A method for preparing dual-targeted antibacterial and osteogenic nanomaterials for implant modification

Abstract

This invention relates to the field of antibacterial material application technology, specifically a method for preparing dual-targeted antibacterial and osteogenic nanomaterials for implant modification. The nanomaterials are prepared by mixing anhydrous citric acid as a carbon source and polyhexamethylene guanidine hydrochloride and diallyl dimethyl ammonium chloride as heteroatom dopant sources to provide guanidine and ammonium groups, respectively. This invention exerts stable antibacterial efficacy against six common infectious bacteria by inhibiting biofilms, disrupting bacterial membranes, and inhibiting bacterial energy metabolism, avoiding antibacterial failure and bacterial resistance risks caused by biofilms and the closed bone marrow cavity microenvironment. Simultaneously, it promotes stable osteogenic properties by promoting glutathione production, clearing ROS, and promoting mitochondrial metabolism to improve bone regeneration in the infected microenvironment, achieving a dual antibacterial and osteogenic effect, effectively solving the defects of lacking osteogenic function and insufficient biosafety.

Description

Technical Field

[0001] This invention relates to the field of antibacterial material application technology, specifically to a method for preparing dual-targeted antibacterial and osteogenic nanomaterials that can be used for implant modification. Background Technology

[0002] Implant-associated infections (IAIs) have become one of the most challenging clinical problems following bone implantation. Although implants are designed to promote tissue repair through mechanical support and osseointegration, their surface properties can still facilitate latent bacterial colonization and outbreaks. Even very low pathogen loads can cause persistent infections by adhering to the implant surface and forming biofilms. IAIs can induce chronic inflammation, leading to multiple pathological effects: local tissue damage, impaired osteoblast regeneration, and accelerated osteoclast activation-mediated bone resorption, ultimately resulting in catastrophic consequences such as prosthesis loosening, osteolytic lesions, and even systemic sepsis. While antibiotics remain the most commonly used solution for implant modification, biofilm formation significantly enhances bacterial resistance to the host immune system and antibiotics. Simultaneously, the closed medullary cavity microenvironment limits antibiotic efficacy, increasing the risk of bacterial resistance and potentially damaging normal host cells.

[0003] Currently, the most common approach to implant modification is antibiotic loading, but this method has significant drawbacks:

[0004] Biofilms and microenvironmental constraints lead to antimicrobial failure: Biofilm formation significantly enhances bacterial resistance to the host's immune system and antibiotics; at the same time, the closed microenvironment, which allows antibiotics to remain, may also cause bacterial resistance to antibiotics.

[0005] Lack of bone-promoting effects and insufficient biosafety: Most antibiotics do not have a direct bone-promoting effect, and long-term retention poses a certain risk of cytotoxicity. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing dual-targeted antibacterial and osteogenic nanomaterials that can be used for implant modification, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing dual-targeted antibacterial and osteogenic nanomaterials that can be used for implant modification. The nanomaterials use anhydrous citric acid (CA) as a carbon source and polyhexamethylene guanidine hydrochloride (PHMG) and diallyl dimethyl ammonium chloride (DDA) as heteroatom doping sources to provide guanidine and ammonium groups, respectively. The nanomaterials are prepared by mixing anhydrous citric acid, polyhexamethylene guanidine hydrochloride, and diallyl dimethyl ammonium chloride in a mass ratio of 0.4:0.2:2.5, ultimately forming positively charged carbon quantum dots with both ammonium and guanidine functional groups. The average particle size of the nanomaterials is 2.43 nm, and the average height is less than 2.5 nm.

[0008] The method for preparing the nanomaterials includes the following steps:

[0009] S1. Solution preparation: Prepare anhydrous citric acid (CA) and polyhexamethylene guanidine hydrochloride (PHMG), dissolve them in ultrapure water, and after complete dissolution, add diallyl dimethyl ammonium chloride (DDA) and stir until a homogeneous solution is formed. The mass ratio of anhydrous citric acid, polyhexamethylene guanidine hydrochloride, and diallyl dimethyl ammonium chloride is 0.4:0.2:2.5.

[0010] S2, hydrothermal reaction: Transfer the homogeneous solution from step S1 to a reaction vessel, seal it, place it in a heating box, and heat it at a constant temperature of 180℃ for 6 hours.

[0011] S3. Filtration and purification: After the reaction in step S2 is completed and the material in the reactor has cooled to room temperature naturally, the material is filtered using a microporous membrane and the filtrate is collected.

[0012] The filtrate was transferred to a cellulose dialysis bag and placed in ultrapure water for dialysis purification. The total dialysis time was 72 hours, with the ultrapure water being replaced every 2 hours for the first 24 hours and every 6 hours thereafter.

[0013] S4. Freeze-drying: The filtrate purified by dialysis in step S3 is dispensed into centrifuge tubes and freeze-dried to obtain the dual-targeted antibacterial osteogenic material.

[0014] Preferably, the nanomaterial exerts stable antibacterial efficacy against common infectious bacteria through mechanisms such as inhibiting biofilm formation, disrupting bacterial membrane structure, and inhibiting bacterial energy metabolism; the common infectious bacteria include Staphylococcus aureus, Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli, and Enterobacter cloacae.

[0015] Preferably, the nanomaterial is non-cytotoxic and biosafety in the concentration range of 30 μg / mL to 90 μg / mL, and also promotes osteoblast proliferation.

[0016] The osteogenic effect of the nanomaterials is achieved through mechanisms that promote glutathione production, scavenge reactive oxygen species (ROS), and improve mitochondrial metabolism, thereby improving the bone regeneration process in the infection microenvironment.

[0017] Preferably, the nanomaterial has a carbon (C) atom content of 77.2%, a nitrogen (N) atom content of 10.24%, and an oxygen (O) atom content of 12.56% in its elemental composition, and its surface contains alkyl chains, guanidine groups, and quaternary ammonium functional groups.

[0018] Preferably, in step S1, anhydrous citric acid (CA) 0.4g, polyhexamethylene guanidine hydrochloride (PHMG) 0.2g, and diallyl dimethyl ammonium chloride (DDA) 4mL are used.

[0019] In step S1, 16 mL of ultrapure water is used.

[0020] Preferably, in step S3, the pore size of the microporous filter membrane is 0.22 μm, and the molecular weight cutoff of the cellulose dialysis bag is 500-1000 Da.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. This invention breaks the paradox of antibacterial and osteogenic properties in implant materials. Under sterile conditions, it promotes osteogenic properties by stimulating mitochondrial metabolism. In the event of infection, it can also kill bacteria by disrupting the bacterial membrane and inhibiting the respiratory chain. After eliminating bacteria, it creates a favorable regenerative microenvironment, which manifests as downregulation of inflammatory factors and promotion of bone regeneration by promoting mitochondrial function. Furthermore, this nanomaterial can be used to modify common implants, endowing common implant materials such as titanium, nickel, magnesium, tantalum, and polyetheretherketone with stable antibacterial and osteogenic properties. This provides a new synthetic strategy for the development of antibacterial drugs and a transformative solution to the global problem of implant-related infections.

[0023] 2. The preparation method of the dual-targeted antibacterial nanomaterials of the present invention is relatively simple, easy to operate, and low in cost. It can be widely used in the modification of various common implant materials and ultimately applied to the prevention and treatment of implant-related infections. The preparation of modified materials using the present invention is also very easy to operate, making it convenient to promote and apply in implantation surgery. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the overall synthesis of a method for preparing a dual-targeted antibacterial and osteogenic nanomaterial that can be used for implant modification according to the present invention;

[0025] Figure 2 This is a TEM schematic diagram of the nanomaterials of this invention;

[0026] Figure 3This is a schematic diagram of the AFM of the nanomaterials of this invention;

[0027] Figure 4 This is a schematic diagram of the Fourier transform infrared spectrum of the nanomaterial of this invention;

[0028] Figure 5 This is a schematic diagram of the XPS energy dispersive spectroscopy of the nanomaterials of this invention;

[0029] Figure 6 This is a schematic diagram of the antibacterial coating of the nanomaterials of this invention against six types of bacteria;

[0030] Figure 7 This is a schematic diagram of bacterial liveness and death staining after the nanomaterials of this invention intervened in six types of bacteria;

[0031] Figure 8 This is a scanning electron microscope (SEM) image showing the effect of the nanomaterials of this invention on the intervention of six types of bacteria.

[0032] Figure 9 This is a schematic diagram illustrating the antibacterial stability of the nanomaterials of this invention against six types of bacteria;

[0033] Figure 10 This is a schematic diagram of the antibacterial curves of the nanomaterials of this invention after intervention with six types of bacteria;

[0034] Figure 11 This is a schematic diagram illustrating the biocompatibility of the nanomaterials of this invention with osteoblasts;

[0035] Figure 12 This is a schematic diagram illustrating the antibacterial properties and biosafety of the modified titanium, nickel, magnesium, tantalum, and polyether ether ketone nanomaterials of the present invention.

[0036] Figure 13 This is a schematic diagram of the CT scan results after the modified titanium, nickel, magnesium, tantalum and polyether ether ketone nanomaterials of the present invention were implanted into the femur of rats. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Please see Figure 1-13This invention provides a technical solution: a method for preparing a dual-targeting antibacterial and osteogenic nanomaterial that can be used for implant modification. The nanomaterial is prepared by mixing anhydrous citric acid, polyhexamethylene guanidine hydrochloride, and diallyl dimethyl ammonium chloride in a mass ratio of 0.4:0.2:2.5. Ammonium and guanidine groups are provided as heteroatomic doping sources using polyhexamethylene guanidine (PHMG) and diallyl dimethyl ammonium chloride (DDA), respectively, for targeting bacteria. Anhydrous citric acid (CA) is used as the carbon source to synthesize carbon quantum dots with both ammonium and guanidine functional groups, which exhibit positive charge under physiological pH conditions of 7.2-7.4. The dual targeting of the material refers to targeting bacteria to achieve antibacterial activity and targeting mitochondria to promote osteogenic activity.

[0039] The nanomaterials exert stable antibacterial efficacy against six common implant-associated infectious bacteria, including Staphylococcus aureus (ATCC25923), Staphylococcus epidermidis (ATCC12228), methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300), Pseudomonas aeruginosa (ATCC27853), Escherichia coli (ATCC25922), and Enterobacter cloacae (ATCC700323), through mechanisms such as inhibiting biofilm formation, disrupting bacterial membrane structure, and inhibiting bacterial energy metabolism.

[0040] The nanomaterial exhibits no cytotoxicity within a concentration range of 30 μg / mL to 90 μg / mL, ensuring good safety while significantly promoting osteoblast proliferation. Specifically, it exerts stable osteogenic properties by promoting glutathione production, scavenging reactive oxygen species (ROS), and promoting mitochondrial metabolism, thus significantly improving bone regeneration in the infection microenvironment.

[0041] The method for preparing the nanomaterials includes the following steps:

[0042] S1. Solution preparation: Prepare 0.4 g of anhydrous citric acid (CA) and 0.2 g of polyhexamethylene guanidine hydrochloride (PHMG). Dissolve both in 16 mL of ultrapure water. After complete dissolution, add 4 mL of diallyl dimethyl ammonium chloride (DDA) (50% aqueous solution, density 1.04 g / cm³) and stir until a homogeneous solution is formed to ensure that the anhydrous citric acid (CA) and polyhexamethylene guanidine hydrochloride (PHMG) are fully dissolved.

[0043] S2, hydrothermal reaction: Transfer the homogeneous solution from step S1 to a 50mL reaction vessel, seal it, place it in a heating box, and heat it at 180℃ for 6 hours.

[0044] S3. Filtration and purification: After the reaction in step S2 is completed and the material in the reactor has cooled naturally to room temperature (25℃±2℃, protected from light), the material is filtered using a 0.22μm microporous membrane, and the filtrate is collected.

[0045] The filtrate was then transferred to a cellulose dialysis bag with a molecular weight cutoff of 500-1000 Da and placed in ultrapure water for dialysis purification. The total dialysis time was 72 hours, with the ultrapure water being replaced every 2 hours for the first 24 hours and every 6 hours thereafter.

[0046] S4. Freeze-drying: The filtrate purified by dialysis in step S3 was dispensed into centrifuge tubes and freeze-dried at a freezing temperature of -80℃, a vacuum of 5Pa, and a drying time of 24h to finally obtain dual-targeted antibacterial osteogenic materials (NGCDs).

[0047] The dual-targeted antibacterial and osteogenic nanomaterials prepared above can be used for surface modification of common implants. After loading them onto the surface of common implant materials such as titanium (Ti), tantalum (Ta), nickel (Ni), magnesium (Mg) and polyether ether ketone (PEEK) using PVB, the materials can be endowed with stable antibacterial and osteogenic properties for the prevention / treatment of implant-related infections.

[0048] Low drug concentration at the lesion site and the ease with which bacteria can colonize and form biofilms on the implant surface are key reasons why bone tissue infections are difficult to treat, and are also one of the reasons for bacterial resistance. Implanting the aforementioned modified bone implants at the lesion site, or replacing non-modified implants, can locally inhibit / clear biofilms, exert significant antibacterial efficacy, while having good biosafety and promoting osteoblast proliferation. This has very high application value for the treatment of bone tissue infectious diseases.

[0049] Figure 2 The middle right figure is a particle size distribution diagram, and Figure 2 The middle left image is a high-resolution TEM image, such as... Figure 2 As shown, the morphology of NGCDs was observed using transmission electron microscopy (TEM), revealing that NGCDs exhibit a quasi-spherical shape with good dispersion and uniformity. Figure 2 The high-resolution TEM image in the upper left corner shows that the lattice spacing of NGCDs is 0.23 nm, consistent with the structure of graphitic carbon. Figure 2 The particle size distribution map in the lower right corner shows that the average particle size of NGCDs is 2.43 nm, which is used to target mitochondria.

[0050] Figure 3 Atomic force microscopy (AFM) images show that the average height of NGCDs is less than 2.5 nm, proving that the diameter of the nanomaterial NGCDs is likely to pass through the outer membrane pores of mitochondria.

[0051] like Figure 4 As shown, Fourier transform infrared spectroscopy (FTIR) reveals that the NGCDs surface exhibits typical functional group characteristics. The absorption peak at that location is attributed to the quaternary ammonium group. The C–H in-plane shearing vibrations indicate that quaternary ammonium groups are retained in the material. The absorption peak corresponds to the N–H bending vibration in the guanidine group. The absorption peak is attributed to the stretching vibration of the guanidinyl C–N bond, further confirming that NGCDs successfully achieved guanidinization. Furthermore, and The nearby absorption peaks may be related to C–N or C–O stretching vibrations, as well as the out-of-plane bending vibration of =C–H in the allyl structure, indicating that DADMAC-derived structures may remain in the material. Taken together, these characteristic peaks suggest that the NGCDs surface contains alkyl chains, guanidino groups, and quaternary ammonium functional groups.

[0052] like Figure 5 As shown, the elemental composition of NGCDs was analyzed using X-ray photoelectron spectroscopy (XPS). The three main typical peaks were attributed to C1s, N1s, and O1s, with atomic contents of 77.2%, 10.24%, and 12.56%, respectively. This indicates that the elemental composition of the nanomaterials contains 77.2% carbon (C), 10.24% nitrogen (N), and 12.56% oxygen (O).

[0053] like Figure 6 As shown, the antibacterial properties of NGCDs were further verified by antibacterial smears. It was found that the control group was covered with bacteria, while after the addition of NGCDs, none of the six bacteria grew. This shows that NGCDs have good spectral antibacterial properties.

[0054] like Figure 7 As shown, the antibacterial properties of NGCDs were further visually assessed by live and dead staining. The results showed that there was almost no red fluorescence representing dead bacteria in the control group, while after the addition of NGCDs, a large amount of red fluorescence appeared in the staining, and the blue fluorescence representing dead bacteria almost disappeared. This shows that NGCDs have good antibacterial properties.

[0055] The morphological changes and bacterial membrane integrity of six bacteria after NGCDs intervention were observed by scanning electron microscopy. The results are as follows: Figure 8 As shown, after NGCDs intervention, Staphylococcus aureus (ATCC25923), Staphylococcus epidermidis (ATCC12228), methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300), Pseudomonas aeruginosa (ATCC27853), Escherichia coli (ATCC25922), and Enterobacter cloacae (ATCC700323) were all inhibited by NGCDs, and the integrity of the bacterial structure was significantly disrupted, resulting in a large number of bacterial fragments.

[0056] like Figure 9As shown, the antimicrobial stability of NGCDs was tested for 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 weeks of storage. It was found that the minimum inhibitory concentration (MIC) of NGCDs against six common bacteria did not change within 20 weeks of storage, indicating that NGCDs have good storage stability.

[0057] like Figure 10 As shown, the antibacterial curves of six bacteria were detected under NGCDs intervention at concentrations of 0, 1 / 8, 1 / 4, 1 / 2, 1, and 2 times the MIC, respectively. It was found that the OD values ​​of the six bacteria changed significantly within 48 hours under NGCDs intervention, which proved that the antibacterial activity of NGCDs on the six bacteria was concentration-dependent, and a significant bactericidal effect could be seen when the concentration of NGCDs was higher than the MIC.

[0058] like Figure 11 As shown, the safety of NGCDs was verified by CCK8. It was found that NGCDs had good safety in the concentration range of 30-90 ug / ml. At the same time, NGCDs could significantly promote osteoblast proliferation at concentrations of 30 and 60 ug / ml, and NGCDs had the most significant osteoproliferative efficacy at a concentration of 60 ug / ml.

[0059] like Figure 12 As shown, the biosafety of different materials modified with NGCDs at concentrations of 50, 100, and 200 μg / ml was first verified. It was found that the modified materials at concentrations of 50 and 100 μg / ml exhibited good safety while also promoting osteoblast proliferation. The best antibacterial effect was observed at a concentration of 100 μg / ml. Similarly, the antibacterial properties of modified titanium, nickel, magnesium, tantalum, and polyetheretherketone against MRSA were verified at 100 μg / ml. It was found that the NGCDs-modified materials maintained good anti-MRSA properties, with only bacterial debris visible on the material surface.

[0060] like Figure 13As shown, a bone implantation model with MRSA infection was established to verify the in vivo antibacterial and osteogenic properties of the material. At 8 weeks, MicroCT was used to more intuitively assess the femoral infection status and osteogenic formation on the surface of the implanted material in each group of rats. The red cylinders represent the implanted material, while the yellow material on the surface of the cylinders represents new bone. It can be observed that the femoral infection of rats implanted with single-crystal materials Ti, Ta, Ni, Mg, and PEEK was severe, and the bone structure was severely damaged. The cross-sectional diagram shows that there was almost no new bone formation around the material. When reconstructing the implanted material, only a few sporadic new bone formations were visible on the material surface, indicating that the severe infection severely limited the osseointegration performance of the material. In the overall femoral reconstruction image, the infection in the Ti and PEEK groups had broken through the medullary cavity, resulting in large defects in the femur. In the Ta, Ni, and Mg groups, the infection signs were also severe, with irregular abscess-like bulges on the femoral surface, which was in stark contrast to the group implanted with modified materials. Furthermore, in the cross-sectional view of the modified material, uniform new bone can be seen surrounding the material. When the implant is reconstructed and analyzed separately, the surface of the modified material is almost completely covered by yellow new bone tissue. Moreover, the femoral reconstruction shows that the femoral shape is intact and there are no signs of infection. It can be seen that the modified implant has good antibacterial and osteointegration properties.

[0061] In summary:

[0062] The nanomaterials prepared in this invention exhibit stable antibacterial efficacy against six common implant-related infectious bacteria—Staphylococcus aureus, Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter cloacae—through mechanisms such as inhibiting biofilm formation, disrupting bacterial membrane structure, and inhibiting bacterial energy metabolism. (See attached diagram.) Figure 6 The results showed that none of the six types of bacteria grew in the experimental group with added NGCDs, while the control group was covered with bacteria, directly demonstrating its broad-spectrum antibacterial ability; (See attached image) Figure 7 The NGCDs group showed only red fluorescence representing dead bacteria, while the blue fluorescence representing live bacteria almost disappeared, further verifying the bactericidal efficacy; (See attached image) Figure 8 NGCDs intervention resulted in disruption of bacterial structural integrity and the appearance of numerous fragments, directly demonstrating its destructive effect on bacterial membranes and biofilms. Figure 9 The results showed that NGCDs did not change their minimum inhibitory concentration (MIC) against six bacteria within 20 weeks of sealed storage. This not only avoids the problem of antibiotics becoming ineffective with long-term use, but also reduces the risk of bacterial resistance from the root because it does not rely on antibiotic mechanisms.

[0063] Meanwhile, the nanomaterials prepared in this invention, through promoting glutathione production, scavenging reactive oxygen species (ROS), and improving mitochondrial metabolism, can significantly promote osteoblast proliferation while exhibiting antibacterial properties. The osteogenic effect is even better at concentrations of 30-60 μg / mL, with the most significant proliferation efficacy at 60 μg / mL. Figure 11 The results showed that NGCDs had no cytotoxicity within the concentration range of 30-90 μg / mL, and the osteoblast proliferation rate in this concentration range was higher than that in the control group, quantitatively confirming its osteogenic capacity; (See attached image) Figure 13 The results showed that rats implanted with NGCDs modified materials had intact femoral morphology and no signs of infection, with uniform new bone formation around the material; while rats implanted with unmodified materials had severe femoral infection and obvious bone destruction, with no new bone formation around the material. This directly verifies that it can still efficiently promote bone formation in an infected microenvironment, achieving a dual effect of antibacterial and bone-promoting action, and breaking the vicious cycle of "infection-osteogenic disorder".

[0064] The nanomaterials prepared in this invention exhibit no cytotoxicity at concentrations of 30-90 μg / mL and can actively promote osteoblast proliferation; (See attached image) Figure 11 It was confirmed that osteoblast survival remained at normal levels at concentrations of 30-90 μg / mL, and cell proliferation rate was significantly higher in the 30-60 μg / mL group than in the control group; (See attached image) Figure 12 The results showed that titanium (Ti), tantalum (Ta), nickel (Ni), magnesium (Mg), and polyether ether ketone (PEEK) materials modified with NGCDs at concentrations of 50-100 μg / mL could promote osteoblast proliferation, further demonstrating their safety in implant modification scenarios and completely avoiding the risk of long-term antibiotic retention damaging normal host cells.

[0065] The nanomaterials prepared in this invention are directly loaded onto the surface of implants using polyvinyl butyral (PVB). After implantation, they can continuously release active ingredients at the site of infection, without relying on systemic administration or tissue penetration. This ensures that the local drug concentration at the lesion site remains at an effective antibacterial / osteogenic level, thus addressing the core pain point of "insufficient concentration leading to antibacterial failure" from the perspective of drug delivery. Figure 12 The results showed that only bacterial fragments were observed on the surface of various implant materials modified with 100 μg / m LNGCDs, with no intact bacteria surviving, confirming that it can stably exert local antibacterial effects on the implant surface, overcoming the limitations of the closed microenvironment of the medullary cavity.

[0066] Finally, the nanomaterials prepared in this invention can be widely loaded onto the surfaces of common orthopedic implant materials such as titanium (Ti), tantalum (Ta), nickel (Ni), magnesium (Mg), and polyetheretherketone (PEEK). Figure 12 Appendix Figure 13 All studies have verified the modification effects of the above five types of materials, and the compatibility is far superior to single-material modification schemes. The preparation method does not require complex equipment and is easy to operate. The implant modification process can be achieved through PVB loading, without the need for special surgical operations, which is low-cost and easy to promote on a large scale in clinical implantation surgery, thereby reducing the cost of medical applications.

[0067] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0068] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a dual-targeted antibacterial osteogenesis-promoting nanomaterial for implant modification, characterized in that: The nanomaterial is prepared by using anhydrous citric acid as a carbon source, and polyhexamethylene guanidine hydrochloride and diallyl dimethyl ammonium chloride as sources of heteroatom doping, respectively providing guanidine groups and ammonium groups. The nanomaterial preparation method comprises the following steps: S1, solution preparation: prepare anhydrous citric acid and polyhexamethylene guanidine hydrochloride, dissolve them in ultrapure water, and then add diallyl dimethyl ammonium chloride after complete dissolution, stir to form a uniform solution, and the mass ratio of anhydrous citric acid, polyhexamethylene guanidine hydrochloride and diallyl dimethyl ammonium chloride is 0.4:0.2:2.5; S2, hydrothermal reaction: transfer the uniform solution in step S1 to a reaction kettle, seal it, and then place it in a heating box, and heat it at 180℃ for 6h; S3, filtration and purification: after the reaction in step S2 is completed and the material in the reaction kettle is naturally cooled to room temperature, the material is filtered by using a microporous filter membrane, and the filtrate is collected; The filtrate is transferred to a cellulose dialysis bag and dialyzed in ultrapure water for purification, and the total dialysis time is 72h, wherein the ultrapure water is replaced every 2h for the first 24h, and then every 6h after 24h; S4, freeze-drying: the filtrate after dialysis and purification in step S3 is divided into centrifuge tubes and subjected to freeze-drying treatment, and finally a double-targeted antibacterial and bone formation promoting material is obtained.

2. The method of claim 1, wherein the method is for preparing a dual-targeting antibacterial osteogenesis-promoting nanomaterial for implant modification. The nanomaterial has stable antibacterial effect on common infectious bacteria through the mechanisms of inhibiting biofilm formation, destroying bacterial membrane structure and inhibiting bacterial energy metabolism; the common infectious bacteria include Staphylococcus aureus, Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterobacter cloacae.

3. The method of claim 1, wherein the method is characterized by: The nanomaterial has no cytotoxicity in the concentration range of 30μg / mL-90μg / mL, has biological safety, and has the effect of promoting the proliferation of osteoblasts; The nanomaterial promotes bone formation by promoting the generation of glutathione, scavenging reactive oxygen species and improving mitochondrial metabolism, thereby improving the bone regeneration process in the infected microenvironment.

4. The method of claim 1, wherein the method is for preparing a dual-targeting antibacterial osteogenesis-promoting nanomaterial for implant modification. The element composition of the nanomaterial contains 77.2% carbon atoms, 10.24% nitrogen atoms and 12.56% oxygen atoms, and the surface contains alkyl chains, guanidine groups and quaternary ammonium group functional groups.

5. The method of claim 1, wherein the method is for preparing a dual-targeting antibacterial pro-osteogenic nanomaterial for implant modification. In step S1, the anhydrous citric acid is 0.4g, the polyhexamethylene guanidine hydrochloride is 0.2g, and the diallyl dimethyl ammonium chloride is 4mL; In step S1, the ultrapure water is 16mL.

6. The method of claim 1, wherein the method is for preparing a dual-targeting antibacterial pro-osteogenic nanomaterial for implant modification. In step S3, the pore size of the microporous filter membrane is 0.22μm, and the molecular weight cut-off of the cellulose dialysis bag is 500-1000Da.

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