obtain a magnesium-aluminum layered double hydroxide;

[0022] (b) adding the magnesium-aluminum layered double hydroxide prepared in step (a) to the sodium folinate solution and stirring the mixture for reaction. After the reaction is completed, the reaction solution is centrifuged, washed, and vacuum-dried to obtain the sodium folinate-intercalated magnesium-aluminum layered double hydroxide;

[0023] (c) adding dopamine to a Tris-HCl solution for prepolymerization to obtain a prepolymer solution; adding the sodium folinate intercalated magnesium aluminum layered double hydroxide of step (b) to the prepolymer solution for reaction; after the reaction is completed, centrifuging, washing, and vacuum drying the reaction solution to obtain the modified magnesium aluminum layered double hydroxide.

[0024] Preferably, in step (a), the concentration of magnesium nitrate in the mixed solution is 1 mol / L, the concentration of aluminum nitrate is 0.5-1 mol / L; the concentration of the sodium hydroxide solution is 1 mol / L; the temperature of the stirring reaction is 80-90° C., and the stirring reaction time is 18-25 hours.

[0025] Preferably, in step (b), the ratio of the magnesium-aluminum layered double hydroxide to the sodium folinate solution is (0.2-1) g: (50-300) mL; the concentration of the sodium folinate solution is 0.02-0.05 mol / L; and the stirring reaction time is 3-5 h.

[0026] Preferably, in step (c), the dosage ratio of dopamine, sodium folinate intercalated magnesium aluminum layered double hydroxide, and Tris-HCl solution is 1 mg: (6-15) mg: (3-5) mL; the prepolymerization time is 15-20 min; and the reaction time is 3-5 h.

[0027] The second object of the present invention is achieved by adopting the following technical solution:

[0028] The preparation method of the composite modified hydrogel preparation comprises the following steps:

[0029] Weigh the raw materials according to the mass percentages; add the milk lipid microspheres grafted with hyaluronic acid to deionized water and mix evenly; then add the modified magnesium aluminum layered double hydroxide and mix evenly.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The composite modified hydrogel formulation of the present invention contains bovine milk lipid microspheres grafted with hyaluronic acid and modified magnesium-aluminum layered double hydroxide. The combination of bovine milk lipid microspheres grafted with hyaluronic acid and modified magnesium-aluminum layered double hydroxide significantly improves the drug loading capacity and sustained-release performance of the hydrogel formulation.

[0032] 2. This invention first grafts the amino acid ethyl ester onto the hyaluronic acid molecular chain by amidating an α-amino acid ethyl ester containing a side-chain amino group with the carboxyl group of hyaluronic acid. A lithium hydroxide-catalyzed deesterification reaction then converts the ester groups in the amino acid ethyl ester into carboxyl groups. Finally, the newly formed carboxyl groups react with amino groups on the surface of bovine milk lipid microspheres to covalently couple the bovine milk lipid microspheres to the hyaluronic acid backbone, thereby modifying the hyaluronic acid. This modification process introduces bovine milk lipid microspheres, significantly increasing drug loading. This invention also introduces active groups, such as amino groups, into the modified magnesium-aluminum layered double hydroxide (LDH) by intercalating sodium folinate into the LDH. This modification not only enhances the affinity of the LDH for the hyaluronic acid grafted onto the bovine milk lipid microspheres, improving their dispersibility, but also synergizes with the LDH's layered structure through hydrogen bonding between the amino groups and drug molecules, further enhancing drug loading capacity.

[0033] 3. The present invention utilizes α-amino acid ethyl esters containing side chain terminal amino groups as a bridge to stably anchor bovine milk lipid microspheres to the hyaluronic acid backbone through covalent coupling. The resulting hydrogel has a porous spatial three-dimensional structure, which can achieve long-term sustained release of drugs. In addition, sodium folinate is intercalated into the magnesium-aluminum layered double hydroxide through a hydrothermal reaction, and further through in situ polymerization of dopamine, a polydopamine layer is formed on its surface. The surface-coated polydopamine layer not only helps to improve the dispersion stability of LDH, but its unique adhesiveness can also "anchor" drug molecules through intermolecular forces, thereby further improving the sustained release effect of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a TEM image of milk lipid microspheres;

[0035] Figure 2 is a TEM image of milk lipid microspheres grafted with hyaluronic acid prepared in Preparation Example 1;

[0036] Figure 3 is a TEM image of the product obtained in Preparation Example 4;

[0037] Figure 4 This is a diagram of the hydrogel preparation product obtained in Example 1 of the present invention;

[0038] Figure 5 is a SEM image of the composite modified hydrogel preparation of Example 1;

[0039] Figure 6 This is a particle size diagram of milk lipid microspheres grafted with hyaluronic acid prepared in Preparation Example 1;

[0040] Figure 7 This is a particle size diagram of the product obtained in Preparation Example 4;

[0041] Figure 8 This is a diagram of the particle size of milk lipid microspheres;

[0042] Figure 9 This is a graph showing the biocompatibility results of the composite modified hydrogel preparation of Example 1 of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the various embodiments described below or the various technical features can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0044] The bovine milk lipid microspheres of the present invention are obtained by irradiating bovine milk exosomes with a dose of 10 Kgy of cobalt 60 (TEM images of bovine milk lipid microspheres are shown in Figure 1 ).

[0045] (1) Preparation example

[0046] Preparation Example 1

[0047] This preparation example provides a milk lipid microsphere grafted with hyaluronic acid. The specific preparation process is as follows:

[0048]

[0049] (1) To a 4 mg / mL aqueous solution of hyaluronic acid (molecular weight 120 kDa) was added 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (CAS: 25952-53-8) and N-hydroxysuccinimide (CAS: 6066-82-6) at a molar ratio of 1:52:52:52 among hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and L-lysine ethyl ester. The pH was adjusted to 5.5 with 1 mol / L sodium hydroxide solution, and then L-lysine ethyl ester (CAS: 3844-53-9) was added. The reaction was allowed to proceed at room temperature for 42 h, and the pH was adjusted to 7.2 to terminate the reaction. After the reaction was completed, the reaction solution was dialyzed for 3 days and freeze-dried to obtain intermediate 1.

[0050] (2) The intermediate 1 of step (1) was added to a methanol aqueous solution (v / v=2:1) ​​at a molar ratio of 1:2. The concentration of the intermediate 1 in the methanol aqueous solution was 9 mg / mL. Lithium hydroxide was then added and the reaction was carried out at room temperature for 4 days. After the reaction was completed, an appropriate amount of acetone was added to the reaction solution to obtain a precipitate. The precipitate was redissolved in water, the pH was adjusted to 4.5, dialyzed for 3 days, and freeze-dried to obtain intermediate 2.

[0051] (3) The intermediate 2 of step (2) was added to a 1 mg / mL PBS solution of bovine milk lipid microspheres with the dosage ratio of 1 mg: 2.7 mg: 1.6 mg: 0.9 mL of the intermediate 2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and bovine milk lipid microspheres PBS solution. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added and reacted for 14 h. After the reaction was completed, the precipitate was centrifuged and resuspended to obtain the bovine milk lipid microspheres grafted with hyaluronic acid. The TEM image of the obtained bovine milk lipid microspheres grafted with hyaluronic acid is shown in FIG. Figure 2 .

[0052] Preparation Example 2

[0053] This preparation example provides a milk lipid microsphere grafted with hyaluronic acid. The specific preparation process is as follows:

[0054]

[0055] (1) To a 5 mg / mL aqueous solution of hyaluronic acid (molecular weight 150 kDa) was added 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (CAS: 25952-53-8) and N-hydroxysuccinimide (CAS: 6066-82-6) at a molar ratio of 1:55:55:55 among hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and L-ornithine ethyl ester. The pH was adjusted to 6 with 1 mol / L sodium hydroxide solution, and then L-ornithine ethyl ester (CAS: 4189-46-2) was added. The reaction was allowed to proceed at room temperature for 48 h, and the pH was adjusted to 7.5 to terminate the reaction. After the reaction was completed, the reaction solution was dialyzed for 3 days and freeze-dried to obtain intermediate 1.

[0056] (2) The intermediate 1 of step (1) was added to a methanol aqueous solution (v / v=2:1) ​​at a molar ratio of 1:3. The concentration of the intermediate 1 in the methanol aqueous solution was 10 mg / mL. Lithium hydroxide was then added and the reaction was carried out at room temperature for 5 days. After the reaction was completed, an appropriate amount of acetone was added to the reaction solution to obtain a precipitate. The precipitate was redissolved in water, the pH was adjusted to 5, dialyzed for 3 days, and freeze-dried to obtain intermediate 2.

[0057] (3) The intermediate 2 of step (2) was added to a 1 mg / mL PBS solution of bovine milk lipid microspheres with the dosage ratio of 1 mg:2.8 mg:1.7 mg:1 mL of the intermediate 2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and bovine milk lipid microspheres PBS solution. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added and reacted for 15 h. After the reaction was completed, the precipitate was centrifuged and resuspended to obtain the bovine milk lipid microspheres grafted with hyaluronic acid.

[0058] Preparation Example 3

[0059] This preparation example provides a milk lipid microsphere grafted with hyaluronic acid. The specific preparation process is as follows:

[0060]

[0061] (1) To a 3 mg / mL aqueous solution of hyaluronic acid (molecular weight 100 kDa) was added 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (CAS: 25952-53-8) and N-hydroxysuccinimide (CAS: 6066-82-6) at a molar ratio of 1:50:50:50 among hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and L-2,4-diaminobutyric acid ethyl ester. The pH was adjusted to 5 using 1 mol / L sodium hydroxide solution. Then, L-2,4-diaminobutyric acid ethyl ester (CAS: 1758-80-1) was added. The reaction was allowed to react at room temperature for 40 h. The pH was adjusted to 7 to terminate the reaction. After the reaction was completed, the reaction solution was dialyzed for 3 days and freeze-dried to obtain intermediate 1.

[0062] (2) The intermediate 1 of step (1) was added to a methanol aqueous solution (v / v=2:1) ​​at a molar ratio of 1:1. The concentration of the intermediate 1 in the methanol aqueous solution was 8 mg / mL. Lithium hydroxide was then added and the mixture was reacted at room temperature for 3 days. After the reaction was completed, an appropriate amount of acetone was added to the reaction solution to obtain a precipitate. The precipitate was redissolved in water, the pH was adjusted to 4, dialyzed for 3 days, and freeze-dried to obtain intermediate 2.

[0063] (3) The intermediate 2 of step (2) was added to a 1 mg / mL PBS solution of bovine milk lipid microspheres with the dosage ratio of 1 mg:2.5 mg:1.5 mg:0.5 mL of the intermediate 2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and bovine milk lipid microspheres PBS solution. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added and reacted for 12 hours. After the reaction was completed, the precipitate was centrifuged and resuspended to obtain the bovine milk lipid microspheres grafted with hyaluronic acid.

[0064] Preparation Example 4

[0065] The difference between this preparation example and preparation example 1 is that steps (1)-(2) are omitted and hyaluronic acid is directly used to graft bovine milk lipid microspheres, that is, the intermediate 2 in step (3) of preparation example 1 is replaced by hyaluronic acid; the TEM image of the product obtained in preparation example 4 is shown in FIG. Figure 3 .

[0066] Preparation Example 5

[0067] This preparation example provides a modified magnesium-aluminum layered double hydroxide, and the specific preparation process is as follows:

[0068] (a) dissolving magnesium nitrate and aluminum nitrate in deionized water to prepare a mixed solution having a magnesium nitrate concentration of 1 mol / L and an aluminum nitrate concentration of 0.7 mol / L; adjusting the pH of the mixed solution to 9 using a 1 mol / L sodium hydroxide solution; and stirring the solution at 85° C. for 20 hours. After the reaction, the reaction solution is centrifuged, washed, and vacuum-dried to obtain a magnesium-aluminum layered double hydroxide;

[0069] (b) adding the magnesium-aluminum layered double hydroxide prepared in step (a) to a 0.03 mol / L sodium folinate solution at a ratio of 0.5 g of magnesium-aluminum layered double hydroxide to 200 mL of sodium folinate solution, stirring and reacting for 4 h. After the reaction, the reaction solution was centrifuged, washed, and vacuum-dried to obtain a sodium folinate-intercalated magnesium-aluminum layered double hydroxide;

[0070] (c) adding dopamine to a Tris-HCl solution (pH = 8.5) with the amount ratio of dopamine, sodium folinate intercalated magnesium aluminum layered double hydroxide, and Tris-HCl solution being 1 mg:8 mg:4 mL, and prepolymerizing for 16 minutes to obtain a prepolymer solution; adding the sodium folinate intercalated magnesium aluminum layered double hydroxide of step (b) to the prepolymer solution and reacting for 4 hours. After the reaction is completed, the reaction solution is centrifuged, washed, and vacuum-dried to obtain the modified magnesium aluminum layered double hydroxide.

[0071] Preparation Example 6

[0072] This preparation example provides a modified magnesium-aluminum layered double hydroxide, and the specific preparation process is as follows:

[0073] (a) dissolving magnesium nitrate and aluminum nitrate in deionized water to prepare a mixed solution having a magnesium nitrate concentration of 1 mol / L and an aluminum nitrate concentration of 1 mol / L; adjusting the pH of the mixed solution to 9.5 using a 1 mol / L sodium hydroxide solution; and stirring the solution at 90° C. for 18 hours. After the reaction is complete, the reaction solution is centrifuged, washed, and vacuum-dried to obtain a magnesium-aluminum layered double hydroxide;

[0074] (b) adding the magnesium-aluminum layered double hydroxide prepared in step (a) to a 0.05 mol / L sodium folinate solution at a ratio of 1 g of magnesium-aluminum layered double hydroxide to 300 mL of sodium folinate solution, stirring and reacting for 5 h. After the reaction, the reaction solution was centrifuged, washed, and vacuum-dried to obtain a sodium folinate-intercalated magnesium-aluminum layered double hydroxide;

[0075] (c) adding dopamine to a Tris-HCl solution (pH = 8.5) with the amount ratio of dopamine, sodium folinate intercalated magnesium aluminum layered double hydroxide, and Tris-HCl solution being 1 mg:15 mg:5 mL, and prepolymerizing for 20 minutes to obtain a prepolymer solution; adding the sodium folinate intercalated magnesium aluminum layered double hydroxide of step (b) to the prepolymer solution and reacting for 5 hours. After the reaction is completed, the reaction solution is centrifuged, washed, and vacuum-dried to obtain the modified magnesium aluminum layered double hydroxide.

[0076] Preparation Example 7

[0077] This preparation example provides a modified magnesium-aluminum layered double hydroxide, and the specific preparation process is as follows:

[0078] (a) dissolving magnesium nitrate and aluminum nitrate in deionized water to prepare a mixed solution having a magnesium nitrate concentration of 1 mol / L and an aluminum nitrate concentration of 0.5 mol / L; adjusting the pH of the mixed solution to 8.5 using a 1 mol / L sodium hydroxide solution; and stirring the solution at 80° C. for 25 hours. After the reaction, the reaction solution is centrifuged, washed, and vacuum-dried to obtain a magnesium-aluminum layered double hydroxide;

[0079] (b) adding the magnesium-aluminum layered double hydroxide prepared in step (a) to a 0.02 mol / L sodium folinate solution at a ratio of 0.2 g:50 mL of the magnesium-aluminum layered double hydroxide to the sodium folinate solution, stirring and reacting for 3 h. After the reaction, the reaction solution was centrifuged, washed, and vacuum-dried to obtain a sodium folinate-intercalated magnesium-aluminum layered double hydroxide;

[0080] (c) adding dopamine to a Tris-HCl solution (pH = 8.5) with the amount ratio of dopamine, sodium folinate intercalated magnesium aluminum layered double hydroxide, and Tris-HCl solution being 1 mg:6 mg:3 mL, and prepolymerizing for 15 minutes to obtain a prepolymer solution; adding the sodium folinate intercalated magnesium aluminum layered double hydroxide of step (b) to the prepolymer solution and reacting for 3 hours. After the reaction is completed, the reaction solution is centrifuged, washed, and vacuum dried to obtain the modified magnesium aluminum layered double hydroxide.

[0081] Preparation Example 8

[0082] The difference between this preparation example and preparation example 5 is that step (b) is omitted, and the rest is the same as preparation example 5.

[0083] (2) Examples

[0084] Example 1

[0085] This embodiment provides a composite modified hydrogel preparation, which is composed of the following raw materials, calculated by mass percentage: 1% of the milk lipid microspheres grafted with hyaluronic acid of Preparation Example 1, 0.2% of the modified magnesium aluminum layered double hydroxide of Preparation Example 5, and the balance being deionized water.

[0086] This embodiment also provides a method for preparing the composite modified hydrogel preparation, which is as follows:

[0087] Weigh each raw material according to the mass percentage; add the milk lipid microspheres grafted with hyaluronic acid prepared in Preparation Example 1 to deionized water and mix evenly, then add the modified magnesium aluminum layered double hydroxide prepared in Preparation Example 5 and mix evenly; the obtained hydrogel preparation product is shown in FIG. Figure 4 , SEM images see Figure 5 .

[0088] Example 2

[0089] This example provides a composite modified hydrogel preparation, which is composed of the following raw materials, calculated by mass percentage: 1.5% of the milk lipid microspheres grafted with hyaluronic acid of Preparation Example 2, 0.1% of the modified magnesium aluminum layered double hydroxide of Preparation Example 6, and the balance being deionized water.

[0090] This embodiment also provides a method for preparing the composite modified hydrogel preparation, which is as follows:

[0091] Weigh the raw materials according to the mass percentages; add the milk lipid microspheres grafted with hyaluronic acid prepared in Preparation Example 2 to deionized water and mix evenly; then add the modified magnesium aluminum layered double hydroxide prepared in Preparation Example 6 and mix evenly.

[0092] Example 3

[0093] This example provides a composite modified hydrogel preparation, which is composed of the following raw materials, calculated by mass percentage: 0.8% of the milk lipid microspheres grafted with hyaluronic acid of Preparation Example 3, 0.3% of the modified magnesium aluminum layered double hydroxide of Preparation Example 7, and the balance being deionized water.

[0094] This embodiment also provides a method for preparing the composite modified hydrogel preparation, which is as follows:

[0095] Weigh the raw materials according to the mass percentages; add the milk lipid microspheres grafted with hyaluronic acid prepared in Preparation Example 3 to deionized water and mix evenly; then add the modified magnesium aluminum layered double hydroxide prepared in Preparation Example 7 and mix evenly.

[0096] (3) Comparative Example

[0097] Comparative Example 1

[0098] The difference between this comparative example and Example 1 is that the milk lipid microspheres grafted with hyaluronic acid in Preparation Example 4 are used instead of the milk lipid microspheres grafted with hyaluronic acid in Preparation Example 1, and the rest is the same as in Example 1.

[0099] Comparative Example 2

[0100] The difference between this comparative example and Example 1 is that the modified magnesium-aluminum layered double hydroxide of Preparation Example 5 is replaced by Preparation Example 8, and the rest is the same as Example 1.

[0101] (IV) Test Examples

[0102] Test Example 1

[0103] The particle size of the milk lipid microspheres grafted with hyaluronic acid and the milk lipid microspheres prepared in Preparation Example 1 was measured using a laser particle size analyzer. The results are shown in Figure 6 、 Figure 7 and Figure 8 .

[0104] Figure 6 This is a particle size diagram of milk lipid microspheres grafted with hyaluronic acid obtained in Preparation Example 1. Figure 7 This is a particle size diagram of the product obtained in Preparation Example 4. Figure 8 This is the particle size diagram of milk lipid microspheres. Figure 6 It can be seen that the average particle size of the milk lipid microspheres grafted with hyaluronic acid obtained in Preparation Example 1 is 99 nm. Figure 7 It can be seen that the average particle size of the product obtained in Preparation Example 4 is 79.8 nm. Figure 8 It can be seen that the average particle size of milk lipid microspheres is 72 nm.

[0105] Test Example 2

[0106] Indomethacin was added to the preparations of Examples 1-3 and Comparative Examples 1-2, respectively, at a mass ratio of indomethacin to preparation of 4:1 to obtain each group of drugs.

[0107] 5 mg of each drug group was accurately weighed and dissolved in 20 mL of methanol. The supernatant was filtered to obtain the absorbance of each supernatant at a wavelength of 318 nm. The indomethacin loading capacity of the preparation was calculated using the following formula based on the measured free indomethacin concentration in the supernatant. The results are shown in Table 1.

[0108] Drug loading rate (%) = (initial amount of indomethacin input - free indomethacin content) / total mass of preparation × 100%.

[0109] Table 1

[0110]

[0111] As shown in Table 1, the drug loading of the hydrogel preparations of Examples 1-3 of the present invention is maintained above 17%, but the drug loading of Comparative Examples 1-2 is reduced. The above test results show that the hydrogel preparations obtained by the present invention have a high drug loading capacity.

[0112] The present invention first grafts the amino acid ethyl ester onto the hyaluronic acid molecular chain by amidation reaction of an α-amino acid ethyl ester containing a side-chain amino group with the carboxyl group of hyaluronic acid. A lithium hydroxide-catalyzed deesterification reaction then converts the ester groups in the amino acid ethyl ester into carboxyl groups. Finally, the newly formed carboxyl groups react with amino groups on the surface of bovine milk lipid microspheres to covalently couple the microspheres to the hyaluronic acid backbone, thereby modifying the hyaluronic acid. This modification introduces bovine milk lipid microspheres, whose natural lipid bilayer structure can also serve as a drug carrier, significantly increasing drug loading. Furthermore, the present invention introduces active groups, such as amino groups, into magnesium-aluminum layered double hydroxide (LDH) by intercalating sodium folinate into the modified LDH. This modification not only enhances the affinity of LDH for the hyaluronic acid grafted onto the bovine milk lipid microspheres, improving their dispersibility, but also synergizes with the LDH's layered structure through hydrogen bonding between the amino groups and drug molecules, further enhancing drug loading capacity.

[0113] Test Example 3

[0114] The sustained-release effects of the hydrogel preparations obtained in Examples 1-3 and Comparative Examples 1-2 were tested as follows:

[0115] The drug groups obtained in Experimental Example 3 and an equivalent amount of indomethacin API as in the drug group in Example 1 were used as test samples. These samples were placed in pre-treated dialysis bags, which were then placed in an Erlenmeyer flask containing 50 mL of release medium: pH 7.4 phosphate buffer containing 0.2% sodium lauryl sulfate. Using a constant temperature shaker method, the Erlenmeyer flask was placed in a constant temperature water bath shaker at 100 rpm in a 37°C water bath. 1 mL of release medium was dispensed (and an equal amount of release medium was replenished at the same temperature) at 1, 4, 8, 12, 24, and 48 hours. UV absorbance was measured at 320 nm, and the cumulative drug release at each time point was calculated. The results are shown in Table 2.

[0116] Table 2

[0117]

[0118] As can be seen from Table 2, the hydrogel preparation obtained by the present invention has an excellent sustained-release effect. Compared with Example 1, the sustained-release effect of the hydrogel preparation obtained by Comparative Examples 1-2 is poor. The above results show that the combination of bovine milk lipid microspheres grafted with hyaluronic acid and modified magnesium aluminum layered double hydroxide can improve the sustained-release effect of the hydrogel preparation. This may be because: the present invention uses α-amino acid ethyl ester containing side chain terminal amino groups as a bridge to stably anchor the bovine milk lipid microspheres on the hyaluronic acid skeleton through covalent coupling. The formed hydrogel has a porous spatial three-dimensional structure and can achieve long-term sustained release of drugs. In addition, sodium folinate is intercalated into the magnesium aluminum layered double hydroxide through a hydrothermal reaction, and further through in situ polymerization of dopamine, a polydopamine layer is formed on its surface. The surface-coated polydopamine layer not only helps to improve the dispersion stability of LDH, but its unique adhesion can also "anchor" drug molecules through intermolecular forces, thereby further improving the sustained-release effect of the drug.

[0119] Test Example 4

[0120] Weigh 1 g of the hydrogel preparations prepared in Examples 1-3, add 2 mL of 0.1 mol / L phosphate buffer (pH 7.0) and 500 μL of 853 U / mL hyaluronidase solution, respectively. Mix thoroughly and place in a 37°C water bath. After 6, 12, and 24 hours, 500 μL of the mixture was mixed thoroughly with 1 mL of 0.1 mol / L phosphate buffer (pH 7.0). The mixture was then boiled to remove the enzyme and centrifuged to remove the supernatant. The uronic acid content was determined by measuring the absorbance at 530 nm using the carbazole colorimetric method. The hyaluronic acid content was converted to degraded hyaluronic acid content by multiplying by 2.07. This content was then converted to the enzymatic hydrolysis rate. Enzymatic hydrolysis rate = (degraded hyaluronic acid content / initial hyaluronic acid content) × 100%. The results are shown in Table 3.

[0121] Table 3

[0122]

[0123] As shown in Table 3, the hydrogel preparation obtained in the present invention has excellent in vitro resistance to enzymatic degradation.

[0124] Test Example 5

[0125] 100 μL of L929 fibroblasts (5 × 10 4DMEM complete medium (50 μg / mL) was placed in a 96-well plate, DMEM serum-free medium was used as a blank control, and DMEM complete medium supplemented with 5% phenol was used as a positive control. The 96-well plate was cultured in a cell culture incubator at 37°C and 5% CO2 for 12 hours, then the medium was discarded and 100 μL of 50 μg / mL of the composite modified hydrogel preparation of Example 1 was added. The cells were cultured in the cell culture incubator for another 24 hours. After the culture was completed, the cell morphology of each group was observed under a microscope. The results are shown in FIG. Figure 9 .

[0126] Figure 9 The biocompatibility results of the composite modified hydrogel preparation of Example 1 of the present invention are shown in FIG. Figure 9 It can be seen that compared with the blank control group, there is no significant difference in the fluorescence intensity of cells in the field of view of the experimental group (composite modified hydrogel preparation of Example 1). The above results show that the composite modified hydrogel preparation of Example 1 has excellent biocompatibility.

[0127] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A composite modified hydrogel preparation, characterized in that: The composition is composed of the following raw materials by mass percentage: 0.8-1.5% of bovine milk lipid microspheres grafted with hyaluronic acid, 0.1-0.3% of modified magnesium aluminum layered double hydroxide, and the balance is deionized water; The preparation method of the milk lipid microspheres grafted with hyaluronic acid comprises the following steps: (1) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to an aqueous solution of hyaluronic acid and adjusting the pH to 5-6, then adding α-amino acid ethyl ester containing a side chain terminal amino group to react, adjusting the pH to 7-7.5 to terminate the reaction, and after the reaction is completed, dialyzing and freeze-drying the reaction solution to obtain intermediate 1; The structural formula of the α-amino acid ethyl ester containing a side chain terminal amino group is as follows, wherein m=1 or 2 or 3: ； (2) adding the intermediate 1 of step (1) to an aqueous solution of methanol, and then adding lithium hydroxide to react. After the reaction is completed, adding acetone to the reaction solution to obtain a precipitate, adding water to the precipitate to redissolve it, adjusting the pH to 4-5, dialyzing, and freeze-drying to obtain the intermediate 2; (3) irradiating the bovine milk exosomes to obtain bovine milk lipid microspheres; adding the intermediate 2 of step (2) to the PBS solution of the bovine milk lipid microspheres, and then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to react. After the reaction is completed, centrifuging and resuspending the precipitate to obtain the bovine milk lipid microspheres grafted with hyaluronic acid; Sodium folinate is intercalated into a magnesium-aluminum layered double hydroxide through a hydrothermal reaction, and further dopamine is in situ polymerized to form a polydopamine layer on the surface of the magnesium-aluminum layered double hydroxide to obtain the modified magnesium-aluminum layered double hydroxide.

2. The composite modified hydrogel preparation according to claim 1, characterized in that: The molar ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and α-amino acid ethyl ester containing a side chain terminal amino group in step (1) is 1:(50-55):(50-55):(50-55).

3. The composite modified hydrogel preparation according to claim 1, characterized in that: The concentration of hyaluronic acid in the aqueous solution of hyaluronic acid in step (1) is 3-5 mg / mL; the molecular weight of the hyaluronic acid is 100-150 kDa; and the reaction time is 40-48 h.

4. The composite modified hydrogel preparation according to claim 1, characterized in that: The molar ratio of the intermediate 1 to lithium hydroxide in step (2) is 1:(1-3); the concentration of the intermediate 1 in the methanol aqueous solution is 8-10 mg / mL; the methanol aqueous solution is composed of methanol and deionized water in a volume ratio of 2:1; and the reaction time is 3-5 days.

5. The composite modified hydrogel preparation according to claim 1, characterized in that: In step (3), the dosage ratio of the intermediate 2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and the PBS solution of bovine milk lipid microspheres is 1 mg: (2.5-2.8) mg: (1.5-1.7) mg: (0.5-1) mL; the concentration of the PBS solution of bovine milk lipid microspheres is 1 mg / mL; and the reaction time is 12-15 h.

6. The composite modified hydrogel preparation according to claim 1, characterized in that: The preparation method of the modified magnesium-aluminum layered double hydroxide comprises the following steps: (a) adding magnesium nitrate and aluminum nitrate to deionized water to obtain a mixed solution; adjusting the pH of the mixed solution to 8.5-9.5 with a sodium hydroxide solution, stirring the solution for reaction; after completion of the reaction, centrifuging, washing, and vacuum drying the reaction solution to obtain a magnesium-aluminum layered double hydroxide; (b) adding the magnesium-aluminum layered double hydroxide prepared in step (a) to the sodium folinate solution and stirring the mixture for reaction. After the reaction is completed, the reaction solution is centrifuged, washed, and vacuum-dried to obtain the sodium folinate-intercalated magnesium-aluminum layered double hydroxide; (c) adding dopamine to a Tris-HCl solution for prepolymerization to obtain a prepolymer solution; The sodium folinate intercalated magnesium aluminum layered double hydroxide of step (b) is added to the prepolymer solution to carry out a reaction. After the reaction is completed, the reaction solution is centrifuged, washed, and vacuum dried to obtain the modified magnesium aluminum layered double hydroxide.

7. The composite modified hydrogel preparation according to claim 6, characterized in that: In step (a), the concentration of magnesium nitrate in the mixed solution is 1 mol / L, and the concentration of aluminum nitrate is 0.5-1 mol / L; the concentration of the sodium hydroxide solution is 1 mol / L; the temperature of the stirring reaction is 80-90° C., and the stirring reaction time is 18-25 hours.

8. The composite modified hydrogel preparation according to claim 6, characterized in that: In step (b), the ratio of the magnesium-aluminum layered double hydroxide to the sodium folinate solution is (0.2-1) g: (50-300) mL; the concentration of the sodium folinate solution is 0.02-0.05 mol / L; and the stirring reaction time is 3-5 hours.

9. The composite modified hydrogel preparation according to claim 6, characterized in that: In step (c), the dosage ratio of dopamine, sodium folinate intercalated magnesium aluminum layered double hydroxide, and Tris-HCl solution is 1 mg: (6-15) mg: (3-5) mL; the prepolymerization time is 15-20 minutes; and the reaction time is 3-5 hours.

10. The method for preparing the composite modified hydrogel preparation according to any one of claims 1 to 9, characterized in that: The steps include: Weigh the raw materials according to the mass percentages; add the milk lipid microspheres grafted with hyaluronic acid to deionized water and mix evenly; then add the modified magnesium aluminum layered double hydroxide and mix evenly.

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