A poly(ethylene glycol) macroporous crystalline hydrogel containing clay, a preparation method thereof and applications thereof

Abstract

The present invention belongs to the technical field of hydrogel preparation, and particularly relates to a poly(ethylene glycol) macroporous hydrogel containing clay, and a preparation method and application thereof. The hydrogel is prepared from a poly(ethylene glycol) derivative, a multi-amino crosslinking agent and clay. By adding clay to a poly(ethylene glycol) derivative solution, then adding a crosslinking agent, mixing and freezing at -20°C, and then thawing at 2-8°C, the hydrogel is obtained. The hydrogel medium of the present invention has a relatively high modulus, uniform size and can be regenerated.

Description

Technical Field

[0001] The invention belongs to the technical field of crystal gel preparation, and specifically relates to a polyethylene glycol (PEG) macroporous crystal gel containing clay and a preparation method thereof. Background Art

[0002] Hydrogel has great application prospects in the biomedical field due to its excellent biocompatibility. However, its pore size is only at the nanometer level, which limits its application in three-dimensional cell culture, chromatography and other fields. Crystal gel is a porous hydrogel, the most notable feature of which is the interconnected ultra-large pores, which are usually prepared by freezing. The unique structure of crystal gel makes it almost unimpeded to absorb solutes in the diffusion solution, and is not limited by size. Therefore, crystal gel is often used in chromatography, cell culture and other fields. Crystal gel also has good elasticity. When subjected to a certain pressure, even if the crystal gel is compressed to 50% of its original thickness, it can be restored by adsorbing solvents. It can also be stored dry and soaked to recover when it is used; crystal gel can also be used multiple times after rinsing.

[0003] Crystal gel has been studied for use in the fields of tissue repair, cell delivery, wound dressings, and tumor immunotherapy. In this type of application, cells and tissues are usually required to infiltrate / grow inside the material, and the size of the cells is between ten microns and tens of microns. The large pores inside the crystal gel are crucial to the infiltration and growth of cells, and can improve the effect of tissue repair or treatment by promoting nutrient transfer, cell-to-cell interaction, etc. Referring to the patent document with publication number CN113842494B, Fan Daidi et al. of Northwest University used polydopamine, chitosan, collagen, etc. to make crystal gel in a patent on an injectable hemostatic crystal gel that promotes tissue regeneration and its preparation method and application. The stress of the prepared crystal gel in the first 60% of the deformation is less than 5KPa, and its compression modulus is less than 10KPa. Referring to the patent document with publication number CN110551353A, Zhang Xufeng and Sun Lifen of Yunnan Normal University provided a large-volume and non-breakable crystal gel material and its preparation method and application, and the stress of the prepared crystal gel at a strain of about 60% is less than 20KPa.

[0004] In the application process of crystal gel, especially when used as a material for in vivo implantation, the crystal gel material needs to withstand greater physiological pressure. For example, when used as a subcutaneous implant, the pressure on the crystal gel will affect its original spatial dimensions, thereby affecting its function. When repairing cartilage, muscle or bone tissue, the material needs to have a certain ability to withstand mechanical loads. In particular, previous studies have found that the hardness of the gel has an important influence on the formation of bone tissue, and materials with higher hardness can promote the osteogenic differentiation of cells. However, the ultra-large pores of the crystal gel lead to low mechanical strength of the crystal gel. Therefore, the development of crystal gel with both a macroporous structure and excellent mechanical properties has important application value. Summary of the Invention

[0005] The object of the present invention is to overcome the problems existing in the prior art and provide a poly(ethylene glycol) macroporous hydrogel containing clay and a preparation method thereof. The macroporous hydrogel provided by the present invention is based on a polyethylene glycol-clay gel system, and significantly improves the mechanical properties of the hydrogel on the premise of realizing large pore diameters. The compression modulus of the prepared hydrogel can reach 130 kPa.

[0006] The preparation of the macroporous hydrogel comprises the following steps: First, use water as a solvent to prepare a polymer monomer solution with a mass fraction of 3%-5%, and then use the polymer monomer solution to prepare a clay mineral or phyllosilicate solution with a mass fraction of 1%-5%, and stir well to dissolve it as solution A; It should be noted that the clay minerals referred to in the present invention are hydrous aluminum silicate minerals with a layered structure; Then, use water as a solvent to prepare an aqueous crosslinker solution with a mass concentration of 0.31725%, and adjust the pH to 9.03 as solution B; After mixing the above solution A and solution B in equal volumes, place them in a freezer at -20°C for 4 days, and then thaw them at 2-8°C for 1 day to obtain the macroporous hydrogel.

[0007] The polymer monomer is a poly(ethylene glycol) aldehyde derivative, that is, an aldehyde-terminated multi-arm polyethylene glycol, and the chemical structural formula of the aldehyde-terminated multi-arm polyethylene glycol is: Where the number of arms n is a positive integer. Preferably, the number of arms n = 3-8;

[0008] The crosslinker is a polyamino compound, and the polyamino compound is preferably polylysine or polyethyleneimine;

[0009] Preferably, the polymer monomer is octa-armed benzaldehyde polyethylene glycol, that is, the number of arms n = 8;

[0010] Preferably, the specific method for adjusting the pH to 9.03 is: adding an aqueous sodium hydroxide solution to the aqueous crosslinker solution to adjust the pH to 9.03.

[0011] Preferably, the thawing temperature is set at 4°C.

[0012] In the prior art, a prepolymer solution generally consists of polymer monomers, crosslinking agents, and solvents (usually water). The solvents are divided into bound solvents and free solvents. Bound water has a strong interaction with the solvent, while free water does not contain solute molecules. In the present invention, polyethylene glycol derivatives are used as polymer monomers, polyamino compounds are used as crosslinking agents, and clay is added as a mechanical enhancer. The solvent is water. Under freezing conditions, the free water in the prepolymer solution forms ice crystals, and the bound water remains in the liquid phase. Polymer molecules and bound water are squeezed into the voids of the ice crystals, and low-temperature concentration increases the polymer concentration, forming a non-frozen liquid microphase. At a relatively high polymer concentration and appropriate pH, the polymer molecules react rapidly in the bound water and polymerize to form a three-dimensional network, i.e., the polymer monomers crosslink. Then, after thawing, the ice crystals serving as templates gradually melt, leaving interconnected macroporous structures in situ. The present invention selects clay or layered silicate as the nanosheet enhancer. Due to its nanoscale size and no chemical bond connection with the polymer, it does not affect the final pore size. Therefore, compared with the prior art, the macroporous cryogel prepared in this application has a relatively high compressive modulus, can resist higher pressures while maintaining the internal pore structure, and is particularly suitable for use as a tissue support material for subcutaneous implantation, cartilage, muscle, or bone tissue repair material. Description of the Drawings

[0013] Figure 1 SEM photograph of cryogel 1 prepared in the specific embodiment;

[0014] Figure 2 SEM photograph of cryogel 3 prepared in the specific embodiment;

[0015] Figure 3 SEM photograph of cryogel 4 prepared in the specific embodiment. Specific Embodiment

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below with reference to the accompanying drawings. The technical solutions in the embodiments of the present invention are part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are exemplary and are intended to explain the present invention and should not be construed as a limitation to the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0017] The following embodiments of the present invention include the following three embodiments, namely cryogel 2, cryogel 3, and cryogel 4. To demonstrate the advantages of the present invention, six comparative examples, namely cryogel 1, cryogel 5, cryogel 6, cryogel 7, cryogel 8, and cryogel 9, are set up.

[0018] The raw material octa-arm phenylaldehyde-based polyethylene glycol used in the following examples and comparative examples was purchased from Shanghai Pengshuo, polyethyleneimine was purchased from Aladdin, and polylysine was purchased from Aladdin; the clay was purchased from Laponite (BYK, Germany); the universal tensile testing machine involved in the test examples was model HY-0580, from Shanghai Hengyi;

[0019] Crystal gel 1

[0020] First, prepare an octa-arm phenylaldehyde-based polyethylene glycol solution with a mass fraction of 5% using water as solution A. Then, prepare an aqueous polylysine solution with a concentration of 0.31725% using water, and add an aqueous sodium hydroxide solution to adjust the pH to 9.03 as solution B. After mixing the above solutions A and B in equal volumes, place them in a -20°C refrigerator and freeze for 4 days, then place them in a -4°C freezer to thaw for 1 day to obtain the crystal gel medium.

[0021] Crystal gel 2

[0022] First, prepare an octa-arm phenylaldehyde-based polyethylene glycol solution with a mass fraction of 5% using water, and use the above solution to prepare a 1% clay solution, stir well to dissolve, as solution A. Then, prepare an aqueous polylysine solution with a concentration of 0.31725% using water, and add an aqueous sodium hydroxide solution to adjust the pH to 9.03 as solution B. After mixing the above solutions A and B in equal volumes, place them in a -20°C refrigerator and freeze for 4 days, then place them in a -4°C freezer to thaw for 1 day to obtain the crystal gel medium.

[0023] Crystal gel 3

[0024] First, prepare an octa-arm phenylaldehyde-based polyethylene glycol solution with a mass fraction of 5% using water, and use the above solution to prepare a 3% clay solution, stir well to dissolve, as solution A. Then, prepare an aqueous polylysine solution with a concentration of 0.31725% using water, and add an aqueous sodium hydroxide solution to adjust the pH to 9.03 as solution B. After mixing the above solutions A and B in equal volumes, place them in a -20°C refrigerator and freeze for 4 days, then place them in a -4°C freezer to thaw for 1 day to obtain the crystal gel medium.

[0025] Crystal gel 4

[0026] First, prepare an octa-arm phenylaldehyde-based polyethylene glycol solution with a mass fraction of 5% using water, and use the above solution to prepare a 5% clay solution, stir well to dissolve, as solution A. Then, prepare an aqueous polylysine solution with a concentration of 0.31725% using water, and add an aqueous sodium hydroxide solution to adjust the pH to 9.03 as solution B. After mixing the above solutions A and B in equal volumes, place them in a -20°C refrigerator and freeze for 4 days, then place them in a -4°C freezer to thaw for 1 day to obtain the crystal gel medium.

[0027] Crystal gel 5

[0028] First, prepare an octa - arm phenylaldehyde - terminated polyethylene glycol solution with a mass fraction of 5% using water, and use the above - mentioned solution as Solution A. Then, prepare an aqueous polylysine solution with a concentration of 0.31725% using water, and add an aqueous sodium hydroxide solution to adjust the pH to 9.03 as Solution B. After mixing the above - mentioned Solution A and Solution B in equal volumes, place them in a - 20 °C refrigerator and freeze for 1 day, then place them in a - 4 °C environment to thaw for 1 day to obtain the hydrogel medium.

[0029] Hydrogel 6

[0030] First, prepare an octa - arm phenylaldehyde - terminated polyethylene glycol solution with a mass fraction of 5% using water, and use the above - mentioned solution to prepare a clay solution with a mass fraction of 1%, stir well to dissolve, and use it as Solution A. Then, prepare an aqueous polylysine solution with a concentration of 0.31725% using water, and add an aqueous sodium hydroxide solution to adjust the pH to 9.03 as Solution B. After mixing the above - mentioned Solution A and Solution B in equal volumes, place them in a - 20 °C refrigerator and freeze for 1 day, then place them in a - 4 °C environment to thaw for 1 day to obtain the hydrogel medium.

[0031] Hydrogel 7

[0032] First, prepare an octa - arm phenylaldehyde - terminated polyethylene glycol solution with a mass fraction of 5% using water, and use the above - mentioned solution to prepare a clay solution with a mass fraction of 3%, stir well to dissolve, and use it as Solution A. Then, prepare an aqueous polylysine solution with a concentration of 0.31725% using water, and add an aqueous sodium hydroxide solution to adjust the pH to 9.03 as Solution B. After mixing the above - mentioned Solution A and Solution B in equal volumes, place them in a - 20 °C refrigerator and freeze for 1 day, then place them in a - 4 °C environment to thaw for 1 day to obtain the hydrogel medium.

[0033] Hydrogel 8

[0034] First, prepare an octa - arm phenylaldehyde - terminated polyethylene glycol solution with a mass fraction of 5% using water, and use the above - mentioned solution to prepare a clay solution with a mass fraction of 5%, stir well to dissolve, and use it as Solution A. Then, prepare an aqueous polylysine solution with a concentration of 0.31725% using water, and add an aqueous sodium hydroxide solution to adjust the pH to 9.03 as Solution B. After mixing the above - mentioned Solution A and Solution B in equal volumes, place them in a - 20 °C refrigerator and freeze for 1 day, then place them in a - 4 °C environment to thaw for 1 day to obtain the hydrogel medium.

[0035] Hydrogel 9

[0036] First, prepare an octa - arm phenylaldehyde - terminated polyethylene glycol solution with a mass fraction of 3% using water, and use the above - mentioned solution to prepare a clay solution with a mass fraction of 1%, stir well to dissolve, and use it as Solution A. Then, prepare an aqueous polylysine solution with a concentration of 0.31725% using water, and add an aqueous sodium hydroxide solution to adjust the pH to 9.03 as Solution B. After mixing the above - mentioned Solution A and Solution B in equal volumes, place them in a - 20 °C refrigerator and freeze for 1 day, then place them in a - 4 °C environment to thaw for 1 day to obtain the hydrogel medium.

[0037] Effect of the Concentration of Clay and Freezing Time on the Mechanical Properties of the Gelatin Hydrogel

[0038] Using a universal tensile testing machine at a speed of 6 mm / min, the stress-strain curves of Examples 1-4 were tested. The compression modulus was calculated by taking the slope of the curve corresponding to the first 10% of the strain. The results are shown in Table 1. Clay has an obvious enhancing effect on the mechanical properties of the gelatin hydrogel. In the range of low concentrations (1%-3%), the strength of the gelatin hydrogel does not change much with the change in clay concentration. However, when the clay concentration increases to 5%, the compression modulus of the gelatin hydrogel increases by an order of magnitude. Continuing to increase the clay concentration to 8% increases the liquid viscosity, making it easy to form bubbles during mixing and difficult to remove during the preparation of the gelatin hydrogel.

[0039] Table 1: Relationship between the Compression Modulus of the Gelatin Hydrogel and the Clay Concentration

[0040] Cryogel material Clay concentration Freezing time (days) Compression modulus (kPa) Cryogel 1 0％ 4 5.3 Cryogel 2 1％ 4 11.8 Cryogel 3 3％ 4 13.8 Cryogel 4 5％ 4 137.4

[0041] Test Example 2: Effect of Freezing Time on the Mechanical Properties of the Gelatin Hydrogel

[0042] Using a universal tensile testing machine at a speed of 6 mm / min, the stress-strain curves of the above-mentioned Gelatin Hydrogel 2, Gelatin Hydrogel 4, Gelatin Hydrogel 6, and Gelatin Hydrogel 8 were tested. The compression modulus was calculated by taking the slope of the curve corresponding to the first 10% of the strain. The results are shown in Table 2. For the gelatin hydrogel prepared by freezing for one day, even when the clay concentration reaches 5%, its compression modulus does not increase significantly. This shows that the freezing time is a key parameter in the preparation of the gelatin hydrogel, and one day of freezing is not sufficient for the stability of the gelatin hydrogel structure.

[0043] Table 2: Compression Modulus of Gelatin Hydrogels Prepared at Different Freezing Times

[0044] Cryogel material Clay concentration Freezing time (days) Compression modulus (kPa) Cryogel 6 1％ 1 4.2 Cryogel 2 1％ 4 11.8 Cryogel 8 5％ 1 3.9 Cryogel 4 5％ 4 137.4

[0045] Test Example 3: In Vitro Degradation of the Gelatin Hydrogel

[0046] The prepared Gelatin Hydrogels 1-9 were placed in PBS buffer at 5 times the mass and observed for degradation in a 37°C water bath shaker. The gelatin hydrogels were taken out regularly and weighed. The swelling rate was calculated according to the mass change, i.e., swelling rate = (real-time mass of the taken-out gelatin hydrogel - initial mass of the gelatin hydrogel) / initial mass of the gelatin hydrogel, as shown in Table 3. The clay concentration has a great influence on the degradation stability of the gelatin hydrogel. When the clay concentration reaches 5%, the mass of the gelatin hydrogel after 7 days is almost the same as the initial mass. At the same time, the concentration of polyethylene glycol is also a key factor affecting degradation. When the concentration drops to 3%, even when the clay concentration reaches 5%, the gelatin hydrogel degrades significantly within 7 days. As shown in Table 4, when the freezing time increases to 4 days, the degradation rate of the gelatin hydrogel significantly slows down. By adjusting the formula and process, the degradation of the gelatin hydrogel can be adjusted within the time range of several days to several months to meet the requirements of different clinical applications for material degradation.

[0047] Table 3 Influence of the composition of the hydrogel on its degradation

[0048] Cryogel material Clay concentration Polyethylene glycol concentration Mass change after 7 days 5 0％ 5％ -71％ 6 1％ 5％ -58％ 7 3％ 5％ -49％ 8 5％ 5％ -2％ 9 5％ 3％ -51％

[0049] Table 4 Extending the freezing time to 4 days can slow down the degradation of the hydrogel

[0050] Cryogel material Clay concentration Polyethylene glycol concentration Mass change after 7 days Mass change after 35 days 1 0％ 5％ -15.31％ +0.62％ 2 1％ 5％ -35.70％ -25.97％ 3 3％ 5％ -28.98％ -32.93％ 4 5％ 5％ +2.53％ -7.66％

[0051] Test Example 4 Internal pore analysis of the hydrogel

[0052] As Figure 1 、 Figure 2 and Figure 3 shown, the internal structure photos of the above-mentioned hydrogel 1, hydrogel 3, and hydrogel 4 are respectively shown. After measurement and statistics, the average values of the internal pore sizes of the above-mentioned hydrogel 1, hydrogel 3, and hydrogel 4 are shown in Table 5 below;

[0053] Table 5: Hydrogel pore size

[0054] Cryogel material Cryogel pore / μm Relative deviation / μm Cryogel 1 119.7 30.6 Cryogel 3 107.3 27.1 Cryogel 4 104.2 18.3

[0055] Comparing the pore sizes in hydrogel 1, hydrogel 3, and hydrogel 4, it can be seen that: with the increase of the clay content in the hydrogel, the change in the pores of the hydrogel is not significant; it can be considered that clay can increase the modulus of the hydrogel within a certain concentration range and does not affect the pore size and connectivity of the hydrogel.

Claims

1. A poly(ethylene glycol) macroporous crystalline gel containing clay, characterized in that, The macroporous hydrogel is obtained through the following preparation process: First, water is used as a solvent to prepare a polymer monomer solution with a mass fraction of 3% - 5%. Then, the polymer monomer solution is used to prepare a clay mineral or phyllosilicate solution with a mass fraction of 1% - 5%, and the solution is fully stirred and dissolved to obtain Solution A; the clay mineral refers to a hydrated aluminosilicate mineral with a layered structure. Then, water is used as a solvent to prepare an aqueous cross-linking agent solution with a mass concentration of 0.31725%, and the pH is adjusted to 9.03 to obtain Solution B. After mixing the above-mentioned Solution A and Solution B in equal volumes, the mixture is placed at -20°C and frozen for 4 days, and then thawed at 2 - 8°C for 1 day to obtain the macroporous hydrogel; The polymer monomer is a polyethylene glycol aldehyde derivative, namely an aldehyde-terminated multi-arm polyethylene glycol, and the chemical structural formula of the aldehyde-terminated multi-arm polyethylene glycol is: , where the number of arms n is a positive integer; The cross-linking agent is a polyamino compound.

2. A poly(ethylene glycol) macroporous crystalline gel containing clay according to claim 1, characterized in that, The number of arms n = 3 - 8.

3. A polyethylene glycol macroporous crystalline gel containing clay according to claim 1, characterized in that, The polyamino compound is polylysine or polyethyleneimine.

4. A polyethylene glycol macroporous crystal gel containing clay according to claim 1, characterized in that, The polymer monomer is octa-armed phenylaldehyde polyethylene glycol, namely the number of arms n = 8.

5. A polyethylene glycol macroporous crystal gel containing clay according to claim 1, characterized in that, The specific method for adjusting the pH to 9.03 is: adding an aqueous sodium hydroxide solution to the aqueous cross-linking agent solution to adjust the pH to 9.

03.

6. A polyethylene glycol macroporous crystalline gel containing clay according to claim 1, characterized in that, The thawing temperature is set at 4°C.

7. Use of a polyethylene glycol macroporous hydrogel containing clay according to any one of claims 1 to 6 in the preparation of a subcutaneous implant tissue support material.

8. Use of a polyethylene glycol macroporous hydrogel containing clay according to any one of claims 1 to 6 in the preparation of a cartilage, muscle or bone tissue repair support material.

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