Responsive self-assembling polypeptide hydrogel microsphere system, preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a responsive self-assembled polypeptide hydrogel microsphere system, a preparation method and application thereof. The responsive self-assembled polypeptide hydrogel microsphere system comprises GelMA, polypeptide I, an anti-inflammatory active ingredient and a polypeptide hydrogel, the polypeptide I is grafted with the GelMA, the anti-inflammatory active ingredient is loaded on the GelMA to form a GEL+polypeptide I+anti-inflammatory active ingredient microsphere, and the polypeptide hydrogel is used to wrap the GEL+polypeptide I+anti-inflammatory active ingredient microsphere to form the responsive self-assembled polypeptide hydrogel microsphere system. The polypeptide hydrogel as a carrier in the microsphere system is a smart responsive hydrogel, a MMP-2 specific recognition polypeptide sequence is introduced through a molecular engineering technology, and a functionalized GEL+polypeptide I+anti-inflammatory active ingredient microsphere is compounded. The above research results indicate that the responsive self-assembled polypeptide hydrogel microsphere system provided by the present application is expected to be widely applied in anti-inflammatory and / or tissue repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a responsive self-assembled polypeptide hydrogel microsphere system, its preparation method, and its application. Background Technology

[0002] The musculoskeletal system is the core framework for human movement and support, and damage and degeneration of its nerves, muscles, and cartilage are major challenges facing modern medicine. Currently used clinical methods such as conservative treatment and surgical repair both have limitations, making it difficult to achieve complete tissue repair and functional reconstruction. Although biomaterials play an important role in tissue damage repair, they still fall short of achieving complete tissue regeneration and functional reconstruction.

[0003] Tissue damage is often accompanied by characteristic microenvironmental changes such as pH acidification, significantly elevated matrix metalloproteinase (MMP) concentrations, and abnormal local temperature fluctuations, which are key factors affecting repair. Intelligent responsive biomaterials, with their specific sensing capabilities for physical and chemical stimuli, can respond to microenvironmental changes in real time, precisely regulate local microenvironmental homeostasis, and achieve controlled release of active substances such as drugs and growth factors, opening up new pathways for the precision treatment of tissue damage.

[0004] Peptide biomaterials, composed of natural amino acids, possess excellent biocompatibility, biodegradability, and functional designability, making them a research hotspot in the field of skeletal muscle tissue repair. Combining self-assembled peptide materials with intelligent responsive functions can effectively address the challenges of skeletal muscle tissue repair. These materials can achieve slow and sustained drug release based on the dynamic changes in the injury microenvironment, significantly increasing the effective drug concentration at the degenerative injury site, prolonging the duration of action, and thus improving therapeutic efficacy.

[0005] Intervertebral disc degeneration refers to the degenerative changes in the intervertebral disc (including the nucleus pulposus, annulus fibrosus, and cartilaginous endplates) caused by factors such as aging, injury, genetics, or abnormal mechanical loading, resulting in structural damage and functional decline. It is one of the main causes of low back pain and spinal dysfunction. Its pathological process begins with the release of pro-inflammatory factors induced by aging or injury, making clinical treatment challenging. The release of pro-inflammatory factors not only triggers apoptosis / death of nucleus pulposus cells (NPCs) but also creates a positive feedback loop of inflammatory factor secretion, inducing an inflammatory cascade response. This leads to abnormal expression of matrix metalloproteinase-2 (MMP-2) and degradation of the extracellular matrix (ECM), ultimately disrupting the homeostasis of the intervertebral disc microenvironment, resulting in structural damage and functional decline. Abnormal MMP-2 expression further exacerbates ECM degradation and angiogenesis through the Rac1 / PAK1 signaling pathway, forming the core pathological mechanism driving intervertebral disc degeneration. Developing novel polypeptide biomaterials has become a viable research and development direction for addressing this pathogenesis. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the first objective of this invention is to provide a responsive self-assembling peptide hydrogel microsphere system; the second objective is to provide a method for preparing the responsive self-assembling peptide hydrogel microsphere system; and the third objective is to provide applications of the responsive self-assembling peptide hydrogel microsphere system.

[0007] To achieve the first objective, the technical solution adopted by this invention is as follows:

[0008] A responsive self-assembling peptide hydrogel microsphere system includes GelMA, peptide I, an anti-inflammatory active ingredient, and a peptide hydrogel. The peptide I is grafted onto GelMA, and the anti-inflammatory active ingredient is loaded onto GelMA to form GEL@peptide I + anti-inflammatory active ingredient microspheres. The peptide hydrogel encapsulates the GEL@peptide I + anti-inflammatory active ingredient microspheres to form a responsive self-assembling peptide hydrogel microsphere system.

[0009] Wherein, GelMA is methacrylamide gelatin, and the amino acid sequence of polypeptide I is shown in SEQ ID NO.1:

[0010] Mal-KPSSAPTQLN;

[0011] Wherein, Mal- represents the maleimide group;

[0012] The structural formula of polypeptide I is shown below:

[0013] ;

[0014] Polypeptide I is a bioactive substance that plays an important role in the proliferation and repair of NPCs. The maleimide group (Mal-) introduced into polypeptide I makes it easy for polypeptide I to branch with -NH2 in the gelatin structure to form a long chain structure, which increases the stability of GEL@polypeptide I+ anti-inflammatory active ingredient microspheres.

[0015] The polypeptide hydrogel is self-assembled from polypeptides II and III. The amino acid sequence of polypeptide II is shown in SEQ ID NO.2, and the amino acid sequence of SEQ ID NO.2 is shown below:

[0016] CRADARADARADARADAC;

[0017] The amino acid sequence of polypeptide III is shown in SEQ ID NO.3, and the amino acid sequence of SEQ ID NO.3 is as follows:

[0018] Mal-(PEG)n-GPLGLAGK-Mal;

[0019] Wherein, PEG is a repeating unit of polydiol, and n is the number of repetitions of the repeating unit, which is an integer selected from 4 to 6;

[0020] Its structural formula is shown below:

[0021] .

[0022] GPLGLAG in the amino acid sequence of peptide III is a responsive peptide fragment. The release of MMP-2 can lead to allosteric hydrolysis of GPLGLAG, thereby weakening the stress effect of GPLGLAG and exerting its responsive function. Cysteine ​​(C) residues are added to both ends of the responsive peptide fragment GPLGLAG. The -SH in the cysteine ​​readily reacts with the NH in the maleimide group (-Mal). Hydrophilic modification with hydrophilic ethylene glycol (-PEG-) yields peptide III.

[0023] This invention provides a responsive self-assembling peptide hydrogel microsphere system designed for precise intervention in intervertebral disc degeneration. The peptide hydrogel serving as the carrier in this microsphere system is a smart responsive hydrogel. Through molecular engineering, MMP-2-specific recognition peptide sequences are introduced, along with functionalized GEL@peptide I + anti-inflammatory active ingredient microspheres. Therefore, when an inflammatory cascade reaction occurs in the intervertebral disc, the abnormally elevated MMP-2 locally can specifically recognize and cleave the response sites in the peptide hydrogel, triggering the depolymerization of the hydrogel network structure, thereby accelerating the release of functionalized GEL@peptide I + anti-inflammatory active ingredient microspheres loaded with anti-inflammatory and repair-promoting drugs. The released functionalized microspheres exert a triple therapeutic effect: 1. Inhibiting the inflammatory factor cascade reaction through anti-inflammatory active ingredients; 2. Promoting NPC proliferation and extracellular matrix synthesis using repair-promoting active substances (grafted peptide I); 3. Reconstructing intervertebral disc homeostasis by regulating the physicochemical properties of the microenvironment, thereby delaying or even reversing intervertebral disc degeneration.

[0024] Preferably, the anti-inflammatory active ingredient is selected from rhein (the active ingredient of diacerein, RHE).

[0025] Preferably, the mass ratio of the polypeptide I, the anti-inflammatory active ingredient, and GelMA is 1:1:1000.

[0026] Preferably, the particle size of the GEL@peptide I + anti-inflammatory active ingredient microspheres is 160-205 μm.

[0027] Preferably, the molar ratio of polypeptide II to polypeptide III is 1:1.

[0028] Preferably, the mass percentage of GelMA in the GEL@peptide I+ anti-inflammatory active ingredient microspheres is 10% to 30%.

[0029] To achieve the second objective, the technical solution adopted by this invention is as follows:

[0030] A method for preparing a responsive self-assembled peptide hydrogel microsphere system, used to prepare the responsive self-assembled peptide hydrogel microsphere system described in any one of the above claims, comprising the following steps:

[0031] S100: Using GelMA, polypeptide I, and anti-inflammatory active ingredients as raw materials, microfluidic-photocrosslinking technology was used to prepare GEL@polypeptide I + anti-inflammatory active ingredient microspheres.

[0032] S200. Add polypeptide II and polypeptide III to water and shake until polypeptide II and polypeptide III dissolve to obtain a polypeptide mixture.

[0033] S300. Add the GEL@peptide I + anti-inflammatory active ingredient microspheres to the peptide mixture, and add saturated sodium chloride aqueous solution. Shake until the solution forms a gel to obtain a responsive self-assembled peptide hydrogel microsphere system.

[0034] Preferably, in step S100, the photoinitiator used in the microfluidic photocrosslinking technology is selected from acylphosphonate derivatives, and the dispersion medium is selected from fluorinated liquid.

[0035] To achieve the third objective, the technical solution adopted by this invention is as follows:

[0036] Applications of responsive self-assembling peptide hydrogel microsphere systems, as described in any of the preceding claims, including applications in the preparation of anti-inflammatory and / or tissue repair drugs.

[0037] Preferably, the dosage form of the drug includes an injection.

[0038] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0039] The responsive self-assembly peptide hydrogel microsphere system provided by this invention includes GelMA, peptide I, an anti-inflammatory active ingredient, and a peptide hydrogel. Peptide I is grafted onto GelMA, and the anti-inflammatory active ingredient is loaded onto GelMA to form GEL@peptide I + anti-inflammatory active ingredient microspheres. The peptide hydrogel encapsulates the GEL@peptide I + anti-inflammatory active ingredient microspheres to form the responsive self-assembly peptide hydrogel microsphere system. This responsive self-assembly peptide hydrogel microsphere system aims to achieve precise intervention in intervertebral disc degeneration. In this microsphere system, the peptide hydrogel, acting as a carrier, is a smart responsive hydrogel. Through molecular engineering technology, MMP-2 is introduced to specifically recognize the peptide sequence, while simultaneously functionalizing GEL@peptide I + anti-inflammatory active ingredient microspheres. Therefore, when an inflammatory cascade reaction occurs in the intervertebral disc, the abnormally elevated MMP-2 can specifically recognize and cleave the response sites in the peptide hydrogel, triggering the depolymerization of the hydrogel network structure, thereby accelerating the release of functionalized GEL@peptide I + anti-inflammatory active ingredient microspheres loaded with anti-inflammatory and repair-promoting drugs. The released functionalized microspheres exert a triple therapeutic effect: First, they inhibit the cascade reaction of inflammatory factors through anti-inflammatory active ingredients; second, they promote the proliferation of NPCs and the synthesis of extracellular matrix by utilizing repair-promoting active substances (grafted polypeptide I); and third, they rebuild the intervertebral disc homeostasis by regulating the physicochemical properties of the microenvironment, thereby delaying or even reversing intervertebral disc degeneration.

[0040] Biological experimental results: The responsive self-assembly peptide hydrogel microsphere system provided by this invention can effectively reduce LPS-mediated inflammation in NP cells, thereby reducing NP cell apoptosis. The peptide hydrogel can inhibit the production of IL-1β, TNF-α, IL-6, and inflammatory MMP-3, MMP-13 and other metalloproteinases in NPCs, inhibiting inflammatory cell death. Simultaneously, this responsive self-assembly peptide hydrogel microsphere system can promote the production of aggrecan and type II collagen in NPCs, reduce the production of ADisintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS5) containing platelet-reactive protein motifs, and also promote ECM generation and cell repair. These research results suggest that the responsive self-assembly peptide hydrogel microsphere system provided by this invention has the potential for widespread application in anti-inflammatory and / or tissue repair applications.

[0041] The present invention provides a method for preparing a responsive self-assembled peptide hydrogel microsphere system. GEL@peptide I + anti-inflammatory active ingredient microspheres are prepared via microfluidic-photocrosslinking, grafted with peptide I, and loaded with the anti-inflammatory active ingredient, then coated with an MMP-2 responsive self-assembled peptide hydrogel. Research results show that the responsive peptide hydrogel microsphere system provided by the present invention has a stable gel state and self-healing properties, ensuring the injectability requirements of the hydrogel. The GEL@peptide I + anti-inflammatory active ingredient microspheres have a good ability to release the anti-inflammatory active ingredient, and the MMP-2 responsive peptide hydrogel carrier can responsively and sustainably release the anti-inflammatory active ingredient.

[0042] In summary, the responsive self-assembling peptide hydrogel microsphere system provided by this invention has the following three major technical advantages: First, through the MMP-2 concentration-dependent drug release mechanism, it achieves precise spatiotemporal delivery of therapeutic drugs, significantly improving treatment efficiency and reducing systemic toxic side effects; Second, the microsphere drug delivery system can effectively protect the biological / drug active ingredients, maintain the local effective drug concentration through sustained-release characteristics, and ensure the long-term effectiveness and stability of the treatment; Third, the excellent biocompatibility and injectability of the hydrogel microsphere system enable it to precisely reach the lesion site through minimally invasive intervention, thus minimizing tissue damage.

[0043] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0044] Figure 1 The image shows the appearance of GEL@KPS+RHE and its appearance under an inverted microscope, as provided in Embodiment 1 of this invention.

[0045] Figure 2 This is a topographic image of GEL@KPS+RHE electron microscope provided in Embodiment 1 of the present invention.

[0046] Figure 3 This is an appearance morphology diagram of R-GEL@KPS+RHE provided in Embodiment 1 of the present invention.

[0047] Figure 4 This is a graph showing the angular frequency rheological results of R-GEL@KPS-RHE under different proportions of GEL@KPS+RHE microspheres provided in Example 1 of this invention.

[0048] Figure 5 This is a graph showing the R-GEL@KPS-RHE shear strain results under different proportions of GEL@KPS+RHE microspheres provided in Example 1 of this invention.

[0049] Figure 6 This is a graph showing the self-repair performance of R-GEL@KPS-RHE under different proportions of GEL@KPS+RHE microspheres provided in Example 2 of this invention.

[0050] Figure 7 This is a graph showing the results of testing the injectability and self-healing properties of R-GEL@KPS-RHE provided in Example 2 of this invention.

[0051] Figure 8 This is a graph showing the in vitro release results of the GEL@KPS+RHE microspheres provided in Example 3 of this invention.

[0052] Figure 9 This is a graph showing the in vitro release results of the R-GEL@KPS+RHE hydrogel microsphere system provided in Example 3 of this invention.

[0053] Figure 10 This is a comparison chart of the in vitro release rates of GEL@KPS+RHE microspheres and R-GEL@KPS+RHE hydrogel microsphere system provided in Example 3 of this invention.

[0054] Figure 11 This is a graph showing the in vitro degradation of the R-GEL@KPS+RHE hydrogel microsphere system provided in Example 4 of this invention at different temperatures.

[0055] Figure 12 This is a graph showing the angular frequency results of R-GEL@KPS co-cultured with different cells, as provided in Example 5 of this invention.

[0056] Figure 13 This is a graph showing the shear strain results of R-GEL@KPS co-cultured with different cells, provided in Example 5 of this invention.

[0057] Figure 14 This invention detects the expression of MMP-2 protein in NPCs cells under different substance interventions, as provided in Example 5 of this invention.

[0058] Figure 15 This invention detects the in-situ degradation of the non-responsive gel group (NRG) and the responsive hydrogel (RG) provided in Example 5 of this invention.

[0059] Figure 16 This is a statistical chart showing the effects of different substances on different inflammatory factors, as provided in Example 6 of this invention.

[0060] Figure 17 This is a Western Blot result of the anti-inflammatory and repair-promoting indicators of different substances provided in Example 6 of this invention. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0062] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0063] Example 1

[0064] A responsive self-assembling peptide hydrogel microsphere system includes GelMA, peptide I (referred to as KPS), rhein (RHE), and peptide hydrogel. Peptide I is grafted onto GelMA, and RHE is loaded onto GelMA to form GEL@KPS+RHE microspheres. The peptide hydrogel encapsulates the GEL@KPS+RHE microspheres to form a responsive self-assembling peptide hydrogel microsphere system (referred to as R-GEL@KPS+RHE).

[0065] Wherein, GelMA is methacrylamide gelatin, and the amino acid sequence of polypeptide I is shown in SEQ ID NO.1, and its structural formula is shown below:

[0066] ;

[0067] The polypeptide hydrogel is self-assembled from polypeptide II and polypeptide III, and the amino acid sequence of polypeptide II is shown in SEQ ID NO.2;

[0068] The amino acid sequence of polypeptide III is shown in SEQ ID NO.3, and its structural formula is shown below:

[0069] .

[0070] I. The preparation of the above-mentioned responsive self-assembled polypeptide hydrogel microsphere system is as follows:

[0071] (a) Preparation of GeMA, the process is as follows:

[0072] Take 90 mL of pure water, add 5 PBS tablets, and stir to dissolve at 45 °C. Then add 10 g of gelatin and stir to dissolve to obtain a gelatin solution. Then add a 0.13 g / mL NaOH aqueous solution to control the pH at around 8.0. Then add 4 mL of methacrylic anhydride at a dropping rate of 100 μL / min. Use a 0.13 g / mL NaOH aqueous solution to adjust the pH of the reaction system to maintain it between 7.5 and 8.5. After stirring magnetically at 40 °C for 24 h, the solution is dialyzed, purified, and lyophilized to obtain GelMA solid powder.

[0073] (II) Preparation of GEL@KPS+RHE microspheres.

[0074] This application designs and utilizes a peptide (KPSSAPTQLN, KPS), a BMP-7 derivative peptide with the chemical formula Lys-Pro-Ser-Ser-Ala-Pro-Thr-Gln-Leu-Asn, which plays an important role in the proliferation and repair of NPCs. The KPS peptide is modified by adding -Mal to the C-terminus of KPS to form Mal-KPSSAPTQLN. The modified peptide reacts with -NH2 on the gelatin structure to form a long-chain structure, thereby improving the stability of the gelatin.

[0075] The preparation process of GEL@KPS+RHE is as follows:

[0076] Preparation of KPS solution: Take 10 mg of KPS powder and dissolve it in 1 mL of PBS buffer solution to obtain a KPS solution with a concentration of 10 mg / mL;

[0077] Take 10 μL of the KPS buffer solution prepared above and add it to 2 mL of RHE solution with a concentration of 100 μg / mL (PBS as solvent) to obtain RHE-KPS mixture. Then, measure 100 mg of the GeMA solid powder prepared above and add it to RHE-KPS mixture, and add 80 μL of polyethylene glycol diacrylate (PEGDA). After shaking to dissolve, GEL@RHE-KPS solution is obtained.

[0078] Take 5 mg of photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LPP) and add it to GEL@RHE-KPS solution. Shake in the dark to dissolve it and obtain the microsphere reaction system.

[0079] Start the microfluidic device and connect the microsphere reaction system and electronic fluorination liquid HFE7500 (trade name Novec 7500) to the aqueous solution port and oil solution port of the microfluidic chip, respectively. Adjust the pressure of the gas valve to control the fluid speed of the aqueous solution channel and the oil solution channel so that the extruded microspheres are of uniform size. At the same time, irradiate with ultraviolet light to achieve photocrosslinking.

[0080] Finally, the obtained microspheres were collected in centrifuge tubes, centrifuged, the supernatant was discarded, and washing solution was added to reconstitute the microspheres. After repeating the operation 4 times, GEL@KPS+RHE microspheres were obtained.

[0081] After centrifugation and purification of GEL@KPS+RHE, it was dispersed in a small amount of pure water. Visually, the GEL@KPS+RHE microspheres exhibited a fluid-like appearance. Figure 1 As shown in A and B; under an inverted microscope, they appear as uniform spheres, as... Figure 1 As shown in C; SEM observation shows that the microspheres are uniformly dispersed and of uniform size, with no broken or porous structures on the surface of the microspheres. Figure 2 As shown.

[0082] Images of GEL@KPS-RHE microspheres obtained using ImageJ software were obtained and processed. The diameter of the microspheres was measured, with an average diameter (D) of 184.08 ± 11.30 μm, and the minimum particle size D... min =159.75μm, maximum diameter D max =204.58μm.

[0083] (III) Preparation of R-GEL@KPS+RHE, the process is as follows:

[0084] The polypeptide RADA16, with the amino acid sequence RADARADARADARADA from C-terminus to N-terminus, is a structurally stable, biocompatible, and non-cytotoxic short-chain polypeptide. This invention introduces cysteine ​​residues (C) at the C-terminus and N-terminus of this sequence to obtain polypeptide II, with the amino acid sequence CRADARADARADARADAC from C-terminus to N-terminus. The responsive polypeptide sequence designed in this invention is GPLGLAG. The release of MMP-2 can lead to allosteric hydrolysis of GPLGLAG, thereby weakening the stress effect of GPLGLAG and exerting a responsive effect. Introducing maleimide groups (-Mal) at both ends of GPLGLAG facilitates the reaction with the -SH groups at both ends of polypeptide II, and hydrophilic modification is performed through hydrophilic oxyethylene (O-CH2-CH2-) to finally obtain polypeptide III (Mal-PEG4-GPLGLAG-Lys-Mal), the structure of which is shown below:

[0085] .

[0086] After mixing peptide II and peptide III in a 1:1 molar ratio, they were dissolved in 400 μL of pure water. The concentration of peptide II was 20 mg / ml. After thorough shaking, peptide II and peptide III were dissolved to obtain a mixed solution of peptides with alternating polymer chains in units of CRADARADARADARADAC-Mal-PEG4-GPLGLAGK-Mal.

[0087] Then, the GEL@KPS+RHE microspheres prepared above were added to the above peptide mixture, and saturated saline (5 μL) was added. The reaction system was gently shaken to allow the solution to gradually gel, thus obtaining the responsive self-assembled peptide hydrogel microsphere system (R-GEL@KPS+RHE).

[0088] Under transmitted light conditions, R-GEL@KPS+RHE was observed to contain microspheres encapsulated within a transparent gel, such as... Figure 3 As shown.

[0089] Test Example 1: Determination of rheological properties.

[0090] To determine the effect of GEL@KPS+RHE microsphere content on the rheological properties of responsive gels, the GEL@KPS+RHE microsphere content was set into four groups according to the mass percentage of GEL, with a total gel volume of 300 μL for each group. The GEL@KPS+RHE microsphere contents were 0 (RADA16 group), 10 wt% (10% GEL group), 20 wt% (20% GEL group), and 30 wt% (30% GEL group).

[0091] I. Determination of angular frequency rheology.

[0092] Among the angular frequency rheological data of the 0 (RADA group), 10% GEL group, 20% GEL group, and 30% GEL group, at a constant temperature and shear strain, as the angular frequency ω varies within the range of 0.1 - 100 rad / s, the storage modulus (G’) of each group is greater than its loss modulus (G’’), that is, G’>G’’. The results are as Figure 4 shown. This result indicates that the change in angular frequency cannot change the gel properties of R - GEL@KPS+RHE, and it has stability. The storage modulus G’ of the 20% GEL group is higher than that of the 0, 10% GEL group, and 30% GEL group, that is, the 20% GEL group has stronger stability.

[0093] II. Determination of shear strain performance.

[0094] As Figure 5 shown, the change in the content of GEL@KPS+RHE microspheres in each group does not affect the existence of the gel - sol transition point. The 0, 10% GEL group, 20% GEL group, and 30% GEL group can all complete the property change from gel to solution under shear. By quantitatively analyzing the intersection values of G’ and G’’, it is found that the shear forces required for the gel - sol transition points of the 10% and 30% groups are lower than that of the 0 group, while the shear force required for the gel - sol transition point of the 20% GEL group is higher than that of the 0 group. That is, all 4 hydrogels have injectability, and the shear force required for injection of the 20% GEL group is greater than the other 3 groups.

[0095] Test Example 2

[0096] I. Determination of self - repair performance.

[0097] As Figure 6 shown, all 4 hydrogels (0, 10% GEL group, 20% GEL group, 30% GEL group) can show alternating changes of G’<G’’ and G’>G’’ with the change of shear force, that is, they show gel - solution - gel changes with the change of shear force magnitude. High shear force can change the hydrogel from the gel state to the solution state, and as the shear force decreases, the gel in the aqueous solution state can recover to the stable gel state again. This result indicates the influence of the change in the content of GEL@KPS+RHE microspheres on the self - repair property of the hydrogel.

[0098] II. Injectability and self - repair appearance characterization.

[0099] The injection resistance of the R - GEL@KPS+RHE composite hydrogel in the syringe is small, and it still exists in the gel appearance after injection. The results are consistent with the rheological results, as Figure 7 shown.

[0100] Test Example 3 Determination of in vitro drug release.

[0101] I. In vitro release of GEL@KPS+RHE microspheres.

[0102] like Figure 8 As shown, the calculated drug coating efficiency of GEL@KPS+RHE microspheres is approximately 67%; the encapsulated drug can be slowly and continuously released from 1 to 7 days; the total release rate of GEL@KPS+RHE microspheres after 7 days is 63.43%.

[0103] II. Drug release in vitro from the R-GEL@KPS+RHE hydrogel microsphere system.

[0104] like Figure 9 and Figure 10 As shown, the R-GEL@KPS+RHE hydrogel microsphere system can slowly and continuously release drugs. Compared with the drug release of GEL@KPS+RHE, the total effective drug release time of R-GEL@KPS+RHE is extended to 9 days; the total release rate of R-GEL@KPS+RHE hydrogel microsphere system is approximately 65.38% after 9 days. The drug release rate of R-GEL@KPS+RHE hydrogel microsphere system is more gradual than that of GEL@KPS+RHE microspheres. The self-assembled peptides inhibit the early burst release of GEL@KPS+RHE microspheres, thereby prolonging the release time.

[0105] Test Example 4: Determination of the degradation performance of the R-GEL@KPS+RHE hydrogel microsphere system.

[0106] like Figure 11 As shown, the R-GEL@KPS+RHE hydrogel microsphere system exhibited swelling during the 0-10 day period, indicating its water absorption capacity in solution. Slow degradation of the hydrogel was detected at 20 days. Under two different temperature conditions, the hydrogel degradation rate showed no significant difference; over a 90-day time span, the hydrogel degradation was approximately 14%.

[0107] In the figure, RT refers to room temperature, which is 10 to 30°C.

[0108] Response analysis of the R-GEL@KPS+RHE hydrogel microsphere system in Example 5.

[0109] I. Response analysis at the level of material rheology.

[0110] Lipopolysaccharide (LPS) was used to induce inflammatory responses in NPCs (non-inflammatory cells). A normal culture group and a blank control group (culture medium) were set up. The three cell types were co-cultured with R-GEL@KPS (without RHE). After 48 hours of culture, the hydrogel microspheres were removed, and the angular frequency and shear strain of the material were measured. The results are as follows: Figure 12and Figure 13 As shown, the mean value of |G'-G''| in the LPS-induced group was significantly lower than that in the other two groups (p<0.05), indicating that R-GEL@KPS had a weaker ability to maintain the gel state than the other two groups. When the shear strain λ value varied from 0.1% to 100%, the gel-sol transition point of the LPS-induced gel material was significantly earlier than that of the other two groups, indicating that its shear strain resistance was weaker than that of the other two groups. The above results indicate that the responsive gel material has a weaker ability to maintain the gel state and a weaker shear strain resistance under inflammatory conditions. This indirectly reflects that the hydrogel microsphere system can be recognized by MMP-2 and cleave the responsive sites in the hydrogel, thus triggering the depolymerization of the hydrogel network structure.

[0111] The angular frequency detection results of R-GEL@KPS co-cultured with different cells are shown in Table 1; the shear strain detection results of R-GEL@KPS co-cultured with different cells are shown in Table 2.

[0112] Table 1. Angular frequency results of R-GEL@KPS co-cultured with different cell types.

[0113]

[0114] Table 2 Shear strain results of R-GEL@KPS co-cultured with different cell types

[0115]

[0116] II. In vitro cellular level response analysis

[0117] The expression of MMP-2 in NPCs under six different co-culture conditions (Control, LPS, R-GEL, R-GEL@RHE, R-GEL@KPS, and R-GEL@RHE+KPS) was detected by Western blotting. The effects of different components on MMP-2 were also analyzed. Figure 14 As shown, the results indicate that the expression level of MMP-2 in the LPS group was higher than that in the Control group, suggesting that MMP-2 expression is increased during the cellular inflammation induction process. The expression level of MMP-2 in the R-GEL combined with LPS group was lower, indicating that MMP-2 protein was lost after the addition of the responsive composite hydrogel material. Compared with the R-GEL group, the R-GEL@RHE, R-GEL@KPS, and R-GEL@RHE+KPS groups showed differences in MMP-2 protein expression, which may be related to the biological effects of RHE and KPS.

[0118] III. Response analysis at the in vivo degradation level.

[0119] The responsive fragment GPLGLAG was modified and linked to a glutamate peptide to create a non-responsive gel group (NRG), which was then compared with a responsive hydrogel (RG). Both groups of gels (NRG and RG) were implanted into the orthotopic intervertebral discs of rats. Intervertebral disc fluorescence imaging was performed immediately after hydrogel injection, and at four time points: day 5, 10, and 15. The fluorescence intensity of the hydrogels was analyzed, and the in vivo in-situ degradation of the comparative gels was calculated. The results are as follows: Figure 15 As shown, the results indicate that, except for the immediate time point after injection, the signal in the responsive group was weaker than that in the non-responsive group, suggesting that the in-situ degradation rate of R-GEL@KPS-RHE hydrogel is faster than that of the non-responsive hydrogel, indirectly proving the responsive performance of the hydrogel to MMP-2 in the intervertebral disc in vivo.

[0120] Test Example 6: Detection of the therapeutic effect of the R-GEL@KPS+RHE hydrogel microsphere system.

[0121] I. Mechanism of R-GEL@KPS+RHE hydrogel microsphere system in delaying intervertebral disc degeneration.

[0122] The GPLGLAG sequence in the R-GEL@KPS+RHE hydrogel microsphere system responds to the degradation of MMP-2, C-RADA16-C-Mal-PEG4-GPLGLAG-Lys-Mal. The weakening of the self-assembled peptide strength accelerates its degradation, leading to the accelerated release of GEL@KPS-RHE microspheres. Simultaneously, the degradation of GEL@KPS+RHE microspheres and the release of RHE loaded on the microspheres exert an anti-inflammatory effect, while the KPSSAPTQLN grafted onto the microspheres promotes repair, thereby mitigating the intervertebral disc degeneration process.

[0123] II. Anti-inflammatory and repair-promoting therapeutic effects of the R-GEL@KPS+RHE hydrogel microsphere system.

[0124] The expression levels of IL-1β, TNF-α, and IL-6 in NP cells from six groups (Control, LPS, R-GEL, R-GEL@RHE, R-GEL@KPS, and R-GEL@RHE+KPS) were detected using ELISA. This was used to examine the different biological effects of different components in R-GEL@KPS-RHE on LPS-mediated NP cell inflammation. The results are as follows: Figure 16As shown, the results indicate that the expression levels of IL-1β, TNF-α, and IL-6 in the LPS group were higher than those in the Control group (p<0.01), suggesting that the cellular inflammation model was successfully constructed. The expression levels of IL-1β, TNF-α, and IL-6 in the R-GEL group, R-GEL@KPS group, and LPS model group were not significantly different (p>0.05), suggesting that the R-GEL group and R-GEL@KPS group may not have an inhibitory effect on inflammatory factors in cellular effects. Compared with the R-GEL group and R-GEL@KPS group, the expression levels of IL-1β, TNF-α, and IL-6 in the R-GEL@RHE group, R-GEL@RHE+KPS group, and LPS model group were all decreased (p<0.05), suggesting that they can inhibit the inflammatory response.

[0125] The inflammatory markers MMP-3 and MMP-13, the pyroptosis markers C-caspase-1 and NLRP3, ECM degradation, ADAMTS5, Aggrecan, and Collagen II were detected using Western blotting. The results are as follows: Figure 17 As shown, the results indicate that the anti-inflammatory effect of RHE is consistent with the ELISA results, namely, RHE inhibits MMP-3 and MMP-13. Regarding apoptosis markers, the expression levels of C-caspase-1 and NLRP3 proteins in the LPS group were higher than those in the Control group, suggesting significant cell apoptosis in the LPS group. The R-GEL group, R-GEL@RHE, R-GEL@KPS group, and R-GEL@RHE+KPS group all inhibited C-caspase-1 and NLRP3 compared to the LPS group, with the R-GEL@RHE+KPS group showing a more significant inhibitory effect on C-caspase-1 and NLRP3. Among ECM metabolic markers, Aggrecan and CollagenII were significantly more expressed in the R-GEL@RHE+KPS group than in the R-GEL@RHE and R-GEL@KPS groups.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A responsive self-assembling peptide hydrogel microsphere system, characterized in that, The system includes GelMA, peptide I, anti-inflammatory active ingredient, and peptide hydrogel. Peptide I is grafted onto GelMA, and the anti-inflammatory active ingredient is loaded onto GelMA to form GEL@peptide I + anti-inflammatory active ingredient microspheres. The peptide hydrogel encapsulates the GEL@peptide I + anti-inflammatory active ingredient microspheres to form a responsive self-assembled peptide hydrogel microsphere system. Wherein, GelMA is methacrylamide gelatin, and the amino acid sequence of polypeptide I is shown in SEQ ID NO.1, and its structural formula is shown below: ； The polypeptide hydrogel is self-assembled from polypeptide II and polypeptide III, and the amino acid sequence of polypeptide II is shown in SEQ ID NO. 2; The amino acid sequence of polypeptide III is shown in SEQ ID NO.3, and its structural formula is shown below: n is selected from an integer between 4 and 6; The anti-inflammatory active ingredient is selected from rhein.

2. The responsive self-assembling polypeptide hydrogel microsphere system as described in claim 1, characterized in that, The mass ratio of the polypeptide I, the anti-inflammatory active ingredient, and GelMA added is 1:1:1000.

3. The responsive self-assembling polypeptide hydrogel microsphere system as described in claim 1, characterized in that, The particle size of GEL@peptide I+ anti-inflammatory active ingredient microspheres is 160-205 μm.

4. The responsive self-assembling polypeptide hydrogel microsphere system as described in claim 1, characterized in that, The molar ratio of polypeptide II to polypeptide III is 1:

1.

5. The responsive self-assembling polypeptide hydrogel microsphere system as described in claim 1, characterized in that, The mass-volume percentage of GelMA in GEL@peptide I+ anti-inflammatory active ingredient microspheres is 10-30%.

6. A method for preparing a responsive self-assembled polypeptide hydrogel microsphere system, characterized in that, The method for preparing the responsive self-assembled polypeptide hydrogel microsphere system as described in any one of claims 1 to 5 comprises the following steps: S100: Using GelMA, polypeptide I, and anti-inflammatory active ingredients as raw materials, microfluidic-photocrosslinking technology was used to prepare GEL@polypeptide I + anti-inflammatory active ingredient microspheres. S200. Add polypeptide II and polypeptide III to water and shake until polypeptide II and polypeptide III dissolve to obtain a polypeptide mixture. S300. Add the GEL@peptide I + anti-inflammatory active ingredient microspheres to the peptide mixture, and add saturated sodium chloride aqueous solution. Shake until the solution forms a gel to obtain a responsive self-assembled peptide hydrogel microsphere system.

7. The method for preparing the responsive self-assembled polypeptide hydrogel microsphere system as described in claim 6, characterized in that, In step S100, the photoinitiator used in the microfluidic photocrosslinking technology is selected from acylphosphonate derivatives, and the dispersion medium is selected from fluorinated liquid.

8. Application of a responsive self-assembling peptide hydrogel microsphere system, characterized in that, The responsive self-assembling polypeptide hydrogel microsphere system according to any one of claims 1 to 5, wherein the application includes its use in the preparation of anti-inflammatory drugs.

9. The application of the responsive self-assembling polypeptide hydrogel microsphere system as described in claim 8, characterized in that, The dosage form of the drug includes injections.

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