Gallium ion and enzyme co-loaded antibacterial agent, and preparation method and application thereof

Abstract

The application discloses a gallium ion and enzyme co-loaded antibacterial agent and a preparation method and application thereof. The antibacterial agent can break through the influence of a microbial cell wall and a biofilm, further release gallium ions to produce active oxygen, metabolic interference and the like, and cooperatively kill or inhibit microorganisms. The antibacterial agent is different from existing antibiotic therapy, and the problem of microbial drug resistance is weakened. Meanwhile, the gallium ions, enzymes and nano carriers used have obvious safety advantages relative to commonly used metal silver ions and the like antibacterial ions. The antibacterial agent can be further applied to antibacterial treatment of infectious diseases such as a microbial infected skin wound and a microbial infected eye wound, and has excellent treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a gallium ion-co-loaded antibacterial agent with the ability to degrade microbial cell walls or biofilm matrices, its preparation method, and its application. Besides achieving a highly effective combination of multiple antibacterial measures against drug-resistant bacteria, fungi, and other microorganisms, it can also be further applied to the treatment of infectious diseases such as ocular or dermal microbial infections. Background Technology

[0002] Microbial infections, such as bacterial and fungal infections, have become a very challenging problem in public health. While antibiotic use has inhibited microbial proliferation to some extent, it has also led to the development of multidrug resistance. Furthermore, biofilms formed by microorganisms further enhance drug resistance, rendering many antibiotics ineffective. Therefore, finding other low-cost, efficient bactericidal pathways is more important than investing significant research costs and time in developing new antibiotics.

[0003] Metal ions, such as silver and copper ions, have attracted widespread attention for inhibiting or killing microorganisms through various pathways, including disrupting surface proteins, generating reactive oxygen species, and damaging cell membranes. However, while commonly used silver ion antibacterial agents possess strong antibacterial capabilities, their toxicity to normal cells cannot be ignored, necessitating the search for safer metal ion formulations. Research has found that microorganisms have an extremely high uptake of iron ions during growth and development, and interfering with iron ion metabolism is a potential pathway for inhibiting microorganisms. Gallium ions have a highly similar ionic structure to iron ions and can act as a "Trojan horse" for microorganisms to take up. However, because gallium cannot be reduced from a high valence state to a low valence state like iron ions, it leads to microbial death. Gallium ion formulations, such as gallium nitrate, have been approved by the US Food and Drug Administration for use as an injectable treatment for hypercalcemia, and even at high doses, they maintain significant safety.

[0004] While gallium ions possess potential and safe antimicrobial capabilities, infectious diseases, such as skin and corneal wound infections, are often caused by microorganisms forming biofilms. These biofilms contain abundant polysaccharides, proteins, and other matrix materials, which can block the entry of antibiotics or metal ions, significantly reducing the effectiveness of antibacterial agents and increasing microbial resistance.

[0005] In summary, this invention provides a combined strategy of disrupting biofilms and using metal ions to overcome the problem of refractory microbial infections. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a gallium ion and enzyme co-loaded antibacterial agent, its preparation method, and its applications. This antibacterial agent can overcome the influence of microbial cell walls and biofilms, further rapidly releasing gallium ions to generate reactive oxygen species and disrupt metabolism, thereby synergistically killing or inhibiting microorganisms. Besides mitigating the problem of microbial resistance compared to existing antibiotic therapies, this antibacterial agent also offers significant safety advantages over commonly used antibacterial agents such as those using gallium ions, enzymes, and nanocarriers.

[0007] To achieve the above objectives, the present invention employs the following technical measures: a gallium ion and enzyme co-loaded antibacterial agent, its preparation method and application, wherein the technical method is as follows:

[0008] A gallium ion and enzyme co-loaded antibacterial agent, by mass percentage, comprises: 30%–90 wt% nanocarrier, 1%–30 wt% water-soluble gallium salt, 1%–30 wt% enzyme, and 1%–30 wt% chelating agent. The antibacterial agent is an amorphous material, wherein gallium is chelated therein in ionic form, and the enzyme is an enzyme that has the function of degrading or destroying components in the cell wall or biofilm matrix of microorganisms.

[0009] A method for preparing a gallium ion and enzyme co-loaded antibacterial agent, comprising:

[0010] The enzyme is mixed with a nanocarrier in solution, and through interaction, the enzyme is loaded onto the nanocarrier to obtain enzyme-loaded nanoparticles; the enzyme is an enzyme that has the function of degrading or destroying components in the cell wall or biofilm matrix of microorganisms.

[0011] A chelating agent was added to the prepared enzyme-carrying nanoparticles, and a water-soluble gallium salt was added to bind gallium.

[0012] After a period of reaction, excess unreacted material is removed by washing to obtain a gallium ion and enzyme co-loaded antibacterial agent.

[0013] In the above technical solution, the nanocarrier is further selected from one or more of porous silica, porous carbon, liposomes, polylactic acid, chitosan, dendritic polymers, and metals.

[0014] Furthermore, the enzyme is selected from one or more of the following: lysozyme, glucanase, cellulase, mannanase, protease, deoxyribonuclease, and chitinase.

[0015] Furthermore, the chelating agent is selected from one or more of dopamine, sodium hyaluronate, polyvinyl alcohol, chitosan, sodium alginate, calcium alginate, gelatin, and cellulose derivatives.

[0016] Furthermore, the antibacterial agent is composed of nanoparticles with a particle size at the nanoscale.

[0017] Furthermore, the antibacterial agent also includes pharmaceutically acceptable excipients.

[0018] Furthermore, the solution is one or more of water, phosphate buffer, citrate buffer, acetate buffer, and Tris-HCl buffer.

[0019] The present invention also provides an antibacterial and wound-healing agent for infected wounds, comprising the antibacterial agent as described above. The infected wound is a microbial infection, wherein the microorganism is bacteria or fungus.

[0020] Compared with the prior art, the present invention has the following advantages and effects:

[0021] The advantages of this invention lie in providing a gallium ion and enzyme co-loaded antibacterial agent and its preparation method. This antibacterial agent is a non-antibiotic antibacterial preparation that avoids antibiotic resistance by combining multiple antibacterial mechanisms, such as generating reactive oxygen species, interfering with iron metabolism, and disrupting or degrading the microstructure of microorganisms and their biofilms. Simultaneously, by combining enzymes that target the microstructure of microorganisms or the biofilm matrix with gallium ions, the antimicrobial ability of metal ions can be effectively improved, particularly reducing the barrier effect of the biofilm matrix on metal ions. Furthermore, the main components of the antibacterial agent all exhibit significant biocompatibility, with gallium ions showing better safety and lower cytotoxicity compared to traditional antibacterial agents such as silver and copper ions. The key components of the antibacterial agent are presented in nanoparticle form, which can effectively protect the internal active ingredients and improve retention and duration of action at the lesion site during further disease application.

[0022] This invention utilizes a combined strategy of enzymes and metal ions to synergistically disrupt biofilms for the treatment of infections, overcoming the following technical challenges: 1) In biological applications, enzymes and metal ions often lose activity or aggregate when faced with interference from the microenvironment, such as temperature, solution salinity, and protein degradation. Direct use of enzymes or metal ions significantly reduces their antibacterial capabilities. 2) In addition to the inherent reduction in their activity, metal ions also interfere with enzyme activity, preventing both from maintaining high activity simultaneously. Therefore, this invention uses a nanocarrier to simultaneously load gallium ions and enzymes that degrade biofilms or cell walls, combining gallium ions and enzymes in a specific manner. On the one hand, nano-sizing protects both the enzyme and gallium, reducing the impact of the external microenvironment on their activity. On the other hand, by first encapsulating the enzyme and then chelating gallium ions, this invention reduces the probability of direct contact between gallium ions and the internal enzyme, minimizing the destructive effect of gallium ions on enzyme activity. This achieves excellent therapeutic effects against infections caused by drug-resistant bacteria, fungi, and other microorganisms and their biofilms. Furthermore, the present invention uses chelation of gallium ions instead of forming crystalline particles, which increases the ion release rate, reduces the difficulty of antibacterial agent degradation, and improves the safety of antibacterial agent in biological applications. Attached Figure Description

[0023] Figure 1 This is a transmission electron micrograph (TEM) of the gallium ion and enzyme co-loaded antibacterial agent synthesized in Example 1.

[0024] Figure 2 This is a scanning electron microscope (SEM) image of the gallium ion and enzyme co-loaded antibacterial agent synthesized in Example 2.

[0025] Figure 3 The hydrated particle size distribution of the gallium ion and enzyme co-loaded antibacterial agent synthesized in Example 3 is shown in the diagram.

[0026] Figure 4 This is a scanning electron microscope (SEM) image of the gallium ion and enzyme co-loaded antibacterial agent synthesized in Example 1 after co-culturing with fungi.

[0027] Figure 5 This is a diagram illustrating the effect of the gallium ion and enzyme co-loaded antibacterial agent synthesized in Example 2 on the healing of bacterial skin infections in mice.

[0028] Figure 6 The image shows the effect of the gallium ion and enzyme co-loaded antibacterial agent synthesized in Example 1 on fungal corneal wounds in mice.

[0029] Figure 7 The X-ray diffraction pattern is shown for the gallium ion and enzyme co-loaded antibacterial agent synthesized in this invention, with the patterns of standard gallium single crystal and gallium oxide single crystal serving as controls.

[0030] Figure 8 This paper compares the antibacterial ion release rate of the gallium ion and enzyme co-loaded antibacterial agent synthesized in this invention with that of common nano-silver antibacterial agents.

[0031] Figure 9 This invention presents a comparison of the activity of the gallium ion and enzyme co-loaded antibacterial agent, the enzyme, and the enzyme after direct mixing and loading with gallium ions.

[0032] Figure 10 This paper compares the stability of the gallium ion and enzyme co-loaded antibacterial agent synthesized in this invention with that of directly using gallium ions under cell culture medium and fungal culture medium conditions.

[0033] Figure 11 The content of active gallium ions in the ocular lesions of infected mice was measured using gallium nitrate, an antibacterial agent co-loaded with gallium ions and enzymes synthesized in this invention, and a mixture of gallium nitrate and enzymes as eye drops. Detailed Implementation

[0034] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0035] The present invention discloses a gallium ion and enzyme co-loaded antibacterial agent, the preparation method of which includes the following steps:

[0036] (1) The nanocarrier and the enzyme are mixed in solution, and the enzyme is loaded onto the carrier through interaction.

[0037] (2) A chelating agent is added to the enzyme-loaded nanoparticles prepared in step (1), and a water-soluble gallium salt is added to bind gallium. After a period of reaction, excess unreacted material is removed by washing to obtain a gallium ion and enzyme co-loaded antibacterial agent.

[0038] In the present invention, the mass ratio of nanocarrier to enzyme in step (1) can be 0.01:1 to 100:1. The solution in step (1) for mixing the nanocarrier and enzyme includes, but is not limited to, water-soluble solutions such as water, phosphate buffer, citrate buffer, acetate buffer, and Tris-HCl buffer, as well as single or mixed solutions of polar and nonpolar solutions under synthesis conditions. The method for loading the enzyme onto the nanocarrier in step (1) includes, but is not limited to, commonly used physicochemical methods such as ultrasonic vibration, stirring, electrostatic adsorption, covalent modification, and microemulsion. The temperature for loading the enzyme onto the nanocarrier in step (1) includes, but is not limited to, 0-50℃. The time for loading the enzyme onto the nanocarrier in step (1) includes, but is not limited to, 1s-72h.

[0039] More preferably, the order in which the chelating agent is added in step (2) includes, but is not limited to, adding it before the soluble gallium salt, adding it simultaneously with the soluble gallium salt, or adding a portion first and then adding another portion simultaneously with the soluble gallium salt. The reaction time in step (2) includes, but is not limited to, 1 second to 72 hours.

[0040] This invention employs a method of first loading the enzyme onto a nanocarrier to obtain enzyme-loaded nanoparticles, and then chelating gallium ions. Compared to directly loading the enzyme and gallium ions onto the nanocarrier or loading gallium ions first and then loading the enzyme, this invention reduces the probability of direct contact between gallium ions and the enzyme before action, minimizing interference from metal ions on enzyme activity. This maximizes the protection of enzyme activity and ensures the synergistic antibacterial effect of the antibacterial agent.

[0041] The gallium ion and enzyme co-loaded antibacterial agent obtained in this invention is an amorphous material in which gallium is chelated in ionic form. Compared with antibacterial agents made of inorganic crystalline particles using conventional methods, the antibacterial agent obtained in this invention can more readily release gallium ions during subsequent biomedical applications, thus enabling faster degradation and improving its biocompatibility. The antibacterial agent of this invention, by mass percentage, comprises 30%–90 wt% nanocarrier, 1%–30 wt% water-soluble gallium salt, 1%–30 wt% enzyme, and 1%–30 wt% chelating agent. The nanocarrier includes porous silica, porous carbon, liposomes, polylactic acid, chitosan, dendritic polymers, metal nanoparticles, etc.; preferably mesoporous silica, polylactic acid, and liposomes, which have excellent enzyme encapsulation capabilities and biocompatibility; the particle size of the nanoparticles is 5–1000 nanometers. The gallium salt is a water-soluble gallium salt such as gallium nitrate, gallium chloride, gallium acetate, or gallium sulfate. The enzymes mentioned include one or more enzymes that can degrade or destroy components in the cell wall or biofilm matrix of microorganisms, such as lysozyme, glucanase, cellulase, mannanase, protease, deoxyribonuclease, and chitinase. The chelating agent is one or more biopharmaceutical polymers such as dopamine, sodium hyaluronate, polyvinyl alcohol, chitosan, sodium alginate, calcium alginate, gelatin, and cellulose derivatives; preferably dopamine. When dopamine is used as a chelating agent in this invention, the chelation of dopamine with gallium ions will generate an additional Raman spectral signal. The release of gallium ions and the drug distribution at the site of infection can be monitored by monitoring the Raman signal generated by chelation. Therefore, the resulting product is not only an antibacterial agent but also an antibacterial agent that can monitor the release of gallium ions in real time.

[0042] Furthermore, the antibacterial agent of the present invention can be used as an antibacterial and wound-healing agent for infected wounds. This agent may also contain pharmaceutically acceptable excipients, which allow the nano-antibacterial agent to be prepared into any dosage form suitable for clinical use, including but not limited to injections, tablets, powders, granules, capsules, gels, oral preparations, ointments, creams, and sprays. It can be applied to the antibacterial treatment of infectious diseases, including but not limited to skin wounds and eye wounds infected with microorganisms. Experiments have demonstrated that this antibacterial agent has excellent therapeutic effects and can replace existing conventional drugs.

[0043] Example 1:

[0044] A method for preparing a gallium ion and enzyme co-loaded antibacterial agent, comprising the following steps:

[0045] (1) The porous silica nanocarrier and the lysozyme were mixed in an aqueous solution with a mass ratio of 1:0.8. The solution was subjected to ultrasonic vibration with a probe for 5 min under an ice bath at 4°C to load the lysozyme onto the porous silica nanocarrier.

[0046] (2) Dopamine hydrochloride was added to the lysozyme-loaded nanoparticles prepared in step (1) in a Tris-HCl buffer solution at pH 9. After stirring for 2 hours, dopamine hydrochloride and gallium nitrate were added simultaneously to allow gallium to bind. The reaction was then stirred for 6 hours. Finally, excess unreacted material was removed by washing to obtain an antibacterial agent co-loaded with gallium ions and lysozyme.

[0047] Figure 1 The TEM image shows the gallium ion and enzyme co-loaded antibacterial agent, clearly revealing its porous nanoparticle structure. A scanning electron micrograph (SEM) image of the gallium ion and enzyme co-loaded antibacterial agent obtained in this example after co-culturing with fungi is shown below. Figure 4 As shown in the image, after the antibacterial agent was applied, the fungus broke down and leaked, with a large amount of antibacterial agent adhering to its surface. The effect of applying the synthesized gallium ion and enzyme co-loaded antibacterial agent to a fungal corneal wound in mice is shown in the image. Figure 6 As shown, by monitoring the fungal keratitis caused by Candida albicans in mice of different control groups, it can be seen that the gallium ion and enzyme co-loaded antibacterial agent, under single-dose administration, is more effective than amphotericin B, exhibiting the best bactericidal and corneal healing effects. Furthermore, the XRD detection results of the antibacterial agent prepared in this invention are as follows... Figure 7 As shown, this antibacterial agent is an amorphous material, with gallium chelated in the antibacterial agent in ionic form rather than forming inorganic crystal particles. This is more conducive to the rapid release of gallium ions. Compared with conventional nano-silver antibacterial agents, the antibacterial ion release rates of the two are as follows: Figure 8As shown; the stability of the synthesized gallium ion and enzyme co-loaded antibacterial agent compared with that of directly using gallium ions under cell culture and fungal culture conditions is as follows: Figure 10 .

[0048] Figure 11 This invention relates to the co-loaded antibacterial agent of gallium ions and enzymes synthesized in this invention, specifically gallium nitrate, and a mixture of gallium nitrate and enzymes, respectively, and the changes in the content of active gallium ions in infectious mouse ocular lesions as eye drops. The antibacterial agent prepared using the method of this invention can more effectively exert the antibacterial effects of enzymes and gallium ions, achieving excellent therapeutic effects.

[0049] Example 2:

[0050] A method for preparing a gallium ion and enzyme co-loaded antibacterial agent, comprising the following steps:

[0051] (1) Lysozyme was loaded onto polylactic acid-glycolic acid copolymer (PLGA) nanocarriers in a solution of dichloromethane and water under ice bath conditions at 4°C by microemulsion method, wherein the mass ratio of PLGA nanocarriers to lysozyme was 10:1, so that the PLGA nanocarriers were loaded with lysozyme.

[0052] (2) Dopamine hydrochloride was added to the lysozyme-loaded nanoparticles prepared in step (1) in a Tris-HCl buffer solution at pH 9. Gallium chloride was then added to allow gallium to bind, and the reaction was stirred for 12 hours. Finally, excess unreacted material was removed by washing to obtain a gallium ion-lysozyme co-loaded antibacterial agent.

[0053] Figure 2 The SEM image of the gallium ion and enzyme co-loaded antibacterial agent prepared in this example shows that the antibacterial agent has a nanospherical structure. The gallium ion and enzyme co-loaded antibacterial agent prepared in this example was used in a mouse skin bacterial (Pseudomonas aeruginosa) infection healing experiment, and the effect is shown in the image below. Figure 5 By monitoring the healing of skin infected with Pseudomonas aeruginosa in the control group and the wounds infected with bacteria treated with gallium enzyme co-loaded antibacterial agent after infection, it can be seen that gallium ions and enzyme co-loaded antibacterial agent have a good antibacterial and healing-promoting effect.

[0054] Example 3:

[0055] A method for preparing a gallium ion and enzyme co-loaded antibacterial agent, comprising the following steps:

[0056] (1) The porous carbon nanocarrier and glucanase were mixed in an aqueous solution, wherein the mass ratio of porous carbon to glucanase was 1:2. The porous carbon nanocarrier was loaded with glucanase by stirring at 25°C for 24 hours.

[0057] (2) Add dopamine hydrochloride to the dextranase-loaded nanoparticles prepared in step (1) in a pH 9 Tris-HCl buffer solution. Add dopamine hydrochloride and gallium nitrate simultaneously to allow gallium to bind. Stir the reaction for 12 hours. Finally, wash away excess unreacted material to obtain a gallium ion-dextranase co-loaded antibacterial agent.

[0058] The size distribution of the antibacterial agent prepared in this example is as follows: Figure 3 As shown, the antibacterial agent has a nanoscale size (approximately 120 nanometers) and is evenly distributed with no large particles appearing.

[0059] The antibacterial agent of this invention is prepared using a specific method, which effectively protects enzyme activity and gallium ion stability. The synthesized gallium ions and enzyme are co-loaded with the antibacterial agent, and the enzyme activity is compared with that of the enzyme and gallium ions directly mixed and loaded. The results are as follows: Figure 9 .

[0060] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A gallium ion and enzyme co-loaded antibacterial agent, characterized in that, By weight percentage, including: The antibacterial agent comprises 30%–90 wt% nanocarrier, 1%–30 wt% water-soluble gallium salt, 1%–30 wt% enzyme, and 1%–30 wt% chelating agent. The antibacterial agent is an amorphous material in which gallium is chelated in ionic form. The nanocarrier is porous silica, porous carbon, or polylactic acid-glycolic acid copolymer. The chelating agent is dopamine. The enzyme is an enzyme that degrades or destroys components in the cell wall or biofilm matrix of microorganisms, selected from one or more of lysozyme, lysozyme, dextranase, cellulase, mannanase, protease, deoxyribonuclease, and chitinase. The preparation method of the antibacterial agent includes: The enzyme is mixed with the nanocarrier in solution, and through interaction, the enzyme is loaded onto the nanocarrier to obtain enzyme-loaded nanoparticles. A chelating agent was added to the prepared enzyme-carrying nanoparticles, and a water-soluble gallium salt was added to bind gallium. After a period of reaction, excess unreacted material is removed by washing to obtain a gallium ion and enzyme co-loaded antibacterial agent.

2. A method for preparing an antibacterial agent co-loaded with gallium ions and an enzyme, characterized in that, include: The enzyme is mixed with a nanocarrier in solution, and through interaction, the enzyme is loaded onto the nanocarrier to obtain enzyme-loaded nanoparticles; the enzyme is an enzyme that has the function of degrading or destroying components in the cell wall or biofilm matrix of microorganisms. A chelating agent was added to the prepared enzyme-carrying nanoparticles, and a water-soluble gallium salt was added to bind gallium. After a period of reaction, excess unreacted material is washed away to obtain a gallium ion and enzyme co-loaded antibacterial agent; The nanocarrier is porous silica, porous carbon, or polylactic acid-glycolic acid copolymer; the chelating agent is dopamine; and the enzyme is selected from one or more of the following: lysozyme, glucanase, cellulase, mannanase, protease, deoxyribonuclease, and chitinase.

3. The antibacterial agent according to claim 1 or the antibacterial agent obtained by the method according to claim 2, characterized in that, The antibacterial agent is composed of nanoparticles with a particle size at the nanoscale.

4. The antibacterial agent according to claim 1 or the antibacterial agent obtained by the method according to claim 2, characterized in that, The antibacterial agent also includes pharmaceutically acceptable excipients.

5. The preparation method according to claim 2, characterized in that, The solution is one or more of water, phosphate buffer, citrate buffer, acetate buffer, and Tris-HCl buffer.

6. An antibacterial and wound-healing agent for infected wounds, characterized in that, It contains the antibacterial agent as described in claim 1.

7. The drug as described in claim 6, characterized in that, The infectious wound is a microbial infection, and the microorganism is bacteria or fungi.

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