Amino acid modified molybdenum disulfide artificial extracellular matrix and application thereof

Abstract

The application belongs to the field of biomedical engineering, and particularly relates to an amino acid modified molybdenum disulfide artificial extracellular matrix and application. The amino acid modified molybdenum disulfide artificial extracellular matrix is prepared, neuron in vitro model PC12 cell suspension is inoculated on the artificial extracellular matrix, and a nerve cell in vitro differentiation model is constructed. The application constructs different kinds of amino acid modified molybdenum disulfide nanomaterial artificial extracellular matrix for nerve cell PC12 cell artificial extracellular matrix, obtains artificial extracellular matrix with good biocompatibility, the artificial extracellular matrix improves PC12 cell activity and promotes differentiation of nerve cell synapse, constructs a neuron in vitro model with high differentiation and higher activity, and the artificial extracellular matrix can be used for nerve regeneration and nerve engineering.

Description

Technical Field

[0001] This invention belongs to the field of medical biomaterials, and relates to a method for preparing an artificial extracellular matrix to promote the growth and differentiation of PC12 cells, an in vitro model of neurons, specifically involving an amino acid-modified molybdenum disulfide artificial extracellular matrix and its application. Background Technology

[0002] With the rapid development of biomedical engineering and nanotechnology, the development of novel biomaterials to promote the growth and differentiation of nerve cells has become a research hotspot. The growth and differentiation of nerve cells are of great significance in nerve regeneration, nerve injury repair, and the treatment of neurodegenerative diseases. However, current technologies lack a highly efficient, simple, economical, and safe material that can effectively promote the growth and differentiation of nerve cells both in vitro and in vivo.

[0003] In recent years, molybdenum disulfide (MoS2), as a typical two-dimensional transition metal sulfide, has attracted much attention due to its unique physical, chemical, and biocompatibility. Its nanosheet structure possesses abundant active sites, enabling it to interact with cells and provide a favorable substrate for nerve cell growth. However, traditional molybdenum disulfide nanosheets still have some limitations in biomedical applications, such as insufficient surface activity, weak cell interaction, and a lack of specific promoting effects on nerve cell growth and differentiation.

[0004] Patent CN 113694251 A proposes a method for preparing thin films that promote nerve cell growth and differentiation. This method achieves regulation of nerve cell growth and differentiation by preparing composite films of molybdenum disulfide nanosheets with 1T and 2H phases and polydimethylsiloxane (PDMS). However, this method mainly relies on the preparation of composite materials with different phase structures and atomic lattice structures of molybdenum disulfide itself. It does not utilize amino acid functionalization, nor does it involve amino acid modification and intercalation of molybdenum disulfide, and the preparation process is relatively complex.

[0005] Patent CN 112642291 A discloses a method for preparing an amino acid-bonded molybdenum disulfide reverse osmosis membrane, mainly for seawater desalination and wastewater treatment. While this technology achieves increased interlayer spacing and performance optimization of molybdenum disulfide through amino acid bonding, it does not involve the field of bioengineering, does not design an artificial extracellular matrix, and does not utilize the construction of a highly differentiated and active PC12 cell neuron in vitro model. Amino acids, as important organic molecules in living organisms, possess excellent biocompatibility and bioactivity. Although existing research has attempted to apply molybdenum disulfide nanosheets to the biomedical field, most methods still suffer from complex processes and a lack of precise and controllable preparation strategies.

[0006] Therefore, developing a novel artificial extracellular matrix based on amino acid-functionalized molybdenum disulfide to efficiently and easily promote the growth and differentiation of nerve cells, and to construct highly differentiated and more active in vitro neuronal models, has significant scientific and practical value. This artificial extracellular matrix should possess good biocompatibility, the ability to enhance cell activity, and specific regulatory capacity to promote PC12 neuronal synaptic differentiation, potentially leading to breakthroughs in the fields of neural regeneration and neuroengineering medicine. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes an amino acid-modified molybdenum disulfide artificial extracellular matrix and its applications.

[0008] The first technical solution of the present invention is an amino acid-modified molybdenum disulfide artificial extracellular matrix, wherein the artificial extracellular matrix is ​​formed by chemical bonding of molybdenum disulfide nanosheets and amino acids, wherein the amino acids are selected from one or more of glycine, proline, phenylalanine and leucine.

[0009] Furthermore, the amino acid-functionalized molybdenum disulfide nanoparticle bioartificial extracellular matrix, the preparation method of the artificial extracellular matrix includes the following steps:

[0010] Molybdenum disulfide nanosheets and amino acids were added to deionized water at a mass ratio of 5:25-30. The mixture was dispersed at room temperature using an ultrasonic device with a power of 300-500W for 30-60 minutes until it was completely dissolved, forming a homogeneous mixed solution A.

[0011] Mixed solution A was placed in a shaker and shaken at 100-200 rpm for 24-72 hours at room temperature to allow the molybdenum disulfide nanosheets to fully react with the amino acids, forming amino acid-functionalized molybdenum disulfide mixed solution B.

[0012] Using a porous polymer membrane with a pore size of 0.1-0.5 μm as a substrate, the mixed solution B was filtered and separated by a vacuum filtration device, with the vacuum degree controlled at 0.05-0.1 MPa, to obtain preliminarily functionalized molybdenum disulfide;

[0013] After the filtered product has settled naturally for 1-3 hours, it is washed with deionized water 3-5 times to remove unreacted impurities.

[0014] The washed product was placed in a freeze dryer and freeze-dried at -40°C to -60°C for 12-48 hours to obtain amino acid-functionalized molybdenum disulfide.

[0015] Under aseptic conditions, an amino acid-functionalized molybdenum disulfide solution was prepared and dripped onto the surface of a PDMS substrate to prepare an artificial extracellular matrix of amino acid-intercalated molybdenum disulfide nanomaterials.

[0016] The second technical solution of this invention is the application of amino acid-modified molybdenum disulfide artificial extracellular matrix in promoting nerve cell growth and differentiation.

[0017] PC12 neurons were seeded onto the prepared artificial extracellular matrix of amino acid-intercalated molybdenum disulfide nanomaterials and cultured in RPMI-1640 and fetal bovine serum. The cells were placed in a constant temperature cell culture incubator at 37°C and 5% carbon dioxide to construct an in vitro model of highly differentiated and highly active PC12 neuronal cells.

[0018] This invention is also applied in the fields of nerve regeneration and nerve injury repair.

[0019] Compared with the prior art, the present invention has the following advantages: the amino acid-functionalized molybdenum disulfide artificial extracellular matrix of the present invention promotes the growth and differentiation of nerve cells, and as a product for the regeneration and repair of neurons and nerve tissue, it has high practical value.

[0020] The amino acids selected in this invention promote biological growth. Leucine, as a key anabolic signaling compound, efficiently promotes the synthesis of target proteins; glycine provides cell protection while improving overall structural stability and biocompatibility; phenylalanine, as a precursor to various neuroactive substances, expands the functional applications of the product; and proline maintains protein stability and protects cells from environmental stress. This invention significantly improves the biocompatibility of molybdenum disulfide through amino acid modification. Experiments show that the artificial extracellular matrix of this invention exhibits lower cytotoxicity upon contact with nerve cells. Figure 7 As shown, the amino acid-functionalized molybdenum disulfide artificial extracellular matrix performed well in terms of cell viability, significantly improving the safety of the material.

[0021] 1. The artificial extracellular matrix of amino acid-functionalized molybdenum disulfide prepared by this invention effectively improves cell growth efficiency and differentiation degree.

[0022] 2. By detecting the proliferative activity of nerve cells, the expression of related proteins, and the growth of neural synapses, it was demonstrated that nerve cells grown on the artificial extracellular matrix of leucine molybdenum disulfide have good proliferation and differentiation effects and have good application prospects.

[0023] 3. The process design of this invention is ingenious, safe, controllable, and low-cost. Patent application number 202111094942.7 describes the synthesis of high-purity 1T-phase molybdenum disulfide in solution using a microwave-assisted method. A 1 mg / mL MoS2 original solution (10 mL, 1 mg / mL) in 1 mg / mL KOH was irradiated using a single-mode microwave (MAS-II Plus, China) at 200 W and 2.45 GHz. The temperature of the suspension was increased from room temperature to 180°C at a rate of 30°C / min and held at 180°C for 40 minutes. The original molybdenum disulfide was annealed in a quartz tube furnace to obtain 2H-phase MoS2 nanosheets.

[0024] The preparation of amino acid-functionalized molybdenum disulfide artificial extracellular matrix in this invention involves adding molybdenum disulfide nanosheets and various amino acids (glycine, alanine, leucine, and valine) to deionized water in specific proportions, sonicating at room temperature for 1 hour until fully dissolved, and then reacting in a shaker at room temperature for 24 hours. The price of 10g of amino acids ranges from 4 to 90 yuan, making the overall experimental cost significantly lower than that of 2H-phase MoS2 nanosheets. The entire process is conducted at room temperature and atmospheric pressure, eliminating the need for microwave reactors, high-temperature furnaces, or high-pressure systems. This results in low equipment costs, low energy consumption, and reduces safety hazards by avoiding high temperatures, strong alkalis, and microwave radiation. Furthermore, it eliminates the need for treating strongly alkaline waste liquids, making it environmentally friendly. Attached Figure Description

[0025] Figure 1 SEM images of molybdenum disulfide: (a) and (b) are SEM images of the surface of molybdenum disulfide without added amino acids; (c) and (d) are SEM images of the surface of the artificial extracellular matrix of amino acid-functionalized molybdenum disulfide prepared in this invention.

[0026] Figure 2 The results show the cell proliferation activity of the artificial extracellular matrix containing glycine, proline, phenylalanine, leucine molybdenum disulfide prepared in this invention and the PDMS control group.

[0027] Figure 3 Microscopic images showing the growth of PC12 cells on artificial extracellular matrix containing glycine, proline, phenylalanine, leucine molybdenum disulfide, and a PDMS control group.

[0028] Figure 4 Enlarged (a) and thumbnail (b) images of the growth of PC12 cells on glycine, proline, phenylalanine, molybdenum disulfide artificial extracellular matrix and PDMS control group.

[0029] Figure 5To test the differentiation degree of PC12 cells on artificial extracellular matrix containing glycine, proline, phenylalanine, leucine molybdenum disulfide and PDMS control group: maximum synaptic length (a), average synaptic length (b), and cell aspect ratio (c).

[0030] Figure 6 Fluorescence images of PC12 cells at different wavelengths observed by confocal laser scanning microscopy on artificial extracellular matrix containing glycine, proline, phenylalanine, molybdenum disulfide leucine, and PDMS control group: (a) actin, (b) cell nucleus, and (c) superimposed image of actin / nucleus.

[0031] Figure 7 Cytotoxicity data of PC12 cells on artificial extracellular matrix containing glycine, proline, phenylalanine, leucine molybdenum disulfide, and PDMS control group: (a) mitochondrial membrane potential; (b) reactive oxygen species level. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail and in completeness below with reference to the accompanying drawings.

[0033] Amino acid-functionalized molybdenum disulfide nanomaterials: Molybdenum disulfide nanosheets and amino acids were added to deionized water in a specific ratio, sonicated at room temperature for 1 h until fully dissolved, and then reacted in a shaker at room temperature for 24 h. The precipitate was collected by centrifugation, washed three times, and freeze-dried to obtain functionalized molybdenum disulfide. Figure 1 As shown, compared to the pure molybdenum disulfide substrate, the amino acid-functionalized molybdenum disulfide substrate has an orderly arrangement of layers, while the MoS2 layer structure, which is farther from the matrix, becomes relatively random and loose due to the addition of amino acids, forming a rough membrane surface, which provides more surface area and contact sites for the synaptic growth of nerve cells.

[0034] Example 1

[0035] 1. Synthesizing the nanobiomaterials described in this invention:

[0036] Preparation of amino acid-functionalized molybdenum disulfide artificial extracellular matrix: Molybdenum disulfide nanosheets and glycine were added to deionized water at a mass ratio of 5:30, and sonicated at room temperature for 1 h until fully dissolved. Then, the mixture was reacted in a shaker at room temperature for 24 h. After natural precipitation, the mixture was rinsed three times with deionized water, frozen at -40℃, and then placed in a vacuum freeze dryer.

[0037] 2. Preparation method of artificial extracellular matrix to promote nerve cell growth and differentiation:

[0038] Polydimethylsilane PDMS substrates were prepared using Sylgard 184 (Dow Corning, USA) as a curing agent. PDMS and the curing agent were mixed at a ratio of 10:1, stirred for 20 minutes, and cured at 70°C for 2 hours. The cured PDMS was then immersed in 30 µL of anhydrous ethanol in 30 µL of APTES solution for 1 hour. After washing three times with anhydrous ethanol, the substrate was dried at 60°C.

[0039] Under aseptic conditions, 5 mg of glycine molybdenum disulfide was weighed and added to 5 ml of anhydrous ethanol to prepare a 1 mg / mL glycine molybdenum disulfide solution. The solution was then ultrasonically dispersed at 3°C ​​for 30 minutes. The 1 mg / mL glycine molybdenum disulfide solution was diluted to 10 mg / L and then evenly dropped onto a PDMS surface. After drying, the glycine molybdenum disulfide artificial extracellular matrix was obtained.

[0040] 3. Research on the role of artificial extracellular matrix in promoting nerve cell growth:

[0041] The prepared glycine disulfide artificial extracellular matrix was placed in 24-well plates for cell culture comparison with wells of PDMS without glycine disulfide coating. Then, approximately 1 mL of 1.0 x 10⁻⁶ ppm ... 5 PC12 cells were cultured at 37°C for 36 hours. When the cell density reached 90%, the viability of PC12 cells was determined using a cell counting kit (Cell Counting Kit-8, CCK-8). After culture, the cell culture medium was discarded, and the cells were washed three times with phosphate-buffered saline. 10 µL of CCK-8 solution and 100 µL of cell culture medium were added to each well, and the cells were incubated for another 4 hours. The cells were then detected at 450 nm using a microplate reader. Wells containing the appropriate amount of cell culture medium and CCK-8 solution but without cells served as blank controls. Figure 2 It can be observed that artificial extracellular matrix significantly improves cell survival rate.

[0042] Example 2

[0043] 1. Synthesizing the nanobiomaterials described in this invention:

[0044] Preparation of amino acid-functionalized molybdenum disulfide artificial extracellular matrix: Molybdenum disulfide nanosheets and proline were added to deionized water at a ratio of 5:28 and sonicated at room temperature for 1 h until fully dissolved. The mixture was then reacted in a shaker at room temperature for 24 h. After natural precipitation, the mixture was rinsed three times with deionized water, frozen at -60℃, and then placed in a vacuum freeze dryer.

[0045] 2. Preparation method of artificial extracellular matrix to promote nerve cell growth and differentiation:

[0046] Polydimethylsilane PDMS substrates were prepared using Sylgard 184 (Dow Corning, USA) as a curing agent. PDMS and the curing agent were mixed at a ratio of 10:1, stirred for 20 minutes, and cured at 65°C for 2.5 hours. The cured PDMS was then immersed in 30 µL of anhydrous ethanol in a 30 µL LAPTES solution for 1 hour. After washing three times with anhydrous ethanol, the substrate was dried at 60°C.

[0047] Under aseptic conditions, 5 mg of proline molybdenum disulfide was weighed and added to 5 ml of anhydrous ethanol to prepare a 1 mg / mL proline molybdenum disulfide solution. The solution was then ultrasonically dispersed at 5°C for 30 minutes. The 1 mg / mL proline molybdenum disulfide solution was diluted to 15 mg / L and then evenly dropped onto a PDMS surface. After drying, the proline molybdenum disulfide artificial extracellular matrix was obtained.

[0048] 3. Research on the role of artificial extracellular matrix in promoting nerve cell growth:

[0049] The prepared proline-molybdenum disulfide artificial extracellular matrix was placed in 24-well plates and compared with PDMS plates without proline-molybdenum disulfide coating for cell culture. Then, approximately 1 mL of 1.0 x 10⁻⁶ ppm 1% PDMS was added to each well. 5 PC12 cells were cultured at 37°C for 36 hours. When the cell density reached 90%, the viability of PC12 cells was determined using a cell counting kit (Cell Counting Kit-8, CCK-8). After culture, the cell culture medium was discarded, and the cells were washed three times with phosphate-buffered saline. 10 µL of CCK-8 solution and 100 µL of cell culture medium were added to each well, and the cells were incubated for another 4 hours. The cells were then detected at 450 nm using a microplate reader. Wells containing the appropriate amount of cell culture medium and CCK-8 solution but without cells served as blank controls. Figure 2 It can be observed that artificial extracellular matrix significantly improves cell survival rate.

[0050] Example 3

[0051] 1. Synthesizing the nanobiomaterials described in this invention:

[0052] Preparation of amino acid-functionalized molybdenum disulfide artificial extracellular matrix: Molybdenum disulfide nanosheets and phenylalanine were added to deionized water at a ratio of 5:25 and sonicated at room temperature for 1 h until fully dissolved. The mixture was then reacted in a shaker at room temperature for 24 h. After natural precipitation, the mixture was rinsed three times with deionized water, frozen at -50℃, and then placed in a vacuum freeze dryer.

[0053] 2. Preparation method of artificial extracellular matrix to promote nerve cell growth and differentiation:

[0054] Polydimethylsilane PDMS substrates were prepared using Sylgard 184 (Dow Corning, USA) as a curing agent. PDMS and the curing agent were mixed at a ratio of 10:1, stirred for 20 minutes, and cured at 60°C for 3 hours. The cured PDMS was then immersed in 30 µL of anhydrous ethanol in 30 µL of APTES solution for 1.5 hours. After washing three times with anhydrous ethanol, the substrate was dried at 60°C.

[0055] Under aseptic conditions, 5 mg of phenylalanine molybdenum disulfide was weighed and added to 5 ml of anhydrous ethanol to prepare a 1 mg / mL phenylalanine molybdenum disulfide solution. The solution was then ultrasonically dispersed at 4°C for 30 minutes. The 1 mg / mL phenylalanine molybdenum disulfide solution was diluted to 12 mg / L and then evenly dropped onto a PDMS surface. After drying, the phenylalanine molybdenum disulfide artificial extracellular matrix was obtained.

[0056] 3. Research on the role of artificial extracellular matrix in promoting nerve cell growth:

[0057] The prepared phenylalanine disulfide artificial extracellular matrix was placed in 24-well plates and compared with PDMS without phenylalanine disulfide coating for cell culture. Then, approximately 1 mL of 1.0 x 10⁻⁶ ppm ... 5 PC12 cells were cultured at 37°C for 36 hours. When the cell density reached 90%, the viability of PC12 cells was determined using a cell counting kit (Cell Counting Kit-8, CCK-8). After culture, the cell culture medium was discarded, and the cells were washed three times with phosphate-buffered saline. 10 µL of CCK-8 solution and 100 µL of cell culture medium were added to each well, and the cells were incubated for another 4 hours. The cells were then detected at 450 nm using a microplate reader. Wells containing the appropriate amount of cell culture medium and CCK-8 solution but without cells served as blank controls. Figure 2 It can be observed that artificial extracellular matrix significantly improves cell survival rate.

[0058] Example 4

[0059] 1. Synthesizing the nanobiomaterials described in this invention:

[0060] Preparation of amino acid-functionalized molybdenum disulfide artificial extracellular matrix: Molybdenum disulfide nanosheets and leucine were added to deionized water at a ratio of 5:27, and sonicated at room temperature for 1 h until fully dissolved. Then, the mixture was reacted in a shaker at room temperature for 24 h. After natural precipitation, the mixture was rinsed three times with deionized water, frozen at -40℃, and then placed in a vacuum freeze dryer.

[0061] 2. Preparation method of artificial extracellular matrix to promote nerve cell growth and differentiation:

[0062] Polydimethylsilane PDMS substrates were prepared using Sylgard 184 (Dow Corning, USA) as a curing agent. PDMS and the curing agent were mixed at a ratio of 10:1, stirred for 20 minutes, and cured at 70°C for 2 hours. The cured PDMS was then immersed in 30 µL of anhydrous ethanol in 30 µL of APTES solution for 1 hour. After washing three times with anhydrous ethanol, the substrate was dried at 60°C.

[0063] Under aseptic conditions, 5 mg of molybdenum leucine disulfide was weighed and added to 5 ml of anhydrous ethanol to prepare a 1 mg / mL molybdenum leucine disulfide solution. The solution was then ultrasonically dispersed at 4°C for 30 minutes. The 1 mg / mL molybdenum leucine disulfide solution was diluted to 10 mg / L and then evenly dropped onto a PDMS surface. After drying, the artificial extracellular matrix of molybdenum leucine disulfide was obtained.

[0064] 3. Research on the role of artificial extracellular matrix in promoting nerve cell growth:

[0065] The prepared leucine molybdenum disulfide artificial extracellular matrix was placed in 24-well plates and compared with PDMS wells without leucine molybdenum disulfide coating for cell culture. Then, approximately 1 mL of 1.0 x 10⁻⁶ ppm 1% PDMS was added to each well. 5 PC12 cells were cultured at 37°C for 36 hours. When the cell density reached 90%, the viability of PC12 cells was determined using a cell counting kit (Cell Counting Kit-8, CCK-8). After culture, the cell culture medium was discarded, and the cells were washed three times with phosphate-buffered saline. 10 µL of CCK-8 solution and 100 µL of cell culture medium were added to each well, and the cells were incubated for another 4 hours. The cells were then detected at 450 nm using a microplate reader. Wells containing the appropriate amount of cell culture medium and CCK-8 solution but without cells served as blank controls. Figure 2 It can be observed that artificial extracellular matrix significantly improves cell survival rate.

[0066] Research on the effect of the artificial extracellular matrix described in this invention on promoting neural cell differentiation:

[0067] PC12 neurons were seeded onto the prepared artificial extracellular matrix of amino acid-intercalated molybdenum disulfide nanomaterials and cultured in RPMI-1640 and fetal bovine serum. The cells were placed in a constant temperature cell culture incubator at 37°C and 5% carbon dioxide to construct an in vitro model of highly differentiated and highly active PC12 neuronal cells.

[0068] Figure 3 , Figure 4 These are microscopic and confocal images of PC12 cells grown for 36 hours on a molybdenum disulfide artificial extracellular matrix (PDMS) containing glycine, proline, phenylalanine, and leucine. Longer neural synapses are clearly visible in the PC12 cells grown on the artificial extracellular matrix. The maximum synapse length, average synapse length, and cell length-to-width ratio of PC12 cells grown on the molybdenum disulfide artificial extracellular matrix and PDMS were measured using the confocal image processing software ZEN (ZEN blue edition 2.3, Germany) and ImageJ. Figure 5 As shown. Figure 6 This indicates that growing on the artificial extracellular matrix provided by the present invention can effectively promote the differentiation of nerve cells and improve cell culture efficiency. Figure 7 (a) Compared with the PDMS control group, the mitochondrial membrane potential of PC12 cells in the glycine, proline, phenylalanine and leucine treatment groups did not change significantly, and there were no statistically significant differences among the groups (ns). Figure 7 (b) ROS levels: No significant differences were observed in intracellular ROS levels between the amino acid treatment groups and the control group. These results indicate that, under the experimental conditions employed in this invention, the four selected amino acids did not cause significant mitochondrial functional damage or oxidative stress in PC12 cells, demonstrating good biosafety.

[0069] 1. The essential difference between "the culture medium also contains amino acids" and "amino acid-modified molybdenum disulfide artificial extracellular matrix" can be divided into the following aspects:

[0070] The difference lies in their physical form: the homogeneity of the solution and the difference in the local microenvironment at the material interface. In the PC12 cell culture process, RPMI-1640 and fetal bovine serum were used. The amino acids in these solutions are homogeneous components dissolved in the culture medium, used to maintain basal cell metabolism and proliferation, and are the common "background conditions" for all experimental groups. However, the amino acid-modified molybdenum disulfide artificial extracellular matrix of this invention belongs to solid-phase material structure / interface modification. It mainly leads to changes in the interfacial properties of the artificial extracellular matrix, such as surface functional groups, surface charge, hydrophilicity, roughness, and protein adsorption behavior, thereby affecting PC12 cell adhesion, morphological spreading, and changes in neural differentiation-related signals.

[0071] 2. The role of amino acids in cell culture media is "global" and diluted; while the molybdenum disulfide amino acid artificial extracellular matrix in this invention acts on the biological interface effect of cell adhesion. This type of effect is usually not replaced by the presence of amino acids in the culture medium.

[0072] 3. The amino acids contained in the culture medium are more of a nutrient and metabolic substrate; while the molybdenum disulfide amino acid artificial extracellular matrix designed in this invention changes the surface properties to affect the "adsorption protein layer" formed by serum proteins on the surface, thereby changing the adhesion and downstream pathways of cell integrins. This is not the same issue as "whether the culture medium contains amino acids".

[0073] 4. The culture medium composition remained completely consistent across all control / experimental groups; therefore, the amino acid composition of the medium could not explain the "inter-group differences," which could only stem from "differences in the cell substrate." All experimental groups used the same batch of RPMI-1640 with the same proportion of fetal bovine serum, and the culture conditions were identical. Therefore, the amino acids in the culture medium, as a common background condition, had the same effect on all groups and could not lead to "differential differentiation / enhanced synaptic growth observed only in a particular material group."

[0074] This invention further sets up a control group consisting of molybdenum disulfide amino acid artificial extracellular matrix and a blank substrate (PDMS control group, i.e., without molybdenum disulfide amino acid), which also proves that the differentiation-promoting effect mainly comes from the contact interface of the molybdenum disulfide amino acid artificial extracellular matrix rather than the soluble amino acids contained in the culture medium.

[0075] This invention also has material cost advantages: Patent document No. 202111094942.7 describes the synthesis of molybdenum disulfide nanosheets dispersed in KOH solution via microwave synthesis, where the temperature of the suspension rises from room temperature to 170°C during microwave treatment. The process involves heating the material at 180℃ (30℃ / min) for 40 minutes, followed by freeze-drying. This process, which involves going from high temperature to freezing, results in a significant waste of energy and involves a rather complicated experimental procedure.

[0076] This invention reduces material usage and waste through an optimized preparation process. For example, by precisely controlling the concentration and dispersion conditions of amino acid-functionalized molybdenum disulfide, the material can be utilized more efficiently, reducing preparation costs. Furthermore, the selected crosslinking agents are all common and low-cost materials, further reducing overall material costs.

[0077] Patent application number 202111094942.7 states that the process of transforming 1T phase MoS2 into 2H phase nanosheets requires annealing at 300-500 degrees Celsius in a quartz furnace for 3-5 hours, which is complex and costly.

[0078] The preparation method of this invention has been optimized, simplifying the operation steps and reducing equipment requirements and energy consumption. For example, by optimizing the ultrasonic dispersion temperature and PDMS curing conditions, preparation can be completed in a shorter time, improving production efficiency and reducing preparation costs. In addition, although steps such as freeze-drying increase process complexity, they significantly improve the matrix properties and stability, resulting in higher economic value in the long run.

[0079] In summary, the artificial extracellular matrix provided by this invention not only promotes cell survival but also effectively promotes the growth of nerve cell synapses, improves cell differentiation and activity, and constructs a highly differentiated and highly active PC12 cell neuron in vitro model. It also has cost advantages and is environmentally friendly, and is expected to become an ideal artificial extracellular matrix for in vitro or in vivo nerve repair and tissue engineering, with broad application prospects.

Claims

1. A method for preparing an artificial extracellular matrix using amino acid-intercalated molybdenum disulfide nanomaterials, characterized in that, Includes the following steps: Molybdenum disulfide nanosheets and amino acids were added to deionized water at a mass ratio of 5:25-30. The mixture was dispersed at room temperature using an ultrasonic device with a power of 300-500W for 30-60 minutes until it was completely dissolved, forming a homogeneous mixed solution A. Mixed solution A was placed in a shaker and shaken at 100-200 rpm for 24-72 hours at room temperature to allow the molybdenum disulfide nanosheets to fully react with the amino acids, forming amino acid-functionalized molybdenum disulfide mixed solution B. Using a porous polymer membrane with a pore size of 0.1-0.5 μm as a substrate, the mixed solution B was filtered and separated by a vacuum filtration device, with the vacuum degree controlled at 0.05-0.1 MPa, to obtain preliminarily functionalized molybdenum disulfide; After the filtered product has settled naturally for 1-3 hours, it is washed with deionized water 3-5 times to remove unreacted impurities. The washed product was placed in a freeze dryer and freeze-dried at -40°C to -60°C for 12-48 hours to obtain amino acid-functionalized molybdenum disulfide. Under aseptic conditions, an amino acid-functionalized molybdenum disulfide solution was prepared and dripped onto the surface of a PDMS substrate to prepare an artificial extracellular matrix of amino acid-intercalated molybdenum disulfide nanomaterials. The amino acid is selected from proline and leucine.

2. The artificial extracellular matrix of amino acid-intercalated molybdenum disulfide nanomaterials prepared according to claim 1, characterized in that, The artificial extracellular matrix is ​​formed by chemical bonding of molybdenum disulfide nanosheets and amino acids.

3. The application of the amino acid-intercalated molybdenum disulfide nanomaterial artificial extracellular matrix according to claim 2 in promoting nerve cell growth and differentiation for non-therapeutic purposes, characterized in that, PC12 neurons were seeded onto the prepared artificial extracellular matrix of amino acid-intercalated molybdenum disulfide nanomaterials, cultured in RPMI-1640 and fetal bovine serum, and placed in a constant temperature cell culture incubator to construct an in vitro model of highly differentiated and highly active PC12 neuronal cells.

4. The application of the artificial extracellular matrix of amino acid intercalated molybdenum disulfide nanomaterials according to claim 2 in the preparation of medical devices or drugs for nerve regeneration and nerve injury repair.

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