Injectable temperature-sensitive hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable temperature-sensitive hydrogel as well as a preparation method and application thereof, and belongs to the technical field of hydrogel. The preparation method comprises the following steps: dissolving silk fibroin and beta-cyclodextrin in water, and carrying out cross-linking reaction on the silk fibroin and the beta-cyclodextrin to obtain a silk fibroin-beta-cyclodextrin solution; n-isopropylacrylamide monomer powder is added into the silk fibroin-beta-cyclodextrin solution, then a photoinitiator is added, ultraviolet irradiation is carried out, so that N-isopropylacrylamide is subjected to photopolymerization to form poly (N-isopropylacrylamide), poly (N-isopropylacrylamide) and silk fibroin-beta-cyclodextrin form a double-cross-linked network, and the temperature-sensitive hydrogel is obtained. The temperature-sensitive hydrogel is relatively low in viscosity at normal temperature, has injectability, and can be rapidly cured at about 35 DEG C. Experiments prove that the temperature-sensitive hydrogel has good biocompatibility, can be used as a drug release system, and can also be used for loading cells. Meanwhile, the silk fibroin is a material with a tissue repair function, so that the temperature-sensitive hydrogel has huge application potential in the aspect of central nervous injury repair.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to an injectable thermosensitive hydrogel, its preparation method, and its application. Background Technology

[0002] Many neuropsychiatric disorders are often accompanied by damage to the central nervous system, which is typically difficult to repair itself. Utilizing the minimally invasive properties of injectable hydrogels, they can be implanted into living tissue with minimal invasiveness, suitable for irregularly shaped sites. As a drug delivery system, the release of therapeutic factors can be controlled to further promote nerve repair and regeneration.

[0003] Many temperature-sensitive hydrogels have been developed, most of which transform from liquid to solid at 37 degrees Celsius, with solidification times ranging from tens of seconds to tens of minutes. However, not every temperature-sensitive hydrogel is suitable for injection into the body, and injectable hydrogel materials for the repair of central nervous system injuries are still rare. Some existing injectable temperature-sensitive hydrogels require lower temperatures (e.g., 4 degrees Celsius) to maintain low viscosity, thus facilitating injection. This requirement for operation at low temperatures presents certain inconveniences.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an injectable thermosensitive hydrogel, its preparation method and application, in order to solve the problem that some existing injectable thermosensitive hydrogels are easy to inject under low temperature conditions, which leads to inconvenience in operation.

[0006] The technical solution of the present invention is as follows: A first aspect of the present invention provides a method for preparing an injectable thermosensitive hydrogel, comprising the steps of: Silk fibroin and β-cyclodextrin were dissolved in water to carry out a cross-linking reaction between silk fibroin and β-cyclodextrin, resulting in a silk fibroin-β-cyclodextrin solution. N-Isopropylacrylamide monomer powder was added to the silk fibroin-β-cyclodextrin solution, and then a photoinitiator was added. The solution was then subjected to ultraviolet irradiation, which caused N-isopropylacrylamide to photopolymerize to form polyN-isopropylacrylamide. At the same time, it formed a double cross-linked network with silk fibroin-β-cyclodextrin to obtain the thermosensitive hydrogel.

[0007] Optionally, the method for preparing the silk fibroin includes the following steps: Silkworm cocoons were placed in a sodium carbonate solution and boiled at 100°C for half an hour to obtain degummed silk. The degummed mulberry silk was dried, and then dissolved in lithium bromide solution at 60°C to obtain mulberry silk-lithium bromide solution. The silkworm silk-lithium bromide solution was dialyzed, and the purified silk fibroin solution was freeze-dried to obtain freeze-dried purified silk fibroin.

[0008] Optionally, based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the mass concentration of the silk fibroin is 2.5%-10%.

[0009] Optionally, based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the mass concentration of the β-cyclodextrin is 2.5%-10%.

[0010] Optionally, based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the mass concentration of the N-isopropylacrylamide monomer is 5%-15%.

[0011] Optionally, the photoinitiator is LAP (lithium phenyl (2,4,6-trimethylbenzoyl)phosphate). Based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, 100 μL of the photoinitiator was added to every 10 mL of the system.

[0012] Optionally, based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the ultraviolet irradiation time for each 10 mL of the system is 1-2 min.

[0013] In a second aspect, the present invention provides an injectable thermosensitive hydrogel, wherein the injectable thermosensitive hydrogel is prepared by the method described in the present invention.

[0014] A third aspect of the present invention provides the application of the injectable thermosensitive hydrogel described herein in the preparation of drug carriers or cell carriers.

[0015] A fourth aspect of the present invention provides the use of the injectable thermosensitive hydrogel of the present invention in the preparation of a medicament for the repair of central nervous system injury, the medicament comprising a carrier and nerve cells or nerve growth factors loaded within the carrier, wherein the carrier is the thermosensitive hydrogel.

[0016] Beneficial Effects: This invention provides an injectable thermosensitive hydrogel with silk fibroin, β-cyclodextrin, and poly(N-isopropylacrylamide) as the main materials. This thermosensitive hydrogel has low viscosity at room temperature, is injectable, and can rapidly solidify at around 35 degrees Celsius. Experimental verification shows that this thermosensitive hydrogel has good biocompatibility, can be used as a drug delivery system, and can also load cells. Furthermore, silk fibroin itself is a material with tissue repair capabilities; therefore, this thermosensitive hydrogel has great application potential in the repair of central nervous system injuries. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the preparation process of thermosensitive hydrogels.

[0018] Figure 2 These are photographs of the thermosensitive hydrogel at room temperature and 35°C.

[0019] Figure 3 This is the curing time curve of the thermosensitive hydrogel at 35°C.

[0020] Figure 4 Release curve of lamotrigine encapsulated in thermosensitive hydrogel.

[0021] Figure 5 Photographs showing the direct injection of thermosensitive hydrogel into the damaged area of ​​a mouse brain.

[0022] Figure 6 The experimental results of encapsulating PC12 cells in a thermosensitive hydrogel and culturing them in vitro for 4 days are shown in the figure. Detailed Implementation

[0023] This invention provides an injectable thermosensitive hydrogel, its preparation method, and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] Some existing injectable thermosensitive hydrogels require low temperatures (e.g., 4 degrees Celsius) to maintain low viscosity, thus facilitating injection. This requirement for operation in low-temperature environments presents certain inconveniences.

[0025] Based on this, the present invention mainly designs an injectable thermosensitive hydrogel that is liquid at room temperature, injectable, and can solidify at temperatures above 35 degrees Celsius. This thermosensitive hydrogel has good biocompatibility. This thermosensitive hydrogel can be used as a drug delivery system or injected into the body as a cell carrier, and has the potential to promote nerve regeneration and repair. It can provide a novel treatment strategy for central nervous system injuries and has potential translational application prospects.

[0026] This invention provides a method for preparing an injectable thermosensitive hydrogel, comprising the following steps: Silk fibroin and β-cyclodextrin were dissolved in water to carry out a cross-linking reaction between silk fibroin and β-cyclodextrin, resulting in a silk fibroin-β-cyclodextrin solution. N-Isopropylacrylamide monomer powder was added to the silk fibroin-β-cyclodextrin solution, and then a photoinitiator was added. The solution was then subjected to ultraviolet irradiation, which caused N-isopropylacrylamide to photopolymerize to form polyN-isopropylacrylamide. At the same time, it formed a double cross-linked network with silk fibroin-β-cyclodextrin to obtain the thermosensitive hydrogel.

[0027] Existing thermosensitive hydrogels often lack the simultaneous properties of good injectability at room temperature, rapid curing at physiological temperatures, and good biocompatibility and biodegradability. However, the thermosensitive hydrogel of this invention exhibits excellent injectability at room temperature. Furthermore, since mice typically have a body temperature of around 34-35 degrees Celsius, the thermosensitive hydrogel developed in this invention can rapidly cure at approximately 35 degrees Celsius. This thermosensitive hydrogel demonstrates good biocompatibility with neuronal cells, possesses biodegradability, and can also load cells or other nerve growth factors. Injection into the mouse brain can be used for research related to the repair of central nervous system injuries.

[0028] It is important to emphasize that existing methods for preparing hydrogels using N-isopropylacrylamide and silk fibroin typically employ glutaraldehyde, which has a certain degree of toxicity, as a cross-linking agent; while hydrogels formed by cross-linking silk fibroin and β-cyclodextrin lack thermosensitive properties. The thermosensitive hydrogel of this invention integrates these three materials: poly-N-isopropylacrylamide provides thermosensitivity, β-cyclodextrin, as a commonly used drug-releasing material, fulfills the drug loading function, and silk fibroin undertakes the repair function. The resulting hydrogel possesses multiple advantages: it achieves complementary material properties, completes the drug loading and release process, and also possesses self-repairing capabilities and thermosensitivity, making it suitable for in vivo injection. Furthermore, the thermosensitive process of this hydrogel is reversible—it is injectable at room temperature and solidifies at 35°C; if the temperature drops back to room temperature, it will return to an injectable state.

[0029] In one embodiment, the method for preparing the silk fibroin includes the following steps: Silkworm cocoons were placed in a sodium carbonate solution and boiled at 100°C for half an hour to obtain degummed silk. The degummed mulberry silk was dried, and then dissolved in lithium bromide solution at 60°C to obtain mulberry silk-lithium bromide solution. The silkworm silk-lithium bromide solution was dialyzed, and the purified silk fibroin solution was freeze-dried to obtain freeze-dried purified silk fibroin.

[0030] In one embodiment, based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the mass concentration of the silk fibroin is 2.5%-10%, such as 2.5%, 5%, 6%, 7.5%, 8%, 10%, etc.

[0031] In one embodiment, based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the mass concentration of the β-cyclodextrin is 2.5%-10%, such as 2.5%, 5%, 6%, 7.5%, 8%, 10%, etc.

[0032] In one embodiment, based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the mass concentration of the N-isopropylacrylamide monomer is 5%-15%, such as 5%, 6%, 7.5%, 8%, 10%, 12%, 15%, etc.

[0033] In one embodiment, the thermosensitive hydrogel is prepared from the following raw materials at the following mass concentrations: 2.5%-10% silk fibroin, 2.5%-10% β-cyclodextrin, 5%-15% N-isopropylacrylamide monomer, and the balance being water. During the preparation process, the mass concentration of each raw material needs to be precisely controlled to ensure the stability of the thermosensitive hydrogel's performance. Silk fibroin, as an important component, has a mass concentration between 2.5% and 10%, which can be adjusted according to specific needs. β-cyclodextrin is also added at a mass concentration of 2.5% to 10%, synergistically working with silk fibroin to enhance the structural stability of the hydrogel. N-isopropylacrylamide monomer is present at a mass concentration of 5% to 15%, imparting thermosensitive properties to the hydrogel, enabling it to undergo a phase transition at a specific temperature. The balance is water, serving as a solvent and reaction medium to ensure thorough mixing and reaction of the raw materials.

[0034] In one embodiment, the cross-linking reaction time between the silk fibroin and β-cyclodextrin is 1 h.

[0035] In one embodiment, the photoinitiator is LAP.

[0036] In one embodiment, based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, 100 μL of the photoinitiator is added to every 10 mL of the system.

[0037] In one embodiment, based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer, and water, the UV irradiation time is 1-2 minutes per 10 mL of the system. That is, the UV irradiation time will be adjusted accordingly with the amount of system used; specifically, the larger the amount of system used, the longer the required UV irradiation time.

[0038] This invention provides an injectable thermosensitive hydrogel, which is prepared using the method described in this invention.

[0039] This invention provides an application of the injectable thermosensitive hydrogel described in this invention in the preparation of drug carriers or cell carriers.

[0040] This invention provides an application of the injectable thermosensitive hydrogel described in this invention in the preparation of a drug for the repair of central nervous system injury. The drug includes a carrier and nerve cells or nerve growth factors loaded within the carrier, wherein the carrier is the thermosensitive hydrogel.

[0041] The present invention will be further described in detail below through specific embodiments.

[0042] Combination Figure 1 As shown, the method for preparing the injectable thermosensitive hydrogel in this embodiment is as follows: Purification of silk fibroin: Following existing methods, silkworm cocoons are placed in a 0.5% sodium carbonate solution and boiled at 100°C for half an hour to degummify. The degummed silk is then thoroughly dried, and subsequently dissolved in a 9.3M lithium bromide solution at 60°C. This solution is then dialyzed, and the purified silk fibroin solution is freeze-dried to obtain freeze-dried purified silk fibroin (SF).

[0043] The freeze-dried and purified silk fibroin was dissolved together with β-cyclodextrin (β-CD) in pure water, and the cross-linking reaction of silk fibroin and β-cyclodextrin was carried out for 1 h to obtain a silk fibroin-β-cyclodextrin solution; wherein, in the solution composed of silk fibroin, β-cyclodextrin and water, the concentration of silk fibroin was 5% and the concentration of β-cyclodextrin was 5%.

[0044] A certain amount of N-isopropylacrylamide monomer (NIPAM) powder is then added to the silk fibroin-β-cyclodextrin solution and completely dissolved; wherein, the concentration of N-isopropylacrylamide in the solution composed of the N-isopropylacrylamide monomer powder and the silk fibroin-β-cyclodextrin solution is 10%.

[0045] Add 1% photoinitiator LAP to the above solution, then irradiate with ultraviolet (UV) light for a certain period of time (until a white precipitate forms in the solution). During this process, N-isopropylacrylamide photopolymerizes to form polyN-isopropylacrylamide, and simultaneously forms a double cross-linked network with silk fibroin-β-cyclodextrin. Shaking the bottle will cause the white precipitate produced by photopolymerization to disappear, and the overall viscosity of the solution will increase. Finally, a thermosensitive hydrogel is obtained.

[0046] like Figure 2 As shown, this thermosensitive hydrogel can be injected at room temperature, and when injected into warm water at 35 degrees Celsius, it will quickly solidify into a white precipitate.

[0047] Figure 3 The curve shows the curing time of the thermosensitive hydrogel at 35°C. It was obtained by testing the curing process of the thermosensitive hydrogel at 35°C using a rheometer. The testing principle is to determine the transition process of the hydrogel from the sol state to the gel state by monitoring the changes in storage modulus (G') and loss modulus (G'') over time. The intersection of G' / G'' occurs at about 2 seconds, meaning that the thermosensitive hydrogel only needs 2 seconds to cure at 35°C.

[0048] Figure 4 Release curves of lamotrigine encapsulated in a thermosensitive hydrogel are presented. These curves were plotted by simulating an in vivo environment: the thermosensitive hydrogel encapsulating lamotrigine was placed in a buffer solution at 35°C, and samples were taken periodically to detect the concentration of lamotrigine in the solution. As shown in the figure, the release rate of lamotrigine is initially rapid, gradually slowing down over time and eventually stabilizing. This indicates that the thermosensitive hydrogel has a good sustained-release effect on lamotrigine, effectively controlling the drug release rate and providing the possibility for sustained and stable drug release in vivo. This is because the thermosensitive hydrogel can encapsulate cells or drug molecules, and after solidification at 35°C, lamotrigine is encapsulated within it; as the silk fibroin and β-cyclodextrin components in the thermosensitive hydrogel gradually degrade, the encapsulated drug molecules are slowly released.

[0049] Figure 5 This image shows the direct injection of thermosensitive hydrogel into the brain injury site of a mouse. As can be seen, the injected thermosensitive hydrogel has effectively filled the brain wound and cured. Due to its injectability and excellent curing properties, thermosensitive hydrogel can be directly applied to the injury site, providing a novel and effective approach for brain injury repair. Furthermore, its good biocompatibility provides a reliable guarantee for its application in brain repair, effectively reducing adverse reactions such as immune rejection. This characteristic makes thermosensitive hydrogels demonstrate enormous application potential in the field of brain injury repair.

[0050] PC12 cells are a cell line cloned from rat adrenal pheochromocytoma. Their main secretory products are catecholamine neurotransmitters, including dopamine and norepinephrine. This cell line possesses NGF receptors on its cell membrane. Under the induction of physiological levels of NGF, the cells stop dividing and grow neurites, differentiating into cells with sympathetic neuronal characteristics. Therefore, they are often used to analyze the molecular mechanisms of neuronal differentiation and NGF action, and can also be used to explore the mechanisms by which growth factors regulate changes in neuronal gene expression. The PC12 cells used in this experiment were purchased from Huatuo Biotechnology Co., Ltd. After being encapsulated in thermosensitive hydrogel and cultured in vitro for 4 days, the cells were stained using KGI Biotechnology's live / dead cell staining reagent, and then photographed using an Olympus fluorescence microscope. The results are as follows: Figure 6 As shown in the figure. Experimental results showed that after 4 days of in vitro culture, the survival rate of PC12 cells was over 90%, indicating that the thermosensitive hydrogel has good biocompatibility. This verifies that the thermosensitive hydrogel can serve as a carrier for nerve cells, directly injected into the mouse brain, for research and application in the repair of central nervous system injuries.

[0051] In summary, this invention provides an injectable thermosensitive hydrogel, its preparation method, and its applications. The thermosensitive hydrogel exhibits good injectability at room temperature and rapidly solidifies at approximately 35 degrees Celsius, with a solidification time not exceeding 5 seconds. Furthermore, this thermosensitive hydrogel demonstrates good biocompatibility; when PC12 cell suspension is dispersed within it, the cell viability remains above 90% after four days of in vitro culture. This thermosensitive hydrogel can serve as a drug delivery system to release drug molecules. This thermosensitive hydrogel material can also be used as a carrier for nerve cells or drug molecules, directly injected into the mouse brain for research and application in the repair of central nervous system injuries.

[0052] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing an injectable thermosensitive hydrogel, characterized in that, Including the following steps: Silk fibroin and β-cyclodextrin were dissolved in water to carry out a cross-linking reaction between silk fibroin and β-cyclodextrin, resulting in a silk fibroin-β-cyclodextrin solution. N-Isopropylacrylamide monomer powder was added to the silk fibroin-β-cyclodextrin solution, and then a photoinitiator was added. The solution was then subjected to ultraviolet irradiation, which caused the N-isopropylacrylamide monomer to photopolymerize to form polyN-isopropylacrylamide. At the same time, it formed a double cross-linked network with silk fibroin-β-cyclodextrin to obtain the thermosensitive hydrogel.

2. The method for preparing the injectable thermosensitive hydrogel according to claim 1, characterized in that, The preparation method of the silk fibroin includes the following steps: Silkworm cocoons were placed in a sodium carbonate solution and boiled at 100°C for half an hour to obtain degummed silk. The degummed mulberry silk was dried, and then dissolved in lithium bromide solution at 60°C to obtain mulberry silk-lithium bromide solution. The silkworm silk-lithium bromide solution was dialyzed, and the purified silk fibroin solution was freeze-dried to obtain freeze-dried purified silk fibroin.

3. The method for preparing the injectable thermosensitive hydrogel according to claim 1, characterized in that, Based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the mass concentration of the silk fibroin is 2.5%-10%.

4. The method for preparing the injectable thermosensitive hydrogel according to claim 1, characterized in that, Based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the mass concentration of the β-cyclodextrin is 2.5%-10%.

5. The method for preparing the injectable thermosensitive hydrogel according to claim 1, characterized in that, Based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the mass concentration of the N-isopropylacrylamide monomer is 5%-15%.

6. The method for preparing the injectable thermosensitive hydrogel according to claim 1, characterized in that, The photoinitiator is LAP; Based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, 100 μL of the photoinitiator was added to every 10 mL of the system.

7. The method for preparing the injectable thermosensitive hydrogel according to claim 1, characterized in that, Based on the system composed of the silk fibroin, β-cyclodextrin, N-isopropylacrylamide monomer and water, the UV irradiation time for each 10 mL of the system is 1-2 min.

8. An injectable thermosensitive hydrogel, characterized in that, It was prepared using the method for preparing injectable thermosensitive hydrogel according to any one of claims 1-7.

9. The use of the injectable thermosensitive hydrogel of claim 8 in the preparation of a drug carrier or cell carrier.

10. The use of the injectable thermosensitive hydrogel of claim 8 in the preparation of a medicament for the repair of central nervous system injury, the medicament comprising a carrier and nerve cells or nerve growth factors loaded within the carrier, wherein the carrier is the thermosensitive hydrogel.

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