Method for preparing a hydrogel scaffold and use of the scaffold thus obtained

Abstract

The present application relates to a kind of water gel support prepared by self-assembled polypeptide and the use of support obtained therefrom, the preparation method includes: 1) self-assembled peptide sequence is linked nerve growth factor analog peptide by covalent bond to obtain bonded functional polypeptide;2) macrophage is separated and cultured, and is induced to be " alternative activation " anti-inflammatory M2 macrophage, obtain culture supernatant and carry out filtration, obtain filtered cell culture supernatant;3) filtered cell culture supernatant is mixed with bonded functional polypeptide, obtain mixed solution and adjust the concentration of the mixed solution, the bonded functional polypeptide self-assembles and forms water gel support.The present application uses M2 macrophage condition culture supernatant to construct regenerative microenvironment, combines polypeptide functional water gel support, realizes better overall interaction, and prepares the water gel support that can supplement and regulate nerve regeneration, the support can promote nerve cell regeneration behavior, provides new selection for tissue engineering biomaterials.

Description

[0001] This application is a divisional application of patent application No. 202110849059.8, filed on July 27, 2021, entitled "Method for preparing hydrogel scaffolds and use of the scaffolds therefrom". Technical Field

[0002] This invention relates to the field of tissue engineering technology, and more specifically, to a method for preparing hydrogel scaffolds using self-assembled peptides and the uses of the resulting scaffolds. Background Technology

[0003] The main causes of peripheral nerve injury include traffic accidents, mechanical trauma, natural disasters, and surgical complications. Approximately one million new cases of peripheral nerve injury are reported in my country each year. The resulting irreversible disabilities severely impact patients' quality of life and mental health, imposing a heavy economic burden on families and society. Furthermore, the repair efficacy of known artificial nerve implants still lags behind that of autologous nerves. Improving implant function through local regenerative microenvironment regulation is a key issue for achieving clinical application.

[0004] The emergence of tissue engineering technology has provided a technical approach for the repair of damaged nerves. Tissue engineering comprises three elements: scaffolds, seed cells, and signaling factors. Among these, the scaffold not only provides physical connection and support for regenerating tissue but also plays a crucial role in regulating the cellular regeneration environment. Hydrogels, with their three-dimensional network structure, possess high water content and are suitable for cell growth. Furthermore, cross-linking growth factor-mimicking peptides further enhances the cellular growth environment. Therefore, successfully constructing scaffold materials that effectively mimic the in vivo microenvironment is of great significance for the regeneration of damaged nerves.

[0005] Macrophages are widely distributed throughout the body and play a vital role in regulating inflammation. They possess high plasticity and are crucial for the body's development and homeostasis. After macrophages polarize to the M2 phenotype, this polarized subtype can finely regulate and respond to various stimuli, secrete signaling factors, combat chronic inflammation, and promote tissue repair and regeneration, playing a key role in the repair of diseased tissues and organs. Research on constructing hydrogel scaffolds from M2 macrophage-derived extracellular matrix is ​​currently unreported, making this a valuable research area. Summary of the Invention

[0006] One aspect of the present invention is to provide a method for preparing a self-assembled polypeptide hydrogel scaffold, wherein the method includes:

[0007] (1) The self-assembled peptide sequence was covalently bonded to a nerve growth factor mimic peptide to obtain a bonded functional polypeptide;

[0008] (2) Isolate and culture macrophages and induce them to become "alternatively activated" anti-inflammatory M2 macrophages. Obtain cell culture supernatant and filter it to obtain filtered cell culture supernatant.

[0009] (3) The filtered cell culture supernatant is mixed with the bonded functional peptide to obtain a mixture and the concentration of the mixture is adjusted. The bonded functional peptide self-assembles to form a hydrogel scaffold.

[0010] Preferably, the nerve growth factor mimic peptide has the function of promoting the regeneration of nerve and vascular cells.

[0011] Preferably, the self-assembled peptide sequence is covalently linked to the nerve growth factor mimic peptide via an amide bond or a disulfide bond.

[0012] Preferably, the molar ratio of the self-assembled peptide sequence to the nerve growth factor mimic peptide is (1~3):1.

[0013] Preferably, the macrophages are derived from the peritoneal cavity or bone marrow of rats / mice, or from human peripheral blood mononuclear lymphocytes.

[0014] Preferably, the culture in step (2) is carried out at 30℃~40℃ and 3%~10% CO2; more preferably, the culture process includes: growing the macrophages in the culture medium to a confluence of 60%~80%, discarding the culture medium and washing with PBS buffer, then adding fresh culture medium to the washed culture and continuing to culture for more than 36 hours, for example 36~72 hours, preferably 40~50 hours;

[0015] Preferably, the cell culture supernatant is filtered using a filter material with a pore size of 0.2~0.45μm.

[0016] Preferably, the method for preparing M2 macrophage culture supernatant is characterized in that the cell culture inducer is 10 ng / ml IL-4 for induction in step (2).

[0017] Preferably, the polypeptide hydrogel scaffold has a nanofiber structure.

[0018] Preferably, the polypeptide hydrogel scaffold has a hydrogel morphology, and the pH of the adjusted gel is 6.9~7.2, and the concentration is 0.5wt%-4wt%.

[0019] Another aspect of the present invention is to provide the use of the hydrogel scaffold prepared above in the in vitro culture of neural Schwann cells, characterized in that the scaffold promotes the growth and activity of neural Schwann cells.

[0020] The use of a multi-hydrogel scaffold prepared by any one of the methods of the present invention in tissue engineering regeneration, characterized in that the scaffold promotes the regeneration of damaged nerves.

[0021] The beneficial effects of this invention are:

[0022] 1. A polypeptide hydrogel scaffold with more defined components, stronger functionality, and higher safety and efficiency was prepared by chemical modification and self-assembly methods. Its nanostructure is more suitable for the repair of damaged nerves, enhances the regeneration speed of specific nerve cells, and improves the clinical treatment effect.

[0023] 2. Synthesize sequences containing different nerve growth factor mimic peptides, prepare hydrogels using self-assembly technology, and further synthesize hydrogel scaffolds containing M2 macrophage conditioned culture conditions to promote the development of regenerative cell synergistic therapy.

[0024] 3. Different polypeptide carriers and different nerve growth factor mimic peptides can also be selected to design and prepare polypeptide hydrogel scaffolds for different nerve cells.

[0025] 4. The biomimetic scaffold with a suitable regenerative immune environment has good nerve cell adhesion and growth performance, suitable tissue compatibility, and nanofibers that are close to the structure of the extracellular matrix. The scaffold prepared by the method of this invention has a better regenerative microenvironment, laying the foundation for product development. Attached Figure Description

[0026] Figure 1 This diagram illustrates the structural pattern of a self-assembled peptide sequence covalently bonded to a nerve growth factor-mimicking peptide.

[0027] Figure 2 To show the morphological image of induced M2 macrophages under a microscope.

[0028] Figure 3 This is a flow cytometry graph illustrating the maturity of M2 macrophages. The graph shows the percentage of CD206 expression detected by flow cytometry.

[0029] Figure 4 This is a transmission electron microscope (TEM) image of a polypeptide hydrogel scaffold. The structure of the nanofibers is shown.

[0030] Figure 5 This is a diagram illustrating a tilted experiment of the polypeptide hydrogel scaffold.

[0031] Figure 6 This is a microscopic image of the morphology of primary cultured Schwann neural cells.

[0032] Figure 7To illustrate the growth rates of neural Schwann cells cultured in the control group and those cultured on a polypeptide hydrogel scaffold. * indicates P < 0.05. Detailed Implementation

[0033] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

[0034] Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described herein can be used to implement the present invention. In fact, the present invention is not limited to the methods and materials described herein, but various conventional adjustments or modifications can be made based on the spirit of the invention, and the adjusted or modified solutions still fall within the protection scope of the present invention.

[0035] Unless the context explicitly indicates otherwise, the terms “a” and “an” as used herein cover plural objects.

[0036] In this article, the term “approximately” refers to approximate values ​​within the range of errors caused by measurement errors, etc.

[0037] In this article, unless otherwise defined, the term "tumor microenvironment" refers to the internal environment in which tumor cells occur and live, characterized by low oxygen, low pH, and high pressure. These characteristics result in a large number of growth factors, cell chemokines, and various proteolytic enzymes in the tumor microenvironment, which produce an immune inflammatory response that is conducive to tumor proliferation, invasion, adhesion, angiogenesis, and resistance to radiotherapy and chemotherapy, thus promoting the development of malignant tumors.

[0038] In one embodiment, the present invention relates to a method for preparing a self-assembled polypeptide hydrogel scaffold, wherein the method includes:

[0039] (1) The self-assembled peptide sequence was covalently bonded to a nerve growth factor mimic peptide to obtain a bonded functional polypeptide;

[0040] (2) Isolate and culture macrophages and induce them to become "alternatively activated" anti-inflammatory M2 macrophages. Obtain cell culture supernatant and filter it to obtain filtered cell culture supernatant.

[0041] (3) The filtered cell culture supernatant is mixed with the bonded functional peptide to obtain a mixture and the concentration of the mixture is adjusted. The bonded functional peptide self-assembles to form a nano-hydrogel scaffold.

[0042] In this document, the self-assembled peptide sequence is formed by the self-assembly of glutamic acid, lysine, alanine, tryptophan, arginine, etc., in aqueous solution to form a small molecule polypeptide of 10-20 amino acids in length. As a preferred example, the small molecule polypeptide hydrogel includes, but is not limited to, RADARADARADARADA, KWKAKAKAKWK, EWEAEAEAE, FEFEFKFKK, and QQKFQFQFEQQ. These amino acids were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0043] In a preferred embodiment, the nerve growth factor mimic peptide may be any known in the art, for example, derived from one or more of the following: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), neurotrophic factor-3 (NT-3), and neurotrophic factor-4 (NT-4).

[0044] In a preferred embodiment, the self-assembled peptide sequence is covalently linked to a nerve growth factor mimic peptide via chemical bonds in a molar ratio of (1–3):1, for example, from one selected from amide bonds or disulfide bonds. If an amide bond is chosen, solid-phase synthesis is performed; if a disulfide bond or peptide carrier is chosen, solid-phase synthesis is performed by modifying cysteine ​​with an epitope peptide, followed by docking of the two peptides.

[0045] The macrophages mentioned in this article may be derived from the peritoneal cavity or bone marrow of rats / mice, or from human peripheral blood mononuclear lymphocytes, or may be macrophages isolated using conventional methods known in the art.

[0046] In a preferred embodiment of the present invention, the cell culture can be performed using any suitable culture medium known in the art (e.g., see the descriptions below: http: / / www.cellbank.org.cn / peiyang.asp; https: / / www.atcc.org / ), such as, but not limited to, RPMI-1640 medium containing fetal bovine serum, DMEM medium containing fetal bovine serum, F-12 medium containing fetal bovine serum, and DMEM / F-12 medium containing fetal bovine serum. In a further preferred embodiment, the culture is performed at 30°C to 40°C and 3% to 10% CO2. In an even more preferred embodiment, the culture process includes: growing the tumor cells to a confluence of 60% to 80% in the culture medium, discarding the culture medium and washing with PBS buffer, then adding fresh culture medium to the washed culture and continuing to culture for at least 36 hours, for example 36 to 72 hours, preferably 40 to 50 hours.

[0047] In a preferred embodiment, the cell culture supernatant is filtered using a filter material with a pore size of 0.2~0.45 μm. The filtered cell culture supernatant mainly contains: proteins secreted by cells (including various cytokines that are major substances affecting the function of immune cells); non-coding RNA (including small RNA and long RNA, etc.); DNA, etc.

[0048] In a preferred embodiment, the method for preparing the M2 macrophage culture supernatant is characterized in that the cell culture inducer is selected from IL-4, preferably, the cell culture stimulant is 0.1-100 ng / mL, for example, 10 ng / mL IL-4. Culture medium is added for culturing, and the culture is carried out in any suitable culture medium known in the art (e.g., see the descriptions below: http: / / www.cellbank.org.cn / peiyang.asp; https: / / www.atcc.org / ), such as, but not limited to, RPMI-1640 medium containing fetal bovine serum, DMEM medium containing fetal bovine serum, F-12 medium containing fetal bovine serum, and DMEM / F-12 medium containing fetal bovine serum. In a further preferred embodiment, the culture is carried out at 30°C to 40°C and 3% to 10% CO2. In an even more preferred embodiment, the culture is carried out for more than 36 hours, for example, 36 to 72 hours, preferably 40 to 50 hours.

[0049] In a preferred embodiment, the polypeptide hydrogel scaffold is characterized in that the scaffold has a nanofiber structure.

[0050] In a preferred embodiment, the polypeptide hydrogel scaffold is characterized in that the scaffold has a hydrogel morphology, the pH of the adjusted gel is 6.9~7.2, and the concentration is 0.5 wt%-4 wt%, for example 1 wt%.

[0051] In a preferred embodiment, each mL of the hydrogel-containing cell suspension contains 1 × 10 4 ~1×10 7 1, preferably 5×10 4 ~1×10 6 One, for example, 1×10 5 The aforementioned neuroSchwan cells.

[0052] In a preferred embodiment, the predetermined shape of the support may be a three-dimensional cell culture scaffold, cell culture dish, cell culture flask, cell culture microplate, bilayer cell culture plate, or any system known in the art suitable for three-dimensional cell culture, such as a 6-well cell culture microplate, a 12-well cell culture microplate, a 24-well cell culture microplate, a 96-well cell culture microplate, a 24-well bilayer cell culture plate, or any commercially available cell culture microplate in the art, such as commercial three-dimensional cell culture systems provided by Thermofisher, FlexCell, etc.

[0053] In a preferred embodiment, the 3D cell culture is carried out at 30°C to 40°C and 3% to 10% CO2 for 2-72 hours, for example, 3-72 hours or 6-72 hours.

[0054] In one embodiment, the present invention relates to the use of the aforementioned polypeptide hydrogel scaffold in the development of damaged nerve repair. For example, by injecting, coating, or otherwise applying the polypeptide hydrogel scaffold, which has been treated by conventional physical, chemical, or biological methods, into a nerve repair conduit, damaged nerve regeneration is promoted.

[0055] The following examples further illustrate the solution of the present invention, but the scope of protection of the present invention is not limited thereto. Example

[0056] The following examples are for illustrative purposes only and are not intended to limit the scope of protection of this application. Unless otherwise stated, all reagents, materials and equipment used in the following examples are commercially available or can be formulated or obtained according to prior art. Unless otherwise stated, the specific experimental methods involved in the following examples are conventional methods described in prior art (e.g., *Molecular Cloning: A Laboratory Manual* (4th Edition), edited by J. Sambrook et al., translated by He Fuchu, Science Press, 2017; *Medical Immunology* (7th Edition), edited by Cao Xuetao, People's Medical Publishing House, 2018).

[0057] Example 1: Preparation of self-assembled peptide sequence-bonded nerve growth factor mimic peptides

[0058] The self-assembled peptide sequence was covalently linked to a nerve growth factor mimic peptide via chemical bonds in a 1:1 molar ratio. The covalent linking was selected from amide bonds, such as... Figure 1 As shown, two peptides (the self-assembled peptide sequence RADARADARADA and the brain-derived neurotrophic factor mimic peptide GGGIDKRHWNS) were docked through solid-phase synthesis.

[0059] Example 2: Culture of rat peritoneal macrophages

[0060] Extraction and isolation of rat peritoneal macrophages: SD rats aged 2-3 months (approximately 300g) were euthanized by cervical dislocation and immersed in 75% ethanol for 10 min. The rats were then lifted upside down, and 10 mL of DMEM high-glucose basal medium was injected into the peritoneal cavity using a sterile syringe. The rats' abdomens were massaged with fingers for 2 min, and then the rats were placed supine and allowed to rest for 7 min. The peritoneal cavity was then aseptically opened, and approximately 8 mL of peritoneal fluid was aspirated using another sterile syringe. The fluid was centrifuged at 400 g for 10 min, the supernatant was discarded, and the cells were resuspended in 6 mL of high-glucose DMEM complete medium and seeded into T25 culture flasks. The flasks were incubated at 37℃ in a 5% CO2 cell culture incubator, and the medium was changed after 4 h. The adherent cells were the extracted rat peritoneal macrophages.

[0061] Example 3: Preparation of M2 macrophage culture supernatant

[0062] The isolated macrophages were cultured in high-glucose DMEM complete medium at 37℃ and 5% CO2 for 3 days. The original medium was then discarded and replaced with RMPI 1640 medium containing 10 ng / ml IL-4, 10% FBS, and 1% penicillin-dextrose antibody. M2 macrophages were obtained after 24 hours of further culture. Cell growth was observed to be good during the culture process. Figure 2As shown, after confocal microscopy imaging, the cells exhibited clear pseudopodia and normal morphology. M2 macrophages obtained from culture were collected and, using CD206 antibody as a marker, their purity was detected by flow cytometry. Figure 3 As shown, after stimulation with high-glucose DMEM complete medium containing 10 ng / mL IL-4 for 3 days, the double-positive expression rate of CD206 antibody reached over 70%, and the purity of M2 macrophages was ideal. The M2 macrophage culture medium was then replaced with serum-free high-glucose medium. After 24 hours, the supernatant was collected and filtered through a 0.22 μm filter to remove cells and debris for later use.

[0063] Example 4: Preparation and structural characterization of self-assembled hydrogel scaffolds

[0064] The polypeptide sequence obtained in Example 1 was dissolved in the M2 macrophage supernatant obtained in Example 3, and the concentration was adjusted. A hydrogel was formed through spontaneous self-assembly of the polypeptide. When the mass concentration of the assembled product was 10 mg / mL or higher, such as... Figure 4-5 As shown, it exhibits a hydrogel structure with a cross-linked three-dimensional network. Transmission electron microscopy results indicate that the length and composition of polypeptide segments significantly influence the morphology of the polymer nanoassemblies and the solution-gel transition temperature.

[0065] Example 5: Method for isolating and culturing primary rat Schwann cells and 3D cell culture

[0066] Take a red-skinned mouse in a clean bench, spray it with alcohol, and wipe the mouse's tail and body clean. Cut off the head. Adjust the mouse's hind limbs with the right hand, palm facing up, and hold the mouse's hind limbs and tail with the thumb and forefinger of the left hand, respectively. Cut off the skin above the hind limbs and tail. Use curved forceps to make an incision in the depression area of ​​the hind limb, cut twice along the spine to separate the flesh from the spine, remove the flesh above, and expose the sciatic nerve and other tissues to be harvested. Take the sciatic nerve from SD red-skinned cells that have been stored for 1 day and place it in a culture dish of high-glucose DMEM complete medium. Gently aspirate the supernatant, remove the sciatic nerve and place it in an EP tube (5 mL). Add 1 mL of collagenase (for 20 mice) and digest for 30 min. Quickly cut the tissue into small pieces with ophthalmic scissors, gently pipette and mix well, and place in an incubator (37℃, 5% CO2). After 30 min, add an equal volume of 1 mL of 0.25% trypsin (Try), gently pipette and mix well, and incubate for 5 min. After 5 minutes, add at least three times the volume of high-glucose DMEM complete medium to stop digestion (3-4 times), mix well, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant. Add fresh high-glucose DMEM complete medium, mix well, filter, and inoculate cells into culture dishes. The next day, 16 hours after inoculation, change the medium and culture in high-glucose DMEM complete medium containing cytarabine (1:1000). Change the medium every four days and culture in high-DMEM complete medium until confluence. Mix the collected Schwann cells with the above hydrogel scaffold at a volume ratio of 1:3 to adjust the cell concentration to 1×10⁻⁶. 5 Cells per mL were incubated at 37°C and 5% CO2 for 30 minutes to obtain cell suspensions containing gel.

[0067] Example 6: Activity detection of Schwann cells

[0068] After CFSE labeling, Schwann cells were cultured at 37°C and 5% CO2 in high-glucose DMEM complete medium for 3 days, and their proliferation was detected by flow cytometry. Results were... Figure 6 As shown in the figure, the cells appear spherical, with more cells exhibiting protrusions, thus better mimicking the regenerative microenvironment. Flow cytometry analysis revealed that the Schwann cells in the hydrogel scaffold group showed a significantly increased growth rate compared to the control group.

Claims

1. A bonded functional polypeptide comprising a self-assembled peptide sequence and a nerve growth factor mimic peptide bound together by covalent bonds, wherein, The self-assembled peptide sequence is RADARADARADA, the nerve growth factor mimic peptide is GGGIDKRHWNS, and the molar ratio of the self-assembled peptide sequence to the nerve growth factor mimic peptide is 1:

1.

2. The bonding functional polypeptide as described in claim 1, characterized in that, The self-assembled peptide sequence is covalently linked to the nerve growth factor mimic peptide via amide bonds or disulfide bonds.

3. The use of the bonding functional polypeptide according to claim 1 or 2 in the preparation of hydrogel scaffolds, characterized in that, The bonded functional peptides self-assemble in the cell culture supernatant of anti-inflammatory M2 macrophages to form the hydrogel scaffold.

4. The use as described in claim 3, characterized in that, Anti-inflammatory M2 macrophages, which are "alternatively activated," are induced by isolating and culturing macrophages from the peritoneal cavity or bone marrow of rats or mice, or from human peripheral blood mononuclear lymphocytes.

5. The use as described in claim 4, characterized in that, The induction was performed using 10 ng / ml of IL-4.

6. The use as described in any one of claims 3-5, characterized in that, The scaffold has a nanofiber structure.

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