A copper-source heterojunction nano antibacterial agent and its preparation method and application

Abstract

The present invention discloses a copper-source heterojunction nano antibacterial agent, a preparation method and its application. The preparation method includes: 1) dispersing cuprous oxide nanoparticles synthesized by using a polymer template in water; 2) adding a silver nitrate aqueous solution to the solution obtained in 1) to react and obtain a copper-source nano heterojunction particle solution; 3) adding a surface modifier to the obtained solution to perform surface coating modification; 4) reacting to obtain coated copper-source nano heterojunction particles, or mixing them with auxiliary materials to obtain a copper-source heterojunction nano antibacterial agent. The present invention forms a nano heterojunction by forming a nano cuprous oxide with silver, and performs coating modification on the surface thereof, so as to achieve a combination of efficient photothermal, photodynamic, chemical reaction kinetics, silver ion and other multiple antibacterial means for, for example, drug-resistant bacteria, fungi and other microorganisms and biofilm infections formed by them, and can be applied to the treatment of infectious diseases such as eye microorganisms or skin microorganisms, and has excellent therapeutic effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to a multifunctional copper-source heterojunction nano antibacterial agent, and a preparation method and application thereof. By forming a nano heterojunction with nano cuprous oxide and silver, and performing coating modification on the surface, a combination of multiple antibacterial means such as photothermal, photodynamic, chemical reaction kinetics, and silver ions can be achieved to effectively treat infections such as resistant bacteria, fungi and other microorganisms and biofilms formed by them, and the agent can be applied to the treatment of infectious diseases such as eye microbial or skin microbial infections. Background Art

[0002] Microbial infection problems, such as bacterial infection and fungal infection, have become a very thorny issue in the field of public health. At present, antibiotics are the main means of resisting microbial infections. Although the use of antibiotics inhibits the proliferation of microorganisms to a certain extent, it also causes the generation of multidrug-resistant strains. In addition to the enhanced drug resistance of free microorganisms themselves, the biofilms they form further enhance the resistance of microorganisms to drugs. For example, the minimum inhibitory concentration of antifungal drugs in Candida albicans biofilms can be increased to nearly 1,000 times that of free Candida albicans. Therefore, there is an urgent need to invest a lot of research costs and time to develop new and efficient bactericidal pathways and strategies.

[0003] The development of nanotechnology has stimulated the application potential of nanomaterials in the biomedical field. Compared with traditional copper ion and soluble copper compound preparations, nano copper-based particle preparations have more excellent antimicrobial properties due to their unique small size effect, quantum effect and large specific surface area, and thus have attracted widespread attention. This type of nano copper particle can synergistically produce antimicrobial activity through multiple mechanisms such as releasing antimicrobial copper ions to destroy microbial cell membrane proteins and generating reactive oxygen under the action of light, effectively avoiding the problem of multidrug resistance. However, the killing ability of single copper-based nanoparticles is greatly reduced for microbial biofilms with stronger drug resistance and more complex structures.

[0004] At present, the local photothermal effect produced by the interaction between materials and external light sources, especially near-infrared (NIR) with low absorption and scattering of biological tissues and body fluids, can also effectively inhibit microorganisms and their biofilms. This photothermal therapy (PTT) based on the photothermal effect of nanomaterials has been widely used in anti-tumor research, but it is in a period of vigorous development in the field of antimicrobial. However, on the one hand, the optical absorption band of copper-based nanoparticles is generally in the visible-ultraviolet band and cannot directly match NIR light to produce a high photothermal effect, and its morphology, size, etc. need to be further regulated. At the same time, it is also necessary to carry out targeted design for the characteristics that there are a large number of matrix materials such as polysaccharides and proteins inside the biofilm, which will block the entry of antibiotics or metal ions.

[0005] To this end, the present invention provides a copper-source heterojunction nano antibacterial agent, which overcomes the problem of refractory microbial infection by combining multiple antimicrobial and anti-biofilm means with a single nano preparation. Summary of the invention

[0006] The purpose of the present invention is to provide a copper-source heterojunction nano antibacterial agent and its preparation method and application in view of the deficiencies of the prior art. The antibacterial agent can break through the influence of microbial microstructure and biofilm by combining multiple pathways including photothermal, photodynamic, chemical reaction kinetics, silver ion release, etc., and inhibit microorganisms in collaboration with near-infrared laser or alone. In addition to being different from existing antibiotic therapies and reducing the problem of microbial resistance, the antibacterial agent is based on low-cost cuprous oxide, and quickly forms a nano antibacterial agent that can be activated by near-infrared with silver nanoparticles, which has obvious advantages in synthesis cost and simple synthesis process.

[0007] In order to achieve the above-mentioned purpose, the technical method adopted by the present invention is as follows:

[0008] A method for preparing a copper-source heterojunction nano antibacterial agent, comprising:

[0009] 1) uniformly dispersing cuprous oxide nanoparticles synthesized using a polymer template in water;

[0010] 2) adding the silver nitrate aqueous solution to the cuprous oxide nanoparticle solution obtained in 1), stirring at room temperature for mixing reaction, and obtaining a copper source nano heterojunction particle solution;

[0011] 3) adding a surface modifier to the obtained copper source nano heterojunction particle solution to modify the surface coating; 4) after the reaction, centrifuging and washing to remove excess unreacted materials to obtain coating-modified copper source nano heterojunction particles, or mixing them with auxiliary materials to obtain copper source heterojunction nano antibacterial agents.

[0012] In the above technical scheme, further, the preparation method of the cuprous oxide nanoparticles synthesized by the polymer template includes the following steps: dispersing the polymer template in water with a mass concentration of 0.5-2%, then dropping a 0.1-1M soluble copper source solution therein, stirring at 800-1200rpm for at least 30min, dropping a hydrazine hydrate solution with a mass fraction of 85% to react for 1-60min, and washing to obtain the cuprous oxide nanoparticles synthesized by the polymer template by further centrifugation. The copper source is a water-soluble copper salt such as copper nitrate, copper acetate, copper sulfate, etc.

[0013] Furthermore, the polymer template is one or more of polyvinyl pyrrolidone, sodium hyaluronate, porous silica, liposome, and dendrimer.

[0014] Furthermore, the mass concentration ratio of silver to copper is preferably 0.01-5:1.

[0015] Furthermore, the surface modifier is one or more of ethylenediaminetetraacetic acid, tartaric acid, hydroxyethylethylenediaminetriacetic acid, dopamine, sodium hyaluronate, polyvinyl alcohol, chitosan, sodium alginate, calcium alginate, gelatin, and cellulose derivatives.

[0016] The antibacterial agent prepared by the above method contains heterojunction nanoparticles formed by nano cuprous oxide and nano silver, and the antibacterial agent includes, by mass percentage: 1% to 90wt% copper source nano heterojunction particles, 1% to 30wt% surface modifier, and 0% to 90wt% auxiliary materials; the antibacterial agent has near infrared light absorption ability, absorption in the optical region above 600nm, and light-heat conversion ability under NIR laser irradiation, including but not limited to being able to efficiently convert light into heat in a short time at 808nm, 980nm, or 1064nm, and quickly heat up. The auxiliary materials are pharmaceutically acceptable auxiliary materials. Using these auxiliary materials, the nano antibacterial agent can be prepared into any dosage form suitable for clinical use, including but not limited to gel, drops, injections, tablets, powders, granules, capsules, gels, oral agents, ointments, creams, sprays, etc. It can be used in antibacterial treatment of infectious diseases including but not limited to skin wounds infected by microorganisms, eye wounds infected by microorganisms, etc.

[0017] The copper-source heterojunction nano antimicrobial agent can be used to prepare drugs for destroying microbial cell walls or biofilms, killing microorganisms and / or promoting healing of infectious wounds. The infectious wounds are microbial infections, and the microorganisms are bacteria or fungi. It can be applied to antibacterial treatment of infectious diseases including but not limited to skin wounds infected by microorganisms, eye wounds infected by microorganisms, etc. Experiments have shown that the antimicrobial agent has a very excellent therapeutic effect and can replace existing conventional drugs.

[0018] Compared with the prior art, the present invention has the following advantages and effects:

[0019] The antibacterial agent of the present invention combines copper source nano heterojunction particles with surface modification coatings and achieves stable existence through auxiliary materials, fully utilizing the interaction of the modified coatings with the components in the microbial cell wall or biofilm matrix, and combining the optical absorption of the antibacterial agent in the near-infrared region and the heating ability under NIR laser, is a new and efficient antibacterial method; the preparation method of the antibacterial agent is to use specific cuprous oxide as the main body, react with silver nitrate in situ, and form the final preparation by compounding the formed cuprous oxide-silver nano composite heterojunction particles and surface modification agents and medical auxiliary materials, and the method is simple, green, and can be prepared on a large scale. In this scheme, by designing the formation of a cuprous oxide-silver heterojunction, the overall optical absorption is tuned to the NIR band of the biological tissue window, and the heating ability can be efficiently generated under near-infrared laser irradiation. The generated local heating can not only be used as a new type of photothermal antibacterial means, but also can promote the release of silver ions and copper ions. The released copper ions can also mediate a Fenton-like reaction to produce hydroxyl radicals. At the same time, the heterojunction structure brings a new photocatalytic effect and realizes a photodynamic effect. Thus, while achieving the synergistic broad-spectrum bactericidal effects including photothermal, photodynamic, chemical reaction kinetics, silver ions, etc., the overall nanoparticle intake dose is reduced and biosafety is improved. The synergy of the above multiple antibacterial pathways, combined with the surface modification coating of the nanoparticles that has a certain degree of destruction or binding to the biofilm matrix, can effectively reduce the barrier effect of the biofilm matrix on metal ions and improve the destructive effect of the antibacterial agent on the biofilm. On the other hand, the antibacterial agent provided by the present invention can be used as a contrast agent for optical coherence tomography and photoacoustic imaging, and can monitor in real time the retention of the antibacterial agent in the lesion, providing accurate guidance for medical staff on drug administration.

[0020] The present invention utilizes a combination strategy of a nano-heterojunction formed by cuprous oxide and silver and a surface modification coating technology to synergistically destroy microorganisms, especially refractory biofilms to treat infection problems, and overcomes the following technical difficulties: 1) Rapid regulation of optical absorption bands: In order to obtain optical absorption in the near-infrared region, so as to achieve the photothermal therapy or photodynamic therapy capability generated by the near-infrared light response, it is usually necessary to control the morphology in a relatively complex manner or to form a complex core-shell structure with gold nanoparticles. The present invention uses inexpensive cuprous oxide as the main body, and only requires a simple in-situ reduction of a silver nitrate solution to quickly form a nano-heterojunction of cuprous oxide and silver, directly achieving strong optical absorption in the near-infrared region. 2) Combination of multifunctional antibacterial pathways: Most nano-antibacterial agents can only achieve the integration of limited antibacterial means such as photothermal or photodynamic, making the antibacterial ability not prominent enough. The present invention selects a heterojunction formed by cuprous oxide and silver as the main antibacterial component, and realizes the coupling of antibacterial methods including photothermal, photodynamic, chemical reaction kinetics, ion antibacterial, photocatalysis, etc., at one time, greatly improving the antibacterial effect. 3) Highly efficient anti-biofilm capability: There are a large number of polysaccharides and proteins inside the biofilm, which will bind to antibacterial drugs and improve drug resistance. General nano antibacterial agents rely more on local photothermal destruction, but in actual application, excessively high temperatures can cause damage to the wound itself, such as corneal wound infection. The antibacterial agent provided by the present invention also modifies the surface of the nanoparticles with a coating for biofilm, which can interfere with the biofilm matrix, so that subsequent cascade treatment can penetrate deep into the lesion. In contrast, the present invention can produce a highly efficient antibacterial effect under mild photothermal conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a transmission electron microscopy (TEM) photograph of the copper-source heterojunction nano antibacterial agent synthesized in Example 1.

[0022] Figure 2 This is the surface charge distribution diagram of the copper-source heterojunction nano antibacterial agent synthesized in Example 2.

[0023] Figure 3 This is the hydrated particle size distribution diagram of the copper-source heterojunction nano antibacterial agent synthesized in Example 3.

[0024] Figure 4 Transmission electron microscopy (TEM) photos of the copper-source heterojunction nano antibacterial agent synthesized in Example 1 after co-culture with fungi and further irradiation with 808-nm laser.

[0025] Figure 5 This is the antibacterial concentration test after the copper-source heterojunction nano-antibacterial agent in Example 2 was co-cultured with drug-resistant bacteria, with antibiotics used as a comparison.

[0026] Figure 6This is a confocal micrograph of the copper-source heterojunction nano-antibacterial agent synthesized in Example 1 for the destruction of fungal biofilms, with the clinical antibacterial agent amphotericin B as a comparison.

[0027] Figure 7 This is a diagram showing the effect of the copper-source heterojunction nano-antibacterial agent synthesized in Example 1 on fungal-infected wounds of the mouse cornea, with the clinical antibacterial agent amphotericin B used as a comparison.

[0028] Figure 8 This is the X-ray diffraction pattern of the copper-source heterojunction nano-antibacterial agent synthesized in Example 1.

[0029] Fig. 9 The copper-based heterojunction nano antibacterial agent synthesized in Example 1 was compared with the near-infrared photothermal heating of common nano-silver and nano-cuprous oxide antibacterial agents.

[0030] Fig.10 This is a diagram showing the effect of the copper-source heterojunction nano antibacterial agent synthesized by the present invention as a contrast agent. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0032] A copper-source heterojunction nano antibacterial agent of the present invention comprises the following steps:

[0033] 1) The cuprous oxide nanoparticles synthesized by using a polymer template are uniformly dispersed in water; 2) The silver nitrate aqueous solution is added to the cuprous oxide nanoparticle solution, stirred for mixing reaction, and copper source nano heterojunction particles are obtained. 3) The copper source nano heterojunction particles are surface coated. 4) After a period of reaction, the excess unreacted materials are washed to remove the coating to obtain the copper source nano heterojunction particles, and the copper source heterojunction nano antibacterial agent is formed with the auxiliary materials.

[0034] In the above technical scheme, the copper-source heterojunction nano antibacterial agent obtained by the present invention is a nano heterojunction structure formed by cuprous oxide and silver. Compared with the simple nano silver, nano cuprous oxide or complex gold nano particles, or the simple mixture of nano silver and nano cuprous oxide obtained by conventional methods, the antibacterial agent obtained by the present invention can quickly obtain near-infrared optical absorption response performance, and the copper-based material has a low cost. In the subsequent biomedical application, it has obvious transformation prospects.

[0035] The copper-source heterojunction nano antibacterial agent obtained by the present invention comprises, by weight, 1% to 90% copper-source nano heterojunction particles, 1% to 30% surface modification coating, and 0% to 90% dressing. The effective antibacterial component of the antibacterial agent is copper-source nano heterojunction particles formed by cuprous oxide and silver, which can make the antibacterial agent have optical absorption in the near-infrared region, and the surface modification coating is a material that has an interactive function with components in the microbial cell wall or biofilm matrix. The dressing can make the copper-source nano antibacterial agent exist in the form of a stable solution or gel.

[0036] In the above method, the polymer template is preferably one or more of polyvinyl pyrrolidone or sodium hyaluronate. The method using the polymer as a template can form uniform loose spherical nano cuprous oxide particles. This loose nanostructure provides more reaction sites to a certain extent, which is more conducive to the reaction activity of reducing silver nitrate to form nano silver in situ on its surface, thereby improving the formation of a heterojunction structure with uniform combination of cuprous oxide and nano silver. The second aspect: the polymer template will be retained in the structure of cuprous oxide, providing it with better solution dispersibility, which is also conducive to the uniform formation of subsequent in situ nano silver reaction. The uniform heterojunction formation also plays an important role in the regulation of subsequent optical properties. The third aspect is that the loose spherical nanostructure has a higher specific surface area, which is conducive to the subsequent release of silver ions and copper ions. Further, the preferred silver nitrate and cuprous oxide, according to the mass concentration ratio of silver to copper is 0.01-5:1, and the nano heterojunction formed by this ratio has a large optical absorption range and a regular heterojunction morphology. Furthermore, the surface modification coating is preferably selected from one or more of ethylenediaminetetraacetic acid and chitosan, which can destroy microbial cell walls and biofilms by chelation and electrostatic interaction, respectively, thereby improving the antibacterial ability.

[0037] Embodiment 1:

[0038] A method for preparing a copper-source heterojunction nano antibacterial agent, comprising the following steps:

[0039] 1) The cuprous oxide nanoparticles synthesized with polyvinyl pyrrolidone as a template are uniformly dispersed in water; 2) The silver nitrate aqueous solution is mixed and reacted for 4 hours at a mass concentration ratio of silver to copper of 0.5:1 to obtain copper source nano heterojunction particles. 3) 1 mL of 0.1 mg / mL ethylenediaminetetraacetic acid disodium salt aqueous solution is added to 10 mL of the copper source nano heterojunction particle solution for surface coating modification. 4) After reacting for 30 minutes, the excess unreacted material is washed to remove the copper source nano heterojunction particles modified with ethylenediaminetetraacetic acid disodium salt coating, and the copper source heterojunction nano antibacterial agent is formed with 4% sodium hyaluronate dressing.

[0040] Figure 1 This is a TEM photo of a copper-based heterojunction nano antibacterial agent. It can be clearly seen that the antibacterial agent is a heterojunction nanoparticle composed of loose nanoparticles with a lower contrast (cuprous oxide) and round nanoparticles with a darker contrast (silver). Figure 8 Further XRD patterns show that the inorganic components of the antibacterial agent are cuprous oxide and silver. The copper-based heterojunction nano antibacterial agent of the present invention can significantly produce a temperature-raising effect under 808nm laser irradiation, while conventional nano silver and nano cuprous oxide cannot produce photothermal capacity under this wavelength ( Fig. 9 ). This is because ordinary nanosilver and nanocuprous oxide have no optical absorption in the near-infrared region, and even ordinary physical mixing of the two cannot achieve this. However, after the nanoheterojunction is formed by the present invention, the plasma resonance effect between the nanosilver in the heterojunction and the optical absorption of cuprous oxide are coupled with each other, extending the optical absorption to the near-infrared band, thereby mediating further photothermal in the near-infrared and enhancing the photocatalytic ability. The TEM photo of the copper-source heterojunction nanoantibacterial agent after co-incubation with Candida albicans can effectively destroy its cell wall structure. After auxiliary irradiation with 808nm near-infrared laser, the fungus was completely destroyed, and the internal cell fluid flowed out (such as Figure 4 ). Figure 6 The confocal micrographs of copper-based heterojunction nanoantimicrobial agents used to destroy fungal biofilms show that the copper-based heterojunction nanoantimicrobial agent-assisted near-infrared laser irradiation treatment method is more effective in destroying biofilms than the clinical antimicrobial agent amphotericin B. At the same time, for keratitis infected with Candida albicans, the copper-based heterojunction nanoantimicrobial agent-assisted near-infrared laser irradiation treatment method has a better therapeutic effect than the amphotericin B treatment method at the same dose ( Figure 7 ). At the same time, after being dripped into the eye, it can be used as an optical coherence tomography (OCT) and photoacoustic imaging contrast agent (PA) for real-time monitoring (such as Fig.10 ).

[0041] Embodiment 2:

[0042] A method for preparing a copper-source heterojunction nano antibacterial agent, comprising the following steps:

[0043] 1) The cuprous oxide nanoparticles synthesized using sodium hyaluronate as a template are uniformly dispersed in water; 2) The silver nitrate aqueous solution is mixed and reacted for 4 hours at a mass concentration ratio of silver to copper of 1:1 to obtain copper source nano heterojunction particles. 3) 1 mL of a chitosan aqueous solution with a concentration of 10 mg / mL is added to 10 mL of the copper source nano heterojunction particle solution for surface coating modification. 4) After reacting for 12 hours, the excess unreacted materials are removed by washing to obtain copper source nano heterojunction particles modified with chitosan coating to directly form copper source heterojunction nano antibacterial agents.

[0044] Figure 2 The potential distribution diagram of the copper-based heterojunction nano antibacterial agent shows that the surface of the antibacterial agent is positively charged after modification with chitosan, which has a strong electrostatic interaction with negatively charged bacteria and their biofilms. Figure 5 , which also shows that the copper-source heterojunction nano antibacterial agent of the present invention has a lower antibacterial concentration than the clinical antibiotic cefazolin sodium.

[0045] Embodiment 3:

[0046] A method for preparing a copper-source heterojunction nano antibacterial agent, comprising the following steps:

[0047] 1) The cuprous oxide nanoparticles synthesized with polyvinyl pyrrolidone as a template are uniformly dispersed in water; 2) The silver nitrate aqueous solution is mixed and reacted for 4 hours at a mass concentration ratio of silver to copper of 0.2:1 to obtain copper-source nano heterojunction particles. 3) Tris-hcl is added to 100 mL of the copper-source nano heterojunction particle solution to adjust the solution pH to 8.5, and then 5 mL of 10 mg / mL dopamine hydrochloride solution is added for surface coating modification. 4) After reacting for 4 hours, the excess unreacted materials are washed to remove the copper-source nano heterojunction particles modified with dopamine coating, and the copper-source heterojunction nano antibacterial agent is formed with 4% sodium hyaluronate dressing.

[0048] Figure 3 This is the hydrated particle size distribution diagram of the copper-source heterojunction nano antibacterial agent, indicating that the synthesized antibacterial agent has a relatively uniform nanoscale size.

[0049] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a copper-source heterojunction nano antibacterial agent, characterized in that: include: 1) The cuprous oxide nanoparticles synthesized using a polymer template are uniformly dispersed in water; 2) adding the silver nitrate aqueous solution to the cuprous oxide nanoparticle solution obtained in 1), stirring at room temperature for mixing reaction, and obtaining a copper source nano heterojunction particle solution; 3) adding a surface modifier to the obtained copper source nano heterojunction particle solution to perform surface coating modification; 4) After the reaction, centrifuge and wash to remove excess unreacted materials to obtain coated copper-source nano-heterojunction particles, or mix them with auxiliary materials to obtain copper-source heterojunction nano-antibacterial agents; The polymer template is one or more of polyvinyl pyrrolidone and sodium hyaluronate; The surface modifier is selected from one or more of ethylenediaminetetraacetic acid and chitosan.

2. The method for preparing the copper-source heterojunction nano antibacterial agent according to claim 1, characterized in that: The preparation method of the cuprous oxide nanoparticles synthesized using a polymer template includes the following steps: The polymer template is dispersed in water with a mass concentration of 0.5-2%, and then a 0.1-1 M soluble copper source solution is added dropwise. After stirring at 800-1200 rpm for at least 30 min, a hydrazine hydrate solution with a mass fraction of 85% was added dropwise for reaction for 1-60 min, and the mixture was centrifuged and washed to obtain cuprous oxide nanoparticles synthesized by a polymer template.

3. The method for preparing the copper-source heterojunction nano antibacterial agent according to claim 1, characterized in that: The mass concentration ratio of silver to copper is 0.01-5:

1.

4. A copper-source heterojunction nano antibacterial agent, characterized in that: The antibacterial agent is prepared according to the method according to any one of claims 1 to 3, wherein the antibacterial agent contains heterojunction nanoparticles formed by nano-cuprous oxide and nano-silver, and the antibacterial agent has near-infrared light absorption capability, absorption in an optical region above 600 nm, and photothermal conversion capability under NIR laser irradiation.

5. The copper-source heterojunction nano antibacterial agent according to claim 4, characterized in that: The antibacterial agent comprises, by weight percentage, 1% to 90 wt% of copper source nano heterojunction particles, 1% to 30 wt% of a surface modifier, and 0% to 90 wt% of auxiliary materials.

6. The copper-source heterojunction nano antibacterial agent according to claim 4, characterized in that: The NIR laser wavelength is 808 nm, 980 nm or 1064 nm.

7. Use of the copper-source heterojunction nano antibacterial agent according to claim 4 in the preparation of a drug for destroying microbial cell walls or biofilms, killing microorganisms and / or promoting healing of infectious wounds.

8. Use of the copper-source heterojunction nano antibacterial agent according to claim 4 in preparing a contrast agent for optical coherence tomography and / or photoacoustic imaging.

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