Polypeptide derivative-based nanofiber as well as enzymatic self-assembly method and application thereof

Abstract

The invention discloses a nanofiber based on a polypeptide derivative and an enzymatic self-assembly method and application of the nanofiber based on the polypeptide derivative, the sequence of the polypeptide derivative is Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg, and the Gly-Phe-Phe-pTyr is of an L configuration or a D configuration. The self-assembled polypeptide derivative disclosed by the invention contains an alkaline phosphatase restriction enzyme cutting site, and the self-assembled polypeptide derivative is self-assembled at a specific part with high enzyme specificity expression through enzyme catalysis to form nanofibers. The nanofiber hydrogel obtained through enzymatic treatment is carried out under physiological conditions, high temperature or ultraviolet irradiation is not needed, damage of high temperature to bioactive substances is avoided, the nanofiber hydrogel is more suitable for a temperature-sensitive in-vivo environment, toxic chemical cross-linking agents or photoinitiators do not need to be introduced, the cytotoxicity of the material is remarkably reduced, active enzymes or drugs can be embedded in situ, and the nanofiber hydrogel can be used for in-situ treatment. The biological function is maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to a nanofiber based on polypeptide derivatives and an enzymatic self-assembly method and application thereof. Background Art

[0002] The pulp-dentin complex (DDC) plays a vital role in tooth protection and function maintenance. However, this tissue is susceptible to damage from factors such as caries and trauma. Therefore, regeneration of the DDC is crucial for restoring tooth vitality. Current regenerative approaches primarily stimulate the differentiation of resident or transplanted stem / progenitor cells to promote DDC reconstruction.

[0003] Recent studies have shown that bioactive molecules (such as proteins, growth factors, and hormones) have shown great potential in the regeneration of the dental pulp-dentin complex. For example, enamel matrix derivatives (EMDs) extracted from porcine tooth germs have been widely used in regenerative engineering and biomimetic mineralization due to their role in pulp cell differentiation and dentin bridge formation. However, due to the inconsistency of raw materials and extraction methods, the effects of EMDs are often unstable. Amelogenin is the main component of EMDs, among which leucine-rich amelogenin peptide (LRAP) and tyrosine-rich amelogenin peptide (TRAP) have been shown to have the activity of inducing cell differentiation, promoting angiogenesis, and biomimetic mineralization.

[0004] Short peptide bioactive molecules often suffer from susceptibility to enzymatic hydrolysis, short half-lives, low bioavailability, and a lack of native protein spatial structure, limiting their application in mimicking the biological activities of native proteins. To address this challenge, a hydrogel based on the self-assembly of amelogenin peptides was developed. Results showed that this hydrogel effectively activated the MAPK-ERK1 / 2 signaling pathway and, through this pathway and TGF-β / Smad signaling, enhanced the mineralization and odontogenic differentiation of human dental pulp cells (HDPCs), demonstrating its great potential for pulp-dentin complex regeneration. However, this hydrogel suffers from low self-assembly concentration and the potential for damage to heat-sensitive bioactive molecules (such as proteins or cells) during the heating-and-cooling preparation process, limiting its biomedical application. Furthermore, high temperatures can induce uncontrolled degradation of polymer chains, resulting in the production of potentially toxic byproducts. Therefore, a milder and more efficient preparation method is urgently needed.

[0005] Alkaline phosphatase (ALP) is widely present in organisms and is also a key enzyme in the mineralization process. It can catalyze the release of phosphate and provide inorganic phosphate for the mineralization of dentin and bone tissue. Odontoblasts in the dental pulp promote the formation and repair of dentin by secreting ALP. After dental pulp damage or inflammation, the activity of ALP in dental pulp cells increases, participating in the repair of dental pulp tissue and the regeneration of hard tissue. Based on this, the present invention uses amelogenin polypeptide (AMG-P8) as the basic assembly unit and innovatively designs an enzymatic self-assembly strategy. Summary of the Invention

[0006] The purpose of the present invention is to address the technical deficiencies in the prior art and to provide a polypeptide derivative containing an enzyme cleavage site that can achieve enzyme-catalyzed self-assembly.

[0007] Another aspect of the present invention provides a nanofiber based on the polypeptide derivative.

[0008] Another aspect of the present invention provides an enzymatic self-assembly method of nanofibers based on the polypeptide derivative.

[0009] The present invention also provides an application of nanofibers based on the polypeptide derivatives.

[0010] The present invention also provides an application of nanofiber in tissue engineering, drug delivery and biosensing.

[0011] The present invention also provides an application of nanofibers in preparing biological preparations for treating dental caries and dental pulp diseases.

[0012] The technical solution adopted to achieve the purpose of the present invention is:

[0013] A polypeptide derivative containing an enzyme cleavage site capable of realizing enzyme-catalyzed self-assembly, wherein the sequence of the polypeptide derivative is Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg.

[0014] In the above technical solution, Gly-Phe-Phe-pTyr in the polypeptide derivative is in L configuration or D configuration, preferably in L configuration. When Gly-Phe-Phe-pTyr is in D configuration, the structural formula of Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg is:

[0015]

[0016] When Gly-Phe-Phe-pTyr is in the L configuration, the structural formula of Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg is:

[0017]

[0018] Another aspect of the present invention further includes an enzymatic self-assembly method of nanofibers based on the polypeptide derivative, characterized in that it comprises the following steps:

[0019] Step 1, solid phase synthesis of the polypeptide derivative;

[0020] Step 2: Add the polypeptide derivative prepared in step 1 to a PBS solution, heat until completely dissolved, adjust the pH value, and then perform enzyme catalysis to induce self-assembly to form nanofibers of the polypeptide derivative, wherein the nanofibers are in a hydrogel state.

[0021] In the above technical solution, the enzyme in step 2 is an enzyme that can catalyze the formation of peptide bonds from polypeptide derivatives, preferably alkaline phosphatase.

[0022] In the above technical solution, the enzyme catalysis temperature in step 2 is 36-38°C.

[0023] In the above technical solution, the specific steps of solid phase synthesis of polypeptide derivatives in step 1 are:

[0024] Step a, the C-terminus of Fmoc-Arg is bound to the resin;

[0025] Step b, removing the Fmoc protecting group of Fmoc-Arg and washing;

[0026] Step c, adding Fmoc-Ile, coupling the C-terminus of Fmoc-Ile with the N-terminus of Arg coupled to the resin, then removing the Fmoc protecting group of Fmoc-Ile and washing;

[0027] Step d, sequentially coupling Fmoc-Met, Fmoc-Asn, Fmoc-Gln, Fmoc-Tyr, Fmoc-Trp, Fmoc-Lys, Fmoc-pTyr in D or L configuration, Fmoc-Phe in D or L configuration, Fmoc-Phe in D or L configuration, and Fmoc-Gly, after each coupling is completed, removing the Fmoc protecting group and performing the next coupling until the last amino acid is coupled, removing the Fmoc protecting group, and washing;

[0028] Step e, adding 2-naphthyl acetic acid to couple with the N-terminus of the polypeptide derivative on the resin, and washing;

[0029] Step f, removing the polypeptide derivative from the resin to obtain a crude product;

[0030] Step g, purifying the crude product of the polypeptide derivative;

[0031] Step h: freeze-drying to obtain polypeptide derivative powder.

[0032] In the above technical solution, the resin described in step a is 2-cl-Trt or rink resin.

[0033] Another aspect of the present invention also includes nanofibers prepared by the enzymatic self-assembly method.

[0034] Another aspect of the present invention also includes the application of the nanofiber in tissue engineering, drug delivery, and biosensing.

[0035] Another aspect of the present invention also includes the use of nanofibers in preparing biological preparations for treating caries and dental pulp diseases.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The polypeptide derivatives used in the present invention can simulate the biological activity of amelogenin, give cells a "dominant" position, give full play to the synergistic effect between cells and matrix, efficiently induce cell differentiation, promote extracellular matrix mineralization, and achieve precise regeneration of the dental pulp-dentin complex.

[0038] 2. The present invention prepares polypeptide derivatives containing alkaline phosphatase cleavage sites that can achieve enzymatic self-assembly, allowing them to form nanofibers through enzymatic self-assembly at specific sites where the enzyme is highly expressed. The nanofibers obtained by enzymatic treatment are processed under physiological conditions without the need for high temperatures or ultraviolet irradiation, thus avoiding the damage of high temperatures to biologically active substances and being more suitable for the temperature-sensitive in vivo environment. The enzymatic reaction, which relies on natural enzyme catalysis, does not require the introduction of toxic chemical cross-linking agents or photoinitiators, thereby significantly reducing the cytotoxicity of the polypeptide derivative nanofibers and enabling the in situ embedding of active enzymes or drugs to maintain their biological functions.

[0039] 3. The enzyme-catalyzed reaction of the present invention is highly selective, enabling precise cross-linking at the molecular level and forming a more uniform three-dimensional network structure. Existing heating and cooling methods can lead to uneven cross-linking density, affecting material properties. Enzymatic hydrogels can be combined with a cascade catalytic system to continuously regulate the microenvironment after gel formation, providing antibacterial, anti-inflammatory, or repair-promoting functions. Existing heating and cooling methods struggle to achieve this dynamic response. The present invention not only addresses the unstable biological activity of amelogenin polypeptides but also leverages the in situ self-assembly advantages of polypeptide derivative hydrogels, providing new strategies and methods for the regeneration of the dental pulp-dentin complex.

[0040] 4. Given the ability of dental pulp cells to secrete alkaline phosphatase (ALP), and the significantly enhanced enzyme activity induced by amelogenin peptides, the present invention introduces specific cleavage sites into the peptide derivative precursor molecule, enabling it to self-assemble through an enzymatic reaction within the specific region of high enzyme expression, forming a stable nanofiber network structure. This nanofiber network tightly surrounds dental pulp cells, accurately mimicking the natural developmental microenvironment of the dental pulp-dentin complex. During this process, AMG-P8 is slowly released locally and actively taken up by the cells, effectively activating the bidirectional interaction and response between cells and their microenvironment. The core of this strategy is to give cells a "dominant" position, fully leveraging the synergistic effect between cells and the matrix, efficiently inducing cell differentiation, promoting extracellular matrix mineralization, and ultimately achieving precise regeneration of the dental pulp-dentin complex through cell transplantation or cell homing. This invention will provide an important basis for the development of superior biological agents and drug delivery systems. At the same time, by studying the mechanisms of dental pulp cell differentiation and the regulation of matrix mineralization, it will further reveal the underlying mechanisms of tissue regeneration, provide new solutions for the treatment of oral diseases such as caries and pulp disease, and open up broader clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Nap-G in Example 1 of the present invention D F D F D The structural formula of pYKWYQNMIR.

[0042] Figure 2 The structural formula of Nap-GFFpYKWYQNMIR in Example 2 of the present invention.

[0043] Figure 3 Shown is the Nap-G of the present invention D F D F D High-resolution mass spectrum of pYKWYQNMIR.

[0044] Figure 4 Shown is the Nap-G of the present invention D F D F D Photograph of the enzymatic formation of D-gel by pYKWYQNMIR and a schematic diagram of the injectability of D-gel.

[0045] Figure 5 Shown are the CMC values ​​of D-gel and L-gel of the present invention.

[0046] Figure 6 Shown are the CMC values ​​of D-gel' and L-gel' prepared in Comparative Example 1.

[0047] Figure 7 Shown are the CD spectra of D-gel and L-gel of the present invention.

[0048] Figure 8 The CD spectra of D-gel', L-gel' and AMGP8 prepared in Comparative Example 1 are shown.

[0049] Figure 9 Shown are the rheological test results of Nap-GFFpYKWYQNMIR of the present invention.

[0050] Figure 10 Shown is the Nap-G of the present invention D F D F D TEM images of pYKWYQNMIR and Nap-GFFpYKWYQNMIR before and after ALP treatment.

[0051] Figure 11 The figure shows the effect of different concentrations of hydrogel on the activity of HDPCs detected by CCK-8. "***" in the figure indicates P < 0.001.

[0052] Figure 12 Shown are the results of Alizarin red staining and semi-quantitative analysis of hydrogels of different types and concentrations. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0054] Example 1

[0055] A Nap-G based D F D F D pYKWYQNMIR (structural formula is as Figure 1 As shown, that is, Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg, wherein Gly-Phe-Phe-pTyr is in D configuration) nanofibers, the preparation method thereof comprises the following steps:

[0056] Step 1, Nap-G D F D F D The solid phase synthesis of pYKWYQNMIR is as follows:

[0057] 1) Weigh 0.5 mmol of 2-Cl-Trt resin into a solid phase synthesizer, add 10 mL of anhydrous dichloromethane (DCM), and place on a shaker to swell for 10 minutes;

[0058] 2) Use an ear bulb to remove DCM from the solid phase synthesizer;

[0059] 3) Dissolve 0.75 mmol of Fmoc-Arg in 20 mL of anhydrous DCM, add 1.5 mmol of DIPEA, and after sufficient dissolution, transfer to the above-mentioned solid phase synthesizer and react at room temperature for 1 h;

[0060] 4) Blocking: Remove the reaction solution from the solid phase synthesizer with an ear bulb, then wash with 10 mL of anhydrous DCM for 1 min each time, for a total of 4 washes. Add 20 mL of a solution prepared with a volume ratio of anhydrous DCM:DIPEA:methanol = 17:1:2, and react at room temperature for 20 min;

[0061] 5) The reaction solution in the solid phase synthesizer was removed using an ear bulb. The mixture was first washed with anhydrous DCM (10 mL per wash, 1 min per wash), four times. The mixture was then washed with N,N-dimethylformamide (DMF), 10 mL per wash, 1 min per wash, four times. 10 mL of DMF containing 20% ​​piperidine by volume was added and the mixture was reacted for 30 min. The mixture was then washed with DMF, 10 mL per wash, 1 min per wash, four times.

[0062] 6) Weigh 1 mmol of Fmoc-Ile, 2 mmol of HBTU, and 2 mmol of DIPEA, add 10 mL of DMF and sonicate to dissolve. Add the dissolved solution to the solid phase synthesizer and react at room temperature for 2 h.

[0063] 7) Fmoc-Met, Fmoc-Asn, Fmoc-Gln, Fmoc-Tyr, Fmoc-Trp, Fmoc-Lys, D-configuration Fmoc-pTyr, D-configuration Fmoc-Phe, D-configuration Fmoc-Phe, Fmoc-Gly, and 2-naphthylacetic acid were added in sequence for coupling. After each coupling, the Fmoc protecting group was removed and the next coupling was performed until the last amino acid was coupled. The Fmoc protecting group was removed and the reaction mixture was washed with DMF 5 times and DCM 5 times after each coupling.

[0064] 8) 10 mL of a solution composed of 95% TFA, 2.5% TIS, and 2.5% H2O by volume was added to the solid phase synthesizer and allowed to react for half an hour. The product was cut from the 2-cl-Trt resin, concentrated in vacuo, and the solvent removed to obtain a crude product, which was then separated and purified using a high performance liquid chromatograph to obtain the polypeptide derivative Nap-G. D F D F D pYKWYQNMIR, reference Figure 3 , for Nap-G D F D F D The high-resolution mass spectrum of pYKWYQNMIR shows that the main peak is half the peak, and the difference between the molecular weight and the theoretical calculated molecular weight is less than 1, which proves that the peptide derivative Nap-G D F D F D Successful synthesis of pYKWYQNMIR.

[0065] Step 2, Preparation of Nanofibers of Peptide Derivatives

[0066] Weigh 1.8 mg of purified peptide derivative Nap-G D F D F D pYKWYQNMIR was placed in a 2 mL glass bottle, 500 μL of PBS solution (pH = 7.4) was added, and ultrasonic vibration was performed until the compound was completely dissolved. The pH value was adjusted to 7.4 with sodium carbonate solution, and alkaline phosphatase ALP was added to induce self-assembly to form nanofibers Nap-G D F D F D YKWYQNMIR is referred to as D-gel, Figure 4 Whether the gel is formed or not can be determined by methods known in the art, such as by inverting the bottle. If the liquid remains at the bottom of the bottle, it is a hydrogel, and if it flows, it is a liquid. Figure 10 This is an electron projection microscope image of D-gel. It can be seen that the microstructure of D-gel is nanofiber.

[0067] Example 2

[0068] The difference from Example 1 is that the D-configuration Fmoc-Tyr, D-configuration Fmoc-Phe, and D-configuration Fmoc-Phe added in step 7) of the solid-phase synthesis of step 1 are replaced with L-configuration to obtain the polypeptide derivative Nap-GFFpYKWYQNMIR (according to common knowledge in the art, when the amino acid configuration is not limited, it is considered to be L-configuration), the structural formula is as follows: Figure 2As shown (i.e., Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg, wherein Gly-Phe-Phe-pTyr is in the L configuration). In step 2, the preparation of the polypeptide derivative nanofiber Nap-GFFYKWYQNMIR (abbreviated as L-gel) is the same as in Example 1, except that the color of the formed hydrogel is not exactly the same as that in Example 1.

[0069] Comparative Example 1

[0070] Comparative Example 1: According to the paper An amelogenin-based peptide hydrogel promoted theodontogenic differentiation of human dental pulp cells, author: Xinxin Li1, Zhaoxia Yu, Shihui Jiang, Xiaohua Dai, Guanhua Wang, Yue Wang, Zhimou Yang, Jie Gao, Huiru Zou, Nap-G D F D F D YKWYQNMIR adopts the heating and cooling method to prepare synthetic nanofibers, referred to as D-gel', and Nap-GFFYKWYQNMIR adopts the heating and cooling method to prepare synthetic nanofibers, referred to as L-gel'.

[0071] The preparation method of sterile 1× PBS used in the test example is as follows:

[0072] Preparation of sterile 1x PBS:

[0073] Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 and dissolve them in 800ml distilled water. Adjust the pH value of the solution to 7.4 with sodium carbonate and hydrochloric acid solution. Finally, add distilled water to make up the volume to 1L. Sterilize in an autoclave and store at room temperature or in a refrigerator at 4℃.

[0074] Test Example 1

[0075] Performance characterization of nanofibers in Examples 1-2 and Comparative Example 1:

[0076] 1. Use a microplate reader to determine the critical assembly concentration (CMC) value;

[0077] Reference Figure 5, is the critical assembly concentration of D-gel and L-gel prepared in Example 1 and Example 2. The critical assembly concentration CMC measurement shows that the CMC value of D-gel is 63μm and the CMC value of L-gel is 8μm. The critical assembly concentration CMC of L-gel is lower than that of D-gel. Figure 6 The CMC values ​​of L-gel' and D-gel' prepared in Comparative Example 1 were 84.25 μM and 86.62 μM, respectively. After introducing the phosphorylation enzymatic response site, the critical assembly concentration (CMC) of Examples 1 and 2 was significantly reduced (from 84.25 μM for L-gel' to 8 μM for D-gel), indicating an enhanced driving force for molecular self-assembly. A low CMC value indicates earlier gelation initiation. This rapid in situ gelation is attributed to an enzymatically triggered secondary structural transition (e.g., L-gel' transitions from random coil to β-sheet), which enhances the orderliness and mechanical strength of the supramolecular network.

[0078] 2. Circular dichroism CD test:

[0079] Peptide solutions at concentrations ranging from 0.5 to 1000 μM were prepared in PBS (pH 7.4). The peptide diluents were then incubated with rhodamine 6G (5 μM) for 10 minutes. The absorbance of the D-gel and L-gel was measured from 520 to 540 nm using a Tecan Spark 9 microplate reader, and the λmax was recorded and calculated as a function of concentration. The prepared peptides and hydrogels were aspirated separately and placed on glass slides for circular dichroism measurements. The resulting data were imported into plotting software to generate CD spectra.

[0080] Reference Figure 7 、 Figure 8 , respectively, are the CD spectra of D-gel and L-gel prepared in Example 1 and Example 2, and D-gel' and L-gel' prepared in Comparative Example 1. The secondary structure of the hydrogel was analyzed by circular dichroism (CD) to explore the molecular arrangement and driving force. Figure 7It shows that D-gel and L-gel have mirror-image characteristics at 208nm and 222nm, reflecting their opposite chirality. SELCON algorithm analysis shows that the D-gel prepared in Example 1 is mainly α-helix (45-50%), while the L-gel prepared in Example 2 is mainly β-fold (40-45%). The D-gel' prepared in Comparative Example 1 has a β-fold structure, and L-gel' is in a random coil state, indicating that enzymatic treatment can change the secondary structure of the hydrogel. The enzymatic treatment significantly optimizes the secondary structure of the hydrogel. The β-fold of the D-configuration hydrogel under heating and cooling conditions is transformed into an α-helix after enzymatic post-treatment. As a rigid structural unit, the α-helix can enhance the orderliness of the molecular arrangement and provide mechanical support for the material. After enzymatic treatment, the L-configuration hydrogel is transformed from a random coil to a β-fold. The β-fold forms a stable layered structure through a hydrogen bond network, reducing the free energy of the system, thereby improving the thermodynamic stability.

[0081] 3. Rheometer time sweep test:

[0082] 0.5 mL of aqueous solution was loaded onto the plate, ALP was added, and the storage modulus (G') and loss modulus (G") of the system were recorded over time at 37°C at a constant frequency (1 Hz) and strain (1%).

[0083] Figure 9 The rheometer time sweep test of 1mM Nap-GFFpY-AMGP8 (i.e., Nap-GFFpYKWYQNMIR, where KWYQNMIR, referred to as AMGP8) was conducted to study the enzymatically treated hydrogel properties of NAP-GFFpY-AMGP8. After adding ALP to the 1mM NAP-GFFpY-AMGP8 solution, in the initial state, the storage modulus (G') was slightly higher than the loss modulus (G"), and NAP-GFFpY-AMGP8 was in a microgel state but unstable. As the enzymatic dephosphorylation reaction proceeded, G' significantly exceeded G", and the system transformed into an elasticity-dominated solid gel state, showing high viscoelasticity. The Nap-GFFpY-AMGP8 constructed by the present invention reached the gel point after 15 minutes when triggered by alkaline phosphatase (ALP), confirming that it has the characteristics of rapid in situ gelation. Compared with D-gel' and L-gel' prepared in Comparative Example 1, D-gel and L-gel can precisely trigger self-assembly in areas with high expression of alkaline phosphatase (ALP) by introducing an enzyme response mechanism, thereby avoiding clinical risks caused by nonspecific gelation (such as ectopic blockage).

[0084] 4. Transmission electron microscope test micromorphology:

[0085] The prepared hydrogel was aspirated and dropped onto a copper grid to remove excess sample and water. The grid was then washed, stained, washed again, dried overnight, and observed under a transmission electron microscope. Figure 10TEM images of 1 mM Nap-GFFpY-AMGP8 before and after ALP treatment reveal that the microstructure of Nap-GFFpY-AMGP8 after ALP treatment is nanofiber-like, further confirming its enzymatic self-assembly. While the nanofiber structure of Nap-GFFpY-AMGP8 before enzymatic treatment is sparse and irregularly arranged, indicating a pre-organized structure during phosphorylation. When converted to L-gel with ALP, L-gel exhibits long, flexible, regular nanofibers, while D-gel exhibits a looser structure. This difference is attributed to the influence of secondary structure: L-gel is primarily composed of β-sheets, tending to form rigid fibers, while D-gel is primarily composed of α-helices, resulting in less rigid fibers.

[0086] Test Example 2

[0087] 1. Cytotoxicity assay:

[0088] CCK-8 was used to detect the toxic effects of hydrogels of different concentrations on HDPCs. The hydrogels D-gel and L-gel assembled by enzymatic self-assembly were used as the experimental group, and the hydrogels D-gel' and L-gel' assembled by heating and cooling method were used as the control group.

[0089] HDPCs from passages 3 to 5 were seeded in 96-well cell culture plates (approximately 5 × 10 cells per well, 100 μL culture medium), with four replicates per group. The plates were then placed in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was then replaced with fresh culture medium containing different concentrations and dilutions of hydrogels (D-gel, L-gel) according to the experimental group.

[0090] After culturing in a constant temperature incubator for 1, 3, and 5 days, the old culture medium was aspirated and the cells were rinsed twice with PBS. A blank control group (without cells) was added at the same time. Then, 100 μL of new 10% FBS αMEM culture medium was replaced. Then, 10 μL of CCK-8 reagent was added to each well. The well plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated for 3 hours. After confirming that there were no bubbles in each well, the OD value at 450 nm was measured in a microplate reader to calculate the cell viability. The calculation formula is:

[0091] Cell viability (%) = [OD(experimental) - OD(blank)] / [OD(control) - OD(blank)] × 100%

[0092] Reference Figure 11Under high concentration (60 μM) treatment conditions, L-gel and D-gel prepared in Example 1 and Example 2 showed significant cell protection effects in the 48-hour cytotoxicity experiment, and the cell survival rates of the L-gel and D-gel groups were significantly improved compared with those of D-gel' and L-gel' (P < 0.001). This result confirms that compared with D-gel' and L-gel' prepared by the heating and cooling method, the enzymatic response type L-gel and D-gel groups still have outstanding ability to maintain cell activity at high drug concentrations. Under low concentration (15 μM) treatment conditions, D-gel' and L-gel' showed high standard deviations, indicating that their assembly stability decreased, resulting in fluctuations in toxic effects. In contrast, L-gel and D-gel maintained data consistency under each concentration gradient, especially maintaining stable biosafety in the low concentration range. This difference may be due to the difference in the molecular action mechanisms of the two types of materials. The enzymatically treated hydrogels L-gel and D-gel achieve precise control over the release of degradation products through dynamic secondary structure regulation, effectively avoiding the nonspecific release of harmful substances. However, D-gel' and L-gel' prepared by the heating and cooling method lack enzyme response characteristics, and structural depolymerization occurs below the critical assembly concentration, resulting in fluctuations in the release of toxic substances. The above results demonstrate the potential application value of enzymatically treated hydrogels L-gel and D-gel as biocompatible scaffold materials, especially in the reconstruction of dental pulp-dentin complex.

[0093] 2. Hydrogel Alizarin Red Staining and Semi-quantitative Analysis:

[0094] Experimental groups: 10% FBS α-MEM culture medium was used as the negative control group, and different concentrations and configurations of D-gel and L-gel were used as experimental groups.

[0095] After 14 days of culture, the culture medium in the culture plate was discarded, and the plate was rinsed three times with PBS and fixed with 4% paraformaldehyde for 10 min.

[0096] Discard the fixative, wash three times with deionized water, absorb the water and slowly add Alizarin Red S staining solution (pH = 4.2), stain for 30 minutes, discard the dye, and wash three times with deionized water; add appropriate amount of deionized water to each well to prevent the well from drying out, and observe and take pictures under an inverted microscope.

[0097] Reference Figure 12 , are the results of Alizarin Red staining and semi-quantitative analysis of hydrogels of different types and concentrations (scale: 500 μm). It can be seen that different hydrogel configurations (D-gel, L-gel) and hydrogel concentrations have a significant effect on calcium deposition capacity.

[0098] Both D-gel and L-gel demonstrated optimal odontoblastic / osteogenic differentiation induction at low concentrations (15 μM). L-gel exhibited the strongest calcium deposition capacity at both low (15 μM) and high (60 μM) concentrations, suggesting that L-gel may possess superior sustained-release properties and maintain stable drug release kinetics at higher concentrations.

[0099] However, the effect of the concentration (30μM) in L-gel and D-gel hydrogels was relatively weak. It is speculated that this abnormal trend may be related to the transformation of the physicochemical properties of the material at a specific concentration or the bidirectional effect of cell metabolism regulation.

[0100] The above results analysis shows that the D-gel and L-gel prepared by the present invention exhibit multiple advantages: precise design of the secondary structure is achieved through chirality regulation, wherein L-gel forms regular nanofibers dominated by β-folding, while D-gel presents flexible fibers dominated by α-helix, showing better structural properties than D-gel' and L-gel' prepared by the heating and cooling method. In terms of critical assembly concentration, compared with D-gel' and L-gel', the critical assembly concentrations of L-gel (8μM) and D-gel (63μM) prepared by the present invention are significantly lower, which greatly improves the assembly efficiency of the material. In addition, the material has the characteristics of rapid gelation within 15 minutes and excellent mechanical properties. This controllable structure and performance characteristics give it broad application prospects in future dental pulp regeneration projects.

[0101] The above is an exemplary description of the present invention. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by other skilled in the art without expending creative labor falls within the scope of protection of the present invention.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide derivative containing an enzyme cleavage site capable of achieving enzyme-catalyzed self-assembly, characterized in that: The sequence of the polypeptide derivative is Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg.

2. The polypeptide derivative containing an enzyme cleavage site capable of achieving enzyme-catalyzed self-assembly according to claim 1, wherein: In the polypeptide derivative, Gly-Phe-Phe-pTyr is in L configuration or D configuration, preferably in L configuration; When Gly-Phe-Phe-pTyr is in the D configuration, the structural formula of Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg is: When Gly-Phe-Phe-pTyr is in the L configuration, the structural formula of Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg is:

3. A method for enzymatic self-assembly of nanofibers based on the polypeptide derivative according to claim 1, characterized in that: The following steps are involved: Step 1, solid phase synthesis of the polypeptide derivative; Step 2: Add the polypeptide derivative prepared in step 1 to a PBS solution, heat until completely dissolved, adjust the pH value, and then perform enzyme catalysis to induce self-assembly to form nanofibers of the polypeptide derivative, wherein the nanofibers are in a hydrogel state.

4. The enzymatic self-assembly method according to claim 3, wherein The enzyme in step 2 is an enzyme that can catalyze the formation of peptide bonds from polypeptide derivatives, preferably alkaline phosphatase.

5. The enzymatic self-assembly method according to claim 3, wherein The enzyme catalysis temperature in step 2 is 36-38°C.

6. The enzymatic self-assembly method according to claim 3, wherein The specific steps of solid phase synthesis of polypeptide derivatives in step 1 are as follows: Step a, the C-terminus of Fmoc-Arg is bound to the resin; Step b, removing the Fmoc protecting group of Fmoc-Arg and washing; Step c, adding Fmoc-Ile, coupling the C-terminus of Fmoc-Ile with the N-terminus of Arg coupled to the resin, then removing the Fmoc protecting group of Fmoc-Ile and washing; Step d, sequentially coupling Fmoc-Met, Fmoc-Asn, Fmoc-Gln, Fmoc-Tyr, Fmoc-Trp, Fmoc-Lys, Fmoc-pTyr in D or L configuration, Fmoc-Phe in D or L configuration, Fmoc-Phe in D or L configuration, and Fmoc-Gly, after each coupling is completed, removing the Fmoc protecting group and performing the next coupling until the last amino acid is coupled, removing the Fmoc protecting group, and washing; Step e, adding 2-naphthyl acetic acid to couple with the N-terminus of the polypeptide derivative on the resin, and washing; Step f, removing the polypeptide derivative from the resin to obtain a crude product; Step g, purifying the crude product of the polypeptide derivative; Step h: freeze-drying to obtain polypeptide derivative powder.

7. The enzymatic self-assembly method according to claim 6, wherein The resin described in step a is 2-cl-Trt or rink resin.

8. A nanofiber prepared by the enzymatic self-assembly method according to claim 3.

9. Use of the nanofiber as claimed in claim 8 in tissue engineering, drug delivery, and biosensing.

10. Use of the nanofiber according to claim 8 in preparing a biological preparation for treating caries and endodontic disease.

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