An ordered peptide nanofiber hydrogel, preparation method and application

Abstract

The ordered peptide nanofiber hydrogel, preparation method and application of the application relate to the technical field of biomaterials and immunology and tumor, and the hydrogel is formed by regulating the self-assembly process of Fmoc-FFE short peptides in a specific buffer solution, and the inside presents a highly ordered arrangement of nanofibers.The ordered peptide nanofiber hydrogel of the application not only has excellent biocompatibility and adjustable mechanical properties, but also can highly simulate the ordered remodeling of extracellular matrix collagen fibers in the development process of solid tumors in vitro.The ordered hydrogel can significantly promote the proliferation, invasion and directional migration of breast tumor cells; the ordered microstructure constructs a physical and mechanical barrier in the tumor immune microenvironment, significantly inhibits the infiltration and activation of T cells, and induces T cell exhaustion.The application provides an ideal in vitro three-dimensional (3D) model for in-depth study of the complex interaction between the ordered ECM regulated tumor promoting mechanism and the inhibition of immune cells.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials, oncology and immunology, and more specifically, to an ordered peptide nanofiber hydrogel, its preparation method and application. Background Technology

[0002] The extracellular matrix (ECM) plays a crucial role in maintaining tissue morphology and regulating cell behavior. Under normal physiological conditions, the ECM presents a relatively loose and disordered network structure; however, during the development and progression of solid tumors (such as breast cancer), tumor cells and stromal cells secrete and remodel the ECM, leading to highly cross-linked and orderly arrangement of collagen fibers (Desmoplasia). This ordered ECM not only increases tissue rigidity but also provides a rapid pathway for tumor cell invasion. In recent years, the tumor immune microenvironment (TME) has become a research hotspot. Clinically, it has been found that many solid tumors respond poorly to immunotherapy (such as CAR-T and PD-1 antibodies), partly because the highly ordered and dense ECM surrounding the tumor forms a robust physical and mechanical barrier, causing effector T cells (such as CD8+ T cells) to be excluded from the tumor parenchyma (forming so-called immune-excluded tumors). At the same time, abnormal mechanical signals can also induce functional exhaustion in T cells that enter the tumor.

[0003] Currently, in vitro 3D cell culture mainly relies on Matrigel or animal-derived collagen. However, these naturally derived materials are not only complex in composition and vary greatly from batch to batch, but their internal fibrous structures are often disordered, making it difficult to accurately simulate the ordered arrangement of ECM in breast tumor tissue, and even more difficult to precisely explore the regulation of T cell immunosuppression by ordered microstructures.

[0004] Therefore, developing a novel hydrogel material with well-defined components, easy synthesis, and the ability to accurately mimic the ordered ECM of tumors is of great significance for elucidating the mechanisms of tumor promotion and T-cell immunosuppression. Summary of the Invention

[0005] This invention provides an ordered peptide nanofiber hydrogel, its preparation method, and its application, in order to overcome the shortcomings of existing in vitro 3D culture materials that cannot effectively simulate the different degrees of order in ECMs during tumor development.

[0006] According to one aspect of the present invention, an ordered peptide nanofiber hydrogel is provided, the ordered peptide nanofiber hydrogel being formed by the self-assembly of a small molecule peptide Fmoc-FFE (fluorenylmethoxycarbonyl-phenylalanine-phenylalanine-glutamic acid), the small molecule peptide Fmoc-FFE comprising a hydrophobic Fmoc group protecting end, a diphenylalanine (FF) core end that promotes β-sheet self-assembly, and a negatively charged hydrophilic glutamic acid (E) tail end.

[0007] The present invention also provides a method for preparing ordered peptide nanofiber hydrogels as described above, comprising the following steps:

[0008] Step 1: Dissolve Fmoc-FFE small molecule peptide powder in a weakly alkaline (pH≈9) PBS buffer solution to form a homogeneous precursor solution;

[0009] Step 2: Adjust the pH of the precursor solution to pH=7.4 to induce intermolecular... Through stacking and hydrogen bonding, nanofiber hydrogels with spatially ordered arrangements are formed through self-assembly.

[0010] Based on the above scheme, preferably, the concentration of Fmoc-FFE in the precursor solution is 1.0 wt%. At this concentration, the internal fiber network of the self-assembled hydrogel exhibits anisotropic ordered arrangement.

[0011] The present invention also provides an application of the ordered peptide nanofiber hydrogel as described above, wherein the ordered peptide nanofiber hydrogel is used to simulate the ordered microstructure of the extracellular matrix (ECM) in solid tumors.

[0012] The present invention also provides an application of the ordered peptide nanofiber hydrogel as described above, wherein the ordered peptide nanofiber hydrogel, through its ordered three-dimensional topological structure and mechanical signals, mediates and promotes the adhesion, proliferation, orientation, migration and invasion of tumor cells.

[0013] The present invention also provides an application of the ordered peptide nanofiber hydrogel as described above, wherein the hydrogel is used to simulate the ordered ECM of tumors in an in vitro co-culture system and to regulate the behavior of immune cells.

[0014] The present invention also provides an application of the ordered peptide nanofiber hydrogel as described above for promoting epithelial-mesenchymal transition of tumor muscle cells.

[0015] The present invention also provides an application of the ordered peptide nanofiber hydrogel as described above, wherein the ordered arrangement of the ordered peptide nanofiber hydrogel forms physical and mechanical constraints, inhibiting the infiltration of effector T cells (Jurkat cells) into the tumor core region.

[0016] The present invention also provides an application of the ordered peptide nanofiber hydrogel as described above, wherein the ordered peptide nanofiber hydrogel downregulates the expression levels of cell surface markers CD25, CD44 and PD-1 through mechanical conduction pathways, and downregulates the production of intracellular cytokines IL-2, IFN-γ and TNF-α, thereby inhibiting T cell function.

[0017] The present invention also provides an application of the ordered peptide nanofiber hydrogel as described above for high-throughput screening of antitumor drugs and immune checkpoint inhibitors in vitro.

[0018] The present invention also provides an application of the ordered peptide nanofiber hydrogel as described above, for promoting cell attachment to the ordered peptide nanofiber hydrogel and orderly arrangement to form a specific tissue.

[0019] The Fmoc group and FF dipeptide sequence in the ordered peptide nanofiber hydrogel of this invention endow the molecule with extremely strong hydrophobic properties and Stacking ability drives the self-assembly of polypeptide molecules into nanofibers and makes it easy to form highly ordered anisotropic structures; while glutamic acid (E) not only provides good water solubility, but also endows the material with negative charge and responsiveness at physiological pH.

[0020] The ordered arrangement of nanofibers in Fmoc-FFE hydrogels can be controlled, mimicking the dynamic changes in the extracellular matrix (ECM) during tumor development. Fmoc-FFE spontaneously assembles into hydrogels in physiological PBS buffer. By controlling gelation kinetics (e.g., concentration, precursor pH), a highly ordered three-dimensional network of internal nanofibers can be fabricated. This structure not only matches the morphology of remodeled collagen fibers in tumors at the nanoscale but also highly matches the characteristics of solid tumors in terms of mechanical properties. Furthermore, by controlling the doping of Ca... 2+ Concentration can enable a realistic simulation of the dynamic changes in the ECM during tumor development and progression.

[0021] This invention utilizes an in vitro 3D culture model to explore the mechanism of ordered ECM-mediated breast tumor development. Breast cancer cells (such as MCF-7) are loaded into this ordered hydrogel. The ordered fibrous topology significantly activates the mechanotransduction pathways of tumor cells (such as the FAK / YAP pathway), promoting rapid proliferation of breast tumor cells and guiding them to migrate and invade along the fiber direction, thus reproducing the malignant progression of tumors in vivo.

[0022] Furthermore, this model can be pioneered for studying the T cell suppression mechanism in the immune microenvironment. This invention is the first to introduce ordered peptide hydrogels into the study of the immune microenvironment. In a co-culture model containing T cells, it was demonstrated that an ordered microenvironment can significantly inhibit the chemotaxis and spatial infiltration of CD8+ T cells. Simultaneously, compared to peptide hydrogels with low order, the highly ordered hydrogel matrix downregulated the expression levels of T cell surface markers CD25, CD44, and PD-1, and downregulated the production of intracellular cytokines IL-2, IFN-γ, and TNF-α, demonstrating the strong inhibitory effect of a highly ordered ECM on immune cells from both physical blockade and mechanosensing perspectives. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings:

[0024] Figure 1a The structural diagram of the small molecule peptide provided in Example 1 of this invention.

[0025] Figure 1b This is a matrix-assisted laser desorption / ionization time-of-flight mass spectrum of a small molecule peptide provided in Example 1 of the present invention;

[0026] Figure 2 This is a schematic diagram illustrating the preparation of the small molecule peptide fiber hydrogel provided in Example 1 of the present invention;

[0027] Figure 3a This is a physical image of the small molecule peptide nanofiber hydrogel provided in Example 1 of the present invention;

[0028] Figure 3b This is a polarization image of the small molecule peptide nanofiber hydrogel provided in Example 1 of the present invention;

[0029] Figure 4 This is a SEM image of the small molecule peptide nanofiber hydrogel provided in Example 1 of the present invention;

[0030] Figure 5 This is a rheological test diagram of the small molecule peptide nanofiber hydrogel provided in Example 1 of the present invention;

[0031] Figure 6 This is a schematic diagram of the in vitro 3D encapsulation and culture of breast tumor (MCF-7) cells in a small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention;

[0032] Figure 7a is a cell viability diagram of breast tumor (MCF-7) cells in the small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention;

[0033] Figure 7 b is an image showing the time-dependent adhesion efficiency of breast tumor (MCF-7) cells in the small molecule peptide nanofiber hydrogel provided in Example 2 of this invention;

[0034] Figure 7 c is a cell proliferation kinetic image of breast tumor (MCF-7) cells provided by the small molecule peptide nanofiber hydrogel in Example 2 of the present invention;

[0035] Figure 8 a is a orientation diagram of breast tumor (MCF-7) cells on the small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention;

[0036] Figure 8 b is a directional analysis diagram of breast tumor (MCF-7) cells on the small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention;

[0037] Figure 9 This is a schematic diagram illustrating the cell motility test of breast tumor (MCF-7) cells in a small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention.

[0038] Figure 10a The image shows the results of the Transwell migration ability test of breast tumor (MCF-7) cells in the small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention.

[0039] Figure 10b This is a diagram showing the Transwell migration ability test of breast tumor (MCF-7) cells in the small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention.

[0040] Figure 10c The image shows the results of the Transwell invasion ability test of breast tumor (MCF-7) cells in the small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention.

[0041] Figure 10d This is a graph showing the transwell invasion ability test of breast tumor (MCF-7) cells in the small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention.

[0042] Figure 11a This is a scratch healing diagram of breast tumor (MCF-7) cells in a small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention;

[0043] Figure 11bThis is a scratch healing analysis diagram of breast tumor (MCF-7) cells in the small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention;

[0044] Figure 12 This is a diagram of epithelial-mesenchymal transition (EMT) of breast tumor (MCF-7) cells in a small molecule peptide nanofiber hydrogel provided in Example 2 of the present invention.

[0045] Figure 13 This is an image showing the proliferation dynamics of Jurkat cells on a small molecule peptide nanofiber hydrogel provided in Example 3 of the present invention.

[0046] Figure 14 The results and analysis of the motility test of Jurkat cells on the small molecule peptide nanofiber hydrogel provided in Example 3 of the present invention are shown in the figure.

[0047] Figure 15 The expression levels of surface markers CD25, CD44 and PD-1 of immune (Jurkat) cells on the small molecule peptide nanofiber hydrogel provided in Example 3 of the present invention;

[0048] Figure 16 The expression levels of intracellular cytokines IL-2, IFN-γ, and TNF-α on the small molecule peptide nanofiber hydrogel provided in Example 3 of this invention. Detailed Implementation

[0049] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0050] It should be understood that, when used in this specification and the appended claims, the term "comprising" indicates the presence of a descriptive feature, integral, step, operation, element, and / or component, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or sets.

[0051] To keep the drawings concise, only the parts relevant to the invention are shown schematically in each figure, and they do not represent the actual structure of the product. Furthermore, for ease of understanding, in some figures, only one of components with the same structure or function is shown schematically, or only one is labeled. In this document, "one" can mean not only "only one" but also "more than one".

[0052] It should also be further understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, the accompanying drawings described below are merely some embodiments of the present invention. Taking Fmoc-FFE as an example, those skilled in the art can obtain other drawings and other implementation methods based on these drawings without any creative effort.

[0054] The sources and methods of obtaining each raw material are described in the following examples:

[0055] The 2-ChlorotritylChlorideResin resin (resin substitution degree 1.307 mmol / g), phenylalanine with an amino group protected by N-fluorene-9-methoxycarbonyl (Fmoc-Phe-OH), glutamic acid with an amino group and a side group carboxyl group protected by N-fluorene-9-methoxycarbonyl and tert-butyl ester (Fmoc-Glu(OtBu)-OH), benzotriazole-N,N,N',N'-tetramethylureafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBT) used in the embodiments of the present invention were all purchased from Jier Biochemical (Shanghai) Co., Ltd.

[0056] The ninhydrin, trifluoroacetic acid (TFA), and N,N-diisopropylethylamine (DIEA) used in the embodiments of this invention were all purchased from Aladdin.

[0057] The dichloromethane (DCM), triisopropylsilane (TIS), N,N-dimethylformamide (DMF), methanol (MeOH), piperidine, and diethyl ether used in the embodiments of this invention were all purchased from Sinopharm Group.

[0058] Please refer to Figure 1, and combine it with... Figure 2 As shown in Figure 3, the present invention provides an ordered peptide nanofiber hydrogel, which is formed by the self-assembly of small molecule peptides, wherein the small molecule peptides include hydrophobic side chains (phenylalanine F, aromatic hydrophobic), β-sheet amino acids (phenylalanine F), hydrophilic amino acids (glutamic acid E), and N-terminal amino groups.

[0059] This peptide nanofiber hydrogel has high mechanical modulus and adjustable orientation, and can be used as a 3D fiber medium for the establishment of in vitro breast tumor models. Due to the cross-linking, deposition and remodeling of collagen fibers and other components during the development of breast tumors, the extracellular matrix of breast tumor cells exhibits an ordered arrangement. Therefore, this ordered peptide nanofiber hydrogel can effectively simulate the natural extracellular matrix of breast tumor cells.

[0060] The small molecule peptide of the present invention is FFE. The small molecule peptide structure after introducing a fluorenyl methoxycarbonyl (Fmoc) group at its N-terminus is: Fmoc-Phe-Phe-Glu-OH, where F (Phe) is phenylalanine, E (Glu) is glutamic acid, and Fmoc is a 9-fluorenyl methoxycarbonyl protecting group.

[0061] The hydrophobic aromatic tail of the Fmoc-FFE of the present invention is an aromatic structure containing a benzene ring, a nonafluorene methoxycarbonyl group, a naphthalene ring, a pyrene ring, etc., and the β-sheet amino acid is a hydrophobic amino acid with β-sheet that can promote the self-assembly of peptides through the β-sheet secondary structure. The hydrophobic amino acid is one of phenylalanine, and the charged amino acid is one of glutamic acid.

[0062] The small molecule peptide Fmoc-FFE of the present invention, after introducing a nonafluorene methoxycarbonyl (Fmoc) molecule at its N-terminus, forms a small molecule peptide self-assembly that can form an ordered peptide nanofiber hydrogel. The ordered peptide nanofiber hydrogel can promote cell attachment and orderly arrangement of the peptide nanofiber hydrogel to form a specific tissue.

[0063] The ordered peptide nanofiber hydrogel of the present invention has the ability to regulate cell behavior and can significantly enhance the orientation and motility of tumor cells.

[0064] The ordered peptide nanofiber hydrogel of the present invention can mimic the fiber arrangement pattern of the extracellular matrix of breast tumor cells, and achieve the purpose of regulating cell behavior, promoting the malignancy of breast tumors and inhibiting immunity by affecting the signaling pathways of breast tumors and immune cells.

[0065] The ordered peptide nanofiber hydrogel of the present invention can also simulate the ordered arrangement of collagen fibers in the extracellular matrix of tumor cells. Through this simulation process, it affects the cell signaling pathways, thereby regulating cell behavior and significantly enhancing the proliferation, directional migration and invasion capabilities of tumor cells.

[0066] The ordered peptide nanofiber hydrogel of the present invention can also simulate the ordered arrangement of collagen fibers in the extracellular matrix of tumor cells. Through this simulation process, it inhibits the behavior of immune cells and significantly inhibits the expression of surface antigens and cellular functional factors of immune cells, thereby simulating immunosuppression similar to that in clinical practice.

[0067] This invention also provides a method for preparing ordered peptide nanofiber hydrogels. Small molecule peptides are synthesized using a peptide solid-phase synthesis method with Fmoc protection strategy. The carrier resin is 2-ChlorotritylChlorideResin resin. The small molecule peptide chain segments are sequentially extended from the C-terminus to the N-terminus on the carrier resin to obtain amphiphilic small molecule peptides. The amphiphilic small molecule peptides form ordered nanofiber hydrogels through self-assembly.

[0068] Specifically, a 1 wt% concentration of small molecule peptides is added to a PBS solution for complete dissolution and assembly. The pH is adjusted to 7-10, and after heating and cooling, the resulting novel nanofiber hydrogel exhibits an ordered internal fiber arrangement. When the 1 wt% concentration of the small molecule peptides is added to a PBS solution for complete dissolution and assembly, and the pH is adjusted to 7.4 while simultaneously doping with different concentrations of Ca... 2+ The novel nanofiber hydrogels formed after heating and cooling (1 mmol / L-3 mmol / L CaCl2) exhibit disordered internal fiber arrangement, with the degree of disorder increasing with the concentration of CaCl2. 2+ The effect increases with increasing concentration. This invention also provides an application of ordered peptide nanofiber hydrogels in promoting epithelial-mesenchymal transition in tumor muscle cells.

[0069] This invention also provides an ordered peptide nanofiber hydrogel, which demonstrates significant application value in the construction of 3D in vitro tumor models and tissue repair. The ordered peptide nanofiber hydrogel of this invention can effectively simulate the dynamic changes in the orderliness of the extracellular matrix (ECM) during tumor development, providing tumor cells with growth conditions close to the in vivo microenvironment. Furthermore, the ordered structure within the hydrogel inhibits the action of immune cells, which is similar to the malignancy of clinical tumors, making it an ideal choice for constructing in vitro 3D culture models.

[0070] To verify the effectiveness of the present invention, experiments will be conducted using specific embodiments below for detailed explanation.

[0071] Example 1

[0072] This embodiment provides an ordered peptide nanofiber hydrogel with the structural formula: Fmoc-FFE. The specific synthesis steps are as follows:

[0073] 1) First, weigh 1g of chlorinated resin and place it in the polypeptide synthesis column. Add 20mL of N,N-dimethylformamide (DMF) to swell for 30min. Use a circulating water pump to evacuate the solvent, and then wash and filter with DMF 3 times.

[0074] 2) Weigh out 3 molar equivalents of Fmoc-Glu(Otbu)-OH from the resin, add 15 mL of DMF to dissolve it completely, then add 2 mL of DIEA and shake to mix. Add the mixture to the peptide synthesis column and stir slowly for 2 hours. After the reaction is complete, wash the column with DMF 3 times.

[0075] 3) Take a small amount of resin from 2) and put it into a glass test tube. Add an appropriate amount of ninhydrin methanol solution (10 mg / mL). Heat the liquid with a hair dryer until it boils. If the resin does not change color, the reaction is complete.

[0076] 4) Remove the Fmoc protecting group from glutamic acid. Add a total of 20 mL of Piperidine / DMF (V / V=1:4) as a deprotecting agent to the solid-phase synthesis column in two batches, stirring magnetically for 15 min each time. After filtering out the solvent, wash with DMF five times (3 min / time), and then evacuate the solvent.

[0077] 5) Weigh 3 molar equivalents of Fmoc-Phe-OH and 3.6 molar equivalents of HBTU and HOBT from the resin, add 15 mL of DMF to dissolve completely, then add 2 mL of DIEA and shake to mix well. Add the mixture to the peptide synthesis column and stir slowly for 2 hours. After the reaction is complete, wash 3 times with DMF, and then repeat steps 3), 4), and 5).

[0078] 6) Wash the resin three times (3 min each time) with DMF, MeOH and DCM respectively, and then vacuum dry it at room temperature for 12 h.

[0079] 7) Add 20 mL of cleavage agent (TFA / H2O / TIS (V / V=95% / 2.5% / 2.5%)) to the dried resin synthesis column obtained in 6). Stir slowly at room temperature for 2 h. After the reaction is complete, collect the filtered liquid using a circulating water pump filtration device. Concentrate the liquid to a viscous state by rotary evaporation. Then add it dropwise to ice-cold ether. After standing, centrifuge to remove the supernatant and collect the precipitate. Wash the precipitate three times with ice-cold ether by centrifugation. Finally, vacuum dry at room temperature to obtain the product peptide and freeze it for later use.

[0080] The small molecule peptide hydrogel obtained in this embodiment was tested. Figure 1a This is a structural diagram of the small molecule peptide hydrogel obtained in this embodiment. Figure 1b This is the mass spectrum of the small molecule peptide hydrogel obtained in this embodiment; Figure 2 This is a schematic diagram illustrating the preparation of the small molecule peptide hydrogel obtained in this embodiment; Figure 3a This is a photograph of the small molecule peptide hydrogel obtained in this embodiment. Figure 3b This is a polarization image of the small molecule peptide hydrogel obtained in this embodiment.

[0081] To further verify the functional effects of the ordered peptide nanofiber hydrogel of the present invention, the following experiments will be conducted using the ordered peptide nanofiber hydrogel obtained in Example 1.

[0082] Example 2

[0083] This embodiment provides an ordered peptide nanofiber hydrogel for studying its mechanisms of promoting tumor malignancy and immunosuppression. First, the self-assembly behavior of the ordered peptide nanofiber hydrogel was investigated. Figure 4The image shows a SEM image of the small molecule peptide hydrogel provided in Example 2 of this invention. The results show that after introducing palmitic acid, the formed Fmoc-FFE can self-assemble into a fiber nanostructure.

[0084] To examine the modulus of the small molecule peptide nanofiber solution, rheological tests were performed on the nanofiber solution regarding its storage modulus and loss modulus (plate gap: 1 mm; temperature: 298 K; test parameters: angular frequency set to 0.1~100 rad / s; logarithmic scan mode). Figure 5 The rheological test diagram of the small molecule peptide nanofiber PBS solution provided in Example 2 of the present invention shows that its storage modulus is greater than its loss modulus, indicating that the nanofiber solution prepared by dissolving our novel small molecule peptide hydrogel in PBS has gel properties, that is, it is a nanofiber hydrogel.

[0085] In order to build an in vitro 3D culture model Figure 6 This is a schematic diagram of the in vitro 3D encapsulation and culture of breast tumor (MCF-7) cells in the small molecule peptide nanofiber hydrogel provided in Example 1 of the present invention. To test the biocompatibility of the small molecule peptide nanofiber hydrogel, Figure 7 a) describes the cytotoxicity (CCK8 assay) of the small molecule peptide nanofiber hydrogel provided in Example 2 of this invention. The CCK-8 assay essentially reflects cell viability by detecting cellular metabolic activity at 450 nm absorbance. Figure 7 a (left) represents the effect of different concentrations of peptide nanofiber hydrogels on the activity of MCF-7 cells; Figure 7 a (right) shows the effect of four peptide hydrogels with different degrees of nanofiber order on the activity of MCF-7 cells. The data indicate that the hydrogel has good biocompatibility and avoids the toxicity of ordered media to cells. Therefore, this novel nanofiber hydrogel possesses the basic properties for in vitro culture.

[0086] To examine the effect of small molecule peptide hydrogels on the adhesion of breast tumor (MCF-7) cells, Figure 7 b shows the adhesion effect of the small molecule peptide hydrogel provided in Example 2 of this invention on MCF-7 cells. The horizontal axis in the figure represents time, and the vertical axis represents the absorbance of MCF-7 cells adhered to the material surface. The results show that Fmoc-FFE exhibits good interaction with MCF-7 cells, and the adhesion effect is related to the degree of internal order; the ordered group shows significantly higher cell adhesion ability compared to the disordered group.

[0087] To examine the effect of small molecule peptide hydrogels on the activity of breast tumor (MCF-7) cells, Figure 7c represents the proliferation effect of the small molecule peptide hydrogel provided in Example 2 of this invention on MCF-7 cells. The horizontal axis in the figure represents time, and the vertical axis represents the absorbance of MCF-7 cells after incubation with the material. The results show that Fmoc-FFE exhibits a good proliferative effect on MCF-7 cell viability, and the proliferation effect is related to the degree of internal order; the ordered group showed significantly higher cell viability compared to the disordered group.

[0088] This invention employs in vitro 3D encapsulation and culture of cells, followed by Calcein-AM staining, to observe the cell orientation mediated by sequenced peptide nanofiber hydrogels using laser scanning confocal microscopy. Figure 8 a represents the effect of the small molecule peptide hydrogel provided in Example 2 of this invention on the growth of different cell types. According to... Figure 8 As shown in Figure a, it can be seen that the higher the degree of orderliness of the nanofibers in the peptide hydrogel, the more consistent the growth angle of MCF-7 cells. From Figure 8 Further analysis revealed that the ordered small molecule peptide nanofiber hydrogel has a directional effect on breast tumor (MCF-7) cells.

[0089] The present invention uses the Transwell method to detect the motility (cell migration, cell invasion) of MCF-7 cells precultured with peptide hydrogel in Example 2 of the present invention. Figure 9 Figure 10 is a schematic diagram illustrating the effect of the small molecule peptide hydrogel provided in Example 2 of the present invention on the motility of MCF-7 cells. Figure 11 is an analysis diagram of the test results of the small molecule peptide hydrogel provided in Example 2 of the present invention on the motility of MCF-7 cells. Figure 10a , Figure 10b Observation and quantitative analysis of the migration ability of MCF-7 cells after pre-culture in peptide hydrogels showed that the number of cells in the ordered group migrated through the chambers significantly more than that in the disordered group, indicating that breast tumor (MCF-7) cells have stronger migration and movement capabilities in ordered gels. Figure 10c , Figure 10d The observation and quantitative analysis of the invasive ability of MCF-7 cells showed that the number of MCF-7 cells infiltrating through the ordered nanofiber structure was significantly greater, indicating that breast tumor (MCF-7) cells have a stronger invasive and motility ability in the ordered gel.

[0090] This invention uses the scratch healing method to detect the motility of MCF-7 cells pre-cultured with peptide hydrogel in Example 2 of this invention. Figure 11a The effect of peptide nanofiber hydrogel provided in Example 2 of this invention on the motility of MCF-7 cells. Figure 11bThis invention provides a quantitative analysis of the healing rate of ordered peptide nanofiber hydrogels on breast tumor MCF-7 cells, as provided in Example 2. After culturing in four gels with varying degrees of order for 24 hours, the scratch healing rates of MCF-7 cells showed significant differences. The FFE-Ca0 group achieved complete wound healing (approximately 100%) after 48 hours. In contrast, the disordered control group exhibited different healing trends. The healing rates of the FFE-Ca1, FFE-Ca2, and FFE-Ca3 groups were approximately 79%, 56%, and 49%, respectively. While these rates were higher than the 27% wound healing rate of the blank control group, the highly ordered FFE-Ca0 group demonstrated a stronger effect.

[0091] To examine the promoting effect of ordered peptide nanofiber hydrogel on malignant phenotype (epithelial-mesenchymal transition) of breast tumor cells, MCF-7 cells were cultured on the small molecule peptide hydrogel provided in Example 2. Figure 12 The morphology of MCF-7 cells observed under a light microscope after 5 days of pre-culture is shown. The 2D control group cells exhibit typical epithelial-like structures (pebble-like, tightly aggregated); FFE-Ca3 (disordered group) only induces mild EMT; with Ca... 2+ As the concentration decreased and the hydrogel's orderliness increased, cells in the FFE-Ca2, FFE-Ca1, and FFE-Ca0 groups gradually transitioned from an epithelial phenotype to a long spindle / fusiform mesenchymal phenotype. The FFE-Ca0 (highly ordered group) showed the most significant EMT phenotype, confirming that ordered peptide nanofiber hydrogels can efficiently induce epithelial-mesenchymal transition (EMT).

[0092] Example 3

[0093] This embodiment aims to verify the ability of the Fmoc-FFE ordered peptide nanofiber hydrogel of the present invention to simulate the immune microenvironment of immune rejection tumors in vitro. It focuses on examining its regulatory role on the infiltration behavior of Jurkat cells into the tumor region and the immune functional state (activation / depletion phenotype) of Jurkat cells, thereby demonstrating that the ordered gel not only has a physical barrier effect, but can also induce immune suppression and depletion of Jurkat cells through abnormal mechanical topological signals.

[0094] To examine the effect of small molecule peptide hydrogels on the activity of immune (Jurkat) cells, Figure 13 The figure shows the effect of the small molecule peptide hydrogel provided in Example 3 of this invention on the proliferation of Jurkat cells. The horizontal axis represents time, and the vertical axis represents the cell proliferation rate of Jurkat cells after incubation with the material. The results indicate that: Figure 13 (Left) This shows that highly ordered nanofibers in the hydrogel have a significant inhibitory effect on the viability of resting Jurkat cells, and the inhibitory effect is related to the degree of internal order. The cell proliferation capacity of the ordered group is significantly reduced compared with that of the disordered group. Figure 13(Right) This shows that the highly ordered nanofibers in the hydrogel have the same inhibitory effect on the viability of activated Jurkat cells.

[0095] To examine the regulatory effect of ordered peptide nanofiber hydrogels on the behavior of immune (Jurkat) cells, Figure 14 This invention demonstrates the regulatory effect of the small molecule peptide hydrogel provided in Example 3 on the motility of Jurkat cells. Cell motility was detected using the Transwell assay. Figure 14 (Left) shows that the number of resting Jurkat cells passing through the chamber is significantly less in the ordered group than in the disordered group, indicating that the ordered gel significantly inhibits the motility of Jurkat cells, and the resting Jurkat cells have stronger motility in the disordered gel. Figure 14 (Right) This shows that the number of activated Jurkat cells passing through the chambers in the ordered group is significantly less than that in the disordered group, indicating that resting Jurkat cells have stronger motility in the disordered gel. All of the above results demonstrate that ordered peptide nanofiber hydrogels can significantly inhibit the motility of Jurkat cells.

[0096] To assess the impact of ordered gels on the functional status of immune cells, pre-cultured Jurkat cells were recovered from each group of hydrogels, and their immunophenotypic markers were detected by flow cytometry. The results are as follows: Figure 15 , Figure 16 As shown in the results, the expression levels of Jurkat cell surface markers CD25, CD44, and PD-1 were significantly reduced in the gel group with higher degree of order; at the same time, the production and expression of intracellular cytokines IL-2, IFN-γ, and TNF-α were significantly downregulated, suggesting that Jurkat cells were in a more pronounced state of immunosuppression and exhaustion.

[0097] The present invention relates to a peptide nanofiber hydrogel with adjustable degree of order and its application, which has the following technical effects:

[0098] 1. This invention successfully constructed a nanofiber hydrogel by utilizing the self-assembly behavior of the small molecule peptide Fmoc-FFE in PBS (phosphate buffered saline). In a PBS solution with a pH of 7.4, a 1 wt% concentration of the small molecule peptide spontaneously formed an ordered nanofiber hydrogel; by doping with different concentrations of Ca... 2+ The hydrogel exhibits structures with varying degrees of disordered fiber arrangement. This characteristic allows it to easily form peptide gels with internal structures under neutral or mild environments, and the arrangement of its internal fibers can effectively mimic the dynamic changes in the extracellular matrix (ECM) during the development and progression of breast tumors. This provides cells with growth conditions that closely resemble the in vivo microenvironment, making it an ideal choice for in vitro tissue culture models.

[0099] 2. The novel nanofiber hydrogel prepared by this invention exhibits excellent biocompatibility, effectively avoiding the toxic effects that ordered media may have on cells. This characteristic is a fundamental prerequisite for its use as a 3D culture scaffold, ensuring the healthy growth and normal function of cells within the gel.

[0100] 3. The ordered arrangement of fibers within this self-assembled small-molecule peptide nanofiber hydrogel not only mimics the microenvironment of cell arrangement in vivo but also significantly influences tumor cell behavior. Specifically, it enhances cell motility and proliferation, providing strong support for research in oncology and immunology.

[0101] 4. Given the ordered arrangement of fibroblasts in various tissues (such as muscle) and the extracellular matrix of tumor cells, the ordered peptide nanofiber hydrogel of this invention shows broad application prospects in oncology and immunology. This discovery not only broadens the application scope of hydrogel materials but also provides new ideas and methods for the treatment of related diseases.

[0102] 5. The ordered arrangement of fibers within the self-assembled small molecule peptide nanofiber hydrogel not only affects the behavior of immune cells but also significantly inhibits their function, providing a new platform for clinical exploration of immunotherapy targets and screening of immunotherapy drugs.

[0103] Finally, the method described in this application is merely a preferred embodiment and is not intended to limit the scope of protection of this invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. An ordered peptide nanofiber hydrogel, characterized in that, The ordered peptide nanofiber hydrogel is formed through the self-assembly of the small molecule peptide Fmoc-FFE (fluorenylmethoxycarbonyl-phenylalanine-phenylalanine-glutamic acid), wherein the small molecule peptide Fmoc-FFE includes hydrophobic Fmoc group protecting ends and promoting... - Folded self-assembled diphenylalanine (FF) core end and negatively charged hydrophilic glutamate (E) tail end.

2. A method for preparing the ordered peptide nanofiber hydrogel as described in claim 1, characterized in that, Includes the following steps: Step 1: Dissolve Fmoc-FFE small molecule peptide powder in weakly alkaline PBS buffer solution to form a homogeneous precursor solution; Step 2: Adjust the pH of the precursor solution to pH=7.4 to induce intermolecular... Through stacking and hydrogen bonding, nanofiber hydrogels with spatially ordered arrangements are formed through self-assembly.

3. The method for preparing an ordered peptide nanofiber hydrogel as described in claim 2, characterized in that, In the precursor solution, the concentration of Fmoc-FFE is 1.0 wt%. At this concentration, the internal fiber network of the self-assembled hydrogel exhibits anisotropic ordered arrangement.

4. An application of the ordered peptide nanofiber hydrogel as described in claim 1, characterized in that, The ordered peptide nanofiber hydrogel was used to mimic the ordered microstructure of the extracellular matrix (ECM) in solid tumors.

5. An application of the ordered peptide nanofiber hydrogel as described in claim 1, characterized in that, The ordered peptide nanofiber hydrogel, through its ordered three-dimensional topological structure and mechanical signals, mediates and promotes the adhesion, proliferation, orientation, migration and invasion of tumor cells.

6. An application of the ordered peptide nanofiber hydrogel as described in claim 1, characterized in that, The hydrogel is used to simulate the ordered ECM of tumors in an in vitro co-culture system and to regulate the behavior of immune cells.

7. An application of the ordered peptide nanofiber hydrogel as described in claim 1, characterized in that, The ordered arrangement of the ordered peptide nanofiber hydrogel creates physical and mechanical constraints, inhibiting the infiltration of effector T cells (Jurkat cells) into the tumor core region. The ordered peptide nanofiber hydrogel downregulates the expression levels of cell surface markers CD25, CD44, and PD-1 through mechanical conduction pathways, and downregulates the production of intracellular cytokines IL-2, IFN-γ, and TNF-α, thereby inhibiting T cell function.

8. An application of the ordered peptide nanofiber hydrogel as described in claim 1, characterized in that, It is used to promote the epithelial-mesenchymal transition of tumor cells.

9. An application of the ordered peptide nanofiber hydrogel as described in claim 1, characterized in that, Used in high-throughput in vitro screening of antitumor drugs and immune checkpoint inhibitors.

10. An application of the ordered peptide nanofiber hydrogel as described in claim 1, characterized in that, The ordered peptide nanofiber hydrogel is used to promote cell attachment and orderly arrangement to form specific tissues.

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