A composition for improving local tissue microenvironment, and a preparation method and application thereof
By combining polymers and peptides with quaternized benzaldehyde groups and ferrocene groups, a polymer hydrogel was constructed, which solved the problems of insufficient antioxidant capacity and poor neutrophil regulation in existing medical materials, and achieved the improvement of tissue microenvironment and chronic wound repair.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- PEKING UNIV SCHOOL OF STOMATOLOGY
- Filing Date
- 2026-03-12
- Publication Date
- 2026-06-16
Smart Images
Figure CN122212957A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of medical biomaterials, and in particular to a composition for improving the local tissue microenvironment, its preparation method, and its application. Background Technology
[0002] Improving the local tissue microenvironment is crucial for tissue defect repair. In some cases, due to microcirculatory disturbances, oxidative stress, and immune response imbalances at the affected site, patients often develop chronic skin lesions that are difficult to heal, severely impacting patient prognosis and imposing a heavy social and economic burden. Neutrophil overactivation is a key issue in this process: pathogenic factors induce elevated levels of reactive oxygen species, TNF-α, and IL-8, prompting neutrophils to release excessive extracellular neutrophil traps (NETs). Excessive NETs further damage the extracellular matrix, inhibit cell proliferation, and hinder tissue defect repair.
[0003] Current reports typically focus on improving the antibacterial and mechanical properties of medical materials, lacking effective antioxidant and neutrophil-targeting immunomodulatory mechanisms, making it difficult to achieve precise repair of the immune microenvironment and sustained therapeutic effects. Therefore, there is an urgent need to develop novel medical materials that possess self-healing properties, enhanced antioxidant capacity, and the ability to load and sustain-release functional peptides, in order to achieve dual regulation of inflammatory responses and oxidative stress, thereby promoting tissue defect repair. Summary of the Invention
[0004] The purpose of this invention is to provide a composition for improving the local tissue microenvironment, its preparation method and application, so as to at least solve the technical problems of limited antioxidant capacity and poor ability to regulate neutrophils in existing medical materials.
[0005] A first aspect of the present invention provides a monomer for preparing polymers that improve the local tissue microenvironment, said monomer containing a quaternized benzaldehyde group having the structure shown in the following formula: , in, m is 1-20, preferably an integer from 1 to 5; X is a halogen, preferably one of F, Cl, Br, and I, more preferably Cl or Br; R1 and R2 may be the same or different, and each is independently one of C1-C10 alkyl groups, preferably one of C3 alkyl groups; R3 is a hydrogen methyl group.
[0006] In some embodiments, the monomer according to the present invention has the following structure: .
[0007] A second aspect of the present invention provides a composition for improving the local tissue microenvironment, comprising a structural unit containing a quaternized benzaldehyde group, a structural unit containing a ferrocene group, a polymer with a polymethacrylate backbone, and a polymeric material loaded with said polymer.
[0008] In some embodiments, the composition for improving the local tissue microenvironment according to the present invention, wherein the polymer has a structure shown in formula (I): Formula I, in, x is between 1 and 100, preferably an integer between 30 and 90; y is 1-100, preferably an integer between 30-90; z is between 1 and 100, preferably an integer between 30 and 90; n is between 1 and 50, preferably an integer between 5 and 15; m is 1-50, preferably an integer from 1-20; X is a halogen, R1 and R2 can be the same or different, and are independently C1-C10 alkyl groups, and R3 is hydrogen or methyl.
[0009] In some embodiments, the composition for improving the local tissue microenvironment according to the present invention further comprises a polypeptide having a fibronectin type III domain or an active fragment thereof.
[0010] In some embodiments, the composition for improving the local tissue microenvironment according to the present invention, wherein the polypeptide or its active fragment is loaded onto the polymer via a Schiff base bond.
[0011] In some embodiments, the composition for improving the local tissue microenvironment according to the present invention includes a polymer material comprising a natural polymer material or a synthetic polymer material.
[0012] In some embodiments, the composition for improving the local tissue microenvironment according to the present invention includes at least one of chitosan, dextran, sodium alginate, hyaluronic acid, gelatin, or derivatives thereof.
[0013] A third aspect of the present invention provides a method for preparing the composition described in the second aspect, comprising: (1) Prepare polymers containing structural units with quaternized benzaldehyde groups, structural units with ferrocene groups, and polymethyl methacrylate skeletons; (2) Crosslink the polymer with a polymeric material to obtain a polymeric material loaded with the polymer; In some embodiments, according to the preparation method of the present invention, the step prior to crosslinking includes reacting the polypeptide or its active fragment with the polymer to obtain a polymer loaded with the polypeptide or its active fragment.
[0014] In some embodiments, according to the preparation method of the present invention, the preparation steps of the polymer include: (a) A quaternization reaction is carried out on a raw material including a tertiary amine monomer, a halogenated benzaldehyde and a first solvent to obtain a monomer containing a quaternized benzaldehyde group; (b) The monomer containing the quaternized benzaldehyde group, the ferrocene-based polymer monomer and the methacrylate monomer are mixed to obtain a monomer mixture, and an initiator is added to the monomer mixture to carry out the reaction to obtain the polymer; Preferably, the tertiary amine monomer is R1 and R2 can be the same or different, and are independently C1-C10 alkyl groups, preferably C1-C3 alkyl groups, and R3 is hydrogen or methyl. Preferably, the halogenated benzaldehyde is Where m is 1-20, preferably an integer of 1-5, and X is a halogen, preferably F, Cl, Br or I, more preferably Cl or Br; Preferably, the first organic solvent is selected from at least one of N,N-dimethylformamide, acetic acid, acetonitrile, dimethyl sulfoxide, and N,N-dimethylacetamide; Preferably, the initiator is an oily initiator selected from at least one of azobisisobutyronitrile, azobisisobutyronitrile, and benzoyl peroxide.
[0015] A fourth aspect of the invention provides the use of the monomer described in the first aspect or the composition described in the second aspect in the preparation of medical materials for improving the local tissue microenvironment.
[0016] This invention creatively combines natural peptides with polymers to obtain a composition (in an exemplary embodiment, this composition is also named PFB-Irisin, where PFB represents a polymer and Irisin represents an irisin peptide) that not only has enhanced antioxidant capacity but also possesses immunomodulatory and antibacterial properties, thereby effectively promoting tissue repair by improving the local tissue microenvironment. Furthermore, the preparation method provided by this invention is simple, mild, and suitable for mature industrial production. Attached Figure Description
[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0018] Figure 1 (a) is a synthetic route diagram of the antioxidant polymer PFB. Figure 1 (b) is the antioxidant polymer PFB. 1 H-NMR characterization image.
[0019] Figure 2 This is a schematic diagram of the gelation process of PFB-Irisin@Gel hydrogel.
[0020] Figure 3 (a) is a SEM image of the hydrogel (6wt%) provided by the present invention. Figure 3 (b) is a SEM image of the hydrogel (8wt%) provided by the present invention. Figure 3 (c) is a SEM image of the hydrogel (10wt%) provided by the present invention.
[0021] Figure 4 This is a schematic diagram of the self-healing process of hydrogels.
[0022] Figure 5 Hydrogels loaded with irisin peptides for ABTS + • Schematic diagram of clearance ability test, from left to right: blank control, PFB@Gel and PFB-Irisin@Gel.
[0023] Figure 6 The graph shows the relationship between the modulus and strain of an 8wt% solids hydrogel at 25℃.
[0024] Figure 7 The graph shows the modulus of an 8wt% hydrogel under alternating strain (300% and 1%) at 25℃ as a function of time.
[0025] Figure 8 This is a schematic diagram illustrating the self-adaptation and self-healing properties of hydrogels.
[0026] Figure 9 A diagram illustrating the hemolytic safety evaluation of hydrogels loaded with irisin.
[0027] Figure 10 (a) DNA quantification results of NETs content after co-culturing irisin-containing hydrogel with activated human neutrophils. Figure 10(b) Sytox immunofluorescence results of NET content after co-culturing irisin-containing hydrogel with activated human neutrophils. Figure 10 (c) Western blot results of NETs-related proteins after co-culturing irisin-containing hydrogels with activated human neutrophils.
[0028] Figure 11 A schematic diagram and quantitative graph of the healing process of a hydrogel loaded with irisin as a wound dressing in a representative wound of diabetic mice are shown.
[0029] Figure 12 The antibacterial properties of the hydrogel loaded with irisin are shown in Figure a. Figure a shows the number of colonies on the agar plate, Figure b shows the results of crystal violet staining analysis, and Figure c shows the results of scanning electron microscopy (SEM). Detailed Implementation
[0030] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementations of the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.
[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, the numerical ranges in this invention should be understood to specifically disclose the upper and lower limits of the range and every intermediate value between them. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0033] Composition In one aspect, the present invention provides a composition for improving the local tissue microenvironment for the repair of tissue defects, particularly for the repair of skin defects, wherein in one specific embodiment, the skin defect includes a chronic wound.
[0034] In one preferred embodiment, the composition of the present invention comprises a structural unit containing a quaternized benzaldehyde group, a structural unit containing a ferrocene group and a polymethacrylate backbone, and a polymeric material supporting said polymer. In another preferred embodiment, the composition of the present invention comprises a structural unit containing a quaternized benzaldehyde group, a structural unit containing a ferrocene group and a polymethacrylate backbone, a polypeptide or an active fragment thereof linked to said polymer, and a polymeric material supporting said polymer.
[0035] In this invention, the polymer material is not particularly limited; any polymer material suitable for gelation can be used. The polymer material can be a natural polymer or a synthetic polymer. As a non-limiting example, the polymer material can be selected from at least one of chitosan, dextran, sodium alginate, hyaluronic acid, gelatin, or derivatives thereof. In a preferred embodiment, the polymer material is chitosan or a derivative thereof. In a more preferred embodiment, the polymer material is a chitosan derivative, such as hydroxypropyl chitosan.
[0036] In this invention, the polypeptide or its active fragment contains at least a fibronectin type III domain to provide immunomodulatory capabilities. In a preferred embodiment, the polypeptide or its active fragment is an irisin polypeptide, the specific sequence of which is not particularly limited, and any irisin polypeptide or variant thereof known in the art may be used.
[0037] In this invention, the irisin polypeptide is loaded onto the polymer via dynamic Schiff base bonds, while the polymer provides antioxidant properties through its own active groups.
[0038] In a preferred embodiment, the composition of the present invention comprises 1-50 parts by weight (preferably 1-40, more preferably 5-30, even more preferably 5-20, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 parts by weight) of a polymer and 1-50 parts by weight (preferably 1-40, more preferably 1-30, even more preferably 1-20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 parts by weight) of a polymeric material.
[0039] In a preferred embodiment, the composition of the present invention comprises 1-50 parts by weight (preferably 1-40, more preferably 5-30, even more preferably 5-20, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 parts by weight) of a polymer, 0.1-10 parts by weight (preferably 0.1-5, even more preferably 0.1-2.5 parts by weight) of a polypeptide, and 1-50 parts by weight (preferably 1-40, more preferably 1-30, even more preferably 1-20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 parts by weight) of a polymeric material.
[0040] In the composition of this invention, a polypeptide or its active fragment is grafted onto a polymer, and a hydrogel with excellent self-healing ability is constructed based on the dynamic Schiff bond reaction between the benzaldehyde group and the amino group of water-soluble hydroxypropyl chitosan. This ensures that the gel material can firmly cover and protect the tissue defect site; and the dynamic bond between the aldehyde group and the amino group contained in irisin allows for dynamic sustained release of the drug. Furthermore, the polymer contains ferrocene groups, which can exert a highly efficient antioxidant effect, scavenging excess ROS free radicals at the tissue defect site and slowly decomposing upon ROS stimulation, achieving responsive release. The cationic structure of the hydrogel network further enhances the removal of nucleic acid structures in NETs, thereby regulating NET formation and promoting tissue defect repair. In addition, the composition of this invention has excellent antibacterial properties, further improving the microenvironment of the tissue defect site.
[0041] Preparation method In one aspect, the method for preparing the above-described composition also provides a method for preparing the above-described hydrogel dressing, comprising: (1) Prepare polymers containing structural units with quaternized benzaldehyde groups, structural units with ferrocene groups, and polymethyl methacrylate skeletons; (2) Crosslink the polymer with a polymeric material to obtain a polymeric material loaded with the polymer.
[0042] In a preferred embodiment, the preparation method of the present invention includes: Step 1: Weigh out polymer powder (sometimes referred to as PFB in this article), dissolve it in a buffer solution at room temperature, and stir until the polymer powder is completely dissolved to obtain a polymer PFB solution with a concentration of 1-20 wt%, preferably 1-15 wt%, and even more preferably 5-15 wt%, for example 5-10 wt%, 6-10 wt%, 6-8 wt%, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 wt%. Step 2: Take hydroxypropyl chitosan powder, place it in a buffer solution, and stir until completely dissolved to obtain a hydroxypropyl chitosan solution with a concentration of 0.1-20 wt%, preferably 0.1-10 wt%, and even more preferably 0.1-5 wt%, such as 0.5-5 wt%, 0.5-2 wt%, 1-5 wt%, or 1, 2, 3, 4, 5 wt%. Step 3: Take PFB solution and hydroxypropyl chitosan solution, shake and mix for 60-120 s, let stand at room temperature for 1-30 minutes to obtain PFB@Gel.
[0043] In this invention, the buffer solution is not particularly limited, and includes, but is not limited to, PBS, HEPES, Tris (or Tris-HCl), MOPS, MES, etc.
[0044] When irisin polypeptide is added, the concentration of irisin polypeptide in PFB solution is 0.1-10 μg / mL, preferably 0.1-5 μg / mL, and even more preferably 0.1-1 μg / mL, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 μg / mL.
[0045] In another preferred embodiment, the preparation method of the present invention includes: Step 1: Weigh 8-12 parts of the antioxidant polymer PFB powder and dissolve it in 100 parts of 0.01M PBS solution at room temperature. Stir at 250-400 rpm until the antioxidant polymer powder is completely dissolved to obtain an antioxidant polymer PFB solution. Step 2: Take the irisin polypeptide and add it to the antioxidant polymer solution described in Step 1 at a ratio of 0.5-2 μg of irisin polypeptide per 1 ml of solution. Stir well to obtain irisin-antioxidant polymer (PFB@Irisin) solution. Step 3: Take 4 parts of hydroxypropyl chitosan powder and put it into 100 parts of 0.01 M PBS solution. Stir until completely dissolved to obtain a 4 wt% hydroxypropyl chitosan solution. Step 4: Take the irisin-antioxidant polymer solution from Step 2 and the hydroxypropyl chitosan solution from Step 3, shake and mix for 60-120 seconds, and let stand at room temperature for 5 minutes to obtain the antioxidant hydrogel PFB-Irisin@Gel. All components are listed in parts by mass.
[0046] Further, the irisin-antioxidant polymer solution described in step four is mixed with the hydroxypropyl chitosan solution at a volume ratio of 1:(0.5-2).
[0047] Preferably, the volume ratio of the irisin polypeptide-antioxidant polymer solution to the hydroxypropyl chitosan solution is selected from 1:0.5, 1:1, 1:1.5, 1:1.8, 1:2, and any range between any two of the above values. Preferably, the volume ratio is 1:1.
[0048] In a preferred embodiment, the preparation steps of the polymer include: (a) A quaternization reaction is carried out on a raw material including a tertiary amine monomer, a halogenated benzaldehyde and a first solvent to obtain a monomer containing a benzaldehyde group; (b) The monomer containing benzaldehyde group, the ferrocene-based polymer monomer and the methacrylate monomer are mixed to obtain a monomer mixture, and an initiator is added to the monomer mixture to carry out the reaction to obtain the polymer.
[0049] In a more preferred embodiment, the preparation step of the polymer includes: Step A: The tertiary amine monomer, halogenated benzaldehyde and the first solvent are mixed and subjected to a quaternization reaction to obtain the benzaldehyde monomer (BM); The quaternization reaction is carried out at a temperature of 10-100℃, preferably 60-75℃, and for a reaction time of 0.5-24 hours, preferably 2-3 hours. Preferably, the tertiary amine monomer is selected from at least one of dimethylaminoethyl methacrylate, dimethylaminoethyl acrylate, diethylaminoethyl methacrylate, diethylaminoethyl methacrylate, dipropylaminoethyl methacrylate, dipropylaminoethyl methacrylate, dibutylaminoethyl methacrylate, dibutylaminoethyl methacrylate, dipentanaminoethyl methacrylate, and dipentanaminoethyl methacrylate. Preferably, the halogenated benzaldehyde is specifically selected from at least one of 4-(bromomethyl)benzaldehyde, 4-(bromoethyl)benzaldehyde, 4-(bromopropyl)benzaldehyde, 4-(bromobutyl)benzaldehyde, 4-(bromopentyl)benzaldehyde, 4-(chloromethyl)benzaldehyde, 4-(chloroethyl)benzaldehyde, 4-(chloropropyl)benzaldehyde, 4-(chlorobutyl)benzaldehyde, and 4-(chloropentyl)benzaldehyde.
[0050] Preferably, the first solvent is selected from at least one of N,N-dimethylformamide, acetic acid, acetonitrile, dimethyl sulfoxide, and N,N-dimethylacetamide; Step B: 4-(methacryloyloxy)butyl-trimethylammonium bromide (BM), 2-(methacryloyloxy)ethyl 6-methyl-4-ferrocene-2-thio-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ester (FM) and polyethylene glycol methacrylate (PEGMA) are mixed in a molar ratio of 1:(1-5):(1-5) to obtain a monomer mixture. Preferably, the molar ratio of BM, FM, and PEGMA can be from 1:1:1 to 1:1:1.5.
[0051] Add 1 mol% of the total monomer amount of initiator to the monomer mixture, use N,N-dimethylformamide (DMF) as the organic solvent, and react at 70°C for 24 hours to obtain a reaction solution containing benzaldehyde and ferrocene groups; the total amount of the monomer mixture added per 1-1.5 mL of the organic solvent is 1 mmol. Preferably, the initiator is at least one of azobisisobutyronitrile (AIBN), azobisisoheptanenitrile (ABVN), dimethyl azobisisobutyrate, and benzoyl peroxide; Step C: The reaction solution obtained in step B is precipitated with diethyl ether, the precipitate is collected and dried under vacuum to obtain an antioxidant polymer powder with ferrocene and benzaldehyde groups for later use.
[0052] application One aspect of the invention provides the use of the compositions described herein in the preparation of medical materials for improving the local tissue microenvironment. The specific type of medical material is not particularly limited and can be any medical product for tissue defect repair (e.g., skin defect repair, such as chronic wounds), such as implant materials, dressings, microneedles, patches, etc. In some embodiments, the medical material is in the form of a gel, particularly a hydrogel dressing.
[0053] Methods for regulating cell function One aspect of the present invention provides a method for regulating neutrophil function, comprising the step of contacting the composition with neutrophils in vitro. The method of the present invention can be a therapeutic method, i.e., for therapeutic use. In another embodiment, the method is a non-therapeutic method, for example, for commercial experimental purposes.
[0054] In this invention, the regulation includes at least one of the following: (1) Suppress NETs release; (2) Inhibit histone citrullination and / or NETs backbone formation; (3) Reduce the expression level of at least one protein selected from PAD4, H3Cit, and MPO.
[0055] In this invention, the neutrophils are PMA-induced neutrophils.
[0056] Preparation Example 1 1. Preparation of BM monomers 10.0 mmol of dimethylaminoethyl methacrylate (DMAEMA, 1.57 g) and 10.0 mmol of 4-bromomethylbenzaldehyde (4-BrBnCHO, 1.99 g) were weighed and added to a round-bottom flask containing 50 mL of tetrahydrofuran. The mixture was subjected to a quaternization reaction at 70 °C for 2 h. After the reaction, the solvent was removed by rotary evaporation to obtain the target benzaldehyde-containing monomer BM. The ¹H-NMR spectrum confirms that the target benzaldehyde-containing monomer BM was successfully prepared in this embodiment.
[0057] 2. Preparation of PFB polymer Weigh 5.0 mmol of benzaldehyde-containing monomer (BM, 1.86 g), 5.0 mmol of 2-(methacryloyloxy)ethyl 6-methyl-4-ferrocene-2-thio-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ester (FM, 2.58 g), 5.0 mmol of polyethylene glycol methacrylate (PEGMA, Mn ~ 950 g / mol, 4.75 g), and 0.15 mmol of azobisisobutyronitrile (AIBN, 0.025 g, 1 mol% of the total monomers), and add them to a round-bottom flask containing 50 mL of N,N-dimethylformamide (DMF). After sealing with a rubber diaphragm, purge with a nitrogen stream for 30 minutes, and then react in an oil bath at 70 °C for 24 hours. After the reaction is complete, precipitate the reaction solution three times in diethyl ether, and dry under vacuum to obtain the polymer PFB containing benzaldehyde and ferrocene. Figure 1 As can be seen from the ¹H-NMR spectrum in (b), the characteristic peaks confirm that the target polymer PFB was successfully prepared in this embodiment.
[0058] Preparation Example 2 The following illustrates the preparation of PFB@Gel: 1. Hydrogel preparation Add commercially available HPCS powder to 0.01 M PBS solution (pH 7.4) and stir overnight at room temperature to obtain a PBS solution with a mass fraction of 4% HPCS.
[0059] 2. Preparation of PFB@Gel An equal volume of 8 wt% PFB (PBS) and 4 wt% chitosan (HPCS) solution was mixed and allowed to stand at room temperature for 5 minutes. The mixture changed from a fluid state to a gel state.
[0060] Preparation Example 3 The following illustrates the preparation of the Irisin-loaded hydrogel PFB-Irisin@Gel: 1. Hydrogel preparation Add commercially available HPCS powder to 0.01 M PBS solution (pH 7.4) and stir overnight at room temperature to obtain a PBS solution with a mass fraction of 4% HPCS.
[0061] 2. Preparation of PFB@Gel 5 μg of irisin peptide was dissolved in an antioxidant polymer PFB solution (8 wt%, 10 mL) to obtain an irisin-loaded PFB solution, which was then mixed with the above HPCS solution in an equal volume ratio to form an irisin-loaded PFB hydrogel.
[0062] Example 1 This embodiment first verified the effect of different HPCS addition amounts on the hydrogel properties of the reaction product.
[0063] The preparation method of the hydrogel loaded with irisin peptide in this embodiment is basically the same as that in Preparation Example 3, except that three hydrogel samples, Gel-6, Gel-8, and Gel-10, were prepared using PBS solutions with different mass concentrations of PFB (6 wt%, 8 wt%, and 10 wt%). Under a scanning electron microscope, as shown... Figure 3 (a)- Figure 3 As shown in (c), it can be clearly seen that the higher the mass concentration of the HPCS PBS solution, the denser the resulting hydrogel structure and the greater the macroscopic hardness of the hydrogel. Therefore, after multiple experimental verifications, the preferred mass concentration of the PFB PBS solution is 6-10 wt%.
[0064] Example 2 This embodiment qualitatively verifies that the obtained reaction product hydrogel has good self-healing properties.
[0065] Prepare a 20 mm heart-shaped hydrogel sample using 0.5 mL of PFB@Gel. Divide the sample into four parts and then place them together. Figure 4 As shown, the four parts completely healed into a complete heart shape after 15 minutes. The results indicate that the hydrogel, after a period of self-shaping, can re-adhere well, the breakage disappears, and the complete heart shape is restored. This verifies that the hydrogel loaded with irisin peptides provided in this embodiment has excellent self-adaptation and self-repair capabilities, can adapt to wounds of different shapes and sizes, and can adapt to wound deformation without breaking or falling off in wound deformation caused by sports and other scenarios.
[0066] Example 3 This embodiment verifies the antioxidant capacity of the hydrogel loaded with irisin.
[0067] Step 1: Mix 400 μL of 4wt% HPCS solution with 172 μL of ABTS solution with an absorbance of 3.+ The solutions were mixed to obtain a 4 wt% HPCS-ABTS solution with an absorbance of 3 and a pH of 7.4. + ˙Solution.
[0068] Step 2: Further dilute the 8wt% HPCS-ABTS with 4wt% HPCS solution. + The solution was prepared until the absorbance was 1.
[0069] Step 3: Mix the PFB polymer solution and the PFB polymer solution containing irisin with HPCS-ABTS respectively. + Equal volumes of solutions were mixed to obtain a solution containing ABTS. + The hydrogels were labeled PFB@Gel (ferrocene-containing aldehyde polymer gel) and PFB-Irisin@Gel (ferrocene-containing aldehyde polymer gel loaded with irisin); PBS solution was used with HPCS-ABTS + Equal volumes of solutions were mixed to form a blank control group (Control).
[0070] Step 4: Take photos of the three sets of hydrogels in the comparative experiment during mixing and 10 minutes after mixing.
[0071] like Figure 5 As shown, it is evident that the blank control group was darker in color after 10 minutes of mixing, while the hydrogels containing the ferrocene structure were significantly lighter in color. Furthermore, the addition of irisin had no clear effect on the gel color, demonstrating that ABTS... + The effective removal of α-oxidants verifies that the polymer structure has a significant effect on improving antioxidant capacity. Therefore, the ferrocene-containing aldehyde-based polymer hydrogel provided by this invention has excellent antioxidant capacity.
[0072] Example 4 This embodiment verifies the strain resistance of the PFB hydrogel loaded with irisin.
[0073] At 25°C, different strains were applied to hydrogels with varying PFB contents, and the values of G' and G'' (frequency = 1 Hz) were recorded. Figure 6 As shown, the maximum shear strain that can be withstood at the intersection of the storage modulus and the loss modulus in different gels ranges from 200% to 398%. The maximum shear strain decreases with increasing PFB content. When the PFB content is 8 wt%, the maximum shear stress that the gel can withstand is 316%.
[0074] Example 5 This embodiment verifies the rheological properties of the PFB hydrogel loaded with irisin peptide.
[0075] like Figure 7As shown, the modulus of an 8 wt% solids hydrogel changes over time under alternating strains (300% and 1%) at 25℃. The hydrogel did not rupture at 1% strain, but completely ruptured at 300% strain. At 300% strain, after each complete rupture, the hydrogel network recovered to its original strength at 1% strain. This verifies that the hydrogel loaded with irisin peptides possesses excellent rheological properties.
[0076] Example 6 This embodiment demonstrates the adaptive and self-healing properties of hydrogel materials at different fabrication stages and in different morphologies. Specific results are described below: Initial Form Shaping: The leftmost image shows the letter "P" formed by hydrogel. This demonstrates that hydrogel can be molded into specific shapes, showcasing its good plasticity in the initial stage, allowing it to form various complex structures according to mold or design requirements.
[0077] Material Filling and Shaping: The top-middle image shows how solidified "P"-shaped hydrogel material can be crushed with a syringe and filled into a "K"-shaped mold using a tool. After demolding, it retains the shape imparted by the mold, demonstrating the hydrogel's excellent self-healing ability and reshaping properties after mechanical damage. The top-right image shows the process of crushing solidified "K"-shaped hydrogel again with a syringe and filling it into a "U"-shaped mold, while the bottom image shows the demolded hydrogel letter "U". This further verifies the hydrogel's adaptive and self-healing properties.
[0078] Overall, Figure 8 This study demonstrates the hydrogel material from its initial plastic state, as well as its excellent self-healing ability and reshaping properties after mechanical damage, highlighting its superior performance in adapting to different shape requirements. It provides intuitive experimental evidence for the wide application of this hydrogel in fields such as biomedical engineering.
[0079] Example 7 This embodiment verifies the safety evaluation of hydrogels loaded with irisin peptides.
[0080] The hemolysis safety evaluation chart clearly shows that after centrifugation, the supernatant of the red blood cell suspension from the material of this invention is clear and transparent, colorless or very pale yellow, and its appearance is basically the same as that of the negative control group (physiological saline). In contrast, the supernatant of the positive control group (distilled water / deionized water) is bright red due to severe hemolysis. This visual comparison directly demonstrates that the material of this invention can effectively maintain the integrity of the red blood cell membrane structure during contact with red blood cells, without causing cell rupture or hemoglobin leakage, proving its excellent blood compatibility. Quantitative data results show that the hemolysis rate of the material of this invention is below the safety threshold, meeting the standards for clinical application of biomaterials, further confirming that the material of this invention has no red blood cell toxicity and will not cause acute hemolytic reactions, showing broad application prospects in the field of local contact medical devices (such as dressings, implants, and drug carriers). Figure 9 ).
[0081] Example 8 This embodiment verifies that the hydrogel loaded with irisin peptides has the function of sustained release of irisin peptides in a physiological environment, and verifies its immunomodulatory effect by comparing the culture results of human neutrophils with those of irisin peptides.
[0082] Human neutrophils were resuspended at a density of 2 million cells / ml in high-glucose 1640 RPMI medium and cultured in an incubator at 37°C and 5% CO2. Neutrophils were stimulated with PMA (100 nM) as a positive inducer for 4 hours to induce NET formation. At the same time, the cells were cultured in a hydrogel loaded with irisin peptide to investigate the effect of the loaded irisin (100 ng / mL) on NET formation. PMA-only induction was used as a control group.
[0083] Giemsa staining was used to detect NET production in the two groups of neutrophils. Neutrophil smears from different groups were fixed with methanol and then co-incubated with Giemsa staining solution at pH 6.4 for 10 minutes. Microscopic observation revealed that the control group cells ruptured and released a large amount of reticular structures, while the irisin-treated cells had more intact cell outlines and significantly reduced extracellular fibrous structures. Further detection of extracellular DNA using SYTOX Green staining involved introducing SYTOX Green-labeled extracellular DNA into both groups of cells. After incubation for 15 minutes, NET formation was observed using a fluorescence microscope. Fluorescence intensity measurements showed that the fluorescence signal in the irisin group was significantly lower than that in the PMA-induced group, suggesting that irisin effectively inhibited NET release. Western blot analysis further confirmed that the loaded irisin reduced the protein expression levels of PAD4 and H3Cit. In summary, the loaded irisin significantly inhibited PMA-induced NET formation by reducing PAD4-mediated histone citrullination levels, demonstrating the potential protective effect of the composition of the present invention in regulating abnormal neutrophil activation and related inflammatory responses. Figure 10 ).
[0084] Example 9 This embodiment verifies the repair effect of PFB@Gel and hydrogel dressing loaded with Irisin peptide on diabetic wounds by continuously monitoring different dressings on the wounds of diabetic mice for 21 days.
[0085] 1. Model Building A diabetes model was established by injecting streptozotocin at a dose of 50 mg / kg / day into 6-week-old C57 / 6J mice. Successful modeling was defined as a blood glucose level above 16.7 mmol / L after 5 days. A full-thickness skin excision was performed on the back of the diabetic mice to create circular lesions with a diameter of 6 mm.
[0086] 2. Evaluation of the efficacy of PFB@Gel and PFB-Irisin@Gel in promoting tissue repair After establishing the diabetic skin lesion model, patients were randomly assigned to three groups: no treatment (Blank), application of 0.5 mL PFB-GEL (Gel group), and application of 0.5 mL PFB-Irisin@Gel. 3M dressings were used to cover the wounds, changing them every two days. Samples were taken immediately post-surgery, and on days 3, 7, 10, and 14 for multi-level evaluation. First, wound healing rate was measured using photography and digital imaging techniques, and granulation tissue formation and skin structure were assessed using HE staining. The results showed that PFB@Gel and PFB-Irisin@Gel have significant repair effects on diabetic wounds and can therefore be used for tissue repair. Figure 11 ).
[0087] Example 10 This embodiment verifies the antibacterial properties of hydrogels loaded with irisin peptides. As an example, this embodiment uses plate count, crystal violet staining, and scanning electron microscopy (SEM) to evaluate the antibacterial activity of the hydrogels. In short, 1 mL and 10 mL of different groups of gels were used... 8 1 mL of bacterial suspension was co-cultured with a concentration of 10^6 / mL. The supernatant was then collected and diluted appropriately. The diluted supernatant was spread onto solid agar plates and incubated for 24 hours. The bactericidal effect was evaluated by colony counting. The diluted supernatant was then incubated in liquid medium for 24 hours. The residual bacterial film was stained with crystal violet and photographed to evaluate its ability to inhibit biofilm formation. The microscopic damage morphology after the bacteria came into contact with the material was directly observed using scanning electron microscopy to clarify its antibacterial mechanism.
[0088] Antibacterial test results as follows Figure 12 As shown in Figure a. Compared with the control group, the number of colonies on the agar plates of the hydrogel-treated group was significantly reduced. Quantitative analysis with crystal violet staining showed ( Figure 12 (Figure b) The amount of biofilm formed in the hydrogel-treated group was significantly lower than that in the control group, indicating that the hydrogel can effectively inhibit the early adhesion and formation of bacterial biofilms. SEM results further revealed the antibacterial mechanism: the bacteria in the control group had intact morphology and smooth surfaces, exhibiting typical spherical or rod-shaped structures; while after hydrogel treatment, a large number of bacterial cell membranes shrank, ruptured, and even leaked their contents, resulting in severely damaged cell morphology. Figure 12 (See Figure c). This result demonstrates that the hydrogel of this invention exerts a powerful antibacterial effect by disrupting the bacterial cell wall / membrane structure, leading to bacterial death.
[0089] In summary, the hydrogel of the present invention not only has a highly efficient ability to kill bacteria, but also effectively inhibits the formation of biofilms, demonstrating its potential as an anti-infective wound dressing or implant material.
[0090] 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A monomer for preparing polymers that improve the microenvironment of local tissues, characterized in that, The monomer contains a quaternized benzaldehyde group and has the structure shown in the following formula: ， in, m is 1-20, preferably an integer from 1 to 5; X is a halogen, preferably one of F, Cl, Br, and I, more preferably Cl or Br; R1 and R2 may be the same or different, and each is independently one of C1-C10 alkyl groups, preferably one of C3 alkyl groups; R3 is a hydrogen methyl group.
2. A composition for improving the local tissue microenvironment, characterized in that, The invention includes structural units containing quaternized benzaldehyde groups, structural units containing ferrocene groups, and polymers with polymethyl methacrylate backbones, as well as polymeric materials supporting said polymers.
3. The composition for improving the local tissue microenvironment according to claim 2, characterized in that, The polymer has the structure shown in formula (I): Equation I, in, x is between 1 and 100, preferably an integer between 30 and 90; y is 1-100, preferably an integer between 30-90; z is between 1 and 100, preferably an integer between 30 and 90; n is between 1 and 50, preferably an integer between 5 and 15; m is 1-50, preferably an integer from 1-20; X is a halogen, R1 and R2 may be the same or different, and are independently C1-C10 alkyl groups, and R3 is hydrogen or methyl.
4. The composition for improving the local tissue microenvironment according to claim 2, characterized in that, Further includes a polypeptide or an active fragment thereof having a fibronectin type III domain; Preferably, the polypeptide or its active fragment is loaded onto the polymer via a Schiff base bond.
5. The composition for improving the local tissue microenvironment according to claim 2, characterized in that, The polymeric materials include natural polymeric materials or artificially synthesized polymeric materials; Preferably, the polymeric material includes at least one of chitosan, dextran, sodium alginate, hyaluronic acid, gelatin, or a derivative thereof.
6. The method for preparing the composition according to any one of claims 2-5, characterized in that, include: (1) Prepare polymers containing structural units with quaternized benzaldehyde groups, structural units with ferrocene groups, and polymethyl methacrylate skeletons; (2) Crosslink the polymer with a polymeric material to obtain a polymeric material loaded with the polymer.
7. The preparation method according to claim 6, characterized in that, The step prior to crosslinking includes reacting the polypeptide or its active fragment with the polymer to obtain a polymer loaded with the polypeptide or its active fragment.
8. The preparation method according to claim 6, characterized in that, The preparation steps of the polymer include: (a) A quaternization reaction is carried out on a raw material including a tertiary amine monomer, a halogenated benzaldehyde and a first solvent to obtain a monomer containing a quaternized benzaldehyde group; (b) The monomer containing the quaternized benzaldehyde group, the ferrocene-based polymer monomer and the methacrylate monomer are mixed to obtain a monomer mixture, and an initiator is added to the monomer mixture to carry out the reaction to obtain the polymer; Preferably, the tertiary amine monomer is R1 and R2 may be the same or different, and are independently C1-C10 alkyl groups, preferably C1-C3 alkyl groups, and R3 is hydrogen or methyl. Preferably, the halogenated benzaldehyde is Where m is 1-20, preferably an integer of 1-5, and X is a halogen, preferably F, Cl, Br or I, more preferably Cl or Br; Preferably, the first organic solvent is selected from at least one of N,N-dimethylformamide, acetic acid, acetonitrile, dimethyl sulfoxide, and N,N-dimethylacetamide; Preferably, the initiator is an oily initiator selected from at least one of azobisisobutyronitrile, azobisisobutyronitrile, and benzoyl peroxide.
9. The use of the monomer of claim 1, or the composition of any one of claims 2-5, in the preparation of medical materials for improving the local tissue microenvironment.
10. A method for regulating neutrophil function, characterized in that, Includes the step of contacting the composition of any one of claims 2-5 with neutrophils in vitro; Preferably, the regulation includes at least one of the following: (1) Suppress NETs release; (2) Inhibit histone citrullination and / or NETs backbone formation; (3) Reduce the expression level of at least one protein selected from PAD4, H3Cit, and MPO.