Biomimetic frog-derived nerve regeneration peptide modified bioactive hydrogel and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to biomimetic frog-derived nerve regeneration peptide modified bioactive hydrogel and a preparation method thereof. The bioactive hydrogel is prepared from the following raw materials: RADA16, CS, VD11 and deionized water; the PNFs hydrogel is prepared from PNFs, CS and VD11 through self-assembly of RADA16 and deionized water, and the optimal mass concentration ratio of PNFs to CS to VD11 in the prepared bioactive hydrogel is 3.33: 20: 0.135. The preparation method is simple, conditions are easy to realize, the use amount of expensive RADA16 can be reduced, and the preparation method is particularly applied to central nervous system injury repair. The bioactive hydrogel has a three-dimensional space suitable for loading stem cells or drug molecules, is used as a carrier for cell culture and stem cell transplantation, supports attachment and growth of cells and promotes tissue repair and regeneration, and has a very wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel and a preparation method thereof. Background Art

[0002] Peptide-based nanohydrogels with multiple biological activities can mimic the microenvironment of nerve regeneration, regulate the immune inflammatory microenvironment after central nervous system injury, induce endogenous neural stem cells to migrate to the injured area for colonization, differentiate into neurons, and promote nerve regeneration. Synthetic peptides that self-assemble into β-sheet peptide nanofibers (PNFs) in acidic aqueous solution will spontaneously begin to cross-link into a complex three-dimensional hydrogel matrix within a few seconds after contacting blood, interstitial fluid and lymph at physiological pH. It can simulate the formation of the repair microenvironment, functional recovery and healing of the central nervous system, and can be used to support and initiate regeneration synthesis after central nervous system injury. Bioactive hydrogels based on PNFs have been used as drug, cell and gene delivery carriers, and have achieved gratifying results. However, none of them have been translated into clinical treatment, and the neurotropism, insufficient injectability and high price of PNFs hydrogels cannot fully meet the needs of clinical treatment. Continue to develop multifunctional combination peptide hydrogels based on PNFs hydrogel, which are suitable for the microenvironment of central nervous system injury repair, improve neural stem cell differentiation and neural network circuit formation, stimulate axon regeneration and reduce glial scars, and accelerate and promote nerve regeneration after central nervous system trauma.

[0003] Chitosan (CS) is the second most abundant natural polysaccharide on earth. It is inexpensive and has the ability to cross the blood-brain barrier, adhere to mucosa and form hydrogels. It has been widely used in central nervous system repair. CS and its derivatives can promote neuronal differentiation and neurite growth. The thermosensitive CS hydrogels we developed earlier, which are between room temperature and body temperature (37°), can quickly gel after local injection to fill tissue defects and are attractive candidates for central nervous system treatment. We propose the concept of CS combined with PNFs multifunctional hydrogels (double three-dimensional structure hydrogels) for repairing central nervous system injuries, creating a favorable microenvironment for central nervous system repair and reducing the use of expensive PNFs hydrogels.

[0004] Central nervous system injury can restart nerve regeneration in amphibians and fish. Amphibians have the ability to regenerate nerve tissue after central nervous system injury, generate new neurons, differentiate and mature, and participate in the circuit of spinal cord repair. The latest research shows that bionic amphibian (frog) spinal cord derived peptide (VDELWPPWLPC, VD11) can promote the repair of spinal cord injury in rats, which is an important discovery in the treatment of central nervous system injury, but the method of loading VD11 is less studied. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a VD11-modified bioactive hydrogel and a preparation method thereof. The multifunctional combination hydrogel of CS / PNFs enhanced with VD11 has bionics, targeted injection, thermosensitivity, biodegradability and good biocompatibility, and can significantly improve the neurotropism of the hydrogel, thereby effectively promoting the repair of central nervous system injuries. It is an ideal biomaterial for promoting tissue healing and has very broad application prospects and clinical application value.

[0006] The technical solutions adopted are:

[0007] A VD11 (bionic frog-derived nerve regeneration peptide) modified bioactive hydrogel, the raw materials of which include RADA16, CS, VD11 and deionized water; RADA16 and deionized water self-assemble into PNFs hydrogel, and the mass concentration ratio of PNFs, CS and VD11 in the prepared bioactive hydrogel is 1-5:10-30:0.0135-1.35.

[0008] As a further preference, the mass concentration ratio of PNFs, CS and VD11 in the bioactive hydrogel (PCV) is 3.33:20:0.135, that is, (3.33 mg / mL) PNFs+2% (w / v) CS+0.135 mg / mL VD11.

[0009] A method for preparing a VD11-modified bioactive hydrogel comprises the following steps:

[0010] (1) Self-assembly of PNFs hydrogel: RADA16 was added to deionized water, ultrasonically vibrated to fully dissolve it, and then allowed to stand to form PNFs hydrogel;

[0011] (2) Preparation of CS aqueous solution: dissolve CS with acetic acid to prepare CS solution; slowly add NaHCO3 solution in an ice water bath under continuous magnetic stirring;

[0012] (3) synthesizing PNFs / CS composite hydrogel, mixing equal volumes of PNFs hydrogel and CS solution prepared in step (2) to obtain a composite hydrogel;

[0013] (4) adding VD11: adding VD11 to the PNFs / CS composite hydrogel and mixing thoroughly to obtain the PCV hydrogel.

[0014] Preferably, in the step (1), self-assembly is carried out at room temperature and a pH value of 7; wherein the ultrasonic oscillation time is 2 to 10 minutes; and the standing time is 2 to 3 hours.

[0015] Preferably, in step (1), the mass ratio of RADA16 to deionized water is 10 to 20 (mg / mL).

[0016] Preferably, in step (2), the mass fraction of acetic acid is 1-2%, and the mass fraction of the prepared CS is 3-6%wt.

[0017] Preferably, in step (2), a 0.22 M NaHCO3 solution having an equal volume to that of the CS solution is slowly added to the ice water bath.

[0018] Preferably, in step (3), the PNFs hydrogel and the CS solution are mixed in equal volumes and then placed in a 37° C. water bath for 1 to 10 minutes to form a gel.

[0019] Preferably, in step (4), after VD11 is added, mechanical stirring or ultrasonic oscillation is used to mix uniformly.

[0020] Preferably, the stirring temperature is room temperature, and the stirring time is 5 to 60 minutes.

[0021] As the innovation point of the present invention, the present invention introduces CS with thermosensitive effect, which has good fluidity at low temperature, can adapt to the irregular lesions and defects after central nervous system injury in clinical practice, and can quickly form a 3D gel-like "scaffold" at human body temperature, effectively bridging nerve tissue. The present invention is loaded with VD11 with neurotropism, further enhancing the effect of promoting tissue and wound healing, especially promoting the healing of the central nervous system. The present invention is injectable and simple to operate, and is an ideal biomaterial for promoting tissue healing.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The VD11-modified hydrogel with injectability, thermosensitivity and enhanced neurotrophic function described in the present invention has excellent thermosensitivity and neurotropism, reduces the use of expensive RADA16, and is suitable for biomedical applications, especially in the repair of central nervous system injuries.

[0024] The bioactive hydrogel of the present invention has a three-dimensional space suitable for loading stem cells or drug molecules, and serves as a carrier for cell culture and stem cell transplantation to support cell attachment and growth; it can load and release drugs or growth factors to promote tissue repair and regeneration.

[0025] The hydrogel of the present invention can be implanted in the injured part of the rat spinal cord, and supports the regeneration and growth of nerve cells through its three-dimensional structure; the addition of biomimetic VD11 helps promote nerve regeneration and functional recovery. It can be used as a biomaterial in the field of tissue engineering and regenerative medicine to support tissue regeneration and functional recovery.

[0026] The preparation method of the present invention is simple, the conditions are easy to achieve, and the cost is low; the material prepared by the technology of the present invention has good biocompatibility, can realize in situ loading of cells / active molecules and in situ injection in vivo, and has very broad application prospects and clinical application value in the fields of three-dimensional cell culture, tissue engineering biological scaffold materials and drug carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The present invention is a flow chart of the method for preparing the bioactive hydrogel.

[0028] Figure 2 Comparison diagram of composite hydrogels containing 1% (w / v), 1.5% (w / v), and 2% (w / v) CS at different concentrations prepared by the present invention before (a) and after 5 minutes (b) in a 37°C water bath.

[0029] Figure 3 Electron microscope scanning image of the best bioactive hydrogel of the present invention containing (3.33 mg / mL) PNFs+2% (w / v) CS+0.135 mg / mL LVD11.

[0030] Figure 4 Graph showing the change of storage modulus (G') and loss modulus (G") of the bioactive hydrogel of the present invention with temperature.

[0031] Figure 5 This is a diagram of the injection state of the PCV hydrogel prepared in the present invention.

[0032] Figure 6 Figure 2 is the live / dead cell staining and CCK-8 experimental analysis after culturing bone marrow mesenchymal stem cells with PCV hydrogels of different concentrations; Figure a is a representative figure of the live / dead cell staining experiment after culturing bone marrow mesenchymal stem cells with PCV hydrogels of different concentrations, Figure b is a comparison of the survival rates in the live / dead cell staining experiment of bone marrow mesenchymal stem cells, and Figure c is a CCK-8 statistical analysis of bone marrow mesenchymal stem cells. DETAILED DESCRIPTION

[0033] The accompanying drawings are only used for exemplary description. The technical solution of the present invention is described in detail below through embodiments in combination with the accompanying drawings. For those skilled in the art, some well-known structures and their descriptions in the accompanying drawings may be omitted, and therefore, they should not be understood as limitations on the present invention.

[0034] The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturer shall be followed. The raw materials, instruments and equipment used, if the manufacturer is not specified, are all conventional products that can be purchased commercially. Other technical methods not described herein adopt existing technologies.

[0035] Example 1

[0036] like Figure 1 As shown, a method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel comprises the following steps:

[0037] (1) Preparation of PNFs hydrogel: 30 mg of RADA16 was added to 3 mL of deionized water, pH 7, and sonicated for 5 minutes to fully dissolve it. The mixture was allowed to stand at room temperature (20-26°C) for 3 hours to self-assemble into PNFs hydrogel with a concentration of 10 mg / mL.

[0038] (2) Preparation of CS aqueous solution: Preferably, 1% acetic acid is used to dissolve CS to prepare a (3% wt) CS solution, and then an equal volume of 0.22 M NaHCO3 is slowly added to the CS solution under continuous magnetic stirring in an ice-water bath.

[0039] (3) Synthesis of PNFs / CS hydrogel: PNFs hydrogel with an equal volume of CS solution was mixed with the solution obtained in step (2) to obtain a composite hydrogel containing 1% (w / v) CS.

[0040] like Figure 2 As shown, according to the above method, (4.5% wt, 6% wt) CS solutions were prepared respectively, and the obtained composite hydrogels contained 1.5% (w / v) and 2% (w / v) CS. The 1% (w / v), 1.5% (w / v), and 2% (w / v) CS solutions were placed in a 37°C water bath respectively, taken out every 1 minute, and the time to gel was observed. Among them, Figure a shows the state before the water bath, and Figure b shows the state after 5 minutes of water bath. By comparison, it can be seen that the composite hydrogel with 2% (w / v) CS has the best gelation.

[0041] (4) Adding VD11: VD11 was added to the composite hydrogel containing 2% (w / v) CS at a concentration of 0.0135 mg / mL, and mixed thoroughly to form the bioactive hydrogel (PCV), i.e., containing 3.33 mg / mL PNFs + 2% (w / v) CS + 0.0135 mg / mL VD11.

[0042] The obtained product was subjected to live and dead cell staining and CCK-8 verification of bone marrow mesenchymal stem cells.

[0043] Example 2

[0044] A method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel comprises the following steps:

[0045] (1) Preparation of PNFs hydrogel: 30 mg of RADA16 was added to 3 mL of deionized water, pH 7, and sonicated for 5 minutes to fully dissolve it. The mixture was allowed to stand at room temperature (20-26°C) for 3 hours to self-assemble into PNFs hydrogel with a concentration of 10 mg / mL.

[0046] (2) Preparation of CS aqueous solution: Preferably, 1% acetic acid is used to dissolve CS to prepare a (6% wt) CS solution, and then an equal volume of 0.22 M NaHCO3 is slowly added to the CS solution under continuous magnetic stirring in an ice-water bath.

[0047] (3) Synthesis of PNFs / CS hydrogel: Mix the PNFs hydrogel with the CS solution in an equal volume with the solution obtained in step (2) to obtain a composite hydrogel containing 2% (w / v) CS. Place the mixture in a 37°C water bath and take it out after it becomes gel-like for 5 to 10 minutes.

[0048] (4) Adding VD11: VD11 was added to the composite hydrogel containing 2% (w / v) CS at a concentration of 0.135 mg / mL and mixed thoroughly to form bioactive hydrogels (PCV) of different concentrations, i.e., containing (3.33 mg / ml) PNFs + 2% (w / v) CS + 0.135 mg / mL VD11.

[0049] The obtained product was subjected to live and dead cell staining and CCK-8 verification of bone marrow mesenchymal stem cells.

[0050] Other unmentioned contents are the same as those in Example 1.

[0051] Example 3

[0052] A method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel, which is different from Example 2 in that VD11 is added to a composite hydrogel containing 2% (w / v) CS at a concentration of 1.35 mg / mL and mixed thoroughly to form bioactive hydrogels (PCV) of different concentrations, namely, (3.33 mg / mL) PNFs+2% (w / v) CS+1.35 mg / mL VD11.

[0053] The obtained product was subjected to live and dead cell staining and CCK-8 verification of bone marrow mesenchymal stem cells.

[0054] Other unmentioned contents are the same as those in Example 1.

[0055] The concentration of each raw material in the formed PCV was (3.33 mg / mL) PNFs + 2% (w / v) CS + 1.35 mg / mL VD11.

[0056] Other unmentioned contents are the same as those in Example 1.

[0057] like Figure 3 As shown, the optimal concentration of PCV hydrogel is (3.33 mg / mL) PNFs + 2% (w / v) CS + 0.135 mg / mL LVD11. The three-dimensional space test measured by electron microscopy scanning was found to have a loose and uniform three-dimensional structure, which is suitable for loading stem cells or drug molecules.

[0058] like Figure 4 As shown, the storage modulus G' and loss modulus G" are functions of temperature (frequency 1rads). When the temperature increases from 20°C to 45°C (1°C / in), G' is first lower than G". When the temperature is very close to the gel temperature of 37°C, G' and G" increase sharply with increasing temperature. Finally, G' is higher than G", indicating that an elastic gel network is formed.

[0059] like Figure 5 As shown, the PCV hydrogel prepared by the present invention is injectable. The PCV hydrogel stored at 4°C is easily aspirated by a 10 mL syringe and is very smooth during injection.

[0060] Comparative Example 1

[0061] A method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel comprises the following steps:

[0062] (1) Preparation of PNFs hydrogel: 30 mg of RADA16 was added to 3 mL of deionized water, pH 7, and sonicated for 5 minutes to fully dissolve it. The mixture was allowed to stand at room temperature (20-26°C) for 3 hours to self-assemble into PNFs hydrogel with a concentration of 10 mg / mL.

[0063] (2) Preparation of CS aqueous solution: Preferably, CS is dissolved in 1% acetic acid to prepare a CS (6% wt) solution, and then 0.22 M NaHCO3 is slowly added in an equal volume to the CS solution under continuous magnetic stirring in an ice-water bath.

[0064] (3) Synthesis of PNFs / CS hydrogel: Mix the PNFs hydrogel with the same volume as the CS solution with the solution obtained in step (2) to obtain a composite hydrogel containing 2% (w / v) CS. Place it in a 37°C water bath and take it out after it becomes gel-like for 5 to 10 minutes.

[0065] like Figure 6The figure shows the live / dead cell staining and CCK-8 experimental analysis after culturing bone marrow mesenchymal stem cells with PCV hydrogels of different concentrations, where (a) shows the live / dead cell staining experimental results of bone marrow mesenchymal stem cells after culturing with bone marrow mesenchymal stem cells with VD11 hydrogels of different concentrations for 3 days, and the composite image of bone marrow mesenchymal stem cells (green is live cells, red is dead cells). From left to right, A: control group PBS; B: PNFs / CS (3.33mg / mL PNFs + 2% (w / v) CS); C: PCV (0.0135mg / mL VD11); D: PCV (0.135mg / mL VD11); E: PCV (1.35mg / mL VD11). The optimal concentration of PCV hydrogel is (3.33mg / mL) PNFs + 2% (w / v) CS + 0.135mg / mL VD11, with the most surviving cells.

[0066] Figure 6 Figure (b) shows the survival rate of bone marrow mesenchymal stem cells in the live / dead cell staining experiment. The survival rate of bone marrow mesenchymal stem cells in each group was greater than 95%. A: control group PBS; B: PNFs / CS (3.33 mg / mL PNFs + 2% (w / v) CS); C: PCV (0.0135 mg / mL VD11); D: PCV (0.135 mg / mL VD11); E: PCV (1.35 mg / mL VD11). PCV hydrogels containing (0, 0.0135 mg / mL, 0.135 mg / mL, 1.35 mg / mL) of VD11 were non-cytotoxic, and PCV hydrogels containing 0.135 mg / mL VD11 had better biocompatibility.

[0067] Figure 6 Figure (c) is the CCK-8 statistical analysis of bone marrow mesenchymal stem cells. The products referred to by A, B, C, D, and E are as described in Figures (a) and (b). After culturing bone marrow mesenchymal stem cells for 1, 2, and 3 days, it was shown that the PCV hydrogels containing (0, 0.0135 mg / mL, 0.135 mg / mL, and 1.35 mg / mL) of VD11 were non-toxic and could promote the growth of bone marrow mesenchymal stem cells. The PCV hydrogel containing 0.135 mg / mL VD11 had the most obvious effect in promoting cell growth.

[0068] Verified by live-dead cell staining of bone marrow mesenchymal stem cells and CCK-8 experiments, the optimal concentration of PCV hydrogel is (3.33 mg / mL) PNFs+2% (w / v) CS+0.135 mg / mL VD11.

[0069] The meanings of the English abbreviations used in the present invention are:

[0070] RADA16: (arginine-alanine-aspartate-alanine) 4;

[0071] PNFs: peptide nanofibers;

[0072] CS: Chitosan

[0073] VD11: Bionic spinal cord derived peptide (VDELWPPWLPC), i.e. bionic frog-derived neural regeneration peptide;

[0074] PCV: Bioactive Hydrogel;

[0075] PBS: phosphate buffered saline;

[0076] CCK-8: Cell counting kit-8.

[0077] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel, characterized in that: The raw materials include RADA16, CS, VD11 and deionized water; RADA16 and deionized water are self-assembled into PNFs hydrogel, and the mass concentration ratio of PNFs, CS and VD11 in the prepared bioactive hydrogel is 1-5:10-30:0.0135-1.

35.

2. The biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel according to claim 1, characterized in that: The mass concentration ratio of PNFs, CS and VD11 in the bioactive hydrogel is 3.33:20:0.

135.

3. The method for preparing the bioactive hydrogel modified with the biomimetic frog-derived nerve regeneration peptide according to any one of claims 1 or 2, characterized in that: The steps include: (1) Self-assembly of PNFs hydrogel: RADA16 was added to deionized water, ultrasonically vibrated to fully dissolve it, and then allowed to stand to form PNFs hydrogel; (2) Preparation of CS aqueous solution: dissolve CS with acetic acid to prepare CS solution; slowly add NaHCO3 solution in an ice water bath under continuous magnetic stirring; (3) synthesizing PNFs / CS composite hydrogel, mixing equal volumes of PNFs hydrogel and CS solution prepared in step (2) to obtain a composite hydrogel; (4) adding VD11: adding VD11 to the PNFs / CS composite hydrogel and mixing thoroughly to obtain the PCV hydrogel.

4. The method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel according to claim 3, characterized in that: In the step (1), self-assembly is carried out at room temperature and a pH value of 7; wherein the ultrasonic oscillation time is 2 to 10 minutes; and the standing time is 2 to 3 hours.

5. The method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel according to claim 3, characterized in that: In the step (1), the mass ratio of RADA16 to deionized water is 10 to 20 (mg / mL).

6. The method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel according to claim 3, characterized in that: In the step (2), the mass fraction of acetic acid is 1-2%, and the mass fraction of the prepared CS is 3-6%wt.

7. The method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel according to claim 3, characterized in that: In step (2), a 0.22 M NaHCO3 solution having an equal volume to that of the CS solution is slowly added to the mixture in an ice water bath.

8. The method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel according to claim 3, characterized in that: In the step (3), the PNFs hydrogel and the CS solution are mixed in equal volumes and then placed in a 37° C. water bath for 1 to 10 minutes to form a gel.

9. The method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel according to claim 3, characterized in that: In the step (4), after VD11 is added, it is mixed evenly by mechanical stirring or ultrasonic oscillation.

10. The method for preparing a biomimetic frog-derived nerve regeneration peptide-modified bioactive hydrogel according to claim 9, characterized in that: The stirring temperature is room temperature, and the stirring time is 5 to 60 minutes.

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