Two-dimensional hydrogen germanene nanosheets, their preparation method and applications

Abstract

This invention discloses a two-dimensional hydrogen germanene nanosheet, its preparation method, and its applications. The two-dimensional hydrogen germanene nanosheet has an ultrathin layered structure, with covalently modified hydrogen atoms on its surface, a lateral dimension of 100–300 nm, and a thickness of 5–10 nm. When activated by a specific wavelength laser, the two-dimensional hydrogen germanene nanosheet can photoexcite and generate singlet oxygen to destroy bacterial biofilms and kill bacteria. Simultaneously, it consumes local oxygen to create a staged hypoxic microenvironment, thereby inhibiting excessive inflammatory responses. After the light exposure ends, as the local oxygen level naturally recovers, it can further enhance the infection clearance function of immune cells, achieving continuous clearance of residual infection. This invention, through staged dynamic regulation of oxygen partial pressure, synergistically integrates direct bactericidal and immune clearance processes, ensuring antibacterial strength while reducing the risk of tissue damage and infection recurrence. It is suitable for the safe and effective treatment of implant-related biofilm infections.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a two-dimensional hydrogen germanene nanosheet, its preparation method, and its application. Background Technology

[0002] In orthopedic implant-associated infections, bacteria readily colonize the implant surface and form biofilms. Biofilms and their associated biofilm infection microenvironment (BIM) can create multiple barriers involving structure, metabolism, and immunity: on the one hand, they hinder the penetration of antimicrobial drugs, and on the other hand, they help bacteria evade clearance by the immune system. This results in a lack of treatment options, high recurrence rates, and currently, effective targeted therapies against BIM characteristics remain limited.

[0003] In recent years, anti-infection strategies based on "oxygen regulation" have become an important research direction. Oxygen, as a key factor in bacterial and host cell metabolism, is dynamically distributed in the biofilm infection microenvironment. Regulating oxygen partial pressure can affect biofilm stability, bacterial survival, and immune cell function. Existing technologies mainly revolve around two pathways: one is to convert oxygen into reactive oxygen species, directly destroying the biofilm structure and killing bacteria; the other is to enhance the oxidative metabolism and phagocytic capacity of immune cells (such as neutrophils) by regulating oxygen levels.

[0004] However, this strategy of "continuously increasing oxygen levels—continuously enhancing reactive oxygen species" still has significant limitations in the complex biofilm infection microenvironment, mainly in the following three aspects: 1) Excessive reactive oxygen species lead to tissue damage and a narrow therapeutic safety window: Existing technologies drive the generation of large amounts of reactive oxygen species by increasing oxygen levels, but their intensity and duration are difficult to control precisely in vivo. Excessive reactive oxygen species can easily cause oxidative damage to surrounding normal tissues while killing bacteria, exacerbating local inflammation, making it difficult to balance efficacy and safety. 2) Uncontrollable post-immune effects induced by PAMPs released from bacterial lysis: The enhanced bactericidal process leads to a large amount of bacterial lysis, releasing pathogen-associated molecular pattern components such as lipoteichoic acid, which may trigger excessive inflammatory responses or even immune storms. In addition, high-intensity stimulation can cause immune cells to enter a state of functional suppression or tolerance, weakening their ability to clear residual biofilms and increasing the risk of infection recurrence. 3) Ignoring the "protective immunomodulatory value" of early hypoxia: Existing strategies generally regard hypoxia as an unfavorable condition and strive to maintain a high-oxygen environment. However, in BIM, the oxygen partial pressure itself has phased characteristics, and immune cell function is highly sensitive to oxygen levels. If there is a lack of tiered design for the needs of different stages of treatment (such as early suppression of adverse post-inflammatory effects and later recovery / enhanced immune clearance), problems such as "effective bactericidal effect at the front end but insufficient clearance at the back end" or "relapse due to post-immune effect" are likely to occur, causing the overall efficacy to fluctuate greatly in complex infection environments.

[0005] In summary, while existing oxygen-regulated anti-infection strategies can reduce bacterial load in the short term by increasing oxygen supply and reactive oxygen species generation, they still face challenges such as high risk of tissue damage, uncontrollable post-immune effects, and insufficient understanding of the regulatory value during the hypoxic phase. These limitations restrict their reliable application in the treatment of implant-related biofilm infections. Therefore, there is an urgent need to develop a new strategy that can dynamically regulate oxygen partial pressure and synergistically integrate bactericidal and immune clearance processes to achieve safer and more thorough treatment of complex biofilm infection microenvironments. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a two-dimensional hydrogen germanene nanosheet, its preparation method, and its applications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide a two-dimensional germanene nanosheet, which has an ultrathin layered structure and is covalently modified with H atoms on its surface. The lateral dimension of the two-dimensional germanene nanosheet is 100-300 nm and the thickness is 5-10 nm.

[0008] The second aspect is to provide a method for preparing two-dimensional hydrogen germanene nanosheets, including the following steps: Step 1, Precursor Synthesis: Under an inert atmosphere, calcium powder and germanium powder are mixed in stoichiometric ratio, sealed, and reacted at high temperature and slowly cooled to prepare a layered CaGe2 crystal precursor. Step 2, Deintercalation and Exfoliation: The CaGe2 crystal precursor is ground and then placed in a low-temperature concentrated hydrochloric acid aqueous solution for stirring and reaction. The calcium layer is removed and initially exfoliated by topological deintercalation and liquid phase etching. Subsequently, the obtained solid product is subjected to ultrasonic treatment to further exfoliate and obtain a two-dimensional hydrogen germanene nanosheet dispersion. Step 3, purification and preservation: The dispersion is centrifuged to separate the supernatant containing the two-dimensional hydrogen germanene nanosheets. After washing until neutral, the supernatant is dispersed in an aqueous medium for preservation.

[0009] Furthermore, in step one, the conditions for the high-temperature reaction are: heating to 1000°C at a rate of 10°C / min, and annealing at this temperature for 24 hours; the slow cooling is furnace cooling to room temperature for 3 days.

[0010] Furthermore, in step two, the concentration of the concentrated hydrochloric acid aqueous solution is 5 mol / L, the low temperature environment is 4°C, the stirring reaction time is 14 days, and the ultrasonic treatment conditions are: ultrasonication at a power of 600W for 24 hours under ice-water bath conditions.

[0011] Furthermore, in step three, the washing is performed by multiple centrifugation washes with ethanol and deionized water; the aqueous medium is deionized water or phosphate buffer; the purified two-dimensional hydrogen germanene nanosheet dispersion is stored in the dark at 4°C.

[0012] The third aspect is to provide the application of the aforementioned two-dimensional hydrogen germanene nanosheets in the preparation of a drug for treating biofilm infections associated with orthopedic implants, wherein the drug, after laser activation, can achieve the following synergistic effects at the site of infection: (1) Photoexcitation generates singlet oxygen to destroy biofilms and kill bacteria, while consuming local oxygen to form a staged hypoxic microenvironment; the staged hypoxic microenvironment can inhibit the excessive inflammatory response caused by pathogen-associated molecular patterns released by bacterial lysis. (2) After the light exposure ends, as local oxygen naturally recovers, it promotes the enhanced function of immune cells in clearing infection, so as to completely clear residual infection.

[0013] Furthermore, the wavelength of the laser is 808nm, and it is irradiated for 10 minutes with a power density of 1.5W / cm².

[0014] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: The two-dimensional hydrogen germanene nanosheets prepared in this invention can perform a synergistic anti-infection method by dynamically regulating oxygen partial pressure in stages under laser activation at a specific wavelength. This method first generates reactive oxygen species through photodynamic catalysis, rapidly disrupting biofilm structures and killing bacteria, thereby significantly reducing bacterial load in the early stages of treatment. This process simultaneously consumes local dissolved oxygen, creating a staged hypoxic window, which helps regulate the intensity of the immune response, reduces the risk of excessive inflammation caused by PAMPs (pathogen-associated molecular patterns) from bacterial lysis products, and preserves the function of immune cells. After phototherapy, as oxygen levels gradually recover, the effector function of immune cells is restored and shows an enhanced clearance trend, further clearing residual bacteria, biofilm debris, and potential recurrence lesions, achieving a staged synergistic closed-loop treatment strategy of "early structural destruction and sterilization—late-stage immune regulation and deep clearance."

[0015] Through the above process, the two-dimensional hydrogen germanene nanosheets of the present invention can improve treatment stability while ensuring antibacterial strength in implant-related biofilm infection scenarios, reduce the risk of residue and recurrence, and promote infection control and tissue repair improvement. Attached Figure Description

[0016] Figure 1 Characterization of two-dimensional hydrogen germanene (H-Germanene) nanosheets; where a is a scanning electron microscope image of the CaGe2 precursor; b is a double aberration-corrected transmission electron microscope image of the CaGe2 precursor; c is the elemental distribution map corresponding to b; d is a transmission electron microscope (TEM) image of the H-Germanene nanosheets; e is the elemental distribution map corresponding to d; f is the Raman spectra of the H-Germanene nanosheets; and g is the transmission mode Fourier transform infrared (FTIR) spectra of the H-Germanene nanosheets.

[0017] Figure 2 The properties and verification of photoactivated oxygen electronic state conversion by H-Germanene nanosheets are shown; where a is the UV–vis–NIR absorption spectrum of H-Germanene nanosheets with different concentrations; b is the degradation of H-Germanene nanosheets after photoexcitation; and c is the degradation of methylene blue catalyzed by reactive oxygen species generated by H-Germanene nanosheets after photoexcitation.

[0018] Figure 3The in vitro antibacterial and anti-biofilm effects of H-Germanene nanosheets are shown. Among them, a is a representative image of bacterial CFU plates under different treatment conditions; b is the statistical result of CFU count in a; c is the quantitative result of OD595 of biofilm stained with crystal violet; d is the relative value of biofilm activity (CCK-8) in each group; e is a digital photograph of biofilm formed under different treatment conditions after crystal violet staining; f is the flow cytometry result of bacteria after PI / SYTO9 co-staining; g is the statistical result in f; h is the flow cytometry statistical result of ROS generation measured by DCFH-DA; i is a schematic diagram of the biofilm destruction and sterilization process of H-Germanene phototherapy strategy in BIM; j is a confocal three-dimensional image of biofilm under different treatment conditions (scale bar 200 μm).

[0019] Figure 4 This study demonstrates the role of H-Germanene nanosheets in activating BIM-specific immune cells through oxygen-mediated processes. Specifically, a) is a schematic diagram illustrating how oxygen acts as an "immune switch" to regulate the H-Germanene phototherapy-induced neutrophil immune function transition; b) shows the subcutaneous infection status of animals in different treatment groups; c) shows Western blot images of CD86 and CD206 protein expression in macrophages; d) shows the statistical results of CD86 and CD206 protein expression levels in macrophages in c (n = 3); e) shows the cell viability curves of neutrophils over time under different treatment conditions (n ​​= 5); f) shows the oxygen level detection under different treatment conditions; g) shows immunofluorescence staining images of neutrophil phagocytic activity under normoxic and hypoxic conditions (scale bar 50 μm); and h) shows the oxygen consumption rate (OCR) and glycolytic acidification rate (ECAR) detection results of neutrophils cultured under different oxygen content conditions.

[0020] Figure 5 The therapeutic effect of H-Germanene nanosheets in an implant-associated osteomyelitis model is shown. Specifically, a) is a schematic diagram of the H-Germanene phototherapy treatment process in the implant-associated osteomyelitis model; b) is the weight change curve of mice in different treatment groups (n = 5); c) is the quantitative analysis of bacterial load (CFU) in femoral tissue (n = 5); d) is the bacterial load (CFU) count results of the biofilm on the implant surface (n = 5); e) is the Ly6G... + Ly6C + Statistical results of flow cytometry analysis of CD11b^high MDSCs (n = 3); f represents Ly6G in bone marrow cells. + Ly6C + Flow cytometry analysis results of CD11bhigh MDSCs; g represents CD86 in bone marrow cells. + Flow cytometry analysis results of M1 macrophages. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0022] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0023] Example 1 This embodiment provides a method for preparing two-dimensional hydrogen germanene (H-Germanene) nanosheets, which employs a process combining precursor solid-phase synthesis, low-temperature acid deintercalation and exfoliation, and ultrasonic dispersion and purification.

[0024] 1. Synthesis of precursor CaGe2 crystal In a glove box protected by an inert gas atmosphere, metallic calcium powder and germanium powder are thoroughly mixed at a stoichiometric ratio (Ca:Ge = 1:2). The mixed powder is then placed into a clean quartz tube, and a vacuum is applied until the pressure is below 10. -3 After Pa, the quartz tube was sealed. The sealed quartz tube was placed in a high-temperature tube furnace and heated to 1000℃ at a rate of 10℃ / min, and annealed at this temperature for 24 hours. After the reaction was completed, the furnace was slowly cooled to room temperature for about 3 days to obtain a layered CaGe2 crystal precursor.

[0025] 2. Deintercalation and preparation of H-Germanene nanosheets The synthesized CaGe2 crystal precursor was ground into a fine powder and transferred to a reaction vessel containing a pre-cooled concentrated hydrochloric acid aqueous solution (approximately 5 mol / L). The reaction system was maintained at a low temperature of 4°C and continuously stirred for 14 days to remove the Ca layer through topological extraction and liquid-phase etching, and to initially achieve interlayer exfoliation. After the reaction was completed, solid-liquid separation was performed by centrifugation, and the solid product was collected.

[0026] The obtained solid product was redispersed in deionized water and ultrasonically treated using an ultrasonic homogenizer in an ice-water bath at a power of 600W for 24 hours to further exfoliate and obtain ultrathin two-dimensional nanosheets. Subsequently, the dispersion was differentially centrifuged, and the supernatant was collected to remove incompletely exfoliated thick particles.

[0027] 3. Purification and Preservation The collected supernatant was washed repeatedly by centrifugation with ethanol and deionized water until the pH of the supernatant was close to neutral to completely remove residual acid and reaction byproducts. Finally, the purified H-Germanene nanosheets were redispersed in deionized water or phosphate buffer to form a stable dispersion, which was stored at 4°C in the dark for subsequent use.

[0028] 4. Material Characterization To ensure the two-dimensional morphology and compositional purity of the materials prepared by the above method, the morphology was observed by transmission electron microscopy (TEM) / scanning electron microscopy (SEM), the elemental mapping was used to confirm the removal of Ca, and the layer thickness was measured by AFM. The structure and bonding characteristics were verified by XRD / XPS / Raman / FTIR.

[0029] Characterization results as follows Figure 1 As shown: Figure 1 In the image, 'a' is a scanning electron microscope image of the CaGe2 precursor. Figure 1 Image b is a double aberration-corrected transmission electron microscope image of the CaGe2 precursor, which shows the structural features of Ca and Ge atoms arranged in a regular alternation along the layered direction, exhibiting a typical Zintl phase layered crystal structure. Figure 1 In the diagram, c represents the elemental distribution of the CaGe2 precursor corresponding to b, showing that Ca and Ge elements are uniformly distributed in the material with an atomic ratio close to 1:2, indicating that the precursor has good stoichiometry and structural integrity. Figure 1 Image d in the middle is a transmission electron microscope image of two-dimensional hydrogen germanene (H-Germanene) nanosheets. It can be seen that the obtained material exhibits a distinct two-dimensional sheet morphology with clear sheet outlines and high electron permeability, indicating that the material has ultrathin characteristics. Figure 1 In Figure 'e', ​​which corresponds to the elemental distribution diagram of 'd', only the Ge element signal was detected. No obvious Ca element residue was observed, indicating that the Ca layer was effectively removed during the low-temperature wet chemical exfoliation process, and the conversion from CaGe2 to two-dimensional hydrogen germanene was successfully achieved. Figure 1 The image shows the Raman spectrum of H-Germanene nanosheets. A characteristic peak corresponding to the in-plane vibration mode appears at approximately 302 cm⁻¹, and a characteristic peak corresponding to the out-of-plane vibration mode appears at approximately 228 cm⁻¹, indicating that the obtained material has vibrational characteristics consistent with those of hydrogenated germanene. Figure 1 The transmission mode Fourier transform infrared spectrum of the H-Germanene nanosheets in the image shows characteristic absorption peaks related to the Ge-H bond, further verifying the successful introduction of hydrogen termination structures onto the surface of germanium atoms.

[0030] In summary, the characterization results demonstrate from multiple perspectives, including morphology, elemental composition, and chemical bonding, that the method of this invention can stably prepare two-dimensional hydrogen germanene nanomaterials with a two-dimensional sheet structure, effective removal of calcium, and well-defined surface hydrogenation modification.

[0031] Example 2 This embodiment aims to verify that the H-Germanene nanosheets prepared in this invention can undergo an energy / electron transfer process under specific wavelength light irradiation, thereby reducing the amount of triplet oxygen in the environment (…). 3 O2 is excited and converted into singlet oxygen (O2) 1 O2), and accompanied by the consumption of local dissolved oxygen, thereby creating a phased low-oxygen microenvironment while achieving the bactericidal function.

[0032] Figure 2 In Figure a, the UV-Vis-NIR absorption spectra of H-Germanene nanosheets with different concentrations are shown. It can be seen that the material exhibits broad-spectrum absorption characteristics in the UV to NIR band, indicating that it has good photoresponse capability and is conducive to electronic state transformation under illumination. Figure 2 Figure b shows the stability changes of H-Germanene nanosheets under photoexcitation conditions. The results indicate that the material can maintain good structural stability during illumination and is suitable for photoactivated catalysis applications. Figure 2 In the figure, c represents the result of H-Germanene nanosheets catalyzing the degradation of methylene blue under photoexcitation conditions. By monitoring the change of the dye absorption peak over time, it was demonstrated that the material can generate reactive oxygen species under light conditions and exhibits significant photocatalytic degradation ability.

[0033] The above results indicate that H-Germanene nanosheets can achieve efficient electronic state conversion under photoexcitation conditions and further induce the generation of reactive oxygen species, thereby endowing them with good photoactivated catalytic performance.

[0034] Example 3 This embodiment systematically verifies the phased synergistic anti-infection mechanism proposed in this invention through a combination of in vitro and in vivo experiments. The core findings demonstrate that: 1) photoactivation of the material effectively breaks down membranes and kills bacteria; 2) this process simultaneously consumes local oxygen, creating a protective hypoxic window; and 3) subsequent oxygen recovery induces training-like enhancement of immune cells, achieving deep clearance of residual infection.

[0035] 1. First-stage verification: Photogenerated singlet oxygen ( 1 O2-mediated membrane disruption and bactericidal effects Experimental Design: An in vitro biofilm model of Staphylococcus aureus or Staphylococcus epidermidis was constructed. The biofilm was co-incubated with H-Germanene nanosheets and then irradiated with a 660nm laser (power 1.5W / cm²). 2 (Time: 10 min). Grouping: Control group, GeNSs group, GeNSs phototherapy group, vancomycin group.

[0036] Figure 3 Image a shows representative images of bacterial colony-forming unit (CFU) plates under different treatment conditions. The results show that the number of bacterial colonies was significantly reduced after treatment with photoactivated H-Germanene nanosheets. Figure 3 In the figure, b represents the corresponding CFU count statistics, which further indicates that H-Germanene nanosheets have a significant bactericidal effect under photoactivation conditions, while the bactericidal ability of the un-photoactivation group is significantly reduced. Figure 3 In the middle, c represents the quantitative result of OD595 of the biofilm after crystal violet staining, indicating that photoactivated H-Germanene nanosheets can effectively inhibit the formation of biofilms. Figure 3 In the figure, d represents the relative activity of biofilms in different treatment groups, showing that the overall activity of biofilms was significantly reduced after photoactivation treatment. Figure 3 In the image above, e is a digital photograph of each group of biomembranes after being stained with crystal violet. It can be directly observed that photoactivated H-Germanene nanosheets have a significant destructive effect on the biomembrane structure.

[0037] Further analysis of bacterial survival status and oxidative stress levels was conducted using flow cytometry. Figure 3 The image in f shows the flow cytometry results of bacteria co-stained with PI / SYTO9. Figure 3 The corresponding statistical results in the figure show that the bacterial mortality rate increased significantly after photo-activated H-Germanene nanosheet treatment. Figure 3 The figure in h represents the flow cytometry results of reactive oxygen species (ROS) generation detected by the DCFH-DA probe, indicating that photoactivated H-Germanene nanosheets can induce a high level of reactive oxygen species generation in the system. Figure 3 The diagram in Figure i illustrates the process of H-Germanene disrupting and killing bacteria in a biofilm infection model. It shows that H-Germanene induces the conversion of oxygen electronic states through photoactivation and generates reactive oxygen species, thereby weakening the biofilm barrier and killing bacteria. Figure 3 The image in middle j shows the three-dimensional imaging results of the biofilm under different treatment conditions using confocal laser scanning microscopy. It can be observed that the biofilm structure in the photoactivated H-Germanene nanosheet treatment group is significantly loose or even disintegrated, while the biofilm in the control group still maintains a dense structure.

[0038] The above results indicate that the H-Germanene nanosheets prepared in this invention can exert significant antibacterial and anti-biofilm effects simultaneously under photoactivated conditions. The effect is closely related to the generation of reactive oxygen species induced by photoactivation, and is suitable for in vitro intervention of biofilm-related infections.

[0039] 2. Second-phase validation: Early treatment oxygen consumption, formation of a hypoxic window, and its immunoprotective effect. Experimental Design: Neutrophils or macrophages, light-treated bacterial lysate supernatant, and H-Germanene nanosheets were co-incubated in an in vitro co-culture system. Local oxygen partial pressure changes were monitored in real time using a hypoxia fluorescent probe (Pimonidazole).

[0040] Figure 4 Figure 'a' is a schematic diagram of how oxygen acts as an "immune switch" to regulate the transformation of neutrophil immune function induced by H-Germanene phototherapy. This illustrates that the present invention dynamically regulates the function of immune cells at different treatment stages by adjusting the local oxygen environment, thereby achieving synergistic clearance of biofilm infections. Figure 4 Figure b shows the comparison of subcutaneous infection in animals in different treatment groups. It can be observed that the infection degree in the H-Germanene phototherapy group was significantly reduced, indicating that the system can effectively improve infection control in the in vivo environment. Its efficacy depends not only on direct bactericidal action but also on its immunomodulatory effect.

[0041] To assess changes in immune cell function, macrophages and neutrophils were further analyzed. Figure 4 In the middle (c), the Western blot results show the expression of CD86 and CD206 proteins in macrophages. Figure 4 In the figure, d represents the corresponding quantitative statistical results. The results show that macrophage polarization changes in the oxygen-regulated environment induced by H-Germanene phototherapy, suggesting that their inflammatory response level is regulated, which helps to avoid the adverse effects of excessive inflammation on tissue repair. Figure 4 Figure e shows the cell viability curves of neutrophils over time under different treatment conditions. The results indicate that neutrophils have good survival stability under H-Germanene phototherapy, which helps maintain the immune defense capacity of the infection site. Figure 4 Figure f shows the results of local oxygen levels under different treatment conditions, indicating that the H-Germanene phototherapy system can create a controllable oxygen environment in the infected area, providing a basic condition for immune function regulation.

[0042] Furthermore, Figure 4The results, presented in Figure g, show the immunofluorescence staining results of neutrophil phagocytic activity under normoxic and hypoxic conditions. The results indicate that changes in oxygen levels significantly affect the phagocytic function of neutrophils, demonstrating that oxygen plays a crucial role in the regulation of immune function. Figure 4 The values ​​of h represent the oxygen consumption rate (OCR) and glycolytic acidification rate (ECAR) of neutrophils cultured under different oxygen content conditions. This indicates that changes in oxygen levels can significantly affect the energy metabolism of neutrophils, thereby regulating their immune function.

[0043] The above results indicate that the H-Germanene phototherapy system constructed in this invention can regulate the function of immune cells in stages by controlling the oxygen level in the infection microenvironment, acting as an "immune switch": in the early stage of treatment, it can effectively kill bacteria and destroy biofilms by combining with photoactivation, and in the later stage, it can promote the transformation of immune cell function and continuously clear residual infection by restoring the oxygen environment, thereby achieving a highly efficient and synergistic therapeutic effect on biofilm-related infections.

[0044] 3. Phase III Validation: Training-like Enhancement of Immune Cells and Clearance of Residual Infection After Oxygen Restoration Experimental Design: A mouse model of biofilm infection associated with tibial implants was established. Patients received local injection of H-germanene followed by phototherapy. Samples were collected at 1, 3, 5, 7, and 14 days post-treatment to systematically evaluate infection control efficacy and immune cell function.

[0045] like Figure 5 As shown in Figure a, a mouse model of femoral implant-related osteomyelitis was established. Figure 5 As shown in Figure b, the weight change curves of mice in different treatment groups reflect their overall health status during treatment. The results showed that the weight of mice treated with H-germanene phototherapy gradually recovered after treatment, and the recovery trend was significantly better than that of the control group and the traditional antibiotic treatment group. This suggests that in the later stages of infection, the H-germanene phototherapy strategy can effectively alleviate persistent infection and inflammatory burden, which is beneficial to the recovery of the body's overall condition. Figure 5 As shown in Figures c and d, the bacterial load of the femoral tissue and implant surface biofilm was detected. The results showed that the number of bacteria in the tissue of the H-germanene phototherapy group was significantly reduced, indicating that after the initial sterilization and biofilm destruction, the enhanced immune function induced by the oxygen recovery phase can sustainably clear residual bacterial infection in the tissue and avoid the formation of chronic or recurrent osteomyelitis.

[0046] Further analysis of bone marrow-derived immune cells revealed that, with the gradual restoration of the oxygen environment, the immune cells exhibited "training-like" functional enhancement characteristics. Flow cytometry results showed ( Figure 5Following H-germanene phototherapy, the proportion of immunosuppressive myeloid-derived suppressor cells (MDSCs) in the bone marrow decreased, while the proportion of effector immune cells with pro-inflammatory and anti-infective functions relatively increased, indicating that the immunosuppressive state was alleviated. Simultaneously, the number of pro-inflammatory macrophages (M1 type) and functional neutrophils in the bone marrow increased, indicating that during the oxygen recovery phase, immune cells transitioned from an early restricted state to an activated state with stronger anti-infective capabilities. This phased recovery of immune function helps to continuously clear residual bacteria and prevent recurrence of infection.

[0047] In summary, the H-Germanene phototherapy strategy proposed in this invention achieves synergistic treatment of implant-associated biofilm infections through a phased process of "photoactivation—hypoxia regulation—oxygen restoration": in the early stage of treatment, the photoactivated antibacterial effect of the material itself rapidly reduces the bacterial load; in the later stage, the restoration of the oxygen environment induces immune cells to produce a training-like enhancement effect, thereby achieving continuous clearance of residual infection and significantly improving the overall treatment effect of implant-associated osteomyelitis.

[0048] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A two-dimensional hydrogen germanene nanosheet, characterized in that, The two-dimensional germanene nanosheets have an ultrathin layered structure and are covalently modified with H atoms on their surface. The lateral dimensions of the two-dimensional germanium nanosheets are 100–300 nm, and the thickness is 5–10 nm.

2. A method for preparing two-dimensional hydrogen germanene nanosheets as described in claim 1, characterized in that, Includes the following steps: Step 1, Precursor Synthesis: Under an inert atmosphere, calcium powder and germanium powder are mixed in stoichiometric ratio, sealed, and reacted at high temperature and slowly cooled to prepare a layered CaGe2 crystal precursor. Step 2, Deintercalation and Exfoliation: The CaGe2 crystal precursor is ground and then placed in a low-temperature concentrated hydrochloric acid aqueous solution for stirring and reaction. The calcium layer is removed and initially exfoliated by topological deintercalation and liquid phase etching. Subsequently, the obtained solid product is subjected to ultrasonic treatment to further exfoliate and obtain a two-dimensional hydrogen germanene nanosheet dispersion. Step 3, purification and preservation: The dispersion is centrifuged to separate the supernatant containing the two-dimensional hydrogen germanene nanosheets. After washing until neutral, the supernatant is dispersed in an aqueous medium for preservation.

3. The method for preparing two-dimensional hydrogen germanene nanosheets according to claim 2, characterized in that, In step one, the conditions for the high-temperature reaction are: heating to 1000°C at a rate of 10°C / min and annealing at that temperature for 24 hours; the slow cooling is cooling to room temperature in the furnace for 3 days.

4. The method for preparing two-dimensional hydrogen germanene nanosheets according to claim 2, characterized in that, In step two, the concentration of the concentrated hydrochloric acid aqueous solution is 5 mol / L, the low temperature environment is 4°C, the stirring reaction time is 14 days, and the ultrasonic treatment conditions are: ultrasonication at 600W power for 24 hours under ice-water bath conditions.

5. The method for preparing two-dimensional hydrogen germanene nanosheets according to claim 2, characterized in that, In step three, the washing process involves multiple centrifugal washes with ethanol and deionized water; the aqueous medium is deionized water or phosphate buffer; and the purified two-dimensional hydrogen germanene nanosheet dispersion is stored at 4°C in the dark.

6. The use of the two-dimensional hydrogen germanene nanosheets as described in claim 1 in the preparation of a medicament for treating biofilm infections associated with orthopedic implants, characterized in that, After laser activation, the drug can achieve the following synergistic effects at the site of infection: (1) Photoexcitation generates singlet oxygen to destroy biofilms and kill bacteria, while consuming local oxygen to form a staged hypoxic microenvironment; the staged hypoxic microenvironment can inhibit the excessive inflammatory response caused by pathogen-associated molecular patterns released by bacterial lysis. (2) After the light exposure ends, as local oxygen naturally recovers, it promotes the enhanced function of immune cells in clearing infection, so as to completely clear residual infection.

7. The application according to claim 6, characterized in that, The laser has a wavelength of 808nm and is irradiated for 10 minutes at a power density of 1.5W / cm².

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