Preparation method of AI-assisted design of light-cured hydrogel targeting TGF-beta lysosomal endocytosis peptide enrichment

Abstract

The application discloses a preparation method of an AI-assisted design targeted TGF-beta lysosome endocytosis peptide enrichment photocured hydrogel, and relates to the technical field of biomedical materials, and comprises the following steps: introducing a lysine residue at the N terminal of an endocytosis peptide which has lysosome targeting ability and can specifically combine with TGF-beta, and covalently connecting a 5-hexyne acyl group to the side chain amino group of the additional lysine residue to obtain an N terminal modified endocytosis peptide derivative; and covalently integrating the AI-assisted design targeted TGF-beta lysosome endocytosis peptide into a photocured GelMA hydrogel network through an MMP-2 responsive linker, realizing the functions of rapid in-situ forming in a spinal cord injury microenvironment, no need of an external light initiator, and on-demand release of active polypeptides, the constructed hydrogel can efficiently release the endocytosis peptide under the condition of high expression of MMP-2, the endocytosis peptide is taken up by cells, targets lysosomes and mediates TGF-beta degradation, so that the formation of a glial scar is inhibited, nerve regeneration is promoted, and the hydrogel has good biocompatibility.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials technology, and in particular to a method for preparing an AI-assisted design for enriching photocurable hydrogels containing TGF-β lysozyme-targeted in vivo phagocytosed peptides. Background Technology

[0002] Biomedical materials technology refers to a cutting-edge interdisciplinary field that designs, develops, and applies biocompatible natural or synthetic materials for the diagnosis, treatment, repair, or replacement of human tissues, organs, or their functions. These materials can interact with living organisms without causing harmful reactions and are widely used in drug delivery systems, tissue engineering scaffolds, implantable devices (such as artificial joints and vascular stents), wound dressings, and intelligent responsive treatment platforms. Their core objectives are to promote tissue regeneration, improve disease treatment outcomes, and enhance patients' quality of life.

[0003] Following spinal cord injury, TGF-β is secreted in large quantities and continuously activates astrocytes, which is a key driver of glial scar formation. Traditional small molecule inhibitors or antibodies have problems such as difficulty in penetrating the blood-brain barrier, high systemic toxicity, and short duration of action, and cannot achieve precise and efficient TGF-β clearance at the injury site. Moreover, the pathological microenvironment after spinal cord injury is characterized by dynamic changes. Traditional hydrogels or sustained-release systems are mostly non-responsive or have slow response speeds, and cannot release therapeutic drugs on demand and rapidly during the golden window of MMP-2 high expression, resulting in missed treatment opportunities or insufficient efficacy. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides an AI-assisted design method for preparing photocurable hydrogels that enrich lysozyme-based peptides targeting TGF-β. This addresses the issue that after spinal cord injury, the massive secretion of TGF-β and its continuous activation of astrocytes are key drivers of glial scar formation. Traditional small molecule inhibitors or antibodies suffer from problems such as difficulty in penetrating the blood-brain barrier, high systemic toxicity, and short-lasting effects, making it impossible to achieve precise and efficient TGF-β clearance at the site of injury.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides a method for preparing an AI-assisted design of a photocurable hydrogel for enriching TGF-β lysozyme-targeted lysozyme-containing peptides, comprising: An N-terminal modified endocytic peptide derivative was obtained by introducing an additional lysine residue at the N-terminus of an endocytic peptide that has lysosomal targeting ability and can specifically bind TGF-β, and then covalently linking a 5-hexynyl group to the side chain amino group of the additional lysine residue. The N-terminal modified endocytic peptide derivative is combined with a peptide linker with an ε-amino group modified by an azide group, which has an MMP-2 restriction site and contains a cysteine ​​residue at the N-terminus and a lysine residue at the C-terminus, to form an endocytic peptide-linker conjugate through a copper-catalyzed azide-alkyne cycloaddition reaction. The endocytic peptide-linker conjugate was mixed with methacrylamide gelatin (GelMA), and under 405 nm ultraviolet light irradiation, the thiol groups of cysteine ​​residues in the conjugate reacted with the methacrylamide groups in GelMA to form the hydrogel in situ within 15 seconds through a thiol-ene click reaction.

[0007] As a preferred embodiment of the preparation method of the AI-assisted design for enriching photocurable hydrogels containing TGF-β lysozyme-mediated endocytosis peptides according to the present invention, wherein the amino acid sequence of the endocytosis peptide is MEEAQRLLEEWEFQERVDELARKYGERVKEVGRQIIQDPDPEVRRKMLDILERIYREAGGGSTSLDASIIWAMMQN.

[0008] As a preferred embodiment of the preparation method of the AI-assisted design for enriching TGF-β lysozyme-based photocurable hydrogels, the amino acid sequence of the polypeptide linker is Cys-Gly-Gly-Ser-Gly-Pro-Leu-Gly-Leu-Ala-Gly-Gly-Gly-Ser-Lys.

[0009] As a preferred embodiment of the preparation method of the AI-assisted design for enriching TGF-β lysozyme-mediated photocurable hydrogel, wherein the ε-amino group of the C-terminal lysine residue of the polypeptide linker is modified with an azide group for click chemical coupling with the alkynyl group on the endocytic polypeptide derivative.

[0010] As a preferred embodiment of the preparation method of the AI-assisted design for enriching TGF-β lysozyme-based photocurable hydrogels, wherein the degree of methacrylation substitution of the methacryloyl gelatin (GelMA) is 40%–90%.

[0011] As a preferred embodiment of the preparation method of the AI-assisted design for enriching TGF-β lysozyme-based photocurable hydrogels, the final concentration of the endocytic peptide-linker conjugate in the hydrogel precursor solution is 1–100 μM.

[0012] As a preferred embodiment of the preparation method of the AI-assisted design for enriching TGF-β lysozyme-based photocurable hydrogels, the photocuring process does not require an external photoinitiator and can be triggered by rapid cross-linking with only 405 nm visible light.

[0013] As a preferred embodiment of the preparation method of the AI-assisted design for enriching TGF-β lysozyme-mediated in vivo peptides using photocurable hydrogel, the hydrogel is in-situ cured within 15 seconds under 405 nm ultraviolet light irradiation, and the resulting network structure has a storage modulus G' of 100–5000 Pa.

[0014] As a preferred embodiment of the preparation method of the AI-assisted design for enriching TGF-β lysozyme-mediated photopolymerizable hydrogel, the hydrogel releases ≥50% of endocytic peptides within 72 hours in a microenvironment with an MMP-2 concentration ≥10 ng / mL.

[0015] As a preferred embodiment of the preparation method of the AI-assisted design for enriching TGF-β lysozyme-mediated photopolymerizable hydrogel, the endocytic peptides are released by MMP-2 enzyme cleavage and can be endocytosed and targeted to lysosomes to mediate the degradation of TGF-β protein.

[0016] As a preferred embodiment of the preparation method of the AI-assisted design for the enrichment of TGF-β lysozyme-based photocurable hydrogels, the preparation method is applicable to spinal cord injury repair, especially to the subacute or chronic stage with high local MMP-2 expression from day 3 to day 14 after injury.

[0017] The beneficial effects of this invention are as follows: By covalently integrating AI-assisted designed TGF-β lysosomal endocytokines into a photocurable GelMA hydrogel network via MMP-2 responsive linkers, the invention achieves rapid in-situ molding in the spinal cord injury microenvironment, eliminates the need for exogenous photoinitiators, and releases active peptides on demand. The constructed hydrogel can efficiently release endocytokines under conditions of high MMP-2 expression. After being taken up by cells, the endocytokines target lysosomes and mediate TGF-β degradation, thereby inhibiting glial scar formation and promoting nerve regeneration. It also possesses good biocompatibility, mechanical tunability, and clinical translational potential. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. 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.

[0019] Figure 1 A schematic diagram of the chemical reaction for constructing the EnpMGelMA hydrogel.

[0020] Figure 2 This is a schematic diagram of GFAP immunofluorescence.

[0021] Figure 3 A bar chart for the quantitative statistical analysis of GFAP fluorescence intensity.

[0022] Figure 4 This is a line graph showing the temporal changes in MMP-2 protein expression in the damaged area.

[0023] Figure 5 Line graph showing the time-series changes in BMS scores for hind limb motor function in mice.

[0024] Figure 6 Representative micrographs of immunofluorescence staining in the spinal cord injury area.

[0025] Figure 7 A bar chart showing the quantitative statistical analysis of CSPG and NF200 fluorescence intensity 8 weeks after spinal cord injury. Detailed Implementation

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0027] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0028] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0029] Example 1, the first embodiment of the present invention, provides the preparation of EnpMGelMA hydrogel, comprising: Peptide synthesis and modification: The endocytic peptide EndoP144 was synthesized using a solid-phase synthesis method. Its amino acid sequence is: MEEAQRLLEEWEFQERVDELARKYGERVKEVGRQIIQDPDPEVRRKMLDILERIYREAGGGSTSLDASIIWAMMQN.

[0030] An additional lysine (K) residue was introduced at the N-terminus of EndoP144, and a 5-hexynoyl group was covalently linked to the ε-amino group of the side chain of the lysine residue to obtain an N-terminal modified endocytic peptide derivative, denoted as K¹(5-hexynoyl)-EndoP144.

[0031] Synthesis of MMP-2 responsive linkers: A polypeptide linker with an MMP-2 restriction site was synthesized, with the following amino acid sequence: Cys-Gly-Gly-Ser-Gly-Pro-Leu-Gly-Leu-Ala-Gly-Gly-Gly-Ser-Lys (abbreviated as C-GGS-GPLGLAG-GGS-K).

[0032] The ε-amino group of the C-terminal lysine (K) residue of the linker was modified with an azide group (N3) to obtain an azide linker.

[0033] Preparation of endocytotoxic peptide-linker conjugates: The above K¹(5-hexynoyl)-EndoP144 and azide linker were dissolved in phosphate buffer (PBS, pH 7.4).

[0034] A copper-catalyzed azido-yne cycloaddition reaction (Click Chemistry) was carried out at room temperature for 2 hours with the addition of a copper catalyst (such as CuSO4) and a reducing agent (such as sodium ascorbate).

[0035] After the reaction was completed, the endocytosed peptide-linker conjugate (EndoP144-Linker) was obtained by dialysis or high performance liquid chromatography (HPLC).

[0036] Preparation and photocuring of EnpMGelMA hydrogel precursor solution: Methacrylamide gelatin (GelMA) was dissolved in PBS to prepare an 8 wt% solution.

[0037] The purified EndoP144-Linker conjugate was added to GelMA solution to a final concentration of 10 μM and mixed thoroughly to obtain the hydrogel precursor solution.

[0038] The precursor solution is transferred to a mold or injected directly into the target location. Under irradiation with visible light at a wavelength of 405 nm (light intensity of about 5 mW / cm²), it can be photocured in situ within 15 seconds to form a stable EnpMGelMA hydrogel.

[0039] Example 2, the first embodiment of the present invention, provides in vitro bioactivity verification of EnpMGelMA hydrogel, including: Primary astrocyte culture: Primary astrocytes were isolated and cultured from the cerebral cortex of newborn mice.

[0040] Establishment and intervention of cell activation model: Cells were divided into three groups: control group (no treatment), TGF-β activation group (addition of 10 ng / mL TGF-β1), and EnpMGelMA intervention group (addition of 10 ng / mL TGF-β1 and 10 μM EnpMGelMA hydrogel extract).

[0041] Cell samples were collected after 48 hours of culture.

[0042] Effectiveness evaluation: The expression level of glial fibrillary acidic protein (GFAP), a marker of astrocyte activation, was detected by immunofluorescence staining and Western blotting.

[0043] The results showed that, compared with the TGF-β activation group, the expression of GFAP in the EnpMGelMA intervention group was significantly reduced, indicating that the EndoP144 peptide can effectively attenuate the abnormal activation of astrocytes induced by TGF-β.

[0044] Example 3, the first embodiment of the present invention, provides the therapeutic application of EnpMGelMA hydrogel in a mouse spinal cord injury model, including: Animal model establishment: A mouse model of spinal cord contusion in the thoracic segment (T9-T10) was established using standard methods.

[0045] Hydrogel implantation: After spinal cord resection surgery, the EnpMGelMA hydrogel precursor solution (containing 10 μMEndoP144-Linker) prepared in Example 1 was precisely injected into the spinal cord injury cavity of mice using a microsyringe.

[0046] Immediately irradiate the injection site with a 405 nm fiber optic light source for 15 seconds to allow the hydrogel to solidify rapidly in situ within the body.

[0047] Treatment efficacy assessment: Motor function recovery: The hind limb motor function of mice was assessed weekly using the Basso Mouse Scale (BMS) scoring system after surgery. The results showed that, compared with the untreated injury control group, the BMS scores of mice in the EnpMGelMA hydrogel treatment group increased significantly from 4 weeks after surgery and continued to improve until 8 weeks, indicating that their motor function was effectively restored.

[0048] Histological analysis: Mice were sacrificed 8 weeks post-surgery, and tissue samples from the spinal cord injury area were collected for staining analysis. Chondroitin sulfate proteoglycan (CSPG) staining showed that the area and density of glial scars in the treatment group were significantly lower than those in the control group. Simultaneously, neurofilament 200 (NF200) immunostaining showed that the treatment group exhibited more axonal regeneration and extension in the injury area, suggesting that EnpMGelMA hydrogel successfully promoted the reconstruction of neural circuits.

[0049] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing an AI-assisted design for enriching TGF-β lysozyme-mediated photopolymerizable hydrogel, characterized in that: include: An N-terminal modified endocytic peptide derivative is obtained by introducing an additional lysine residue at the N-terminus of an endocytic peptide that has lysosomal targeting ability and can specifically bind TGF-β, and then covalently linking a 5-hexynyl group to the side chain amino group of the additional lysine residue. The N-terminal modified endocytic peptide derivative is combined with a peptide linker having an MMP-2 restriction site and containing a cysteine ​​residue at the N-terminus and an azido-modified lysine residue at the C-terminus, and the endocytic peptide-linker conjugate is formed by a copper-catalyzed azido-alkyne cycloaddition reaction. The endocytic peptide-linker conjugate was mixed with methacrylamide gelatin (GelMA), and under 405 nm ultraviolet light irradiation, the thiol groups of the cysteine ​​residues in the conjugate reacted with the methacrylamide groups in the GelMA to form the hydrogel in situ within 15 seconds through a thiol-ene click reaction.

2. The method for preparing AI-assisted design of photocurable hydrogels for enriching TGF-β lysozyme-mediated in vivo peptides as described in claim 1, characterized in that: The amino acid sequence of the endocytic polypeptide is MEEAQRLLEEWEFQERVDELARKYGERVKEVGRQIIQDPDPEVRRKMLDILERIYREAGGGSTSLDASIIWAMMQN.

3. The method for preparing AI-assisted design of photocurable hydrogels for enriching TGF-β lysozyme-mediated in vivo peptides as described in claim 1 or 2, characterized in that: The amino acid sequence of the polypeptide linker is Cys-Gly-Gly-Ser-Gly-Pro-Leu-Gly-Leu-Ala-Gly-Gly-Gly-Ser-Lys, and its C-terminal lysine residue is modified with an azide group at the ε-amino level.

4. The method for preparing AI-assisted design photocurable hydrogels for enriching TGF-β lysozyme-mediated in vivo peptides as described in any one of claims 1–3, characterized in that: The degree of methacrylation substitution of the GelMA is 40%–90%.

5. The method for preparing AI-assisted design photocurable hydrogels for enriching TGF-β lysozyme-mediated in vivo peptides as described in any one of claims 1–4, characterized in that: The final concentration of the endocytic peptide-linker conjugate in the hydrogel is 1–100 μM.

6. A photocurable hydrogel for enriching lysozyme-β targeting TGF-β, prepared by the method described in any one of claims 1-5, characterized in that: In the hydrogel, endocytic peptides are covalently coupled to the GelMA backbone via MMP-2 responsive linkers, and can release endocytic peptides with lysosomal targeted degradation activity against TGF-β in the presence of MMP-2.

7. The photocurable hydrogel as described in claim 6, characterized in that: The hydrogel is photocured in situ within 15 seconds under 405nm ultraviolet light irradiation, and the storage modulus G' after curing is 100–5000 Pa.

8. Use of the photocurable hydrogel as described in claim 6 or 7 in the preparation of medical materials for treating spinal cord injuries.

9. The use as described in claim 8, characterized in that: The spinal cord injury referred to is a spinal cord injury that has progressed from the acute phase to the subacute or chronic phase, at which time the expression level of MMP-2 in the injured area is elevated.

10. An implantable sustained-release material for spinal cord injury repair, comprising the photocurable hydrogel as described in any one of claims 6–7.

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