Temperature-sensitive hydrogel for resisting bacteria and promoting repair and preparation method of temperature-sensitive hydrogel

Abstract

The invention provides a temperature-sensitive hydrogel for resisting bacteria and promoting repair and a preparation method thereof, the temperature-sensitive hydrogel comprises PEG-NB, a matrix metalloproteinase degradable peptide cross-linking agent, gelatin, indocyanine green and levofloxacin, and the temperature-sensitive hydrogel is prepared by photo-crosslinking. According to the temperature-sensitive hydrogel provided by the invention, the structure and the performance of the hydrogel are optimized through the synergistic effect of the gelatin and the PEG-NB, and the mechanical strength, the biocompatibility, the degradability and the like of the hydrogel are remarkably improved; meanwhile, the photo-thermal antibacterial function of the photo-thermal agent indocyanine green and the chemical antibacterial function of levofloxacin are integrated, and near-infrared light irradiation is combined, so that the antibacterial and skin repair promoting effects of the hydrogel are synergistically improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a thermosensitive hydrogel for antibacterial and repair-promoting purposes and its preparation method. Background Technology

[0002] Skin tissue is the largest and outermost exposed organ in the human body, playing a vital role in protecting the body from external environmental damage. After injury, bacteria from the surrounding skin and environment rapidly accumulate at the wound site. When the bacterial count fails to stabilize at a certain level, exceeding the host's immune clearance capacity, the wound will suppurate and ulcerate, becoming difficult to heal and leading to clinically relevant infections. The primary lifestyle of bacteria within infected wounds is that of biofilms. Biofilms are bacterial communities attached to the extracellular matrix. Biofilm formation is considered one of the reasons for drug resistance in antibiotic-resistant bacteria and a key factor in the transition of wounds to a chronic, non-healing state.

[0003] To date, various biomaterials, including living cell tissues, nanofibers, hydrogels, and sponges, have been developed to promote the repair of bacterial-infected wounds. Hydrogels, capable of absorbing large amounts of water and maintaining a swollen state, possess excellent hydrophilicity and biocompatibility, making them widely used in drug delivery. Thermosensitive hydrogels, due to their unique responsive properties, show promising applications in wound dressings. However, most current hydrogel-based wound healing materials often suffer from an imbalance between antibacterial properties and repair-promoting functions, limiting their effectiveness in both antibacterial and skin-repair-promoting applications.

[0004] Therefore, how to improve the antibacterial properties and skin repair effects of hydrogels, thereby promoting their application in the field of medical technology, is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a thermosensitive hydrogel for antibacterial and wound healing purposes and its preparation method. The thermosensitive hydrogel is prepared using norbornene (NB)-modified 8-arm PEG (i.e., PEG-NB), a matrix metalloproteinase-degradable peptide cross-linking agent, gelatin, indocyanine green, and levofloxacin. The purpose of this invention is twofold: firstly, to improve the biodegradability, biocompatibility, and drug release effect of the thermosensitive hydrogel; and secondly, to enhance its antibacterial properties and wound healing effects when used for skin wound treatment.

[0006] To achieve the above objectives, the present invention employs the following technical solution: On the one hand, the present invention provides an antibacterial and repair-promoting thermosensitive hydrogel, comprising PEG-NB, gelatin and a drug.

[0007] Furthermore, the drug includes indocyanine green and levofloxacin.

[0008] The thermosensitive hydrogel provided by this invention uses a three-dimensional porous network formed by cross-linking natural polymer gelatin and synthetic polymer PEG-NB as the framework. Through physical and chemical reactions, it loads the small molecule photothermal reagent indocyanine green (ICG) and the chemical drug levofloxacin, forming a smart medical material with better biocompatibility and stronger repair activity, which has extremely high clinical translation potential.

[0009] The thermosensitive hydrogel prepared in this invention utilizes the thiol (-SH) groups at the ends of matrix metalloproteinase-degradable peptide chains to form covalent crosslinks with norbornene (NB) at the ends of PEG-NB chains via thiol-ene click chemistry. This provides a stable chemical crosslinking framework for the network, ensuring the hydrogel's gel strength and anti-swelling properties at body temperature. The added gelatin molecular chains bind to PEG segments through non-covalent forces such as hydrogen bonds and hydrophobic interactions, forming physical crosslinking points. These, together with the covalent crosslinking points of PEG-NB, construct a three-dimensional porous network structure. The synthetic polymer (PEG-NB) ensures the gel's mechanical strength, while the natural polymer (gelatin) imparts temperature responsiveness and promotes cell adhesion, addressing the issues of insufficient bioactivity and poor tissue adhesion in pure PEG-NB hydrogels. By adjusting the ratio of PEG-NB to gelatin, the mechanical strength of the composite network can be significantly improved after combining the two, overcoming the defects of pure PEG-NB hydrogels, such as easy deformation and loss in vivo.

[0010] Meanwhile, the composite network structure formed by PEG-NB and gelatin provides a space for indocyanine green and levofloxacin, trapping the two small molecule reagents and drugs within the pores. The uniform pore distribution of the composite network structure ensures that indocyanine green and levofloxacin are evenly distributed within the hydrogel, thus guaranteeing the consistency of antibacterial and photothermal effects. Indocyanine green, as a small molecule photothermal reagent, can efficiently absorb light energy and convert it into heat energy under near-infrared (NIR) irradiation, raising the local temperature of the hydrogel and surrounding infected tissue to approximately 45°C. This directly disrupts the bacterial cell membrane or denatures enzymes or proteins within the bacteria, thereby achieving photothermal sterilization. Simultaneously, the localized high temperature generated by the photothermal effect causes a gel-sol phase transition in the gelatin within the hydrogel, accelerating the release of levofloxacin by enhancing the hydrogel's fluidity. Furthermore, increasing the temperature enhances the permeability of bacterial cell membranes, promoting the entry of the chemical drug levofloxacin into the bacteria, thereby improving the utilization rate of the chemical drug and exerting a broad-spectrum bactericidal effect. The combination of indocyanine green and levofloxacin achieves a synergistic effect of chemical and photothermal antibacterial action, significantly improving antibacterial efficiency. The added gelatin also promotes cell proliferation and migration, providing nutrients for wound repair, thus enhancing the antibacterial effect of the drug, accelerating tissue repair, and synergistically promoting wound healing.

[0011] Further, the concentration of PEG-NB is 5-10% w / v, the concentration of gelatin is 5-10% w / v, the concentration of indocyanine green is 25-50 μg / ml, and the concentration of levofloxacin is 5-10 μg / ml.

[0012] Preferably, the concentration of PEG-NB is 5% w / v, the concentration of gelatin is 5% w / v, the concentration of indocyanine green is 25 μg / ml, and the concentration of levofloxacin is 5 μg / ml.

[0013] Furthermore, it also includes a matrix metalloproteinase-degradable peptide and a photoinitiator LAP, wherein the amino acid sequence of the matrix metalloproteinase-degradable peptide is Ac-GCRDGPQGIWGQDRCG-NH2.

[0014] Furthermore, the present invention provides a method for preparing the thermosensitive hydrogel as described above, comprising the following steps: Step (1): Prepare PEG-NB solution; Step (2): Mix the PGB-NB solution and the matrix metalloproteinase-degradable peptides in the LAP solution to form a preliminary solution; Step (3): Add gelatin, indocyanine green and levofloxacin to the preliminary solution to obtain a thermosensitive hydrogel precursor solution; Step (4): Thermosensitive hydrogel precursor solution is irradiated with ultraviolet light to obtain thermosensitive hydrogel.

[0015] Further, the PGB-NB solution and matrix metalloproteinase-degradable peptides in step (2) are mixed in a thiol-ene ratio of 8:5.

[0016] In another aspect, the present invention provides the use of a hydrogel for preparing reagents with improved antibacterial effects, the hydrogel comprising PEG-NB, gelatin, indocyanine green, and levofloxacin.

[0017] Furthermore, the hydrogel also includes a matrix metalloproteinase-degradable peptide and a photoinitiator LAP, wherein the amino acid sequence of the matrix metalloproteinase-degradable peptide is Ac-GCRDGPQGIWGQDRCG-NH2.

[0018] Further, the concentration of PEG-NB is 5-10% w / v, the concentration of gelatin is 5-10% w / v, the concentration of indocyanine green is 25-50 μg / ml, and the concentration of levofloxacin is 5-10 μg / ml.

[0019] In another aspect, the present invention provides the use of the thermosensitive hydrogel described above in the preparation of dressings that promote the repair of skin wounds, including burns, cuts, and surgical incisions.

[0020] In another aspect, the present invention provides the use of the thermosensitive hydrogel described above in the preparation of reagents for treating infectious bone defects or oral and maxillofacial bacterial infections, wherein the oral and maxillofacial bacterial infections are selected from periodontitis, peri-implantitis, and postoperative infections of the maxillofacial region.

[0021] The research results of this invention fully demonstrate that the hydrogel prepared by the optimal method provided by this invention has significant and excellent effects in improving antibacterial, anti-inflammatory and skin wound repair. The hydrogel provided by this invention can be applied to the treatment of infected skin wounds (used as a wound dressing for burns, cuts or surgical incisions, including but not limited to burns, cuts or surgical incisions), infected bone defects (including but not limited to osteomyelitis, open fractures), and oral and maxillofacial bacterial infections (including but not limited to periodontitis, peri-implantitis, postoperative infections of the maxillofacial region), and also has excellent effects in promoting repair. Applying the hydrogel of this invention to these fields is still within the protection scope of this invention.

[0022] The present invention has the following beneficial effects: 1. A three-dimensional porous network structure of temperature-sensitive hydrogel was constructed using PEG-NB and gelatin. The synergistic effect between gelatin and PEG-NB was utilized to optimize the structure and properties of the hydrogel, significantly improving its mechanical strength, biocompatibility, and biodegradability. 2. By utilizing the transformation of gelatin molecules from a gel state to a liquid state at high temperatures, the fluidity of the hydrogel is improved, thereby promoting the release of drug molecules, enhancing the hydrogel's ability to promote cell adhesion, cell proliferation and migration, and improving the hydrogel's effect on promoting skin repair. 3. Photothermal antibacterial effect is achieved by combining the small molecule photothermal agent indocyanine green with near-infrared light irradiation, and at the same time, the chemical antibacterial effect of the chemical drug levofloxacin is combined to achieve a synergistic antibacterial effect and promote skin repair. 4. By synergistically combining synthetic polymer PEG-NB with natural polymer gelatin, a balance is achieved in the hydrogel's temperature-sensitive controllability, mechanical strength, and bioactivity. Functionally, it relies on the dual synergistic bactericidal effect of "levofloxacin chemical antibacterial + indocyanine green photothermal antibacterial" to efficiently eliminate infection. Various molecules support each other and work together to exert a significant antibacterial and skin-repairing effect. Attached Figure Description

[0023] Figure 1 The 1H NMR spectrum of the 8-arm PEG-NB is shown.

[0024] Figure 2 To assess the photocrosslinking and injectability of different hydrogels.

[0025] Figure 3 Rheological properties of P hydrogel and PGI@L hydrogel.

[0026] Figure 4 The mechanical properties of the three hydrogels are described.

[0027] Figure 5 Cryo-scanning electron microscopy images of three hydrogels.

[0028] Figure 6 The swelling properties of the three hydrogels are shown.

[0029] Figure 7 The degradation properties of the three hydrogels are shown.

[0030] Figure 8 The photothermal effect of PGI hydrogels at various concentrations.

[0031] Figure 9 The photothermal stability of PGI-25 hydrogel.

[0032] Figure 10 Macroscopic images showing the temperature sensitivity of the hydrogel.

[0033] Figure 11 The rheology of hydrogels is temperature-sensitive.

[0034] Figure 12 The drug release rates of the three hydrogels are given.

[0035] Figure 13 The results of quantitative analysis of CCK-8 in L929 cells after co-culturing with different hydrogels.

[0036] Figure 14 Live / dead staining results of L929 cells after co-culturing with different hydrogels.

[0037] Figure 15 The adhesion of HUVEC cells and L929 cells on different hydrogels.

[0038] Figure 16 To investigate the effects of different hydrogels on promoting HUVEC cell migration.

[0039] Figure 17 The values ​​represent the OD values ​​of different hydrogels co-cultured with bacteria.

[0040] Figure 18 Count the colonies of bacteria co-cultured with different hydrogels.

[0041] Figure 19 The results of live / dead staining in co-culture of bacteria with different hydrogels.

[0042] Figure 20 SEM images of different hydrogels co-cultured with bacteria.

[0043] Figure 21The effects of different hydrogels on inhibiting biofilm.

[0044] Figure 22 To investigate the antibacterial and skin repair effects of different hydrogels in a rat skin infection model.

[0045] Figure 23 To investigate the antibacterial and skin-repairing effects of different hydrogels in a diabetic rat skin infection model.

[0046] Figure 24 GO enrichment was obtained from the RNA sequencing results of wound regeneration tissue from the PGI@L+NIR group hydrogel.

[0047] Figure 25 KEGG analysis of RNA sequencing results from wound regeneration tissues obtained from PGI@L+NIR group hydrogels. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to embodiments. It should be noted that the following embodiments are only used to explain and illustrate this invention and are not intended to limit this invention.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0050] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0051] Example 1: Preparation of a thermosensitive hydrogel for antibacterial and repair-promoting purposes The preparation steps of the thermosensitive hydrogel PGI@L for antibacterial and repair-promoting purposes provided in this embodiment are as follows: (1) 200 mg of 8-arm PEG-COOH, 153.4 mg of EDC, and 92.07 mg of NHS were dissolved in 15 ml of DCM and magnetically stirred for 3 hours at room temperature. Then, 98.56 mg of NB-NH2 was added, and the reaction continued for 72 hours with magnetic stirring. After the reaction was complete, the DCM solvent was removed using a rotary evaporator. The resulting solution was dissolved in 20% THF solution and placed in a dialysis bag (MWCO: 3000 Da). Gradient dialysis was used to remove excess impurities. During dialysis, 20%, 15%, 10%, and 5% THF solutions were used for 6 hours each, followed by dialysis in deionized water for 3 days, with water changes made periodically. Finally, the purified solution was filtered through a 0.8 μm filter to obtain a purified PEG-NB solution. This solution was then rapidly frozen in liquid nitrogen and freeze-dried at -80℃. The resulting polymer (PEG-NB) was finally identified by 1H-NMR. Figure 1 ).

[0052] (2) PEG-NB and matrix metalloproteinase degradable peptide were mixed and dissolved in a thiol-ene ratio of 8:5 in a 0.05% photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) solution to form a preliminary solution. The amino acid sequence of the matrix metalloproteinase degradable peptide was Ac-GCRDGPQGIWGQDRCG-NH2. (3) Add gelatin, indocyanine green (ICG) and levofloxacin (Levo) to the preliminary solution and mix them so that the concentrations of PEG-NB, gelatin, indocyanine green and levofloxacin are 5% w / v, 5% w / v, 25 μg / ml and 5 μg / ml, respectively, to obtain the thermosensitive hydrogel precursor solution. (4) The precursor solution was irradiated with 365 nm ultraviolet light for 30 seconds to form PGI@L thermosensitive hydrogel.

[0053] To verify the excellent antibacterial and repair-promoting effects of PGI@L thermosensitive hydrogel, this embodiment also prepared the following four hydrogels according to the above preparation steps: P hydrogel: The preparation steps are basically the same as above, except that step (3) is not performed (i.e., gelatin, indocyanine green and levofloxacin are not added to the precursor solution). PG hydrogel: The preparation steps are basically the same as above, except that only gelatin is added to the precursor solution in step (3) for physical mixing, and indocyanine green and levofloxacin are not added; PGI hydrogel: The preparation steps are basically the same as above, except that gelatin and indocyanine green are added to the precursor solution in step (3), and levofloxacin is not added; PI@L hydrogel: The preparation steps are basically the same as above, except that indocyanine green and levofloxacin are added to the precursor solution in step (3), and gelatin is not added.

[0054] Example 2: Performance Characterization of Thermosensitive Hydrogel This embodiment provides a detailed characterization of the key properties of the P hydrogel, PG hydrogel, and PGI@L hydrogel prepared in Example 1, including their injectability, porous structure, rheological behavior, and degradation performance. First, a portion of the precursor solution of each of the three hydrogels was taken, irradiated with 365 nm ultraviolet light for 30 seconds, and then the mold was removed. The hydrogels were observed to have formed, exhibiting good photocrosslinking and injectability. Figure 2 Subsequently, the rheological properties of the hydrogel precursor solution were scanned using a rheometer, and the hydrogel was crosslinked using 365 nm ultraviolet light 3 seconds after the scan. Figure 3The rheological curves show that after UV irradiation, the storage modulus of the PGI@L hydrogel precursor solution rapidly increased and exceeded the loss modulus, indicating that the hydrogel had transformed into a gel state and reached a stable state after 20 seconds. Further analysis of the mechanical properties of the hydrogels revealed that all three hydrogels exhibited uniform stiffness. The Young's moduli of P, PG, and PGI@L hydrogels were 30.15±1.80 kPa, 44.72±1.59 kPa, and 45.75±1.62 kPa, respectively, indicating that the addition of gelatin enhanced the mechanical strength of the hydrogels. Figure 4 ).

[0055] To evaluate the microstructure of the hydrogels, this invention further employed cryo-scanning electron microscopy (cryo-SEM) to observe the morphology and pore structure of the hydrogels. The images show that the P hydrogel, PG hydrogel, and PGI@L hydrogel all exhibit a typical three-dimensional porous network structure, with interconnected sponge-like porous structures inside. The average pore size of the P hydrogel is approximately 14 μm; the average pore sizes of the PG and PGI@L hydrogels are approximately 7.5 μm and 7.3 μm, respectively. Figure 5 This uniform pore size distribution provides a favorable microenvironment for the transport of biological substances and cell proliferation and adhesion, indicating that the prepared hydrogel has good internal structure controllability and biocompatibility.

[0056] Further hydrogel swelling experiments showed that the swelling rate of P hydrogel in PBS after 48 hours was 19.52±1.66%, while the swelling rates of PG and PGI@L hydrogels were 42.38±2.19% and 43.22±1.44%, respectively. This indicates that the addition of gelatin significantly increased the water retention of the hydrogels. Figure 6 To further evaluate the degradation performance of the hydrogels, in this embodiment, the three hydrogels were immersed in a 1 U / ml type I collagenase solution and cultured in a constant temperature shaker. The experimental results showed that the degradation rates of PG hydrogel and PGI@L hydrogel were significantly higher than those of P hydrogel after 14 days, indicating that the physical mixture of gelatin in the hydrogels was degraded more rapidly by type I collagenase. Figure 7 ).

[0057] Example 3: Concentration screening of photothermal agent indocyanine green To investigate suitable concentrations of the photothermal agent indocyanine green (ICG), this example evaluated the photothermal properties of different concentrations of ICG in hydrogels. Following the preparation method of PGI gel in Example 1, the concentrations of ICG in the hydrogels were 25 μg / ml, 50 μg / ml, and 100 μg / ml, respectively. PGI-25, PGI-50, and PGI-100 hydrogels were formed by UV irradiation, with PBS and PG hydrogels serving as control groups. The hydrogels were heated using 808 nm near-infrared (NIR) light. The results showed that the PGI-25 group reached 44°C within 5 minutes and stabilized at approximately 47°C after 10 minutes; in contrast, the hydrogels of the PGI-50 and PGI-100 groups reached 60–70°C after 10 minutes. Figure 8 This indicates that PGI-25 hydrogel is suitable for photothermal therapy without damaging healthy tissue. Furthermore, after five heating and cooling cycles, the photothermal performance of PGI-25 remained stable without significant degradation. Figure 9 ).

[0058] To characterize the temperature sensitivity of hydrogels, a temperature sensitivity verification experiment was conducted on P hydrogel and PG hydrogel. Both hydrogels were placed on a heatable platform and heated to 45°C for 3 minutes. The PG hydrogel showed significant liquid exudation. Figure 10 The P-hydrogel showed no significant change. The process was characterized using a rheometer. Figure 11 As can be seen, compared to the P hydrogel, the PG hydrogel shows a significant decrease in storage modulus (G') starting at 39℃, which corresponds to the liquefaction and exudation of gelatin in the hydrogel.

[0059] Meanwhile, in the drug release experiment, PGI@L hydrogel released approximately 20% of the drug without near-infrared light irradiation. After five cycles of near-infrared irradiation with heating and cooling, PGI@L hydrogel released approximately 76% of the drug; while PI@L hydrogel released approximately 53% of the drug. This indicates that adding gelatin to the precursor solution significantly improved the drug release capacity of the hydrogel. Figure 12 ).

[0060] Example 4: Biocompatibility of Thermosensitive Hydrogel To verify the biocompatibility of the PG, PGI, PI@L, and PGI@L hydrogels prepared in Example 1, this example assessed their biocompatibility through a co-culture experiment of cells and hydrogels. The PGI, PI@L, and PGI@L hydrogels were irradiated with 808 nm near-infrared light (NIR). The effects of each hydrogel group on the proliferation and activity of L929 cells were detected using a CCK-8 assay kit and AM / PI staining agent, respectively. Figure 13 and Figure 14It can be observed that there is no difference in cell survival rate and cell activity among the treatment groups, indicating that neither the hydrogel itself nor near-infrared irradiation has a significant inhibitory effect on cell growth, demonstrating that the hydrogel has good cell compatibility.

[0061] Furthermore, in this embodiment, HUVECs and L929 cells were seeded on the surfaces of P, PG, and PGI@L hydrogels, respectively. The cytoskeleton was labeled using F-actin fluorescence staining, and the cell adhesion on different hydrogel surfaces was observed. Figure 15 The experimental results showed that cell adhesion on the surface of the P hydrogel was poor, and cells failed to adhere effectively, indicating that the hydrogel surface had a weak promoting effect on cell adhesion. In contrast, cell adhesion on the surfaces of PG and PGI@L hydrogels was significantly improved, with cells exhibiting obvious tentacle-like protrusions and significant extension of the cytoskeleton, indicating that these two hydrogels had a significant promoting effect on cell adhesion. This demonstrates that adding gelatin to the hydrogel solved the problems of insufficient bioactivity and poor tissue adhesion of pure PEG-NB hydrogel.

[0062] In the HUVECs cell scratch assay, the relative migration rate of cells in the PGI@L+NIR group was significantly higher than that in the PGI+NIR and PI@L+NIR groups, indicating that the gelatin added to the hydrogel can promote cell migration. Furthermore, the relative migration rate of the PGI@L+NIR group was also significantly higher than that of the PGI@L group, suggesting that near-infrared light irradiation and heat treatment further promoted the release of gelatin from the hydrogel, thereby accelerating the cell migration process. Figure 16 ).

[0063] Example 5: Study on the antibacterial properties of thermosensitive hydrogels This embodiment further verifies the antibacterial properties of the hydrogel through a co-culture experiment with Escherichia coli and Staphylococcus aureus. The experimental method is as follows: 200 μl of each group of hydrogels and 2 ml of bacterial culture medium suspensions were prepared (10... 4 The mixture (CFU / ml) was mixed and subjected to NIR treatment according to the grouping, followed by incubation at 37°C for 6 hours. The culture medium was then used for OD value determination, plate plating, and subsequent live / dead staining experiments.

[0064] Plate plating colony count and bacterial culture OD value analysis showed that ( Figure 17 , Figure 18The control group and PG group had the highest colony count and OD value, with no significant difference. However, the colony count and OD value of the PGI+NIR group, PGI@L group, PI@L+NIR group, and PGI@L+NIR group were significantly lower, showing a gradual decreasing trend. The PGI@L+NIR group exhibited significantly better antibacterial effect than the other groups, demonstrating the best antibacterial performance. Further fluorescent staining of the co-cultured bacteria was performed using bacterial live / dead staining (SYTO 9 and PI). The fluorescence image results (…) Figure 19 Consistent with the above colony count and OD value analysis, the PGI@L+NIR group showed the weakest fluorescence of live bacteria and the most dead bacteria, further supporting the excellent antibacterial performance of the PGI@L+NIR group.

[0065] Electron microscopy also showed that the Escherichia coli and Staphylococcus aureus in the PGI@L+NIR group suffered more thorough damage. Compared with other groups, the bacterial cell walls showed more severe damage and cavitation, and even leakage of cell contents. Figure 20 These results indicate that the combined levofloxacin and near-infrared light bactericidal strategy employed in the PGI@L+NIR group exhibits significant antibacterial effects, far superior to the PGI+NIR, PGI@L, and PI@L+NIR groups. This is because the treatment method combining PGI@L hydrogel with near-infrared light achieves two key benefits: firstly, by adding gelatin to the hydrogel to enhance its temperature response and biocompatibility, promoting drug release; and secondly, by utilizing indocyanine green under near-infrared light irradiation to achieve photothermal sterilization. Simultaneously, the localized high temperature generated by the photothermal effect enhances bacterial cell membrane permeability, facilitating the entry of the chemical drug levofloxacin into the bacteria and improving drug utilization. Therefore, adding gelatin, indocyanine green, and levofloxacin to the P hydrogel and combining it with near-infrared light irradiation can achieve synergistic antibacterial effects.

[0066] This embodiment further evaluated the inhibitory effect of hydrogel on bacterial biofilm formation by co-culturing hydrogel with bacterial suspension to promote natural biofilm formation, and then staining the biofilm with crystal violet. The results showed ( Figure 21 Whether it was Escherichia coli or Staphylococcus aureus, the biofilms in the control group and PG group were relatively thick and intact, while the biofilm formation in the PGI@L+NIR group was significantly reduced, with only a very small amount of biofilm appearing. This indicates that the PGI@L+NIR group is significantly better than the PI@L+NIR group and other groups in inhibiting biofilm formation, and can quickly clear the source of infection in a short time. This also further demonstrates that the combination of PGI@L hydrogel and near-infrared light has a significant synergistic antibacterial effect.

[0067] Example 6: In vivo antibacterial and repair-promoting study of thermosensitive hydrogels To evaluate the antibacterial and repair-promoting effects of different hydrogels in vivo, this example established two full-thickness skin defect infection models: one in SD rats and the other in diabetic GK rats. We created full-thickness defects with diameters of 15 mm and 10 mm on the backs of SD and GK rats, respectively, and infected them with Staphylococcus aureus for 24 hours. Then, the hydrogel prepared in Example 1 was injected into the wound site. After filling the defect area, it was irradiated with 365 nm ultraviolet light to gel, followed by irradiation with 808 nm near-infrared light (NIR). The wounds were monitored and analyzed within two weeks post-surgery.

[0068] from Figure 22 and Figure 23 The results showed that at 14 days, the wounds of the SD and GK rats in the control group and PG group had healed compared to 1 day, but there were still large wounds. The wounds of the PGI+NIR group, PGI@L group, PI@L+NIR group and PGI@L+NIR group showed significant healing. Among them, the wound healing of the PGI@L+NIR group was significantly better than that of the other groups, almost reaching complete healing. This indicates that PGI@L hydrogel combined with near-infrared light irradiation can effectively fight bacteria and significantly promote skin repair. Based on the structure and properties of the hydrogel, it can be inferred that the reason for its effectiveness lies in the simultaneous addition of gelatin, indocyanine green, and levofloxacin to the PGI@L hydrogel. Norbornene on the PEG forms covalent cross-linking points through thiol-ene click chemistry, providing a stable "chemical cross-linking framework" for the network and ensuring the hydrogel's gel strength and anti-swelling properties at body temperature. The addition of gelatin promotes cell migration and adhesion, accelerating wound healing. Simultaneously, indocyanine green exerts a photothermal bactericidal effect upon near-infrared light irradiation, synergistically fighting bacteria with levofloxacin to prevent inflammation caused by infection, thus providing a safe environment for tissue regeneration. Therefore, the synergistic effect between PEG-NB, gelatin, indocyanine green, and levofloxacin significantly enhances the hydrogel's skin repair effect.

[0069] Furthermore, this invention also verified the repair effects of the above-mentioned groups of hydrogels by constructing a bone defect model. The results showed that the PGI@L+NIR group hydrogel also had a significant and excellent effect in promoting bone defect repair. Its effect in promoting bone tissue regeneration and healing was significantly better than that of the other groups, indicating that the thermosensitive hydrogel prepared by PEG-NB, gelatin, indocyanine green and levofloxacin also has significant advantages in promoting bone defect repair.

[0070] Example 7: Study on the mechanism of thermosensitive hydrogel in promoting skin repair To further elucidate the mechanism by which PGI@L+NIR hydrogel promotes skin repair, this invention performed transcriptome RNA sequencing on skin regeneration tissues from the control group and the PGI@L+NIR group, and conducted GO enrichment and KEGG analyses on differentially expressed genes. The GO enrichment analysis results showed that ( Figure 24 The differentially expressed genes were mainly enriched in processes related to immune response regulation, angiogenesis, cell proliferation and migration, and cell adhesion. This suggests that treatment in the PGI@L+NIR group may promote tissue regeneration by regulating these cellular functions. In particular, the enrichment of genes related to angiogenesis and cell proliferation and migration may enhance the speed of vascular reconstruction and tissue repair during skin repair.

[0071] KEGG enrichment analysis ( Figure 25 Further studies revealed significant enrichment of the PGI@L+NIR group in several important pathways. In the environmental information processing (EIP) pathway, viral infection, cytokine signaling, PI3K-Akt signaling, and ECM-receptor interaction pathways were significantly enriched, suggesting that the PGI@L+NIR group enhances immune responses, angiogenesis, cell function, and extracellular matrix remodeling by regulating these signaling pathways, thereby directly promoting wound repair and tissue regeneration. In the cellular processes (CP) pathway, the enrichment of the MAPK and cytokine signaling pathways further supports the important role of cell proliferation and migration in wound healing.

[0072] Example 8: Selection of Thermosensitive Materials This embodiment further investigates the effect of different temperature-sensitive materials on the hydrogel properties through a co-culture experiment of temperature-sensitive hydrogels and Staphylococcus aureus. The preparation method of the hydrogels is basically the same as in Example 1, except that the temperature-sensitive materials used are natural gelatin molecules, PF127, and PNIPAM, respectively, and the control group is P hydrogel (i.e., without the addition of gelatin, indocyanine green, and levofloxacin). The hydrogels are irradiated with 808 nm near-infrared light (NIR) to test the antibacterial properties of different hydrogels, and the OD of the bacterial culture medium of different hydrogel test groups is measured. 600 The results are shown in Table 1.

[0073] Table 1. Antibacterial properties of hydrogels made of different temperature-sensitive materials

[0074] The test results show that, compared with the control group, the OD of the culture medium after co-culturing the hydrogel with the added temperature-sensitive material and bacteria is higher. 600 The value decreased significantly, indicating that the addition of temperature-sensitive materials to the hydrogel can achieve excellent antibacterial effects. Among them, the OD value of test group 1 was significantly lower. 600The lowest value indicates that hydrogels prepared from natural gelatin molecules have the best antibacterial effect. This is because natural gelatin molecules are in a gel state at room temperature, and undergo a phase transition (from gel to liquid state) upon heating, enhancing the fluidity of the hydrogel and allowing the gelatin to carry out more drug, thus promoting drug release. In contrast, PF127 and PNIPAM have opposite thermosensitive properties. PF127 is liquid at low temperatures, but rapidly forms a stable gel when the temperature rises to body temperature (approximately 37°C); PNIPAM expands at lower temperatures, and the hydrogel volume shrinks upon heating, compressing and releasing the drug. The characteristics of these two materials mean that hydrogels prepared from them cannot effectively release the drug, resulting in low efficacy. Therefore, natural gelatin molecules are preferred as the thermosensitive material for preparing thermosensitive hydrogels.

[0075] Example 9: Selection of Photothermal Agent This invention further evaluated the effect of photothermal agents on the antibacterial effect of thermosensitive hydrogels to screen for the photothermal agent with the best antibacterial effect for preparing thermosensitive hydrogels. In this embodiment, thermosensitive hydrogels were prepared according to the method of Example 1, wherein the photothermal agents used were indocyanine green, gold nanoparticles, and graphene, respectively, while the control group did not add any photothermal agent. The bacterial culture OD was determined by co-culturing the hydrogel with Staphylococcus aureus. 600 The values ​​characterize the antibacterial effect of each hydrogel, and the hydrogels were irradiated with 808 nm near-infrared light (NIR). The test results are shown in Table 2.

[0076] Table 2. Antibacterial properties of hydrogels with different photothermal agents

[0077] As shown in Table 2, the hydrogels prepared with different photothermal agents all exhibited certain antibacterial effects. Among them, the hydrogel with added indocyanine green showed the highest OD value in the co-culture medium with bacteria. 600 The lowest value was found in the photothermal conversion efficiency of indolecyanine green (ICB), followed by graphene, indicating that the hydrogel prepared with ICB had the best antibacterial effect. This is because ICB has a high photothermal conversion efficiency, which can efficiently absorb light energy and convert it into heat energy, achieving effective photothermal antibacterial activity. Simultaneously, the photothermal effect induced by ICB improves the drug utilization efficiency of levofloxacin, promoting the effect of chemical antibacterial activity and achieving a synergistic effect between photothermal and chemical antibacterial agents. In contrast, metal nanoparticles tend to aggregate in solution, affecting the uniformity of the photothermal effect, and are expensive and have certain biotoxicity. While graphene-prepared hydrogels also have good antibacterial effects, their synthesis is complex and may also pose toxicity issues. Therefore, ICB is the preferred choice.

[0078] Example 10: Optimization of matrix metalloproteinase-degradable peptide sequences This embodiment further compares the antibacterial properties of thermosensitive hydrogels prepared with matrix metalloproteinase-degradable peptides of different sequences, in order to optimize the sequence of matrix metalloproteinase-degradable peptides. The preparation method of the thermosensitive hydrogel is basically the same as in Example 1, wherein the matrix metalloproteinase-degradable peptides used are the four peptide sequences shown in Table 3. The antibacterial effect without thermosensitive hydrogel is evaluated by co-culturing hydrogels with Staphylococcus aureus. The hydrogels are irradiated with 808 nm near-infrared light (NIR), and the test results are shown in Table 3.

[0079] Table 3. Antibacterial properties of hydrogels containing different matrix metalloproteinase-degradable peptides

[0080] As shown in Table 3, when the sequence of the matrix metalloproteinase-degradable peptide was designed as Ac-GCRDGPQGIWGQDRCG-NH2, the OD of the prepared hydrogel and the culture medium after co-culturing with bacteria was [not specified]. 600 The lowest OD value indicates the best antibacterial effect of the hydrogel. This may be related to the structural characteristics of the sequence and the arrangement of functional groups. The Ac-GCRDGPGQIWGQDRCG-NH2 sequence has certain hydrophilicity and degradability, which can effectively promote the interaction between the hydrogel and the bacterial surface, further enhancing the antibacterial activity. These functional groups can effectively bind to and disrupt the bacterial cell membrane structure, inhibiting bacterial growth and reproduction. In comparison, other sequences such as GPLGVRG, CKGPGQIWGQGGK, and PLGLAG have lower OD values. 600 A higher value indicates that their antibacterial effect is relatively weak. This may be because these sequences have low hydrophilicity or low degradation rate, failing to fully exert their ideal antibacterial effect.

[0081] Example 11: Optimization of heating method for thermosensitive hydrogel This invention also investigated the effect of heating methods on the properties of thermosensitive hydrogels during use. Thermosensitive hydrogels were prepared using essentially the same method as in Example 1. The hydrogels were then co-cultured with Staphylococcus aureus, and subsequently heated using 808 nm near-infrared (NIR) light irradiation, microwave heating, ultrasonic heating, and electromagnetic field heating, respectively. The control group received no heating treatment. The OD values ​​of the bacterial culture were then measured. 600 The values ​​and test results are shown in Table 4.

[0082] Table 4. Effect of heating method on the antibacterial properties of hydrogels

[0083] The test results in Table 4 show that the antibacterial properties of hydrogels treated with different heating methods vary significantly. Hydrogels irradiated with 808 nm near-infrared light exhibit the best antibacterial effect, followed by those heated by electromagnetic fields, while hydrogels heated by ultrasound show the worst antibacterial effect. This is because indocyanine green in the hydrogel can achieve photothermal sterilization under near-infrared light irradiation. Simultaneously, the localized high temperature generated by the photothermal effect can promote the entry of levofloxacin into the bacteria, improving drug utilization. Microwave heating, ultrasonic heating, and electromagnetic field heating cannot achieve photothermal sterilization, and temperature control is difficult, resulting in uneven heating effects. This may affect the structure and composition of the hydrogel, leading to the degradation or inactivation of some components, thus reducing the antibacterial effect. Therefore, heating hydrogels with near-infrared light irradiation not only improves their antibacterial properties but also simplifies experimental procedures, offering significant advantages.

[0084] Example 12: Wavelength optimization of near-infrared light This invention also evaluated the effect of near-infrared light wavelength on hydrogel properties. Thermosensitive hydrogels were prepared using a method essentially the same as in Example 1. The hydrogels were then irradiated with near-infrared light at 808 nm (near-infrared I region) and 1064 nm (near-infrared II region), respectively. The OD values ​​of the bacterial culture medium after co-culturing the hydrogel with Staphylococcus aureus were measured. 600 Values ​​are used to characterize the properties of hydrogels.

[0085] The results showed that the OD of the hydrogel co-culture medium with bacteria after irradiation with 808 nm near-infrared light was significantly higher. 600 The value was only 0.07, while the OD value of the hydrogel and bacterial co-culture medium after near-infrared light irradiation in zone II was much higher. 600 The value can reach 0.30 because the maximum absorption peak of indocyanine green is in the range of 780-820 nm. Therefore, it can only achieve photothermal therapy under near-infrared I region photoexcitation and cannot be adapted to the near-infrared II region.

[0086] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and alterations made by those skilled in the art without departing from the spirit and scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An antibacterial pro-rep airive thermosensitive hydrogel, characterized in that, This includes PEG-NB, gelatin, and pharmaceuticals.

2. The temperature-sensitive hydrogel of claim 1, wherein, The drugs include indocyanine green and levofloxacin.

3. The temperature-sensitive hydrogel of claim 2, wherein, The concentration of PEG-NB is 5-10% w / v, the concentration of gelatin is 5-10% w / v, the concentration of indocyanine green is 25-50 μg / ml, and the concentration of levofloxacin is 5-10 μg / ml.

4. The thermosensitive hydrogel as described in claim 4, characterized in that, It also includes matrix metalloproteinase-degradable peptides and photoinitiator LAP, wherein the amino acid sequence of the matrix metalloproteinase-degradable peptides is Ac-GCRDGPQGIWGQDRCG-NH2.

5. The method for preparing the thermosensitive hydrogel according to any one of claims 1-4, characterized in that, Includes the following steps: Step (1): Prepare PEG-NB solution; Step (2): Mix the PGB-NB solution and the matrix metalloproteinase-degradable peptides in the LAP solution to form a preliminary solution; Step (3): Add gelatin, indocyanine green and levofloxacin to the preliminary solution to obtain a thermosensitive hydrogel precursor solution; Step (4): Thermosensitive hydrogel precursor solution is irradiated with ultraviolet light to obtain thermosensitive hydrogel.

6. The preparation method according to claim 5, characterized in that, The PGB-NB solution and matrix metalloproteinase-degradable peptides in step (2) are mixed at a thiol-ene ratio of 8:

5.

7. The use of a hydrogel in the preparation of reagents with enhanced antibacterial effects, characterized in that, The hydrogel comprises PEG-NB, gelatin, indocyanine green, and levofloxacin.

8. The use as described in claim 7, characterized in that, The hydrogel also includes a matrix metalloproteinase-degradable peptide and a photoinitiator LAP, wherein the amino acid sequence of the matrix metalloproteinase-degradable peptide is Ac-GCRDGPQGIWGQDRCG-NH2.

9. The use of the thermosensitive hydrogel according to any one of claims 1-4 in the preparation of dressings that promote skin wound repair, characterized in that, The skin traumas include burns, cuts, and surgical incisions.

10. The use of the thermosensitive hydrogel according to any one of claims 1-4 in the preparation of a reagent for treating infectious bone defects or bacterial infections of the oral cavity and maxillofacial region, characterized in that, The oral and maxillofacial bacterial infections are selected from periodontitis, peri-implantitis, and postoperative infections of the maxillofacial region.

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