Neuroinflammation regulating material based on fusion protein controlled release and preparation method and application thereof

Abstract

The application relates to a nerve inflammation regulation material based on a fusion protein controlled release and a preparation method and application, and belongs to the technical field of biomedical materials. The nerve inflammation regulation material based on the fusion protein controlled release comprises: a type I collagen protein matrix from a mammal under 6 months old; a CBD-TIMP-GDNF fusion protein comprising a collagen binding domain, an MMP cleavable TIMP peptide segment and a glial cell-derived neurotrophic factor; wherein the CBD-TIMP-GDNF fusion protein is combined with the collagen protein matrix to form an MMP responsive controlled release system, and the glial cell-derived neurotrophic factor is released by cleavage of the TIMP peptide segment. The nerve inflammation regulation material can promote polarization of microglia from pro-inflammatory M1 type to anti-inflammatory M2 type, and effectively treat nerve inflammation.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and in particular to a neuroinflammatory regulatory material based on controlled release of fusion protein, its preparation method and application. Background Technology

[0002] Neuroinflammation, as a common pathological hub for central nervous system diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke, and traumatic brain injury, affects the quality of life of tens of millions of patients worldwide. Current clinical treatment mainly relies on drugs such as nonsteroidal anti-inflammatory drugs, glucocorticoids, and immunosuppressants. However, these treatment methods have the following limitations: (1) The blood-brain barrier restricts the entry of drugs into the central nervous system, resulting in insufficient drug concentration in brain tissue; (2) Systemic administration causes serious side effects, including immunosuppression, increased risk of infection, and endocrine disorders; (3) Lack of targeting, making it impossible to precisely regulate the inflammatory response in specific brain regions; (4) Difficulty in achieving long-term stable anti-inflammatory effects, requiring repeated administration.

[0003] In recent years, therapeutic strategies based on microglia polarization regulation (such as direct injection of IL-4 / IL-13 cytokines, statin intervention, gene vector delivery, or M2 cell transplantation) have theoretically possessed the potential to regulate M1 / M2 phenotypic switching. However, due to the extremely short half-life of cytokines requiring frequent injections, poor selectivity and significant side effects of small molecule drugs, safety risks associated with gene therapy, and immune rejection caused by cell transplantation, none of these strategies have achieved a breakthrough in clinical translation.

[0004] Biomaterial-mediated regulation of neuroinflammation offers a novel approach to addressing these challenges. Collagen, with its excellent biocompatibility and adaptability to neural tissue, is an ideal carrier, especially collagen derived from young animals, which can preserve a more complete extracellular matrix structure and function. However, existing collagen-based materials generally lack the ability to actively respond to the inflammatory microenvironment, hindering intelligent controlled drug release. Although matrix metalloproteinase (MMP)-based responsive materials have been explored in the field of oncology, their application in neuroinflammation scenarios is almost nonexistent, and existing MMP-responsive designs do not synergize with microglial polarization mechanisms. While glial cell-derived neurotrophic factor (GDNF) has been shown to drive microglial polarization towards the M2 type and inhibit neuroinflammation, its short in vivo half-life (on the order of minutes) and lack of targeted delivery systems make it difficult to achieve sustained therapeutic effects when administered alone. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the objectives of this application include providing a neuroinflammatory regulatory material based on controlled release of fusion proteins, its preparation method, and its application, in order to achieve precise targeted regulation of microglial cell polarization and thereby achieve effective treatment of neuroinflammatory diseases.

[0006] In a first aspect, embodiments of this application provide a neuroinflammatory regulatory material based on controlled release of a fusion protein, comprising: a type I collagen matrix derived from mammals under 6 months of age; a CBD-TIMP-GDNF fusion protein, including a collagen-binding domain, a cleavable TIMP peptide segment of MMP, and glial cell-derived neurotrophic factor; wherein the CBD-TIMP-GDNF fusion protein binds to the collagen matrix to form an MMP-responsive controlled release system, releasing glial cell-derived neurotrophic factor through TIMP peptide cleavage.

[0007] This application primarily aims to construct an MMP-responsive microglia polarization regulation system based on a CBD-TIMP-GDNF fusion protein. This system precisely regulates the polarization transition of microglia from M1 to M2, thereby establishing an anti-inflammatory protective microenvironment. Utilizing the pathological characteristic of significantly upregulated MMP-2 and MMP-9 activity during neuroinflammation, the system achieves site-specific release of GDNF, promoting the M1-to-M2 polarization transition of microglia and establishing a long-term stable anti-inflammatory microenvironment. Specifically, this application prioritizes tissues from animals under 6 months of age as the source of type I collagen matrix. This young extracellular matrix (ECM) possesses higher bioactivity and better neurocompatibility; furthermore, young collagen maintains an intact triple-helix structure, containing abundant cell-binding sites and growth factor-binding domains, providing a suitable microenvironmental support for microglia polarization. Subsequently, a CBD-TIMP-GDNF fusion protein was constructed. The collagen-binding domain (CBD) enables this fusion protein to stably bind to the type I collagen matrix, forming an MMP-responsive controlled-release system. The TIMP peptide, acting as a specific substrate for MMP-2 and MMP-9, is cleaved in the inflammatory microenvironment. GDNF, as an effector molecule, regulates microglial polarization. During neuroinflammation, activated microglia and astrocytes secrete large amounts of MMP-2 and MMP-9, with local concentrations 8-15 times higher than normal. These high concentrations of MMP bind to and cleave the TIMP peptide, releasing free GDNF (achieving site-specific release of GDNF), thus achieving a positive correlation between the severity of inflammation and the amount of drug released.

[0008] In some embodiments of this application, the molecular weight of the CBD-TIMP-GDNF fusion protein is 32-36 kDa.

[0009] In some embodiments of this application, the molecular weight of the type I collagen matrix is ​​280-320 kDa.

[0010] In some embodiments of this application, the neuroinflammation modulating material is a three-dimensional network structure with a porosity of 75-85%; and / or, the tensile strength of the neuroinflammation modulating material is 0.05-0.5 MPa, and the elastic modulus is 5-50 kPa.

[0011] In some embodiments of this application, the dosage forms of the neuroinflammatory modulating material include injectable hydrogel, sustained-release microspheres, and implantable scaffolds; wherein the concentration of the injectable hydrogel is 5-15 mg / ml, and the diameter of the sustained-release microspheres is 10-100 μm.

[0012] Secondly, this application provides a method for preparing the above-mentioned neuroinflammatory regulatory material based on controlled release of fusion protein, comprising: taking mammalian tissues under 6 months of age, extracting and purifying type I collagen matrix to obtain type I collagen powder; constructing a recombinant expression vector pET-CBD-TIMP-GDNF containing a collagen-binding domain, a TIMP peptide, and a glial cell-derived neurotrophic factor coding sequence, transforming it into Escherichia coli BL21 or transfecting mammalian cells for protein expression, and purifying it to obtain CBD-TIMP-GDNF fusion protein; dissolving the type I collagen powder in PBS buffer to obtain a collagen solution; and subsequently adding the CBD-TIMP-GDNF fusion protein to obtain the neuroinflammatory regulatory material.

[0013] This preparation method can rapidly and effectively construct a CBD-TIMP-GDNF fusion protein, thereby obtaining an MMP-responsive microglia polarization regulation system based on the CBD-TIMP-GDNF fusion protein. This system can precisely regulate the polarization transition of microglia from M1 to M2, thereby establishing an anti-inflammatory protective microenvironment.

[0014] In some embodiments of this application, the extraction and purification steps include a dilute acetic acid extraction method, wherein the concentration of dilute acetic acid is 0.08-0.3 mol / L, the extraction temperature is 4-15℃, and the extraction time is 48-72 h; the protein retention rate of the purified type I collagen matrix is ​​≥85%.

[0015] In some embodiments of this application, the concentration of the collagen solution is 8-20 mg / mL.

[0016] In some embodiments of this application, the preparation method includes adding CBD-TIMP-GDNF fusion protein to a final concentration of 30-150 μg / mL.

[0017] Thirdly, the embodiments of this application provide the application of the above-mentioned neuroinflammatory regulatory material based on controlled release of fusion protein in the preparation of drugs or medical devices for treating neuroinflammatory diseases, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, post-stroke inflammation, and traumatic brain injury.

[0018] The neuroinflammatory modulatory material provided in this application is suitable for preparing drugs or medical devices for various neuroinflammatory diseases, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, post-stroke inflammation, and traumatic brain injury. For example, for Alzheimer's disease, the neuroinflammatory modulatory material can be precisely delivered to the lesion areas such as the hippocampus and cortex via stereotactic injection or intraventricular administration. For Parkinson's disease, it can be injected into the substantia nigra-striatal pathway to protect dopamine neurons and inhibit inflammation caused by α-synuclein aggregation. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a graph showing the amino acid composition analysis of Experimental Example 1 of this application; Figure 2 The results of thermal denaturation analysis for Test Example 1 of this application; Figure 3 The hydroxyproline retention rate of Experimental Example 1 in this application; Figure 4 This is a plasmid map of the CBD-TIMP-GDNF fusion protein from Experimental Example 2 of this application; Figure 5 This is the dose-response curve of GDNF in Test Example 2 of this application; Figure 6 The retention rate of GDNF bioactivity in Test Example 2 of this application; Figure 7 The results of the hydrogel rheological property analysis for Experiment Example 3 of this application; Figure 8 The MMP-responsive GDNF release curve for Test Example 3 of this application; Figure 9 The results of the cytokine secretion level analysis for Experiment Example 4 of this application; Figure 10 The results of gene expression analysis of microglia M1 type in Experiment Example 4 of this application; Figure 11 The results of the microglia polarization regulation function verification experiment in Experiment Example 4 of this application (microglia M2 type gene expression analysis results); Figure 12 The results are from the balance beam test in Example 5 of this application; Figure 13 The results of the rotator test in Example 5 of this application; Figure 14 The pro-inflammatory cytokine levels in Experiment Example 5 of this application; Figure 15 The anti-inflammatory cytokine levels of Experimental Example 5 in this application. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] Microglia can polarize into M1 (pro-inflammatory) or M2 (anti-inflammatory) types under different stimuli. M1 microglia express markers such as iNOS and CD86, and secrete pro-inflammatory factors such as TNF-α, IL-1β, and IL-6, which aggravate nerve damage. M2 microglia express markers such as Arg1 and CD206, and secrete anti-inflammatory and neurotrophic factors such as IL-10, TGF-β, and GDNF, which exert neuroprotective effects.

[0023] The following is a detailed description of a neuroinflammatory regulatory material based on controlled release of a fusion protein, its preparation method, and its application, according to an embodiment of this application.

[0024] This application provides a neuroinflammatory regulatory material based on controlled release of a fusion protein, comprising: a type I collagen matrix derived from mammals under 6 months of age; a CBD-TIMP-GDNF fusion protein, including a collagen-binding domain, an MMP-cleavable TIMP peptide, and glial cell-derived neurotrophic factor; wherein the CBD-TIMP-GDNF fusion protein binds to the collagen matrix to form an MMP-responsive controlled release system, releasing glial cell-derived neurotrophic factor through TIMP peptide cleavage.

[0025] This application primarily aims to construct an MMP-responsive microglia polarization regulation system based on a CBD-TIMP-GDNF fusion protein. This system precisely regulates the polarization transition of microglia from M1 to M2, thereby establishing an anti-inflammatory protective microenvironment. Utilizing the pathological characteristic of significantly upregulated MMP-2 and MMP-9 activity during neuroinflammation, the system achieves site-specific release of GDNF, promoting the M1-to-M2 polarization transition of microglia and establishing a long-term stable anti-inflammatory microenvironment. Specifically, this application prioritizes tissues from animals under 6 months of age as the source of type I collagen matrix. This young extracellular matrix (ECM) possesses higher bioactivity and better neurocompatibility; furthermore, young collagen maintains an intact triple-helix structure, containing abundant cell-binding sites and growth factor-binding domains, providing a suitable microenvironmental support for microglia polarization. Subsequently, a CBD-TIMP-GDNF fusion protein was constructed. The collagen-binding domain (CBD) enables this fusion protein to stably bind to the type I collagen matrix, forming an MMP-responsive controlled-release system. The TIMP peptide, acting as a specific substrate for MMP-2 and MMP-9, is cleaved in the inflammatory microenvironment. GDNF, as an effector molecule, regulates microglial polarization. During neuroinflammation, activated microglia and astrocytes secrete large amounts of MMP-2 and MMP-9, with local concentrations 8-15 times higher than normal. These high concentrations of MMP bind to and cleave the TIMP peptide, releasing free GDNF (achieving site-specific release of GDNF), thus achieving a positive correlation between the severity of inflammation and the amount of drug released.

[0026] The TIMP peptide includes the common recognition sequence PLGLAG of MMP-2 and MMP-9; the molecular weight of the CBD-TIMP-GDNF fusion protein is 32-36 kDa. As a specific substrate for MMP-2 and MMP-9, the TIMP peptide is specifically cleaved in the neuroinflammatory microenvironment, achieving on-demand release of GDNF. The MMP-responsive controlled-release mechanism of this neuroinflammatory regulatory material is based on the pathological characteristics of the dynamic changes in MMP activity during neuroinflammatory processes. Under normal physiological conditions, the activities of MMP-2 and MMP-9 are low, and TIMP forms a stable complex with MMP, effectively sealing GDNF. When neuroinflammatory occurs, microglia, astrocytes, and infiltrating peripheral immune cells secrete large amounts of MMP-2 and MMP-9, with local concentrations reaching 8-15 times the normal level. High concentrations of MMP bind to the TIMP peptide, undergoing proteolysis at the specific site PLGLAG, releasing free GDNF; the Michaelis constant Km for the cleavage reaction is 2.1 × 10⁻⁶. -6 M, with a catalytic efficiency of 3.8 × 10⁻⁶ kcat / Km. 5 M -1 s -1The release rate of GDNF is positively correlated with the severity of inflammation, enabling adaptive regulation of the inflammatory state. Released GDNF activates the GFRα1 / RET receptor complex and TrkB receptor on the surface of microglia, activating downstream PI3K / Akt and MAPK / ERK signaling pathways, promoting the nuclear translocation of transcription factors STAT6 and IRF-4, and upregulating the expression of M2 marker genes Arg1, CD206, and IL-10. In other words, when inflammation occurs in the central nervous system, activated microglia and astrocytes secrete large amounts of MMP-2 or MMP-9, cleaving TIMP peptides and releasing GDNF at the specific site PLGLAG. The released GDNF specifically activates the TrkB receptor and PI3K / Akt signaling pathway, promoting microglia polarization from pro-inflammatory M1 to anti-inflammatory M2, secreting anti-inflammatory factors such as IL-10 and TGF-β, and inhibiting NF-κB inflammatory pathway activation.

[0027] The GDNF in this application is a recombinant human glial cell-derived neurotrophic factor with a molecular weight of approximately 15 kDa, containing 134 amino acid residues and possessing the typical four-helix bundle structure of the TGF-β superfamily. GDNF plays multiple roles in the regulation of neuroinflammation: activating microglia's TrkB receptors and promoting cell polarization from M1 to M2; upregulating the expression of anti-inflammatory factors IL-10, TGF-β, and IL-4, and downregulating the secretion of pro-inflammatory factors TNF-α, IL-1β, and IL-6; protecting neurons from inflammatory damage and improving neuronal survival; promoting oligodendrocyte survival and myelin repair, and improving nerve conduction function.

[0028] In this application, the type I collagen matrix is ​​derived from the tendon, skin, or bone matrix of animals under 6 months of age; the molecular weight of the type I collagen matrix is ​​280-320 kDa, and the α1 / α2 chain ratio is 2:1; the degradation cycle of the type I collagen matrix is ​​6-12 weeks, and the degradation products are amino acids or small peptides. The type I collagen matrix, derived from the tendon, skin, or bone matrix of young animals under 6 months of age, can maintain the advantageous characteristics of young ECM, namely, maintaining the natural triple helix structure and bioactivity of collagen, with a protein retention rate ≥85%. The molecular weight of the type I collagen matrix is ​​concentrated in the 280-320 kDa range, and the α1 / α2 chain ratio remains at the natural 2:1 ratio, which can improve the structural stability of collagen molecules. This type I collagen matrix has good neurocompatibility, supporting neuronal adhesion and neurite extension, while providing a suitable polarization microenvironment for microglia; the matrix surface carries a negative charge, which is conducive to forming stable electrostatic interactions with GDNF. Moreover, the degradation rate of this type I collagen matrix matches the neuroinflammation repair timeline. It is gradually degraded in vivo by collagenase and MMP, with a degradation cycle of 6-12 weeks. The degradation products are amino acids and small peptides, which are completely biocompatible and non-immunogenic.

[0029] In this application, the neuroinflammation-regulating material has a three-dimensional network structure with a porosity of 75-85%; its tensile strength is 0.05-0.5 MPa, and its elastic modulus is 5-50 kPa. The neuroinflammation-regulating material provided in this application forms a three-dimensional network structure under physiological conditions, with a porosity of 75-85%, which facilitates nutrient exchange and the clearance of inflammatory factors, providing a long-term protective microenvironment for neurons. Simultaneously, this neuroinflammation-regulating material possesses optimized physicochemical properties and long-term stability, with mechanical strength adapted to the physiological environment of the central nervous system; its tensile strength of 0.05-0.5 MPa and elastic modulus of 5-50 kPa are close to the biomechanical properties of brain tissue.

[0030] In this application, the dosage forms of the neuroinflammation modulating material include injectable hydrogel, sustained-release microspheres, and implantable scaffolds; wherein the concentration of the injectable hydrogel is 5-15 mg / mL, and the diameter of the sustained-release microspheres is 10-100 μm. This neuroinflammation modulating material can be prepared in different dosage forms to meet different clinical needs, including injectable hydrogel, sustained-release microspheres, and implantable scaffolds; the injectable hydrogel has a concentration of 5-15 mg / mL, and the gelation time is controlled within 3-10 min; the sustained-release microspheres have a diameter of 10-100 μm and can achieve continuous release for 1-3 months; the implantable scaffold can be designed in the form of a thin film, porous sponge, etc., suitable for different implantation sites.

[0031] The type I collagen matrix in this neuroinflammatory regulation material forms a fibrous network after gelation, which mimics the natural extracellular matrix of nerve cells. The fiber diameter is 50-200 nm, and the pore size is suitable for cell migration and nutrient diffusion.

[0032] The neuroinflammatory regulatory material provided in this application exerts neuroprotective function by regulating microglia polarization. The released GDNF can reprogram the metabolic state of microglia, shifting from pro-inflammatory glycolytic metabolism to anti-inflammatory oxidative phosphorylation metabolism. M2 microglia secrete neurotrophic factors BDNF, NGF, and IGF-1, promoting neuronal survival and maintaining synaptic plasticity. Simultaneously, they secrete anti-inflammatory factors IL-10 and TGF-β to form a local immunosuppressive microenvironment, blocking the inflammatory cascade. Among these, M2 microglia also have a scavenging function, efficiently phagocytosing Aβ protein, tau protein aggregates, and cell debris, reducing the accumulation of neurotoxic substances. They can also secrete the matrix metalloproteinase inhibitor TIMP-1, stabilizing the integrity of the blood-brain barrier; promoting oligodendrocyte precursor cell differentiation and myelin regeneration, and repairing myelin damage caused by inflammation.

[0033] The preparation method of the above-mentioned neuroinflammatory regulatory material based on controlled release of fusion protein is described below.

[0034] The preparation method of neuroinflammatory modulation materials based on controlled release of fusion proteins includes the following steps: (1) Extraction and purification of type I collagen matrix Tissues from mammals under 6 months of age were collected, and type I collagen matrix was extracted and purified to obtain type I collagen powder.

[0035] In the extraction and purification steps, the extraction method includes dilute acetic acid extraction. For example, tendon tissue from healthy young animals under 6 months of age is selected, and after removing fat and blood vessels, it is extracted with dilute acetic acid at 4-15°C for 48-72 hours. The tissue is then purified through steps such as salt precipitation, dialysis desalting, and freeze drying to obtain a type I collagen matrix with a purity of ≥95% and a protein retention rate of ≥85%.

[0036] The concentration of dilute acetic acid is 0.08-0.3 mol / L. For example, the concentration of dilute acetic acid includes, but is not limited to, 0.08 mol / L, 0.09 mol / L, 0.1 mol / L, 0.11 mol / L, 0.12 mol / L, 0.13 mol / L, 0.14 mol / L, 0.15 mol / L, 0.16 mol / L, 0.17 mol / L, 0.18 mol / L, 0.19 mol / L, 0.2 mol / L, 0.21 mol / L, 0.22 mol / L, 0.23 mol / L, 0.24 mol / L, 0.25 mol / L, 0.26 mol / L, 0.27 mol / L, 0.28 mol / L, 0.29 mol / L, and 0.3 mol / L.

[0037] (2) Expression and purification of CBD-TIMP-GDNF fusion protein A recombinant expression vector pET-CBD-TIMP-GDNF containing the coding sequences of collagen-binding domain (CBD), TIMP peptide, and glial cell-derived neurotrophic factor (GDNF) was constructed. The vector was transformed into Escherichia coli BL21 or transfected into mammalian cells for protein expression. After purification, the CBD-TIMP-GDNF fusion protein was obtained.

[0038] Among the usable mammalian cells are 293T cells.

[0039] The purification process includes steps such as His-tag affinity chromatography, cation exchange chromatography, and gel filtration chromatography, achieving a purity of ≥90% and maintaining ≥85% bioactivity.

[0040] In the CBD-TIMP-GDNF fusion protein, the amino acid sequence of CBD is shown in SEQ ID NO.1, the amino acid sequence of the TIMP peptide is shown in SEQ ID NO.2, the amino acid sequence of GDNF is shown in SEQ ID NO.3, and the complete amino acid sequence of CBD-TIMP-GDNF is shown in SEQ ID NO.4.

[0041] (3) Material composite and molding The type I collagen powder prepared in step (1) was dissolved in PBS buffer to obtain a collagen solution; then, the CBD-TIMP-GDNF fusion protein prepared in step (2) was added to obtain a neuroinflammatory regulatory material.

[0042] The concentration of the collagen solution is 8-20 mg / mL. For example, the concentration of the collagen solution includes, but is not limited to, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, and 20 mg / mL.

[0043] As one implementation method, the type I collagen powder prepared in step (1) is dissolved in PBS buffer to obtain a collagen solution (concentration of 8-20 mg / mL); then, the CBD-TIMP-GDNF fusion protein prepared in step (2) (final concentration of 30-150 μg / mL) is added; and the mixture is gelled in a 37°C incubator for 20-40 min to form a neuroinflammatory regulatory material with MMP responsiveness.

[0044] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0045] Example 1 This embodiment describes the extraction of young type I collagen, including the following steps: The tail tendon of a 3-month-old healthy SD rat was selected as the collagen source. The tendon tissue was removed under sterile conditions, and surrounding fat and blood vessels were removed. The tissue was then thoroughly washed with pre-cooled physiological saline. The processed tendon tissue was cut into 1-2 mm pieces and placed in 0.5 M acetic acid solution. The mixture was gently stirred and extracted at 4°C for 72 h. Undissolved tissue residue was removed by filtration through multiple layers of gauze to obtain a clear collagen solution. Subsequently, the collagen was precipitated by salting out with 3.5 M sodium chloride solution. The collagen precipitate was collected by centrifugation and redissolved in 0.1 M acetic acid solution. The solution was dialyzed with deionized water through a dialysis bag for 96 h, with the dialysate changed every 12 h, until the conductivity was <50 μS / cm. The collagen solution after dialysis was freeze-dried to obtain type I collagen powder.

[0046] Example 2 This embodiment describes the construction of a CBD-TIMP-GDNF fusion protein, including: A fusion gene containing coding sequences for CBD, TIMP, and GDNF was designed and synthesized. The CBD sequence, derived from the collagen-binding domain of Clostridium collagenase A, is 330 bp in length and encodes 110 amino acids. The TIMP sequence, containing the common recognition site PLGLAG for MMP-2 and MMP-9, is 60 bp in length. The GDNF sequence is mature human GDNF, 402 bp in length and encodes 134 amino acids. The functional domains are connected by a flexible linker GSG.

[0047] The fusion gene was cloned into the pET-28a expression vector to construct the recombinant expression vector pET-28a-CBD-TIMP-GDNF; the vector was transformed into E. coli BL21(DE3) competent cells, and positive clones were screened on LB agar plates containing kanamycin; single clones were picked and inoculated into LB liquid medium and cultured at 37°C with shaking until OD. 600 =0.8, add 1mM IPTG to induce protein expression, and incubate at 30°C with shaking for 6h.

[0048] The His-tagged fusion protein was purified using a Ni-NTA affinity chromatography column. The column was equilibrated with binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0), and the protein solution was loaded. The protein was washed with wash buffer (20 mM Tris-HCl, 500 mM NaCl, 60 mM imidazole, pH 8.0) and eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 300 mM imidazole, pH 8.0). The eluent was further purified by cation exchange chromatography to remove endotoxins and other contaminating proteins. Finally, aggregates were removed by gel filtration chromatography to obtain monodisperse CBD-TIMP-GDNF fusion protein.

[0049] Example 3 This embodiment describes the preparation of neuroinflammatory modulatory materials (construction of an MMP-responsive controlled-release system), including: The type I collagen powder prepared in Example 1 was dissolved in sterile PBS buffer to prepare a collagen solution of 12 mg / mL, and the pH was adjusted to 7.2. The CBD-TIMP-GDNF fusion protein prepared in Example 2 (final concentration 80 μg / mL) was added, and the resulting mixture was gently stirred at 4°C for 4 h to ensure sufficient binding between the fusion protein and collagen. The mixture was then transferred to a 37°C incubator and allowed to stand for 30 min to form a collagen hydrogel. The prepared hydrogel was transparent or translucent, with appropriate elasticity and viscosity.

[0050] Experimental Example 1 This experimental example is for characterizing the type I collagen powder prepared in Example 1, including: 1. SDS-PAGE analysis of molecular weight distribution showed that the molecular weights of the α1 and α2 chains were 129 kDa and 118 kDa, respectively, while the molecular weight of the β chain (dimer) was 250 kDa. This molecular weight distribution is consistent with the characteristics of type I collagen. Figure 1 Amino acid composition analysis showed that the glycine content was 33.8%, and the total content of proline and hydroxyproline was 22.1%, which is close to the theoretical value.

[0051] 2. The thermal denaturation temperature (Td = 38.7°C) was determined using differential scanning fluorometry (DSF), a highly efficient and sensitive classic technique for assessing protein thermal stability. This experiment was performed on a real-time quantitative PCR instrument. Its core principle is based on a fluorescent dye (such as SYPRO Orange) that is highly sensitive to hydrophobic environments. At low temperatures, the protein structure is compact, and the dye cannot bind to the internal hydrophobic regions, resulting in a weak fluorescence signal. As the temperature is increased at a programmed, uniform rate (e.g., from 25°C to 95°C), the protein gradually unfolds and denatures, exposing the previously encapsulated hydrophobic amino acid residues, which then bind to the dye, leading to a significant increase in fluorescence intensity. The instrument acquires fluorescence signals at each degree, ultimately obtaining a temperature-dependent fluorescence change curve. The temperature corresponding to the inflection point is defined as the thermal denaturation temperature Tm (referred to as Td in this study). Figure 2 The thermal denaturation analysis results show that Td = 38.7°C, indicating that the protein's thermal stability is better than that of human body temperature (37°C), thus providing crucial data support for maintaining its intact structure and effective function under physiological conditions from the perspective of conformational stability.

[0052] 3. The determination of hydroxyproline content employed an acid hydrolysis-spectrophotometric method, the gold standard for the specific quantification of collagen and its derivatives, as hydroxyproline is a highly characteristic amino acid in the collagen family. The detection procedure begins with rigorous acid hydrolysis of the sample: the protein sample is hydrolyzed for 16-24 hours under concentrated hydrochloric acid (e.g., 6M HCl), high temperature (110°C), and anaerobic conditions, thereby completely breaking down the protein into free amino acid components, including hydroxyproline. After hydrolysis, the hydrolysate is neutralized and diluted, then subjected to specific colorimetric reactions sequentially with an oxidizing agent (e.g., chloramine-T) and a chromogenic agent (e.g., p-dimethylaminobenzaldehyde). Hydroxyproline is quantitatively converted into a rose-red product with strong absorption at a specific wavelength (typically around 560 nm). Finally, the absorbance of the solution is measured using a UV-Vis spectrophotometer, and the absolute content of hydroxyproline in the sample can be accurately calculated by comparing it with a standard curve of known concentrations of hydroxyproline. By comparing this content with the theoretical value or the content of the initial raw materials, its retention rate during the preparation process can be calculated. This indicator directly reflects the purity of the collagen product and the degree to which its unique amino acid structure is retained during the processing. Figure 3 As can be seen from the data, the hydroxyproline retention rate of the type I collagen prepared in Example 1 of this application is 89.1%, indicating that the structure of the type I collagen molecule is highly stable.

[0053] Experimental Example 2 This experimental example is for characterizing the CBD-TIMP-GDNF fusion protein constructed in Example 2, including: 1. SDS-PAGE analysis showed that the fusion protein had a molecular weight of approximately 34 kDa and a purity of >92%; Western blot analysis using the His tag confirmed that the protein was expressed correctly.

[0054] 2. Plasmid map of CBD-TIMP-GDNF fusion protein as shown below. Figure 4As shown, the origin (ori) is the sequence necessary for the plasmid to replicate itself in the host cell; AmpR (ampicillin resistance gene) is the ampicillin resistance gene, which allows bacteria containing this plasmid to grow in a selective medium containing ampicillin, used to screen for successful transformants; this gene is driven by the AmpR promoter. CMV enhancer and CMV promoter are the cytomegalovirus enhancer and promoter, respectively, used to drive transcription of downstream genes at high expression levels, typically used for gene expression in mammalian cells; the IL-2 signal sequence is an IL-2 signaling sequence immediately following the CMV promoter, used to guide the secretion of the fusion protein extracellularly. CBD-TIMP-GDNF is the core fusion protein coding sequence; this plasmid will express a fusion protein containing a collagen-binding domain (CBD), the metalloproteinase inhibitor tissue factor (TIMP), and glial cell-derived neurotrophic factor (GDNF). 6×His represents a 6-histidine tag, commonly used for fusion protein purification. The β-globin poly(A) signal, located downstream of the fusion protein coding sequence, is a β-globin polyadenylation signal used to ensure proper termination and stability of mRNA transcripts.

[0055] 3. Validation of GDNF's in vivo biological function, i.e., the half-maximal effective concentration (EC50). 50 The determination of the chemiluminescence activity (EC5) was achieved through a cell-level TrkB receptor-activated reporter gene assay. This assay first required culturing an engineered cell line stably expressing the TrkB receptor and a luciferase reporter gene specifically activated by the downstream signaling pathway of GDNF. During the experiment, cells were seeded in culture plates and, after adhesion, stimulated for a period of time with different concentration gradients of the test fusion protein sample, a positive control (standard GDNF), and a negative control to activate the downstream signaling pathway and induce reporter gene expression. After stimulation, a luciferase substrate was added to the cells. This substrate, catalyzed by luciferase, produced a chemiluminescent signal, the intensity of which was proportional to the bioactivity of GDNF. Finally, a multi-functional microplate reader with chemiluminescence detection capabilities was used to read the signal values, and Python was used for logistic curve fitting to calculate the EC5 of the sample. 50 Value. By comparing the EC values ​​of the sample and the standard. 50 Value (EC) 50 = 0.11 ng / mL), from which the bioactivity retention rate of GDNF can be calculated to be 87.3% (see Figure 5 and Figure 6 ).

[0056] Experimental Example 3 This experimental example is a functional verification of the neuroinflammation modulation material prepared in Example 3, including: 1. Rheological testing and analysis using a rheometer to evaluate the mechanical properties of hydrogels, such as... Figure 7 As shown, the storage modulus G' is 286 Pa, the loss modulus G" is 34 Pa, and tanδ=0.12, indicating that the hydrogel has good elastic characteristics.

[0057] 2. In vitro MMP responsive release assay: Hydrogels were treated with different concentrations of recombinant MMP-2 and MMP-9. The experiment was divided into four groups: control group (no MMP), low concentration group (50 ng / mL MMP), medium concentration group (200 ng / mL MMP), and high concentration group (800 ng / mL MMP). The gels were incubated at 37°C, and samples were taken periodically to detect the released GDNF content.

[0058] from Figure 8 The test results showed that the cumulative release of GDNF in the control group was only 5.7% within 72 hours, indicating that the fusion protein was stably bound to the collagen matrix; the cumulative release in the low-concentration MMP group was 23.4%; the medium-concentration group was 58.7%; and the high-concentration group reached 81.2%. The release rate showed a good positive correlation with the MMP concentration, indicating that the neuroinflammation modulating material had good MMP response performance.

[0059] Test Example 4 This experimental case validates the function of microglia in regulating polarization, including: In vitro polarization regulation experiments were conducted using the BV-2 microglia cell line (donated by the Academy of Military Medical Sciences). Cells were seeded in 24-well plates at a density of 2 × 10⁶ cells / well. 5 Cells / well were cultured in DMEM medium for 24 hours until adherence. The experiment was divided into five groups: normal control group, LPS activation group (1 μg / mL LPS, to establish the M1 polarization model), GDNF treatment group (10 ng / mL free GDNF), material treatment group (neuroinflammatory regulation material prepared in Example 3), and material + MMP group (neuroinflammatory regulation material + 200 ng / mL MMP-2 pretreatment).

[0060] After 48 hours of treatment, the expression of microglial polarization marker genes was detected by qRT-PCR. M1 marker genes included iNOS, TNF-α, IL-1β, and CD86; M2 marker genes included Arg1, CD206, IL-10, and TGF-β. Figure 10 and Figure 11 It can be seen that the expression of M1 marker genes was significantly upregulated in the LPS-activated group, while the expression of M2 marker genes was downregulated (see...). Figure 10 The GDNF treatment group can partially reverse this change; the material treatment group has limited effectiveness due to the limited release of GDNF (see...). Figure 10The material + MMP group showed the most significant effect in promoting M2 polarization, with Arg1 expression upregulated by 4.2-fold, CD206 by approximately 3.5-fold, IL-10 by 3.8-fold, and TGF-β by approximately 3.9-fold. (See...) Figure 11 ).

[0061] ELISA is used to detect the secretion levels of cytokines in cell culture supernatants, such as... Figure 9 As shown, the secretion levels of anti-inflammatory factors IL-10 and TGF-β in the material + MMP group increased by 5.3 times and 3.7 times, respectively, compared with the control group, while the secretion levels of pro-inflammatory factors TNF-α and IL-6 decreased by 67% and 72%, respectively. These results confirm that the neuroinflammatory regulatory material provided in this application can effectively regulate the polarization of microglia towards the M2 type.

[0062] Experimental Example 5 This experimental case studies evaluate the neuroprotective effects in an animal model, including: To establish an LPS-induced neuroinflammation mouse model and evaluate the in vivo effects of the material, forty 8-10 week old male C57BL / 6 mice were randomly divided into four groups: normal control group, LPS model group, material treatment group, and positive control group (dexamethasone treatment), with 10 mice in each group. The LPS model group was established by intraventricular injection of LPS (5 μg, dissolved in 5 μL of physiological saline); the material treatment group received an injection of the neuroinflammation-regulating material prepared in Example 3 (20 μL, containing 2 μg of GDNF) at the same site 1 hour after LPS injection; and the positive control group received an intraperitoneal injection of dexamethasone (2 mg / kg).

[0063] Behavioral assessments were performed on postoperative days 3, 7, 14, and 28, including a balance beam test (to assess motor coordination) and a tumbler test (to assess motor learning ability and balance function).

[0064] The experimental steps for the balance beam test are as follows: ① Place the mouse at the starting end of the balance beam. ② Place a strong beam of light behind the mouse to lure it across the balance beam until it reaches the finish line. ③ Before completing step ②, conduct 3 days of training to enable the mouse to reach the finish line from the starting end of the balance beam. ④ After the mouse reaches the finish line, let it rest in the dark box at the finish line for 30 seconds. ⑤ Repeat steps ②-④, 3 times a day, until the mouse can pass the balance beam smoothly at a speed of 20 cm / s. ⑥ Record the time required for the mouse to reach the finish line from the starting point of the balance beam. ⑦ Start testing and record the time from day 4. ⑧ Repeat steps ②-⑦ until the test is complete.

[0065] The experimental steps for the rotator bar test are as follows: ① For the first 3 days of the experiment, the mice were trained at a speed of 150 rpm / min to gradually adapt to the instrument. The training was conducted 3 times a day with a 10-minute interval between each session. ② During training, if a mouse falls off the spinning bar, it should be immediately put back on the spinning bar and training should continue until the training is completed. ③ During the final training session on day 3, mice that could stay stably on the rotundus for about 150 seconds were selected and assigned to the experimental group.

[0066] ④ On the experimental day, the initial speed of the rotor was 5 rpm / min, and then increased to a maximum speed of 40 rpm / min within 5 minutes.

[0067] ⑤ Record the time each mouse can stay on the spinner before falling, repeat 3 times, calculate the average time each mouse stays on the spinner, and then perform subsequent statistical analysis.

[0068] The results of the balance beam test are as follows Figure 12 As shown, the results of the swivel test are as follows: Figure 13 As shown in the figure, in the rotarod test, the normal control group had the longest dwell time, indicating that its motor and balance abilities were well maintained; in contrast, the dwell time of the LPS model group was significantly shorter, while the material treatment group was still able to maintain a relatively long dwell time, indicating that the neuroinflammation modulation material provided in this application has a good neuroprotective effect. Similarly, consistent results were observed in the balance beam experiment: the control group had the highest score, the LPS model group had the lowest score, and the material treatment group had a significantly higher score than the LPS model group, indicating that the neuroinflammation modulation material provided in this application can effectively promote the functional recovery of animal models of nerve injury.

[0069] Animals were euthanized 28 days post-surgery, and brain tissue was collected for analysis. ELISA was used to detect the levels of inflammatory factors in the brain tissue. In the material treatment group, the levels of TNF-α, IL-1β, and IL-6 were reduced by 58%, 60%, and 57%, respectively, compared to the model group. IL-10 (see...) Figure 14 The levels of α and TGF-β increased by 3.7-fold and 4.4-fold, respectively (see [link to article]). Figure 15 These results confirm the efficacy and safety of the neuroinflammation modulating material provided in this application in vivo.

[0070] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A neuroinflammatory regulatory material based on controlled release of a fusion protein, characterized in that, include: Type I collagen matrix derived from mammals under 6 months of age; The CBD-TIMP-GDNF fusion protein includes a collagen-binding domain, an MMP-cleavable TIMP peptide, and glial cell-derived neurotrophic factor. The CBD-TIMP-GDNF fusion protein binds to the type I collagen matrix to form an MMP-responsive controlled-release system, which releases the glial cell-derived neurotrophic factor through the cleavage of the TIMP peptide. The neuroinflammation modulation material has a three-dimensional network structure and an elastic modulus of 5-50 kPa. The TIMP peptide includes the common recognition sequence PLGLAG of MMP-2 and MMP-9; In the CBD-TIMP-GDNF fusion protein, the amino acid sequence of CBD is shown in SEQ ID NO.1, the amino acid sequence of the TIMP peptide is shown in SEQ ID NO.2, the amino acid sequence of GDNF is shown in SEQ ID NO.3, and the complete amino acid sequence of CBD-TIMP-GDNF is shown in SEQ ID NO.

4. The molecular weight of the CBD-TIMP-GDNF fusion protein is 32–36 kDa; The functional domains of CBD, TIMP peptide, and GDNF are connected by GSG flexible connectors. The neuroinflammation regulating material is a collagen hydrogel, wherein the concentration of type I collagen in the collagen hydrogel is 12 mg / mL, the concentration of the CBD-TIMP-GDNF fusion protein is 80 μg / mL, and the pH of the collagen hydrogel is 7.

2. The collagen hydrogel is formed by stirring the type I collagen and the CBD-TIMP-GDNF fusion protein at 4°C for 4 h and then letting it stand at 37°C for 30 min. The tensile strength of the neuroinflammation modulating material is 0.05-0.5 MPa, the elastic modulus is 5-50 kPa, and the neuroinflammation modulating material forms a three-dimensional network structure under physiological conditions with a porosity of 75-85%. The application of the neuroinflammatory regulatory material based on controlled release of fusion protein in the preparation of drugs or medical devices for treating neuroinflammatory diseases, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, post-stroke inflammation, and traumatic brain injury.

2. The neuroinflammation regulation material based on controlled release of fusion protein according to claim 1, characterized in that, The molecular weight of the type I collagen matrix is ​​280-320 kDa.

3. The neuroinflammation regulation material based on controlled release of fusion protein according to any one of claims 1-2, characterized in that, The dosage forms of the neuroinflammatory modulating materials include injectable hydrogels, sustained-release microspheres, and implantable scaffolds; The concentration of the injectable hydrogel is 5-15 mg / mL, and the diameter of the sustained-release microspheres is 10-100 μm.

4. A method for preparing a neuroinflammatory regulatory material based on controlled release of a fusion protein as described in any one of claims 1-2, characterized in that, include: Tissues from mammals under 6 months of age were extracted and purified to obtain type I collagen powder. A recombinant expression vector pET-CBD-TIMP-GDNF containing a collagen-binding domain, a TIMP peptide, and a glial cell-derived neurotrophic factor coding sequence was constructed. The protein was expressed by transforming Escherichia coli BL21 or transfecting mammalian cells, and the CBD-TIMP-GDNF fusion protein was obtained after purification. The type I collagen powder was dissolved in PBS buffer to obtain a collagen solution; subsequently, the CBD-TIMP-GDNF fusion protein was added to obtain the neuroinflammation regulation material.

5. The preparation method according to claim 4, characterized in that, In the extraction and purification steps, the method for extracting the type I collagen matrix includes dilute acetic acid extraction, wherein the concentration of the dilute acetic acid is 0.08-0.3 mol / L, the extraction temperature is 4-15℃, and the extraction time is 48-72 h; after purification, the protein retention rate of the type I collagen matrix is ​​≥85%.

6. The preparation method according to claim 4, characterized in that, The concentration of the collagen solution is 8-20 mg / mL.

7. The preparation method according to claim 4, characterized in that, include: Add the CBD-TIMP-GDNF fusion protein to a final concentration of 30-150 μg / mL.

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