Visual multifunctional injectable hydrogel with endogenous and endogenous stimulation responsiveness as well as preparation method and application of visual multifunctional injectable hydrogel

Abstract

The invention discloses a visual multifunctional injectable hydrogel with endogenous and endogenous stimulation responsiveness as well as a preparation method and application of the visual multifunctional injectable hydrogel. The hydrogel is mainly prepared from oxygen vacancy molybdenum oxide nanoparticles Ce6-coated PEG-MoOx, transglutaminase and gelatin, wherein the oxygen vacancy molybdenum oxide nanoparticles Ce6-coated PEG-MoOx are loaded with a photosensitizer chlorin e6 and are modified by polyethylene glycol. The hydrogel disclosed by the invention can be applied to preparation of anti-inflammatory, anti-infection or wound repair promoting medicines, especially medicines for treating periodontitis or oral infection. The visible multifunctional injectable hydrogel with endogenous and endogenous stimulus responsiveness, which is constructed by the invention, can be developed through photoacoustic imaging, can play a role of a contrast agent, presents a morphological structure of an area in which the hydrogel is located, can eliminate negative oxidative stress of tissues, can efficiently and quickly kill bacteria, and can be used for preparing a medical dressing for treating endogenous and endogenous stimulus responsiveness. The inorganic signal molecules are controlled in a long-acting and controlled-release manner to play anti-inflammatory and angiogenesis promoting roles, so that tissue healing is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of injectable hydrogels, and particularly relates to an exogenous and endogenous stimulus-responsive visual multifunctional injectable hydrogel, a preparation method thereof and an application thereof, in particular to an antibacterial, anti-inflammatory, tissue regeneration-promoting and visual multifunctional injectable hydrogel capable of responding to the tissue microenvironment in the human body and external light stimulation, a preparation method thereof and an application thereof. Background Art

[0002] In recent years, hydrogels have become star materials in the fields of wound dressings, drug delivery and tissue engineering due to their unique performance advantages, and significant progress has been made in the application research, especially in the treatment of irregularly shaped tissue defects or infections. Currently, hydrogels mainly play antibacterial, anti-inflammatory and tissue regeneration and repair promoting roles.

[0003] (1) Antibacterial hydrogels kill by direct contact of the hydrogel. To avoid drug resistance and ensure the treatment effect, photodynamic therapy (PDT), chemodynamic therapy (CDT), and sonodynamic therapy (SDT) can be used in the formulation of drug-loading schemes. These methods can all generate toxic reactive oxygen species (ROS), which can directly lyse cell membranes, damage genetic material, interfere with energy metabolism, etc., with good effects and no drug resistance. Photothermal therapy (PTT) can directly cause thermal damage under light irradiation, melting cell membranes and inactivating proteins by denaturation. These schemes can make the treatment effect particularly efficient and controllable by artificially regulating the activation parameters of the drug (such as laser power, ultrasonic power, action duration, etc.), and adjust the parameters intuitively in real time according to the treatment progress, which not only ensures the treatment effect but also protects the surrounding normal tissue cells. However, for deep-seated and complex-shaped lesions, these treatment methods have problems such as attenuation of light and ultrasonic doses, which weaken the treatment effect.

[0004] (2) Eliminating tissue inflammation and relieving cell oxidative stress are the key to repairing tissue damage, promoting wound healing, and restoring normal body functions. In recent years, with the development of molecular medicine technology, a series of inorganic signaling molecules have also begun to be used for anti-inflammatory treatment, especially some essential trace elements for the human body, which play an important role in the metabolism, growth, chemotaxis, differentiation and other behavioral functions of tissue cells, and can promote the transformation of pro-inflammatory macrophages (M1 type) to pro-regenerative macrophages (M2 type), inhibit the release of inflammatory factors, and clear local excess ROS. Among them, NSAIDs and hormonal anti-inflammatory drugs have potential tissue damage, such as dermatitis, capillary dilation, tissue atrophy and degeneration, which may lead to the degeneration of normal organ functions, thus limiting their application. Stem cells and growth factors are biologically active, and there are problems such as cumbersome and complex processes such as preparation, storage, and transportation, high technical costs, and potential immune rejection. In recent years, with the development of molecular medicine technology, a series of inorganic signaling molecules have also begun to be used in anti-inflammatory treatment, especially some essential trace elements for the human body, which play an important role in the metabolism, growth, chemotaxis, differentiation and other behavioral functions of tissue cells, and can promote the transformation of pro-inflammatory macrophages (M1 type) to pro-regenerative types (M2 type), inhibit the release of inflammatory factors, and clear local excess ROS.

[0005] (3) Hydrogels are used to promote tissue regeneration, including bone tissue and soft tissue, which both require blood vessels to supply nutrients. Especially in the regeneration and reconstruction of large tissue defects, blood vessels play an extremely important role. At present, hydrogels mainly play the role of scaffolds for cell growth, and can coordinate the drugs they carry to regulate cell adhesion, proliferation and differentiation. They can also dynamically adapt to the microenvironment (pH, enzymes, mechanical signals, etc.) to control drug delivery. In recent years, the induction and promotion effects of inorganic signal molecules on soft and hard tissue cells such as vascular tissue and bone tissue have also been discovered. They promote tissue cell regeneration by directly improving mitochondrial metabolism, promoting the expression of genes related to cell proliferation and differentiation, and activating signal pathways to promote the synthesis and secretion of regeneration-related proteins.

[0006] (4) With the development of molecular medical diagnosis technology, in recent years, some experts and students have also started to endow hydrogels with diagnostic functions. Non-invasive and accurate diagnosis is an important development direction for disease diagnosis and treatment. To overcome the limitations of traditional diagnostic techniques, current research mainly focuses on three major technical paths: early molecular diagnosis based on biomarkers (such as inflammatory factors like IL-1β and MMP-8 in tissue secretions and qPCR detection of pathogenic microorganisms); quantification of the degree of tissue defect and destruction, as well as the size of neoplasms, using three-dimensional CT, MRI and other reconstruction techniques; and improvement of diagnostic efficiency by integrating multi-modal clinical data through artificial intelligence deep learning algorithms. However, due to problems such as high cost, complex operation and insufficient standardization, the clinical transformation of these technologies faces challenges. Photoacoustic imaging technology (PAI) has characteristics such as non-invasiveness, high spatio-temporal resolution and real-time imaging, eliminating the subjective bias of traditional probing, and deeply conforming to the concept of "high-definition medicine", which is expected to bring a revolutionary breakthrough to clinical diagnosis. Summary of the Invention

[0007] The purpose of the present invention is to provide a visually multifunctional injectable hydrogel responsive to internal and external stimuli.

[0008] The present invention also aims to provide a preparation method of the above injectable multifunctional hydrogel.

[0009] The last purpose of the present invention is to provide the application of the above hydrogel in the preparation of anti-inflammatory, anti-infective or wound repair-promoting drugs, especially in the preparation of drugs for treating periodontitis or oral infections.

[0010] The above first purpose of the present invention can be achieved by the following technical solution: a visually multifunctional injectable hydrogel responsive to internal and external stimuli, mainly composed of polyethylene glycol-modified molybdenum oxide nanocrystals with oxygen vacancies loaded with photosensitizer chlorin e6 (Ce6@PEG-MoO x , transglutaminase and gelatin. In the hydrogel, the concentration of the polyethylene glycol-modified molybdenum oxide nanocrystals with oxygen vacancies loaded with photosensitizer chlorin e6 (Ce6@PEG-MoO x is 62.5 - 500 μg / mL, the concentration of the transglutaminase is 10 - 80 mg / mL, and the concentration of the gelatin is 100 - 300 mg / mL.

[0011] Preferably, in the hydrogel, the concentration of the polyethylene glycol-modified molybdenum oxide nanocrystals with oxygen vacancies loaded with photosensitizer chlorin e6 (Ce6@PEG-MoO x is 125 - 250 μg / mL, the concentration of the transglutaminase is 12 - 60 mg / mL, and the concentration of the gelatin is 150 - 250 mg / mL.

[0012] More preferably, in the hydrogel, the concentration of the polyethylene glycol-modified molybdenum oxide nanocubes with oxygen vacancies loaded with the photosensitizer chlorin e6, Ce6@PEG-MoO x is 125 μg / mL, the concentration of the transglutaminase is 50 mg / mL, and the concentration of the gelatin is 200 mg / mL.

[0013] Preferably, the polyethylene glycol-modified molybdenum oxide nanocubes with oxygen vacancies loaded with the photosensitizer chlorin e6 of the present invention, Ce6@PEG-MoO x is prepared by the following method: ammonium molybdate and polyethylene glycol are dissolved in an ethanol solution according to a mass ratio of 40-45:50, the pH value is adjusted to ≤3, and after mixing, the mixture is kept at a constant temperature in a high-pressure reactor at 150-200 °C for 12-24 h. After cooling, centrifuging and washing, the precipitate is dried to obtain the polyethylene glycol-modified molybdenum oxide nanocubes with oxygen vacancies, PEG-MoO x , and the photosensitizer chlorin e6 and PEG-MoO x are mixed at a feeding ratio of 3-5:1 to obtain Ce6@PEG-MoO x nanoparticles.

[0014] To better obtain Ce6@PEG-MoO x nanoparticles, the photosensitizer chlorin e6 and PEG-MoO x can be mixed at a feeding ratio of 3-5:1, stirred, centrifuged, washed with water, centrifuged again. After repeating the centrifugation-washing several times, the precipitate is dried to obtain a powder, which is Ce6@PEG-MoO x nanoparticles.

[0015] More preferably, in the preparation process of the polyethylene glycol-modified molybdenum oxide nanocubes with oxygen vacancies loaded with the photosensitizer chlorin e6, Ce6@PEG-MoO x : ammonium molybdate and polyethylene glycol are dissolved in an ethanol solution according to a mass ratio of 43:50, the pH value is adjusted to 3, and after mixing, the mixture is kept at a constant temperature in a high-pressure reactor at 180 °C for 18 h. After cooling, centrifuging and washing, the precipitate is dried to obtain the polyethylene glycol-modified molybdenum oxide nanocubes with oxygen vacancies, PEG-MoO x , and the photosensitizer chlorin e6 and PEG-MoO x are mixed at a feeding ratio of 4:1, stirred, centrifuged, washed with water, centrifuged again. After repeating the centrifugation-washing several times, the precipitate is dried to obtain a powder, which is Ce6@PEG-MoO x nanoparticles. For convenience of preparation, this step can also be omitted, and Ce6 and PEG-MoO x are simply mixed.

[0016] The present invention designs a multifunctional injectable hydrogel that exhibits a photoacoustic effect within the wavelength range of the first near-infrared region, can respond to the internal microenvironment of the human body and external light stimulation, and simultaneously has antibacterial, anti-inflammatory, and tissue regeneration and repair-promoting effects. The present invention uses porcine skin extract gelatin as the hydrogel substrate and transglutaminase as the cross-linking agent to construct an edible hydrogel carrier that blocks the downward movement of bacteria, has excellent adhesion sealing and flexibility, and a drug-controlled release effect, and loads molybdenum-containing oxygen vacancy composite nanoparticles, namely Ce6@PEG-MoO x , a multifunctional diagnosis and treatment system with accurate photoacoustic imaging diagnosis, strong sterilization, and tissue regeneration and repair-promoting effects. Furthermore, the core concept of the present invention is spawned: to construct a "diagnosis and treatment integration" multifunctional system for periodontitis that combines accurate diagnosis and efficient treatment.

[0017] The above second object of the present invention can be achieved by the following technical solution: The preparation method of the exogenous and endogenous stimulus-responsive visual multifunctional injectable hydrogel includes the following steps: Take polyethylene glycol-modified molybdenum oxide nanocrystals with oxygen vacancies loaded with photosensitizer chlorin e6, Ce6@PEG-MoO x , transglutaminase, and gelatin, mix them and let them stand still to obtain the exogenous and endogenous stimulus-responsive visual multifunctional injectable hydrogel.

[0018] Preferably, the standing time is 1 to 3 minutes.

[0019] More preferably, the standing time is 2 minutes.

[0020] The hydrogel prepared by the method of the present invention is an injectable multifunctional hydrogel that can respond to the tissue microenvironment in the human body and external light stimulation, has antibacterial, anti-inflammatory, cell growth-promoting, and visual properties.

[0021] The above last object of the present invention can be achieved by the following technical solution: The application of the above hydrogel in the preparation of anti-inflammatory, anti-infective, or wound repair-promoting drugs, especially in the preparation of drugs for treating periodontitis or oral infections.

[0022] The present invention provides a polyethylene glycol-modified molybdenum oxide nanocrystal with oxygen vacancies, PEG-MoO x , photosensitizer chlorin e6 (Ce6), and transglutaminase (TG) cross-linked gelatin-based multifunctional visual hydrogel (Gelatin-based TG enzyme crosslinked multifunctional hydrogel), wherein both gelatin and TG enzyme are of biological origin, and after cross-linking, they have good biological safety and adhesion; the singlet oxygen generated by the photosensitizer Ce6 through light irradiation can quickly kill bacteria or tumor cells; polyethylene glycol-modified molybdenum oxide nanocrystals with oxygen vacancies, PEG-MoO xIt can be developed by photoacoustic imaging in the first near-infrared region, acting as a contrast agent, and having enzyme-like activity. It can respond to the weakly acidic microenvironment of human inflammation or tumors, catalyze the generation of O2 from endogenous H2O2 in tissues, enhance the photodynamic therapy effect of Ce6, and relieve local tissue oxidative stress and reduce inflammatory responses. Mo, as an inorganic signaling factor, can activate angiogenesis-related factors in tissues and promote the regeneration of hard and soft tissues. The Ce6@PEG-MoO x loaded TGase-crosslinked gelatin-based hydrogel of can optimize its physicochemical properties by adjusting the proportion of each component to meet the requirements of photodynamic therapy and anti-inflammatory and tissue regeneration.

[0023] Therefore, the injectable multifunctional hydrogel of the present invention has the following functions: 1) Adhere to and seal the wound, construct an immediate barrier, firmly attach and function; 2) Clearly develop through photoacoustic imaging, providing accurate and repeatable three-dimensional size values such as area and depth; 3) Remove H2O2 in the inflammatory and tumor microenvironments, relieve oxidative stress; 4) Quickly and effectively kill bacteria or tumors; 5) Degrade and slowly release the inorganic signaling molecule Mo to promote the regeneration of hard and soft tissues; 6) Have strong adhesion ability in oral environments such as wetness, chewing and swallowing; 7) Have appropriate mechanical properties and slowly degrade to match the ingrowth of hard and soft tissues.

[0024] The present invention has the following advantages:

[0025] (1) The PEG-MoO x in the present invention has oxygen vacancies and a hollow structure, as well as a coexisting system of multiple valence states of molybdenum, which endows excellent near-infrared absorption ability. High-precision photoacoustic imaging can be achieved in the first near-infrared region, providing a material basis for the non-invasive diagnosis and real-time monitoring of diseases. At present, most reports on the diagnostic role of hydrogels are very limited. However, the hydrogel of the present invention can visualize the local structure through photoacoustic imaging after being injected into the lesion, objectively and standardly present the lesion size value, and has high repeatability, solving the problems that the current diagnosis completely depends on the doctor's experience and the detection of some complex regions is inaccurate;

[0026] (2) The PDT bactericidal effect depends on three major factors: light, photosensitizer, and oxygen. After the hydrogel of the present invention is injected into the lesion, it in-situ responds to the endogenous weakly acidic microenvironment in the inflammatory region, catalyzes the in-situ decomposition of accumulated H2O2 in tissues to generate O2, relieves local inflammatory stress injury, and plays an anti-inflammatory role. Under the irradiation of a near-infrared laser with a wavelength of 660 nm, the photosensitizer Ce6 can quickly utilize the O2 generated by the catalysis of PEG-MoO x in response to exogenous stimuli to generate x O2, destroying the plaque biofilm and tumors, solving the problem that insufficient light in deep tissues leads to insufficient activation of Ce6 and limited PDT bactericidal effect; 1 O2, destroying the plaque biofilm and tumors, solving the problem that insufficient light in deep tissues leads to insufficient activation of Ce6 and limited PDT bactericidal effect;

[0027] (3) The killing effect of PDT is activated by laser. In the present invention, Ce6 generates cytotoxicity in response to laser irradiation during illumination. 1 O2 scavenges plaque biofilm, and PEG-MoO x responds to the tissue microenvironment to enhance the enzyme catalytic effect. When the laser is turned off, PDT can be terminated to achieve precise control. PEG-MoO x itself does not cause ROS chemical damage or photothermal damage. Therefore, the present invention also solves the problem of inevitable damage to normal tissue cells by antibacterial hydrogels.

[0028] (4) In the present invention, Mo released by the degradation of the hydrogel can be used as an inorganic signaling molecule, which can promote the regeneration and reconstruction of tissue blood vessels, further promote the regeneration and repair of tissues and organs after antibacterial and anti-inflammatory effects, quickly and efficiently. The regulation of inorganic signaling molecules avoids the problems of complex protocols, high costs, and induction of immune responses caused by traditional transplanted cells or added cytokines.

[0029] (5) Generally speaking, the endogenous and exogenous stimulus-responsive visual multifunctional injectable hydrogel constructed in the present invention can be developed by photoacoustic imaging, play the role of a contrast agent, present the morphological structure of its location area, eliminate the oxidative stress of tissue burden, kill bacteria efficiently and quickly, and release inorganic signaling molecules in a long-term controlled manner to play anti-inflammatory and angiogenesis-promoting effects, thereby promoting tissue healing. Brief Description of the Drawings

[0030] Figure 1 is the synthesis process of PEG-MoO nanoparticles with photoacoustic imaging performance and enzyme-like activity in Example 1. x

[0031] Figure 2 is PEG-MoO nanoparticles in Example 2 and Ce6@PEG-MoO formed after loading Ce6. x xRelated physicochemical characterization of nanoparticles. In Figure A, SEM is scanning electron microscopy and TEM is transmission electron microscopy. Figure B is a dynamic light scattering diagram, where the abscissa Size (nm) represents size and the ordinate Number (%) represents the percentage of quantity. Figure C is EDS element calibration, where the abscissa Energy (keV) is energy and the ordinate Intensity (a.u.) represents intensity. Figures D and E are X-ray photoelectron spectroscopy analyses, where the abscissa Binding energy (eV) is binding energy and the ordinate Intensity (a.u.) represents intensity. Figure F is an ESR electron spin resonance spectrum, where the abscissa Magnetic field (mT) is magnetic field strength and the ordinate Intensity (a.u.) is the corresponding intensity. Figure G is the X-ray diffraction XRD detection result. Figure H is FTIR Fourier transform infrared spectroscopy analysis where the abscissa Wavenumber (cm -1 ) is wavelength and the ordinate Transmittance is transmittance. Figure I is the Zeta potential detection result, where the ordinate Zeta potential (mV) represents the Zeta potential;

[0032] Figure 3 is the detection of the catalase (CAT) activity and peroxidase (POD) activity of the PEG-MoO x nanoparticles in Example 2. In Figure A, a dissolved oxygen meter is used to detect the situation of O2 generated by adding different concentrations of PEG-MoO x nanoparticles to the H2O2 solution. The abscissa Time (min) represents different time points, and the ordinate O2 concentration (mg L -1 ) represents the oxygen concentration detected by the dissolved oxygen meter; In Figure B, H2O2 is added to the solution every 8 minutes to detect the continuously generated oxygen concentration; In Figure C, a dissolved oxygen meter is used to detect the situation of O2 generated by adding PEG-MoO x nanoparticles to the H2O2 solution in different pH environments in vivo. In Figure D, the MB and TMB probes are adjusted to different pH values, PEG-MoO x is added and dissolved, then H2O2 is added, and laser irradiation at a wavelength of 660 nm (1 W / cm 2 , 1 min) is used to observe the color changes of the MB and TMB probes;

[0033] Figure 4 is the comparison of the CAT activity and photodynamic efficacy before and after loading Ce6 in Example 2. In Figure A, a dissolved oxygen meter is used to detect PEG-MoO x and Ce6@PEG-MoO xhas an approximate catalytic effect on the generation of O2 from H2O2, where the ordinate O2 concentration (mg L -1 ) represents the oxygen concentration. Figure B shows the ability of the SOSG probe to detect the generation of O2 under light, where the ordinate Generation of SOSG (a.u.) represents 1 the ability to generate O2; 1

[0034] Figure 5 is the related characterization of loading Ce6@PEG-MoO x nanoparticles into TG-crosslinked gelatin-based to construct the internal and external stimulus-responsive visual multifunctional injectable hydrogel in the present invention. Figure A in it is the scanning electron microscope characterization of the hydrogel morphology. In Figure A, Blank hydrogel represents the empty hydrogel, and Hydrogel loading Ce6@PEG-MoO x represents the Ce6@PEG-MoO x -loaded hydrogel. Figure B shows that the Ce6@PEG-MoO x -loaded hydrogel has injectability. In Figure B, With / Without TGase represents gelatin with / without TGase. Figure C shows that the Ce6@PEG-MoO x -loaded hydrogel has good toughness and pigskin adhesiveness. Figure D is the adhesiveness between the extracted tooth and pig gingiva detected by a universal mechanical detector. The ordinate Stress refers to the tensile fracture strength. Figures E and F are rheological detections. The abscissa Ang. frequency is the angular frequency. Figure G is the detection of the release of Mo in the Ce6@PEG-MoOx-loaded hydrogel in artificial saliva by ICP-MS inductively coupled plasma mass spectrometer. The ordinate Release of Mo (%) is the release rate of Mo. Figure H is that the hydrogel is immersed in artificial saliva and weighed at different time periods to evaluate its in vivo degradation;

[0035] Figure 6 is the photothermal situation of the hydrogels loaded with different material components in Example 2. Figure A is the photothermal photo of the hydrogels containing different concentrations of PEG-MoO x under 660 nm wavelength laser irradiation. Figure B is the temperature increase of the hydrogels containing different concentrations of PEG-MoO x under 660 nm wavelength laser irradiation over time. Figure C is the temperature change of the hydrogel after irradiation with different power density light intensities. Figure D is the photothermal cycle of the hydrogel containing PEG-MoO x . Figures E and F are the photothermal heating-up situations of the hydrogels with different components;

[0036] Figure 7 is different concentrations of PEG-MoO in Example 2​x , Ce6, Ce6@PEG-MoO x Antibacterial effect of Ce6, Ce6@PEG-MoO nanoparticles under light irradiation. Figure A shows the photos of colony formation on the plate; Figures B, C, and D show the colony counting statistics. Note: E.coli is Escherichia coli, a representative of Gram-negative bacteria, and S.aureus is Staphylococcus aureus, a representative of Gram-positive bacteria; NIR- represents no light irradiation, and NIR+ represents light irradiation; CFU mL -1 is the number of colonies. The abscissa Concentration represents the concentration of each material (μg mL -1 ).

[0037] Figure 8 is the verification of the antibacterial effect of Ce6@PEG-MoO x in Example 2 and its corresponding concentration of Ce6. Figure A shows the verification of the antibacterial effect against periodontitis-related pathogenic anaerobic bacteria, Figure B shows the colony statistics, and Figure C shows the disruption of plaque biofilms by different hydrogels. Note: P.gingivalis is Porphyromonas gingivalis, F.nucleatum is Fusobacterium nucleatum, E.coli is Escherichia coli, S.aureus is Staphylococcus aureus, CFU mL -1 is the number of colonies. NIR- represents no light irradiation, NIR+ represents light irradiation, Blank represents the empty hydrogel, that is, the cross-linked hydrogel containing only TGase and gelatin, With Ce6 represents the Ce6-containing hydrogel, that is, the cross-linked hydrogel containing only Ce6, TGase, and gelatin, With Ce6@PEG-MoO x represents the injectable multifunctional hydrogel containing Ce6@PEG-MoO x in the present invention;

[0038] Figure 9 is the photoacoustic imaging effect of injecting different components of hydrogels into the agarose model in Example 2;

[0039] Figure 10 is the promotion of the migration and angiogenesis of human umbilical vein endothelial cells (HUVEC) by Ce6, PEG-MoO x , Ce6@PEG-MoO x . Figure A shows the detection of cell migration by the Transwell experiment, Figure C shows the evaluation of cell migration by the scratch experiment through observing the scratch healing, Figure E shows the angiogenesis experiment with Matrigel by evaluating the number of nodes, and Figures B, D, and F show the statistics. Note: The ordinate Migration numbers in Figure B is the number of migrated cells, the ordinate Scratch healing(%) in Figure D is the scratch healing area, and the ordinate Number of nodes in Figure F is the number of vascular nodes;

[0040] Figure 11 It is the statistical results of the micro-CT three-dimensional images, two-dimensional images and related numerical values of the rat jawbones in Example 2. In the three-dimensional images, the blue lines represent the cementoenamel junction and the alveolar crest top, the green lines represent the measured distance from the cementoenamel junction to the alveolar crest top (CEJ-ABC Distance (nm)), the red dotted line frame is the detected bone density (BV / TV (%)), Positive control untreated rats is the positive control, representing rats without any treatment, and Negative control periodontitis is the negative control, representing rats with only a periodontitis model constructed and untreated;

[0041] Figure 12 is the biological safety detection of the Ce6@PEG-MoO x hydrogel. Among them, Figure A is the hemolysis test of PEG-MoO x , Ce6 and Ce6@PEG-MoO x compared with PBS (negative control) and Triton X-100 (positive control). The ordinate Hemolysis ratio in Figure A refers to the hemolysis rate. Figure B is the evaluation of the proliferation activity of HUVEC cells after treatment with PEG-MoO x and PEG-MoO x @Ce6 for 24 h and 72 h. Among them, the abscissa Concentration of PEG-MoO x refers to the concentration of PEG-MoO x , and the ordinate Cell viability refers to the cell viability. Figure C is the H&E staining of the main organs (heart, liver, spleen, lung and kidney) of the animals sacrificed on the 7th day and 28th day after treatment with PEG-MoO x @Ce6. Detailed implementation manners

[0042] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only for illustrative purposes and should not be construed as limitations to the present invention. Unless otherwise specified, the raw materials and equipment used in the following embodiments are obtained through conventional commercial channels.

[0043] Example 1

[0044] The visual multifunctional injectable hydrogel with internal and external stimulus responsiveness provided in this example is prepared as follows:

[0045] (1) Adjust the properties such as Mo valence state, particle size, hollow mesoporous structure, and oxygen vacancy concentration by feed ratio, pH, reaction temperature, etc., and then regulate and optimize PEG-MoOx In order to study the water solubility, ROS scavenging ability, CAT / SOD-like activity, etc. of the present invention, ammonium molybdate (430 mg) and polyethylene glycol (500 mg) were dissolved in an ethanol solution, and the pH was adjusted to ≤3 (pH=3 in the present embodiment), and then mixed and reacted in a high-pressure reactor at 160-180° C. (180° C. was selected in the present embodiment) for 12-24 h (16 h was selected in the present embodiment), cooled, centrifuged, washed, and the precipitate was dried to obtain a blue-black PEG-MoO x Nanoparticle powder, with a yield of 555.3 mg per time, has good water solubility and can be evenly dispersed in water to form a blue-black solution (10 mg / mL) ( Figure 1 );

[0046] (2) Ce6 and PEG-MoO x The mixture was mixed at a ratio of 4:1, stirred, centrifuged at high speed, washed with water, and centrifuged at 16000 rpm for 20 min. The centrifugation-washing was repeated several times, and the powder obtained after precipitation and drying was Ce6@PEG-MoO x Nanoparticles;

[0047] (3) Weighing gelatin powder and dissolving it in water, stirring it thoroughly to prepare a hydrogel gelatin base (200 mg / mL), solidifying it into a jelly at room temperature, and heating it each time it is taken to quickly melt it into a hydrogel precursor solution for preparing a hydrogel preparation;

[0048] (4) Weigh glutamine aminotransferase (TG) and dissolve it in water, mix well to prepare a cross-linking agent (500 mg / mL), and mix it with the gelatin precursor solution at a ratio of TG:gelatin = 1:10 (volume ratio) to achieve cross-linking. Heating can accelerate the cross-linking speed.

[0049] (5) Weigh Ce6@PEG-MoO x After the nanoparticles are fully mixed with gelatin and TG enzyme, the visualized multifunctional injectable hydrogel responsive to endogenous and exogenous stimuli of the present invention (TG enzyme-catalyzed cross-linked Ce6@PEG-MoO x Nanoparticles in gelatin-based hydrogels).

[0050] Example 2

[0051] Characterization and test results of the visualized multifunctional injectable hydrogel responsive to endogenous and exogenous stimuli prepared in Example 1

[0052] 1) The present invention synthesizes PEG-MoO rich in oxygen vacancies x Nanoparticles have good water solubility and Ce6 is successfully loaded on them:

[0053] The visualized multifunctional injectable hydrogel responsive to endogenous and exogenous stimuli prepared in Example 1 was analyzed by scanning electron microscopy, transmission electron microscopy, dynamic light scattering, EDS element calibration, X-ray photoelectron spectroscopy analysis, and ESR electron spin resonance spectroscopy. The results are as follows: Figure 2 As shown, from Figure 2 It can be seen that:

[0054] Figure 2 The scanning electron microscopy and transmission electron microscopy results in Figure A show that PEG-MoO x Nanoparticles are hollow spheres of uniform size with a rough surface, which are suitable for drug loading;

[0055] Figure 2 Figure B shows that the dynamic light scattering results of nanoparticle size characterization show that PEG-MoO x The particle size is consistent with that in electron microscopy, which is about 200 nm;

[0056] Figure 2 The EDS elemental analysis results of Figure C show that PEG-MoO x The nanoparticles are composed of Mo, O, and C elements;

[0057] Figure 2 The XPS photoelectron spectroscopy analysis results of Figures D and E show that PEG-MoO x Mo is composed of multiple valence states, including Mo 4+ (accounting for 24.55%), Mo 5+ (intermediate state, accounting for 29.93%), Mo 6+ (accounting for 45.52%);

[0058] Figure 2 The ESR electron spin resonance monitoring results of Figure F show that PEG-MoO x Contains abundant oxygen vacancies (g = 2.003 indicates oxygen vacancies);

[0059] Figure 2 The X-ray diffraction (XRD) results of Figure G show that PEG-MoO x It is a non-crystalline structure;

[0060] Figure 2 The FTIR Fourier transform infrared spectroscopy analysis of the H graph in the figure shows that Ce6@PEG-MoO x In the PEG-MoO x Ce6 is successfully loaded;

[0061] Figure 2 The Zeta potential test results of the I graph show that PEG-MoO x With Ce6@PEG-MoO x The surface is negatively charged.

[0062] The above detection results indicate that the present invention successfully synthesized PEG-MoO x nanoparticles rich in oxygen vacancies, which have good water solubility, and successfully loaded Ce6 thereon.

[0063] 2) Detection of enzyme-like activity

[0064] Detection of CAT-like enzyme activity: Different amounts of PEG-MoO x nanoparticles were added to a 5 mM H2O2 solution to make the final concentrations of PEG-MoO x nanoparticles be 0, 25, 50, 100, 200 μg / mL respectively. The dissolved oxygen meter continuously detected the concentration of O2 generated in the solution; the amount of O2 generated by the catalysis of 25 μg / mL PEG-MoO x nanoparticles was also detected, and the H2O2 solution was continuously added every 8 minutes for continuous detection; H2O2 solutions with pH = 5.5, 7.4, and 8.5 were respectively prepared to detect the amount of O2 generated.

[0065] Detection of photocatalytic activity: The MB probe was adjusted to different pH values, added with PEG-MoO x and dissolved, irradiated with a 660 nm wavelength laser (1 W / cm 2 , 1 min), and the color change of the MB probe was observed.

[0066] Detection of POD-like enzyme activity: The TMB probe was adjusted to different pH values of 5.5, 7.4, and 8.5 respectively, added with PEG-MoO x and dissolved, and then H2O2 was added, and the color change of the TMB probe was observed.

[0067] Detection of SOD-like enzyme activity: The SOD detection buffer, WST-8, and the enzyme solution were uniformly mixed to prepare the WST-8 / enzyme working solution. After melting and mixing the reaction initiation solution in the kit, PEG-MoO x , Ce6@PEG-MoO x , the WST-8 / enzyme working solution, and the reaction initiation working solution were successively added to a 96-well plate, thoroughly mixed, incubated in an incubator at 37 °C for 30 min, and the absorbance value at 450 nm was detected by an enzyme-labeled instrument.

[0068] Figure 3 The result of Figure A in x shows that the dissolved oxygen meter detected that PEG-MoO x can catalyze the decomposition of H2O2 to generate O2, and the amount of O2 generated is positively correlated with the concentration of PEG-MoO x , indicating that PEG-MoO

[0069] Figure 3The results of Figure B show that when H2O2 is added every 8 minutes, O2 can still be continuously catalyzed and generated by PEG-MoO x , verifying that PEG-MoO x has persistent CAT activity, which can be used to continuously scavenge local ROS in inflamed tissues and relieve oxidative stress for a long time;

[0070] Figure 3 The results of Figure C show that the CAT activities of PEG-MoO x are different in different pH environments in vivo;

[0071] Figure 3 The results of Figure D show that PEG-MoO x has no peroxidase (POD activity) or photocatalytic activity in different pH environments in vivo, indicating that hydroxyl radicals (·OH) or superoxide anions (O2 - ) will not be formed in tissues, thus avoiding uncontrollable chemical kinetic damage.

[0072] It can be seen from Figure 3 that PEG-MoO x nanoparticles have good CAT activity under different pH environments in vivo, can respond to the microenvironment of inflamed tissues, catalyze the generation of O2 from local endogenous H2O2, relieve tissue hypoxia and oxidative stress, and can still continuously play the role of catalyzing the production of O2 when adding H2O2 every 8 minutes, indicating that it can continuously consume newly generated H2O2 in the inflammatory reaction, continuously relieve inflammation, and can also enhance the PDT function of Ce6; PEG-MoO x nanoparticles have good SOD enzyme activity, can effectively scavenge intracellular reactive oxygen species, play an anti-inflammatory role, and are beneficial for cells to restore normal physiological metabolism and promote tissue repair; PEG-MoO x nanoparticles have no photocatalytic performance and no POD activity, indicating that hydroxyl radicals (·OH) or superoxide anions (O2 - ) will not be formed in tissues, thus avoiding uncontrollable chemical kinetic damage and not causing damage to normal tissues.

[0073] 3) Verification of the enzyme-like activity of the composite component: Using a dissolved oxygen meter, according to the method of "2) Enzyme-like activity detection" above, use a dissolved oxygen meter to detect the ability of 25 μg / mL of PEG-MoO x and 125 μg / mL of Ce6@PEG-MoO x nanoparticles to catalyze the production of O2 from 5 mM of H2O2 solution, and use the SOSG probe to detect the O2 x generation ability of 25 μg / mL of PEG-MoO x , 100 μg / mL of Ce6 and 125 μg / mL of Ce6@PEG-MoO 1 under light.

[0074] PEG-MoO x and Ce6@PEG-MoO x for their CAT-like activities and production of 1 O2 are as Figure 4 shown. It can be seen from Figure 4 that:

[0075] Figure 4 The result in Figure A of x shows that the dissolved oxygen meter detected that PEG-MoO x and Ce6@PEG-MoO x had similar effects on catalyzing H2O2 to generate O2, indicating that the composite nanoparticles Ce6@PEG-MoO x still maintained the CAT activity of the PEG-MoO

[0076] Figure 4 The result in Figure B of 1 shows the ability of the SOSG probe to detect the generation of O2 under light irradiation, showing that Ce6@PEG-MoO x had a stronger ability to produce x O2 than PEG-MoO 1 and Ce6. PEG-MoO x had no ability to produce 1 O2, but could promote Ce6 to produce 1 O2.

[0077] The results show that:

[0078] The Ce6@PEG-MoO x composite nanoparticles did not affect the performance of its single components, that is: PEG-MoO x still maintained its CAT activity; Ce6 still maintained its PDT function of producing 1 O2, and obtained the enhancement effect of PEG-MoO x

[0079] Loading Ce6 did not affect the CAT activity of PEG-MoO x and the O2 produced by PEG-MoO x catalyzing H2O2 could directly serve as the raw material for Ce6 to respond to exogenous light stimulation to exert PDT antibacterial effect, promoting 1 the production of O2, and a synergistically enhanced PDT sterilization effect could be obtained for periodontitis antibacterial.

[0080] 4) The visually multifunctional injectable hydrogel with endogenous and exogenous stimulus responsiveness constructed by the present invention has the properties of injectability, adhesiveness, flexibility, and sustained and slow release of drugs required for periodontal pocket drug delivery.

[0081] Weigh Ce6@PEG-MoO x nanoparticles and prepare a 1250 μg / mL solution. Also prepare a 500 mg / mL gelatin solution and a 300 mg / mL TGase solution. Take 0.1 mL of Ce6@PEG-MoOx, 0.8 mL of gelatin, and 0.1 mL of TGase solution and mix them well. Quench the hydrogel with liquid nitrogen and observe the cross-sectional morphology using a scanning electron microscope. For easy observation, the hydrogels in Figures B and C are stained with crystal violet. After injecting to form the word "SYSU" on the tabletop, place it in an incubator at 37 °C for 2 minutes, stick it to the wall parallel to the horizontal plane, and judge whether crosslinking is successful according to whether the gelatin still has fluidity and can flow down. Bend the hydrogel in all directions after injecting it onto the surface of pig skin and observe the state of the hydrogel. Use a universal mechanical detector to detect the adhesion force between the hydrogel adhered to the neck of an extracted tooth and pig gingiva, and use a rheological mechanical detector to detect the storage modulus and loss modulus of the hydrogel to evaluate the toughness and adhesion force of the hydrogel. Immerse the hydrogel in artificial saliva, and take the artificial saliva extraction solution on the 2nd, 3rd, 4th, 5th, 9th, and 12th days to detect the release of Mo from the Ce6@PEG-MoO x hydrogel in artificial saliva, and take out the hydrogel to weigh;

[0082] TGase-crosslinked gelatin-based hydrogel and its results of loading and delivering Ce6@PEG-MoO x are as follows Figure 5 shown:

[0083] Figure 5 In Figure A of : The results of scanning electron microscopy characterization of the hydrogel morphology show that the hydrogel crosslinked by Gelatin and TGase presents a loose and porous structure, which is suitable for drug loading, and Ce6@PEG-MoO x does not affect the structural morphology of the hydrogel;

[0084] Figure 5 In Figure B of : The results show that the Ce6@PEG-MoO x -loaded hydrogel has injectability, and TGase can effectively promote crosslinking and curing;

[0085] Figure 5 In Figure C of : The results show that the Ce6@PEG-MoO x -loaded hydrogel has good toughness and pig skin adhesiveness, and does not fall off or break when bent in all directions;

[0086] Figure 5 In Figure D of : The adhesion between the extracted tooth and pig gingiva detected by a universal mechanical detector shows that the Ce6@PEG-MoO x -loaded hydrogel has good adhesion ability in the periodontal pocket and is not affected by the loaded Ce6@PEG-MoOx Effect;

[0087] Figure 5 In Figure E and Figure F: The rheological test results show that for the Ce6@PEG-MoO-loaded x hydrogel and the blank hydrogel, they have approximate storage modulus and loss modulus, indicating that the Ce6@PEG-MoO-loaded x does not affect the toughness and adhesion of the hydrogel;

[0088] Figure 5 In Figure G: The results of ICP-MS (Inductively Coupled Plasma Mass Spectrometer) detecting the release of Mo from the Ce6@PEG-MoO-loaded x hydrogel in artificial saliva show that the hydrogel can slowly release Mo, and the release rate reaches 90% on the 12th day;

[0089] Figure 5 In Figure H: The hydrogel is immersed in artificial saliva, and it is taken out and weighed at different time intervals to evaluate its in vivo degradation situation. The results show that for the Ce6@PEG-MoO-loaded x hydrogel and the blank hydrogel, both degrade slowly in artificial saliva and are almost completely degraded on the 12th day.

[0090] The results show that the Ce6@PEG-MoO-loaded x does not affect the properties of the hydrogel, and preliminary evaluation proves that it is suitable for injection into the periodontal pocket and can play a role in continuously delivering drugs in the complex oral environment.

[0091] 5) The endogenous and exogenous stimulus-responsive visual multifunctional injectable hydrogel constructed by the present invention has low photothermal performance and is used for drug delivery in the periodontal pocket. While activating the PDT antibacterial effect by laser irradiation, it has no stimulating effect on the root pulp.

[0092] PEG-MoO with concentrations of 8, 4, 2, 1, 0.5, and 0.25 mg / mL are respectively prepared x , and a 500 mg / mL gelatin solution and a 300 mg / mL TG enzyme solution are prepared. 0.1 mL of PEG-MoO x , 0.8 mL of gelatin, and 0.1 mL of TG enzyme solution are respectively taken, fully mixed in an EP tube, and then adjusted to a power density of 1 W / cm 2 with a 660 nm wavelength laser and irradiated for 10 min, and the temperature change is detected by an infrared camera. Take the hydrogel with a final concentration of 25 μg / mL of PEG-MoO x , use a 660 nm wavelength laser, and adjust the power density to 1, 0.8, 0.6, and 0.4 W / cm 2 respectively, irradiate for 10 min, and the temperature change is detected by an infrared camera. Take PEG-MoOx The hydrogel with a final concentration of 25 μg / mL was irradiated with a laser at a wavelength of 660 nm and a power density of 1 W / cm 2 , and the laser was turned off after 1 min of irradiation to allow it to cool naturally to room temperature, which took about 4 min. Then the laser was turned on again for 1 min of irradiation and then turned off again. This process was repeated 5 times to detect the photothermal cycling effect of the hydrogel. Prepare 1 mg / mL Ce6@PEG-MoO x nanoparticles, and prepare 500 mg / mL gelatin solution and 300 mg / mL TG enzyme solution. Take 0.1 mL of Ce6@PEG-MoO x , 0.8 mL of gelatin, and 0.1 mL of TG enzyme solution, mix well. Take 0.1 mL of Ce6, 0.8 mL of gelatin, and 0.1 mL of TG enzyme solution, mix well, and irradiate with a laser at a wavelength of 660 nm and a power density of 1 W / cm 2 for 1 min, and use an infrared camera to detect the temperature change.

[0093] The photothermal properties of PEG-MoO x -loaded and Ce6@PEG-MoO x -loaded hydrogels are as shown in Figure 6 , and it can be seen from Figure 6 :

[0094] Figure 6 Figures A-C in x show that the infrared camera observation results show that the photothermal effect of PEG-MoO x -loaded hydrogel is positively correlated with the concentration of PEG-MoO Figure 6 , the light power, and the light irradiation duration; x Figure D in

[0095] Figure 6 shows that the PEG-MoO x -loaded hydrogel has a good photothermal cycling effect;

[0096] 6) The Ce6@PEG-MoO x composite nanoparticles of the present invention have a strong antibacterial effect

[0097] Select Escherichia coli (E. coli, ATCC 25922) and Staphylococcus aureus (S. aureus, ATCC 25923) as representatives of Gram-negative (G - ) and Gram-positive (G + ) bacteria respectively. Dilute the bacteria in the logarithmic growth phase to an appropriate concentration (such as OD600 = 0.1), and mix them with PEG-MoO at gradient concentrations respectivelyx , Ce6, Ce6@PEG-MoO x After mixing the solutions, they were either not irradiated with light or irradiated with 660 nm wavelength laser, then plated and cultured. By reading the number of colonies, the antibacterial effects of each group of solutions were evaluated, and the minimum bactericidal concentration (MBC) of Ce6 and Ce6@PEG-MoO x was obtained, and the antibacterial performance of Ce6@PEG-MoO x was preliminarily evaluated (Table 1);

[0098] The in vitro PDT antibacterial effect is as Figure 7 shown. It can be seen from Figure 7 that:

[0099] Figure 7 Figures A - D in x show that Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were cultured with different concentrations of PEG-MoO x , Ce6, Ce6@PEG-MoO 2 under light irradiation (660 nm, 1 W / cm x ², 1 min) / without light irradiation, and the colony-forming counts were obtained. The results showed that Ce6@PEG-MoO x had the most potent PDT sterilization effect, which was stronger than that of the single-component Ce6, while the single-component PEG-MoO

[0100] Table 1. Minimum bactericidal concentration (MBC) of Ce6 and Ce6@PEG-MoO x against E. coli and S. aureus

[0101]

[0102] Porphyromonas gingivalis (P. gingivalis, ATCC 33277) and Fusobacterium nucleatum (F. nucleatum, ATCC 25586) were selected as representatives of anaerobic pathogenic bacteria causing periodontitis. Bacteria in the logarithmic growth phase were diluted to an appropriate concentration (such as OD600 = 0.1). Using the MBC of Ce6 and Ce6@PEG-MoO x obtained from the aerobic bacteria experiment, the solutions of Ce6 and Ce6@PEG-MoO x were mixed with the two anaerobic bacteria respectively, then irradiated with light / without light irradiation, plated and cultured. By reading the number of colonies, the sterilization effects of Ce6 and Ce6@PEG-MoO x were verified, and the sterilization effect of Ce6@PEG-MoO x was comprehensively evaluated, and the Ce6@PEG-MoO determined by the bacteria experimentx The MBC concentration of [substance] was used as the detection concentration for subsequent experiments. The bacterial suspension in the logarithmic growth phase was diluted to an appropriate concentration (such as OD600 = 0.1) with a culture medium. 200 μL of the bacterial suspension was added to each well of a 96-well plate and cultured at 37 °C. After the bacteria formed a biofilm at the bottom of the wells, a culture medium containing PEG-MoO x , Ce6, and Ce6@PEG-MoO x was added. After illumination, it was cultured in an environment at 37 °C for 24 h, and then bacterial live / dead fluorescence staining was performed. The damage of the material to the biofilm was observed.

[0103] The antibacterial effects of Ce6@PEG-MoO x and Ce6 were verified as Figure 8 shown. It can be seen from Figure 8 that:

[0104] Figure 8 Figures A and B in [[reference]] show the colony counting of the culture of the bacterial suspension after treatment with drugs at the MBC concentration of periodontal-related pathogenic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum under illumination / non-illumination conditions. The results show that at the MBC concentration of Ce6@PEG-MoO x (125 μg / mL, containing 100 μg / mL of Ce6), Ce6 had no killing effect on both groups of bacteria under illumination / non-illumination, while Ce6@PEG-MoO x could not kill under non-illumination conditions but could completely kill under illumination, verifying that PEG-MoO x can promote the photodynamic antibacterial effect of Ce6;

[0105] Figure 8 Figure C in [[reference]] shows the live / dead staining of E. coli and S. aureus biofilms after illumination of different component hydrogels. The results show that the blank group hydrogel contained a large number of live bacteria. In the Ce6 treatment group, a large number of bacteria were killed, but there were still a large number of bacteria surviving, while in the Ce6@PEG-MoO x group, almost all bacteria were killed, indicating that Ce6 has a photodynamic anti-bacterial biofilm effect, and PEG-MoO x can enhance this effect.

[0106] The results show that: The optimal concentration of Ce6@PEG-MoO x in the hydrogel constructed in the present invention is 125 μg / mL.

[0107] 7) The visually multifunctional injectable hydrogel with endogenous and exogenous stimulus responsiveness constructed in the present invention has excellent photoacoustic imaging performance

[0108] containing 125 μg / mL of Ce6@PEG-MoOx Hydrogels, containing 25 μg / mL of PEG-MoO x Hydrogels, as well as single-component hydrogels containing 100 μg / mL of Ce6 and blank hydrogels were respectively injected into 4% agarose molds. After crosslinking and curing, the photoacoustic imaging effects of the hydrogels were detected using a photoacoustic device (VEVO 3100LAZR-X, FujiFilm VisualSonics Inc.) with a laser wavelength of 680 nm;

[0109] Ce6@PEG-MoO-loaded x The photoacoustic imaging effects of the Ce6@PEG-MoO-loaded hydrogels are as Figure 9 shown. It can be seen from Figure 9 that when using a photoacoustic device (VEVO 3100LAZR-X, FujiFilm VisualSonics Inc.) to detect the photoacoustic signals of the hydrogels in the agarose model at a wavelength in the first near-infrared region, the results show that both PEG-MoO x -loaded hydrogels and Ce6@PEG-MoO x -loaded hydrogels have the most excellent photoacoustic imaging performance;

[0110] 8) The visually multifunctional injectable hydrogel with endogenous and exogenous stimulus responsiveness constructed in the present invention has good angiogenesis-promoting function

[0111] Transwell cell migration experiment: 10^4 HUVEC cells were added to the upper chamber of the transwell. The upper chamber contained serum-free medium, and the lower chamber contained medium with Ce6, PEG-MoO x , Ce6@PEG-MoO x . After 24 h, the cells were fixed, stained and counted, and compared with the control group to evaluate the effects of each component solution on the migration function of HUVEC.

[0112] Wound scratch assay: After scratching the HUVEC cells seeded in a 6-well plate, Ce6, PEG-MoO x , Ce6@PEG-MoO x were respectively added to the medium. After 48 h, the proportion of the healed area was calculated and compared with the control group to evaluate the effects of each component solution on the migration function of HUVEC.

[0113] Angiogenesis experiment: 5×10^4 HUVEC cells were seeded into a 48-well plate containing Matrigel matrix gel. Ce6, PEG-MoO x , Ce6@PEG-MoO x were respectively added to the medium. After 4 h, the formation of lumens was observed under a light microscope to evaluate the effects of the nanoparticles on the angiogenesis function of HUVEC.

[0114] Ce6@PEG-MoO x The promotion of HUVEC migration and angiogenesis is as follows Figure 10 shown, from Figure 10 it can be seen that:

[0115] Figure 10 Figures A - D in [[ ]] show: The migration of HUVEC was detected by Transwell assay and scratch assay, and the results showed that PEG-MoO x and Ce6@PEG-MoO x treated cells had stronger migration ability, while the migration ability of Ce6-treated cells was weaker than that of the control group, indicating that PEG-MoO x had the function of promoting HUVEC migration;

[0116] Figure 10 Figures E - F in [[ ]]: The results of Matrigel angiogenesis assay showed that PEG-MoO x and Ce6@PEG-MoO x treated cells had stronger angiogenesis ability, while the angiogenesis ability of Ce6-treated cells was weaker than that of the control group, indicating that PEG-MoO x had the function of promoting HUVEC angiogenesis and could effectively promote the tissue repair of the wound.

[0117] 9) The constructed exogenous and endogenous stimulus-responsive visual multifunctional injectable hydrogel of the present invention has a good therapeutic effect in the rat periodontitis model

[0118] The rat periodontitis model was constructed by ligation method. The rats were anesthetized by intraperitoneal injection of 1% sodium barbital at a dose of 40 - 50 mg / kg. The second maxillary molar was ligated with 4-0 nylon thread at the neck for 10 days, and the nylon thread was checked every other day to see if it fell off. If it fell off, it was ligated again in time. After 10 days, the rats were anesthetized, and a large amount of food residues and dental plaque were found to accumulate on the tooth surface and the surface of the nylon thread ligated at the neck of the second maxillary molar, and the gingiva was swollen and the attachment loss was obvious, preliminarily judging that the periodontitis animal model was successfully constructed. After removing the nylon thread, periodontal scaling was performed on the second molar. The beagle dog periodontitis model was also constructed by ligation method. The specific grouping was as follows: Ctrl group: no treatment; Ce6 group: a single-component hydrogel containing Ce6 was injected into the periodontium after periodontitis modeling; PEG-MoO x group: a single-component hydrogel containing PEG-MoO x was injected into the periodontium after periodontitis modeling; Ce6@PEG-MoO x group: a hydrogel containing Ce6@PEG-MoO x was injected into the periodontium after periodontitis modeling; PD group: only sutures were removed and scaling was performed after periodontitis modeling, and no drug was given;

[0119] Ce6@PEG-MoO loaded x The hydrogel has a very significant therapeutic effect on rat periodontitis, such as Figure 11 shown. It can be seen from Figure 11 that: Quantitative analysis of CEJ-ABC distance and bone volume fraction (BV / TV) by micro-CT data shows that the Ce6@PEG-MoO x treatment group has the smallest CEJ-ABC distance, the highest bone density, and superior therapeutic effect on periodontitis.

[0120] 10) Biosafety detection of Ce6@PEG-MoO x loaded hydrogel

[0121] a) Cytotoxicity: Using the CCK8 method, after 10^4 HUVEC cells were seeded in each well of a 96-well plate and adhered, 0, 25, 50, 100, 200 μg / mL of PEG-MoO x solution and Ce6@PEG-MoO x solution containing an equal amount of PEG-MoO x were added respectively. After culturing for 30 min, the cell proliferation activity at 24 and 72 h was detected by the CCK8 method.

[0122] b) Hemolysis experiment: Rabbit whole blood was centrifuged at 1000 rpm for 10 min and washed 3 times with PBS. The blood cells were diluted to 5% (v / v). 50 μL of Ce6@PEG-MoO x solution with a concentration of 125 μg / mL and its corresponding concentrations of PEG-MoO x and Ce6 single-component solutions were added to 1 mL of rabbit blood cell suspension. Incubated at 37 °C for 2 h, centrifuged at 1000 rpm for 10 min, observed for hemolysis, and the OD value of the supernatant at 540 nm was detected. 0.1% Triton was used as the positive control group and PBS as the negative control group. The hemolysis rate was calculated as follows:

[0123] Hemolysis rate (%) = (A x - A PBS ) / (A Triton - A PBS ) × 100%

[0124] (where A x represents the OD value of the sample group to be measured)

[0125] The results are as Figure 12 shown. It can be seen from Figure 12 that:

[0126] Figure 12 Figure A in x shows that PEG-MoO xHemolysis assay compared with PBS (negative control) and Triton X-100 (positive control) showed that PEG-MoO x , Ce6 and Ce6@PEG-MoO x All are non-hemolytic;

[0127] Figure 12 Figure B shows: CCK-8 method to evaluate the PEG-MoO x and Ce6@PEG-MoO x The proliferation activity of HUVEC cells after 24h and 72h of treatment showed that the proliferation activity of HUVEC reached more than 75% of that of the control group;

[0128] Figure 12 Figure C shows that Ce6@PEG-MoO x H&E staining of the main organs (heart, liver, spleen, lung, and kidney) of animals sacrificed on the 7th and 28th days after treatment showed that the control group and Ce6@PEG-MoO x There was no difference between the groups.

[0129] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. A person skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention, and any improvement and deformation based on the concept of the present invention shall fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. A visually multifunctional injectable hydrogel responsive to internal and external stimuli, characterized in that, Mainly composed of polyethylene glycol-modified molybdenum oxide nanocrystals with oxygen vacancies loaded with photosensitizer chlorin e6, Ce6@PEG-MoO x , transglutaminase, and gelatin. In the hydrogel, the concentration of the polyethylene glycol-modified molybdenum oxide nanocrystals with oxygen vacancies loaded with photosensitizer chlorin e6, Ce6@PEG-MoO x is 62.5 - 500 μg / mL, the concentration of the transglutaminase is 10 - 80 mg / mL, and the concentration of the gelatin is 100 - 300 mg / mL.

2. The endogenous and exogenous stimulus-responsive visualizable multifunctional injectable hydrogel according to claim 1, wherein In the hydrogel, the concentration of the polyethylene glycol-modified molybdenum oxide nanocubes with photosensitizer chlorin e6 loaded (Ce6@PEG-MoO x is 125-250 μg / mL, the concentration of the transglutaminase is 12-60 mg / mL, and the concentration of the gelatin is 150-250 mg / mL.

3. The endogenous and exogenous stimulus-responsive visualizable multifunctional injectable hydrogel according to claim 2, wherein In the hydrogel, the concentration of the polyethylene glycol-modified molybdenum oxide nanocubes with photosensitizer chlorin e6 loaded (Ce6@PEG-MoO x is 125 μg / mL, the concentration of the transglutaminase is 50 mg / mL, and the concentration of the gelatin is 200 mg / mL.

4. The visualized multifunctional injectable hydrogel responsive to internal and external stimuli according to any one of claims 1-3, characterized in that, The polyethylene glycol-modified molybdenum oxide nanocubes with oxygen vacancies loaded with photosensitizer chlorin e6, Ce6@PEG-MoO x are prepared by the following method: Ammonium molybdate and polyethylene glycol are dissolved in an ethanol solution according to a mass ratio of 40-45:50, the pH value is adjusted to ≤3, and after mixing, the mixture is subjected to a constant-temperature reaction in a high-pressure reactor at 150-200 °C for 12-24 h. After cooling, centrifugation and washing, the precipitate is dried to obtain polyethylene glycol-modified molybdenum oxide nanocubes with oxygen vacancies, PEG-MoO x . The photosensitizer chlorin e6 and PEG-MoO x are mixed at a feeding ratio of 3-5:1, and the obtained powder is the nanocubes Ce6@PEG-MoO x .

5. The exogenous and endogenous stimulus-responsive visualizable multifunctional injectable hydrogel according to claim 4, wherein Ammonium molybdate and polyethylene glycol were dissolved in an ethanol solution according to a mass ratio of 43:50, the pH value was adjusted to 3, and after mixing, the mixture was kept at a constant temperature of 180 °C in a high-pressure reactor for 18 h. After cooling, centrifugation and washing, the precipitate was dried to obtain polyethylene glycol-modified molybdenum oxide nanoparticles with oxygen vacancies PEG-MoO x . After mixing the photosensitizer chlorin e6 and PEG-MoO x according to a feeding ratio of 4:1, the obtained powder was Ce6@PEG-MoO x nanoparticles.

6. Method for preparing an exogenous and endogenous stimulus-responsive visual multifunctional injectable hydrogel according to any one of claims 1-3, characterized in that including the following steps: Take the polyethylene glycol-modified molybdenum oxide nanocubes with oxygen vacancies loaded with photosensitizer chlorin e6, Ce6@PEG-MoO x , transglutaminase and gelatin, mix them and let them stand still to obtain an endogenous and exogenous stimulus-responsive visual multifunctional injectable hydrogel.

7. Use of the hydrogel according to any one of claims 1-3 in the preparation of an anti-inflammatory, anti-infective or wound repair drug.

8. Use of the hydrogel according to any one of claims 1-3 in the preparation of a drug for treating periodontitis or oral infection.

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