Hydrogel material as well as preparation method, application and use method thereof

Abstract

The invention discloses a hydrogel material as well as a preparation method, application and a use method thereof. The preparation method comprises the following steps: (1) reacting chitosan with a compound containing double bonds to obtain modified chitosan; the substitution degree of the modified chitosan is 25 to 80 percent; (2) reacting the modified chitosan with a chelating ligand to obtain a hydrogel material; the content of the chelating ligand in the hydrogel material is 3-9wt%. The hydrogel material obtained by the preparation method disclosed by the invention has the advantages of moderate degradation time, high photocuring speed and low cytotoxicity.

Description

Technical Field

[0001] The invention relates to a hydrogel material and a preparation method, application and using method thereof. Background Art

[0002] Tumors pose a serious threat to human health, and the development of treatments for them is a key research area in medicine. Brain tumors, due to their complex and critical environment, pose significant challenges to their treatment. Surgery is a common treatment option. While it can directly remove visible tumor tissue, complete excision is difficult due to the unclear boundaries of brain tumors, leaving residual tumor cells as a potential risk of recurrence. Radiotherapy, while capable of killing tumor cells with radiation, is ineffective for larger tumors, and excessive radiation can damage healthy brain tissue. To overcome the limitations of single-agent treatments, combined surgical and radiotherapy approaches have emerged. Radiotherapy, as a postoperative adjuvant therapy, effectively kills residual tumor cells, prevents recurrence and metastasis, and has become a crucial approach for improving the efficacy of brain tumor treatment. On the one hand, local administration of radiotherapy drugs before cranial closure requires ensuring that they effectively cross the blood-brain barrier and reach the target site. On the other hand, administration of radiotherapy drugs after cranial closure requires ensuring that they effectively cross the blood-brain barrier and reach the target site. Otherwise, not only will they fail to achieve effective treatment, but they may also damage more healthy tissue.

[0003] Hydrogel is a material with a three-dimensional network structure, composed of cross-linked water-soluble polymers, which can absorb and retain a large amount of water in water without dissolving. This characteristic makes hydrogel widely used in the fields of biomedicine, environmental engineering, smart materials, etc. CN101550200A discloses a macrocyclic polyamine-coupled chitosan gene carrier, which is substituted with a macrocyclic polyamine compound on the chitosan. The macrocyclic polyamine compound is coupled to the 2-amino group of chitosan with an amide bond, or to the 6-hydroxyl group of chitosan with an ester bond. CN107973866A discloses a tetraazamacrocyclic manganese complex modified chitosan composite material with high SOD enzyme activity, the chemical name of which is tetraazamacrocyclic manganese complex modified chitosan composite material, and the chemical formula is (C6H 10 NO4) 2n (MnC 16 H 26 H4O6) m The above chitosan materials cannot be photocured. Summary of the Invention

[0004] In view of this, one object of the present invention is to provide a method for preparing a hydrogel material. The hydrogel material obtained by this preparation method has a moderate degradation time, a fast photocuring speed, and low cytotoxicity. Another object of the present invention is to provide a hydrogel material. Another object of the present invention is to provide a use of the hydrogel material. Yet another object of the present invention is to provide a method for using the hydrogel material.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions.

[0006] In one aspect, the present invention provides a method for preparing a hydrogel material, comprising the following steps:

[0007] (1) reacting chitosan with a double bond-containing compound to obtain modified chitosan;

[0008] The double bond-containing compound is selected from one or more of acrylic acid, methacrylic acid, acrylic anhydride, methacrylic anhydride, acrylate polyethylene glycol succinimide ester, methacrylate polyethylene glycol succinimide ester, 3-butenoic acid, 2,5-dioxo-1-pyrrolidyl ester;

[0009] The degree of substitution of the modified chitosan is 25 to 80%;

[0010] (2) reacting the modified chitosan with a chelating ligand to obtain a hydrogel material;

[0011] The chelating ligand is selected from one or more of ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, nitrilotriacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and 1,4,7-triazacyclo-1,4,7-triacetic acid;

[0012] The content of the chelating ligand in the hydrogel material is 3-9 wt%.

[0013] According to the preparation method of the present invention, preferably, the chitosan has a deacetylation degree of ≥90% and a weight-average molecular weight of 100-400 kDa.

[0014] According to the preparation method of the present invention, preferably, in step (1), the reaction is carried out in the presence of water, the reaction temperature is 40-70° C., and the reaction time is 1-5 h.

[0015] The preparation method according to the present invention preferably further comprises the following steps:

[0016] The first reaction product obtained by reacting chitosan with the double bond-containing compound is placed in a dialysis bag for dialysis, and then filtered and freeze-dried in sequence to obtain modified chitosan;

[0017] The dialysis bag can pass substances with a molecular weight of 20 kDa or less.

[0018] According to the preparation method of the present invention, preferably, the chelating ligand is a chelating ligand activated by N-hydroxysuccinimide.

[0019] According to the preparation method of the present invention, preferably, the chelating ligand, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are reacted to obtain the chelating ligand activated by N-hydroxysuccinimide.

[0020] According to the preparation method of the present invention, preferably, in the step (2), the reaction is carried out in the presence of water, the reaction temperature is 15 to 35° C., and the reaction time is 8 to 18 hours.

[0021] In another aspect, the present invention provides a hydrogel material, which is prepared by the above preparation method.

[0022] In another aspect, the present invention provides use of the hydrogel material in treating brain tumors.

[0023] In another aspect, the present invention provides a method for using the hydrogel material, comprising the following steps:

[0024] The hydrogel material is mixed with a photoinitiator and then exposed to light.

[0025] The hydrogel material of the present invention has a moderate degradation time, a fast light curing speed, and low cytotoxicity. The hydrogel material of the present invention can be loaded with radionuclides and can be used for the treatment of brain tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a microscopic image of the lung cancer cell line LLC after being cultured in the presence of the hydrogel material of Example 2-3 for 48 hours.

[0027] Figure 2 This is a microscopic image of fibroblast 3T3 cells cultured in the presence of the hydrogel material of Example 2-3 for 48 hours.

[0028] Figure 3 This is a microscope image of microglial cells BV2 after being cultured in the presence of the hydrogel material of Example 2-3 for 48 hours.

[0029] Figure 4 This is a microscopic image of the lung tumor cell line LLC after being cultured in the presence of the hydrogel material of Comparative Example 2-2 for 48 hours.

[0030] Figure 5 This is a microscopic image of fibroblast 3T3 cells cultured in the presence of the hydrogel material of Comparative Example 2-2 for 48 hours.

[0031] Figure 6 This is a microscope image of microglial cells BV2 after being cultured in the presence of the hydrogel material of Comparative Example 2-2 for 48 hours. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0033] <Method for preparing hydrogel material>

[0034] The preparation method of the hydrogel material of the present invention comprises the following steps: (1) double bond modification step; and (2) chelate ligand modification step. Each step is described in detail below.

[0035] Double bond modification steps

[0036] The invention reacts chitosan with a double bond-containing compound to obtain modified chitosan.

[0037] The deacetylation degree of chitosan may be ≥90%. The weight average molecular weight of chitosan may be 100-400 kDa, preferably 150-350 kDa, and more preferably 190-300 kDa.

[0038] The double-bond-containing compound can be selected from one or more of acrylic acid, methacrylic acid, acrylic anhydride, methacrylic anhydride, polyethylene glycol succinimide acrylate, polyethylene glycol succinimide methacrylate, and 3-butenoic acid, 2,5-dioxo-1-pyrrolidyl ester. Preferably, the double-bond-containing compound is selected from one or more of acrylic acid, methacrylic acid, acrylic anhydride, and methacrylic anhydride. According to one embodiment of the present invention, the double-bond-containing compound is acrylic anhydride.

[0039] Chitosan and the double bond-containing compound react in the presence of water. The mass volume ratio of chitosan to water can be 0.8:(70-130) g / mL, preferably 0.8:(80-120) g / mL, and more preferably 0.8:(90-110) g / mL.

[0040] The reaction temperature may be 40 to 70°C, preferably 45 to 65°C, and more preferably 50 to 60°C.

[0041] The reaction time may be 1 to 5 hours, preferably 2 to 4 hours, and more preferably 3 to 4 hours.

[0042] The mass volume ratio of chitosan to the double bond-containing compound can be 0.8:(0.3-1.8) g / mL, preferably 0.8:(0.5-1.5) g / mL, and more preferably 0.8:(0.8-1.2) g / mL, which can ensure that the hydrogel material has a moderate degradation time.

[0043] In certain embodiments, the method further comprises the following steps: placing the first reaction product obtained by reacting chitosan with the double bond-containing compound into a dialysis bag for dialysis, and then sequentially filtering and freeze-drying to obtain modified chitosan.

[0044] The dialysis bag can pass substances with a molecular weight of 20 kDa or less; preferably, the dialysis bag can pass substances with a molecular weight of 14 kDa or less.

[0045] The dialysis can be performed at a temperature of 15 to 35° C., preferably 20 to 30° C. The dialysis time can be 3 to 12 days, preferably 5 to 10 days, and more preferably 7 to 8 days.

[0046] The degree of substitution of the modified chitosan may be 25-80%, preferably 35-75%, and preferably 50-58%, so that the hydrogel material has a moderate degradation time.

[0047] Steps for chelating ligand modification

[0048] The invention reacts the modified chitosan with a chelating ligand to obtain a hydrogel material.

[0049] The chelating ligand is selected from ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, nitrilotriacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and 1,4,7-triazacyclo-1,4,7-triacetic acid, preferably 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0050] Preferably, the chelating ligand is a chelating ligand activated by N-hydroxysuccinimide. Specifically, the chelating ligand, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are reacted to obtain the chelating ligand activated by N-hydroxysuccinimide.

[0051] The molar ratio of the chelating ligand to 1-ethyl-(3-dimethylaminopropyl)carbodiimide may be 1:(0.7-1.9), preferably 1:(0.9-1.7), and more preferably 1:(1.3-1.6).

[0052] The molar ratio of the chelating ligand to N-hydroxysuccinimide may be 1:(0.7-1.9); preferably 1:(0.9-1.7); and more preferably 1:(1.3-1.6).

[0053] The chelating ligand, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are reacted in the presence of water.

[0054] The mass volume ratio of the chelating ligand and water can be (20-100):10 mg / mL; preferably (40-80):10 mg / mL; more preferably (50-70):10 mg / mL.

[0055] The reaction temperature may be 15 to 35° C., preferably 20 to 30° C., and more preferably 22 to 28° C. The reaction may be carried out under stirring.

[0056] The reaction time may be 0.1 to 2 hours, preferably 0.3 to 1.5 hours, and more preferably 0.5 to 1 hour.

[0057] The mass ratio of the chelating ligand to the modified chitosan is (30-90):0.8 mg / g; preferably (40-80):0.8 mg / g; and more preferably (70-80):0.8 mg / g. This can reduce the cytotoxicity of the hydrogel material and improve the hydrogel's ability to chelate radionuclides.

[0058] The chelating ligand and the modified chitosan can react in the presence of water. The mass volume ratio of the modified chitosan to water can be 0.8:(30-70) g / mL, preferably 0.8:(40-60) g / mL.

[0059] The reaction temperature may be 15 to 35° C., preferably 20 to 30° C., and more preferably 22 to 28° C. The reaction may be carried out under stirring.

[0060] The reaction time may be 8 to 18 hours, preferably 10 to 15 hours, and more preferably 12 to 13 hours.

[0061] In certain embodiments, the method further comprises the following steps: placing the second reaction product obtained by the reaction of the modified chitosan with the chelating ligand into a dialysis bag for dialysis, and then filtering and freeze-drying in sequence to obtain a hydrogel material.

[0062] The dialysis bag can pass substances with a molecular weight of 20 kDa or less; preferably, the dialysis bag can pass substances with a molecular weight of 14 kDa or less.

[0063] The dialysis can be performed at a temperature of 15 to 35° C., preferably 20 to 30° C. The dialysis time can be 1 to 5 days, preferably 2 to 4 days, and more preferably 3 to 4 days.

[0064] The content of the chelating ligand in the hydrogel material is 3-9 wt%, preferably 4-8 wt%, and more preferably 6-8 wt%, which enables the hydrogel to have lower cytotoxicity and good radionuclide loading capacity.

[0065] <Hydrogel Materials and Their Uses>

[0066] The hydrogel material of the present invention is prepared by adopting the above method.

[0067] The hydrogel material of the present invention has a moderate degradation time, a fast photocuring speed, and low cytotoxicity. Furthermore, the modified chelating ligand in the hydrogel material of the present invention is capable of chelating radionuclides. Therefore, the hydrogel material of the present invention can be used to treat brain tumors.

[0068] <How to use hydrogel>

[0069] The present invention mixes the hydrogel material with a photoinitiator and then irradiates the mixture with light.

[0070] The photoinitiator can be selected from benzophenone, 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexylphenyl ketone, phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone), phenyl dimethyl ketal. Preferably, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt (LAP).

[0071] The hydrogel material and the photoinitiator can undergo a photocuring reaction in the presence of water or a phosphate buffer solution.

[0072] The amount of the photoinitiator used can be 0.2 wt% to 1.0 wt% of the mass of the hydrogel material; preferably 0.3 wt% to 0.7 wt%; more preferably 0.4 wt% to 0.6 wt%.

[0073] A light source with a wavelength of 200 to 500 nm can be used for irradiation to initiate the curing reaction. Preferably, the wavelength of the light source is 300 to 450 nm. More preferably, the wavelength of the light source is 350 to 420 nm.

[0074] The irradiation time may be 5 to 30 seconds, preferably 5 to 15 seconds.

[0075] Examples 1-1 to 1-3 and Comparative Examples 1-1 to 1-2

[0076] 0.8 g chitosan (deacetylation degree ≥ 90%, M WThe chitosan mixture was mixed with 100 mL of deionized water to obtain a chitosan mixture. Methacrylic anhydride was added to the chitosan mixture, and then reacted at 55° C. for 3 hours to obtain a first reaction product.

[0077] 300 mL of deionized water was added to the first reaction product, which was then placed in an 8-14 kDa dialysis bag and dialyzed at 25°C for 7 days. The contents of the dialysis bag were collected to obtain the dialyzed first reaction product. The dialyzed first reaction product was vacuum filtered and then freeze-dried to obtain modified chitosan (methacryloylated chitosan).

[0078] The substitution degrees of methacrylic anhydride and modified chitosan are shown in Table 1.

[0079] The degree of substitution was tested using the trinitrobenzenesulfonic acid (TNBS) method. This method utilizes the quantitative reaction between primary amino groups and TNBS. TNBS reacts with unmodified primary amino groups (i.e., free amino groups) on chitosan under weakly alkaline conditions to form an intermediate complex containing a chromophore. The number of free amino groups on unmodified and methacryloylated chitosan was determined using a UV-visible spectrophotometer, thereby calculating the degree of substitution at different methacrylic anhydride dosages.

[0080] Table 1

[0081]

[0082]

[0083] Examples 2-1 to 2-5 and Comparative Examples 2-1 to 2-4

[0084] 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and 10 mL of deionized water were mixed and then reacted at 25°C with stirring for 0.5 h to obtain NHS-DOTA.

[0085] NHS-DOTA was mixed with 50 mL of a mixture of modified chitosan and water, and then reacted at 25°C with stirring for 12 hours to produce a second reaction product. The modified chitosan content in the mixture of modified chitosan and water was 0.8 g. The second reaction product was placed in an 8-14 kDa dialysis bag and dialyzed at 25°C for 3 days. The contents of the dialysis bag were collected to obtain the dialyzed second reaction product. The dialyzed second reaction product was filtered and then freeze-dried to obtain a hydrogel material.

[0086] The amounts of DOTA, EDC, and NHS, the types of modified chitosan, and the content of chelating ligands in the hydrogel materials are shown in Table 2 .

[0087] The chelating ligand content was determined using the trinitrobenzenesulfonic acid (TNBS) method. This method utilizes the quantitative reaction between primary amino groups and TNBS. TNBS reacts with unmodified primary amino groups (i.e., free amino groups) on chitosan under weakly alkaline conditions to form an intermediate complex containing a chromophore. The number of free amino groups in the methacryloylated chitosan modified with the chelating ligand is determined using a UV-visible spectrophotometer, thereby calculating the chelating ligand content at different reaction dosages.

[0088] Table 2

[0089]

[0090]

[0091] Experimental example

[0092] 1. The hydrogel material was prepared into a hydrogel solution with a concentration of 250 mg / mL using sterile water for injection. The LAP photoinitiator was prepared into a photoinitiator solution with a concentration of 6 mg / mL using sterile water for injection. 1 mL of the photoinitiator solution was drawn into a disposable sterile syringe and injected into 5 mL of the hydrogel solution, and the mixture was shaken for 10 minutes to obtain a mixture. The mixture was irradiated with a 405 nm light source at a distance of 5 cm from the surface of the mixture. The time required from the start of irradiation to the complete curing of the hydrogel (curing time) was recorded. The results are shown in Table 3.

[0093] 2. The cured hydrogel obtained by the method in 1 above was placed in a lysozyme solution in PBS (lysozyme concentration: 1.5 μg / mL) and incubated at 37°C on a shaker with gentle agitation. The lysozyme solution in PBS was refreshed daily to simulate continuous enzyme activity. The time required for complete degradation of the cured hydrogel (degradation time) was recorded. The results are shown in Table 3.

[0094] Table 3

[0095]

[0096]

[0097] 3. Prepare a 2.5 mg / mL LAP initiator standard solution in phosphate buffer. Add the LAP initiator standard solution to the hydrogel material and shake to fully soak the hydrogel. Heat in a dark water bath at 60-70°C for 30 minutes, shaking several times during heating. Immediately sterilize the suspension using a 0.22 μm sterile syringe filter to obtain a 50 mg / mL hydrogel suspension.

[0098] Collect cells and prepare a cell suspension using resuspension buffer preheated at 37°C. Add 50-100 μL of the cell suspension to each well of a 96-well plate. Irradiate the cell suspension with a 405 nm light source to gel the cell suspension. Add DMEM medium to each well and incubate at 37°C in an incubator for 5 minutes. After washing the sample, remove the medium and add fresh medium for a further 48 hours. Observe the cells under a microscope.

[0099] Figure 1-3 The following is a microscopic image of the lung cancer cell line LLC, fibroblast 3T3 and microglial cell BV2 cultured in the presence of the hydrogel material of Example 2-3 after 48 hours. Figure 1-3 It can be seen that the cells are in good condition after 48 hours of culture.

[0100] Figure 4-6 The following is a microscope image of the lung cancer cell line LLC, fibroblast 3T3 and microglial cell BV2 cultured in the presence of the hydrogel material of Comparative Example 2-2 after 48 hours. Figure 4-6 It can be seen that the hydrogel material of Comparative Example 2-2 exhibits greater cytotoxicity.

[0101] The present invention is not limited to the above-mentioned embodiments. Any modification, improvement, or substitution that can be conceived by those skilled in the art without departing from the essential content of the present invention shall fall within the scope of the present invention.

Claims

1. A method for preparing a hydrogel material, characterized in that: The steps include: (1) reacting chitosan with a double bond-containing compound to obtain modified chitosan; The double bond-containing compound is selected from one or more of acrylic acid, methacrylic acid, acrylic anhydride, methacrylic anhydride, acrylate polyethylene glycol succinimide ester, methacrylate polyethylene glycol succinimide ester, 3-butenoic acid, 2,5-dioxo-1-pyrrolidyl ester; The degree of substitution of the modified chitosan is 25 to 80%; (2) reacting the modified chitosan with a chelating ligand to obtain a hydrogel material; The chelating ligand is selected from one or more of ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, nitrilotriacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and 1,4,7-triazacyclo-1,4,7-triacetic acid; The content of the chelating ligand in the hydrogel material is 3-9 wt%.

2. The preparation method according to claim 1, characterized in that The chitosan has a deacetylation degree of ≥90% and a weight-average molecular weight of 100-400 kDa.

3. The preparation method according to claim 1, characterized in that In step (1), the reaction is carried out in the presence of water, the reaction temperature is 40 to 70° C., and the reaction time is 1 to 5 hours.

4. The preparation method according to claim 1, characterized in that The following steps are also included: The first reaction product obtained by reacting chitosan with the double bond-containing compound is placed in a dialysis bag for dialysis, and then filtered and freeze-dried in sequence to obtain modified chitosan; The dialysis bag can pass substances with a molecular weight of 20 kDa or less.

5. The preparation method according to claim 1, characterized in that The chelating ligand is a chelating ligand activated by N-hydroxysuccinimide.

6. The preparation method according to claim 5, characterized in that The chelating ligand, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are reacted to obtain the chelating ligand activated by N-hydroxysuccinimide.

7. The preparation method according to claim 1, characterized in that In the step (2), the reaction is carried out in the presence of water, the reaction temperature is 15 to 35° C., and the reaction time is 8 to 18 hours.

8. A hydrogel material, characterized in that: The hydrogel material is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the hydrogel material according to claim 8 in treating brain tumors.

10. The method for using the hydrogel material according to claim 8, characterized in that: The steps include: The hydrogel material is mixed with a photoinitiator and then exposed to light.

Images