Nanocomposite and use thereof

Abstract

The application relates to a kind of nanocomposites, comprising: carrier;Silver;Vancomycin (Van);And hemin chloride (Hemin);Wherein, the silver, vancomycin (Van) and hemin chloride (Hemin) are loaded on carrier;Wherein the nanocomposite releases silver ion, vancomycin and hemin chloride under near infrared light irradiation.The nanocomposite of the application uses nanomaterial as carrier, and can achieve the purpose of high-efficiency sterilization by the loading of silver combined with Van in hollow cavity.At the same time, hemin chloride can realize photo-thermal conversion in cooperation with silver, and effectively remove redundant active oxygen.The successful implementation of the nanocomposite will lay a foundation for constructing efficient and safe antibacterial strategy and provide a new idea for pathogen infection treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of preparation and application of antibacterial agents, in particular to a kind of nanocomposite and its application. BACKGROUND

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is one of the six major pathogens that are difficult to treat, causing a public health problem worldwide (Wan Meichen et al., 2021). As the last line of defense against antibiotics, Vancomycin (Van) is the main method for treating MRSA infection in clinical practice (Rodvold et al., 2014). However, the extensive use of Van has not only led to the emergence of Vancomycin-resistant Staphylococcus aureus, but also increased the risk of kidney toxicity (Lodise et al., 2009). Therefore, it is urgent to develop new methods for treating MRSA infection to reduce the use of Van and delay the development of antibiotic resistance. Photothermal therapy (PTT) as a kind of photothermal responsive complex can convert near-infrared light radiation into heat, and can achieve local controllable heating, which has been widely used in antibacterial and antitumor therapy, and is a promising alternative strategy to antibiotics (Huang et al., 2021). However, PTT is usually accompanied by photodynamic, which produces excess reactive oxygen species. Studies have shown that redundant reactive oxygen species can have certain toxic side effects on tissues and induce inflammatory responses in infected areas (Wan et al., 2019).

[0003] Therefore, when using PTT for antibacterial, the impact on the infected microenvironment must also be considered to achieve better therapeutic effect. This urgently needs to design and synthesize a kind of nanocomposite that can not only effectively kill MRSA and treat skin infections caused by it, but also remove redundant reactive oxygen species and prevent their toxic effects on surrounding normal tissue cells. SUMMARY

[0004] To solve the technical problems existing in the prior art, the present application provides a kind of nanocomposite, comprising: carrier;Silver;Vancomycin (Van);And hemin chloride (Hemin);Wherein, the silver, vancomycin (Van) and hemin chloride (Hemin) are loaded on the carrier;Wherein the nanocomposite releases silver ion, vancomycin and hemin chloride under near-infrared light irradiation.

[0005] The nanocomposite as described above, wherein the carrier is a hollow particle containing mesopores.

[0006] The nanocomposite as described above, wherein the carrier particle size is 91 nm-400 nm; preferably 95 nm-300 nm; more preferably 100 nm; and the Zeta potential of the carrier is -13.92±0.47 mV.

[0007] The nanocomposite as described above, wherein the carrier is a silica particle.

[0008] The nanocomposite as described above, wherein the silver loading in the nanocomposite is 36%-50%, preferably 42.1%; the vancomycin loading in the nanocomposite is 20%-29%, preferably 23.5%; and the hematin chloride loading in the nanocomposite is 20%-30%, preferably 24.1%.

[0009] The nanocomposite as described above, wherein the particle size of the nanocomposite is 91 nm-300 nm; preferably 100 nm-200 nm; more preferably 180 nm; and the Zeta potential is -26.13±0.96 mV.

[0010] A nanobactericide comprising the nanocomposite as described in any one of the above.

[0011] Use of the nanocomposite as described in any one of the above in the preparation of a nanobactericide for preventing and / or treating a disease associated with a pathogen.

[0012] The use as described above, wherein the nanobactericide is a near-infrared laser responsive antibacterial agent.

[0013] Use of the nanocomposite as described above or the nanobactericide as described in claim 7 in affecting the morphology of a pathogen biofilm or scavenging reactive oxygen species in a living organism.

[0014] The use as described above, wherein the affecting the morphology of a pathogen biofilm comprises collapsing, shrinking and / or rupturing the biofilm on the surface of the pathogen.

[0015] The use as described above, wherein the affecting the morphology of a pathogen biofilm comprises inhibiting biofilm formation.

[0016] The use as described in any one of the above, wherein the pathogen is methicillin-resistant Staphylococcus aureus (MRSA).

[0017] A method for killing or inhibiting the growth of a pathogen in vitro, comprising administering to the pathogen an effective amount of the nanocomposite as described in any one of the above or the nanobactericide as described above.

[0018] Use of the nanocomposite as described in any of the above or the nano-antibacterial agent as described in claim 7 in the preparation of a medicament for preventing and / or treating a pathogen infection in a mammal.

[0019] Use of the above as described, the infection is a skin infection.

[0020] Use of the above as described, the mammal includes but is not limited to human, primate, rodent, rabbit, canine, livestock.

[0021] Use of the above as described, the mammal includes but is not limited to human, chimpanzee, monkey, mouse, rat, rabbit, dog, pig, cow, sheep.

[0022] The nanocomposite of the present application takes nanomaterial as a carrier, and introduces silver, Van, and hematin chloride to construct a photothermal triggered nanocomposite HAVH. HAVH can achieve high-efficiency sterilization by loading silver and Van in the hollow cavity. At the same time, hematin chloride can cooperate with silver to realize photothermal conversion and effectively remove redundant active oxygen. The successful implementation of the nanocomposite will lay a foundation for constructing an efficient and safe antibacterial strategy and provide a new idea for pathogen infection treatment BRIEF DESCRIPTION OF DRAWINGS

[0023] In the following, the preferred embodiments of the present application will be further described in detail with reference to the accompanying drawings, in which:

[0024] Figure 1 is a characterization diagram of HMSN, HA and HAVH according to an embodiment of the present application; wherein, Figure 1 a is a characterization diagram of HMSN, HA and HAVH under transmission electron microscope according to an embodiment of the present application; Figure 1 b is an energy spectrum characterization diagram of HAVH under transmission electron microscope according to an embodiment of the present application;

[0025] Figure 2 is a characterization diagram of HMSN, HA, HAV and HAVH according to another embodiment of the present application; wherein Figure 2 a is a particle size column chart according to an embodiment of the present application; Figure 2 b is a potential column chart according to an embodiment of the present application;

[0026] Figure 3 is a NIR response release curve of HAVH according to an embodiment of the present application, wherein Figure 3 a is an Ag + release curve according to an embodiment of the present application; Figure 3 b is a Van release curve according to an embodiment of the present application; Figure 3c is a Hemin release response curve according to an embodiment of the present application;

[0027] Figure 4 is a H2O, Hemin, HMSN, HA and HAVH (250 pg / mL) temperature rise curve under near-infrared irradiation NIR conditions according to an embodiment of the present application;

[0028] Figure 5 is a HAVH clearance of three kinds of active oxygen under +NIR conditions with near-infrared irradiation and -NIR conditions without near-infrared irradiation according to an embodiment of the present application; wherein Figure 5 a is a HAVH clearance rate curve of hydrogen peroxide (H2O2) according to an embodiment of the present application; Figure 5 b is a HAVH clearance rate curve of superoxide anion (O2 - ) according to an embodiment of the present application; Figure 5 c is a HAVH clearance rate curve of hydroxyl radical (·OH) according to an embodiment of the present application;

[0029] Figure 6 is a HAVH clearance effect on MRSA under +NIR conditions with near-infrared irradiation and -NIR conditions without near-infrared irradiation, and anti-MRSA effect under different treatment times according to an embodiment of the present application; wherein Figure 6 a is a survival rate of MRSA after PBS, Van, Hemin, HMSN, HA, HAVH according to an embodiment of the present application; Figure 6 b is a survival rate of MRSA under -NIR conditions without near-infrared irradiation or +NIR conditions with near-infrared irradiation under different treatment times according to an embodiment of the present application;

[0030] Figure 7 is an effect of HAVH on MRSA biofilm, morphology and intracellular structure according to an embodiment of the present application; wherein, Figure 7 a is a scanning electron microscope (SEM) diagram of MRSA treated with HAVH_NIR and PBS for 24 h according to an embodiment of the present application; Figure 7 b is a transmission electron microscope (TEM) diagram of MRSA treated with HAVH_NIR and PBS for 24 h according to an embodiment of the present application; Figure 7 c is an inhibitory effect of PBS, Van, Hemin, HMSN, HA, HAVH on biofilm formation after MRSA according to an embodiment of the present application; Figure 7 d is a removal effect of PBS, Van, Hemin, HMSN, HA, HAVH on established MRSA biofilm according to an embodiment of the present application;

[0031] Figure 8 is the experimental design and results of HAVH treatment of skin infection according to an embodiment of the present application; wherein, Figure 8 a is the experimental design of MRSA infection treatment according to an embodiment of the present application; Figure 8 b is the thermal image of PBS_NIR and HAVH_NIR treated mice according to an embodiment of the present application; Figure 8 c is the temperature rise curve of PBS_NIR and HAVH_NIR treated mice according to an embodiment of the present application; Figure 8 d is the body weight of mice after PBS, PBS_NIR, Van, HAVH, HAVH_NIR treatment according to an embodiment of the present application;

[0032] Figure 9 a is the wound photo after PBS, PBS_NIR, Van, HAVH, HAVH_NIR treatment according to an embodiment of the present application; Figure 9 b and Figure 9 c is the LB plate photo and bacterial count collected from infected wound after PBS, PBS_NIR, Van, HAVH, HAVH_NIR treatment according to an embodiment of the present application; Figure 9 d is the wound diameter after PBS, PBS_NIR, Van, HAVH, HAVH_NIR treatment according to an embodiment of the present application;

[0033] Figure 10 a and Figure 10 b is the HE and Masson staining after PBS, PBS_NIR, Van, HAVH, HAVH_NIR treatment according to an embodiment of the present application;

[0034] wherein, in Figures 8-10 N = 5, ***p < 0.01, ***p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION

[0035] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0036] In the following detailed description, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration specific embodiments of the application. In the drawings, like numerals describe substantially similar components throughout the several views. Specific embodiments of the application are described in sufficient detail to enable those skilled in the art to practice the application, and it is to be understood that other embodiments can be utilized and that structural changes can be made without departing from the scope of the present application.

[0037] Bacterial infection is a serious threat to human health. When using antibiotics to treat bacterial infection, due to the effects of body metabolism and biofilm barrier, it is difficult for the antibiotics to accumulate at the infection site, and it is even more difficult to kill intracellular surviving bacteria. Excessive use of antibiotics to achieve good antibacterial effect will lead to serious bacterial drug resistance, and even cause super bacteria to wreak havoc. The nano-antibacterial agent of the present application uses a nanomaterial as a carrier, and introduces silver, vancomycin (Van), and hemin to construct a photo-thermal triggered nanocomposite—HAVH. HAVH can achieve high-efficiency sterilization by loading silver and vancomycin (Van) in the hollow cavity. At the same time, hemin can cooperate with silver to achieve photo-thermal conversion, and effectively eliminate redundant active oxygen.

[0038] In the present application, the terms have the following meanings:

[0039] The "nanocomposite", "Hemin-HAVH@Ag@Van", or "HAVH" as used herein refers to a photo-thermal triggered nanocomposite constructed by introducing silver, Van, and hemin into a hollow mesoporous silica nanoparticle. In some embodiments, the size of the nanocomposite is 180 nm, and the zeta potential is -26.13 ± 0.96 mV. In some embodiments, HMSN can inhibit the formation of pathogen biofilm and eliminate the formed biofilm. In some embodiments, after treating the pathogen with HMSN, the surface of the pathogen can be clearly observed to be collapsed, shrunk, and some ruptured. In some embodiments, the pathogen is pathogenic bacteria, and further, the pathogen can be drug-resistant pathogenic bacteria. In some specific embodiments, the pathogen is methicillin-resistant Staphylococcus aureus (MRSA).

[0040] Further, in some embodiments, the nanocomposite has a near-infrared (NIR) response effect, and is a targeted nanocomposite. Specifically, the nanocomposite can rapidly release drugs under NIR, achieving the purposes of high-efficiency sterilization and active oxygen elimination.

[0041] Further, the present application can synthesize hollow mesoporous silica nanoparticles by simple calcination; then load a certain concentration of silver, vancomycin, and hemin chloride into the interior or surface of the hollow mesoporous silica nanoparticles, centrifugal dry to obtain nanocomposite (hereinafter referred to as HAVH) crystals. In some embodiments, the loading amounts of silver, vancomycin, and hemin chloride in HAVH are 42.1%, 23.5%, and 24.1%, respectively.

[0042] As used herein, "hollow mesoporous silica" or its nanoparticles (Hollow mesoporous silica nanosphere, HMSN for short, hereinafter referred to as HMSN) is composed of SiO2, which is used as a drug delivery carrier in the present application. Hollow mesoporous silica nanostructured material has both hollow and mesoporous properties, can efficiently load drugs and achieve drug release effect, and is a drug delivery carrier with good application prospect. The drug carrier based on HMSN can overcome the biological membrane barrier, deliver antibiotics to the cytoplasm to kill bacteria in cells, thereby reducing the amount of antibiotics used, and has the advantages of low drug resistance, broad spectrum, targeting, etc.

[0043] Photothermal therapy (PTT) refers to the conversion of near-infrared light radiation into heat by photothermal responsive complexes, achieving local controllable heating. PTT has been widely used in antibacterial and antitumor therapy, and is a promising antibiotic replacement strategy (Huang X, Lu Y, Guo M et al. Recent strategies for nano-based ptt combined with immunotherapy: From a biomaterial point of view [J]. Theranostics, 2021, 11: 7546-7569.). However, photothermal agents usually have poor water solubility, easy aggregation, complex synthesis and modification, and non-negligible biological toxicity (Liu Q M, Jin G Q. Research progress of nano-photothermal agents in the integration of tumor diagnosis and treatment [J]. Journal of Medical Postgraduates, 2020, 33: 531-536.). PTT can cause thermal damage to normal tissues, especially when excessive reactive oxygen species (ROS) are generated in cooperation with PDT, which can have toxic side effects on normal tissues and induce inflammation in infected areas (Wan S S, Cheng Q, Zeng X et al. A mn(iii)-sealed metal-organic framework nanosystem for redox-unlocked tumor theranostics [J]. ACS Nano, 2019, 13: 6561-6571.). Therefore, when using PTT for antibacterial therapy, the impact on the infected microenvironment must also be considered to achieve better therapeutic effect.

[0044] Chlorinated hemin or Hemin has the same meaning, which is the active center of hemoprotein, such as cytochrome, peroxidase, myoglobin and hemoglobin, and has peroxidase-like activity similar to peroxidase. Hemin is a heavy metal ion extracted from hemoglobin in mammalian blood (Zhao L P. The effect of chlorinated hemin on the antioxidant stress damage of hUC-MSCs [D]. Lanzhou University. 2022.). Hemin has been widely used in anti-inflammatory and anti-tumor therapy (Lian M, Xue Z, Qiao X et al. Movable hollow nanoparticles as reactive oxygen scavengers [J]. Chem, 2019, 5: 2378-2387.). In summary, Hemin as a ROS scavenger has absolute advantages such as easy availability of materials, low cost and significant effect, which makes it have very great application prospect.

[0045] As used herein, "loading amount" refers to the percentage of the drug in the total mass of the complex. The loading amount is calculated by quantifying the content of different drugs after the complex is stirred in PBS solution for 24 hours. The formula is: loading amount = m 药物 / m 复合物 .

[0046] In some embodiments, the drug can include silver, silver ions, chlorinated hemin, vancomycin, or a collection of any one or more thereof. For example, in some embodiments, the silver loading amount refers to the mass percentage of silver in the HAVH; the chlorinated hemin loading amount refers to the mass percentage of chlorinated hemin in the HAVH; the vancomycin loading amount refers to the mass percentage of vancomycin in the HAVH.

[0047] In some embodiments, the loading amount of Van and Hemin in the HAVH can be calculated using ultraviolet-visible spectrophotometry. In some embodiments, the loading amount of Ag in the HAVH can be determined using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700).

[0048] As used herein, "drug loading amount" refers to the total amount of the loaded drug and the carrier (including the amount of the carrier and the loaded drug). In some embodiments, the drug can include silver, silver ions, chlorinated hemin, vancomycin, or a collection of any one or more thereof.

[0049] The drug loading amount of Ag in the HAVH is determined using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700), and the formula is as follows:

[0050] Drug loading amount = (content of Van, Ag or Hemin wrapped in HAVH / content of HAVH) x 100%.

[0051] As used herein, "release amount" refers to the amount of drug dissolved at different times, expressed as a percentage of the total drug amount in the complex. In some embodiments, the measurement method of the release amount includes, but is not limited to, ultraviolet-visible spectrophotometry and ICP-MS.

[0052] For example, in some embodiments, the silver release amount is the mass percentage of the silver content in the solution at different times to the total silver content in the complex; the hematin chloride negative release amount is the mass percentage of the hematin chloride content in the solution at different times to the total hematin chloride content in the complex; and the vancomycin release amount is the mass percentage of the vancomycin content in the solution at different times to the total vancomycin content in the complex.

[0053] As used herein, the "antibacterial agent" refers to an antibacterial agent comprising the above-mentioned nanocomposite HAVH complex, which has the function of inhibiting and killing pathogenic bacteria, such as pathogenic bacteria, including drug-resistant bacteria, etc. In some embodiments, the antibacterial agent can be a liquid preparation, a freeze-dried preparation, a powder, a tablet, a capsule, etc. comprising the above-mentioned nanocomposite.

[0054] As used herein, the "near-infrared trigger" in the present application refers to the exposure of the mesoporous structure of the HMSN and the massive release of the drug therein under near-infrared irradiation of the HAVH.

[0055] As used herein, "MRSA" or "methicillin-resistant Staphylococcus aureus" is a typical gram-positive bacterium, and its infection is a globally recognized public health problem. The complex recurrent wound and soft tissue infection caused by it is difficult to be completely treated with a single antibiotic. In some embodiments, the infection of the MRSA instrument is a skin infection.

[0056] As used herein, "drug resistance" or "drug resistance" refers to the increased tolerance of bacteria to the action of antibacterial drugs. Once drug resistance occurs, the antibacterial effect of the drug will decrease significantly. Drug resistance can be divided into acquired drug resistance and natural drug resistance according to its cause. Some strains of pathogenic bacteria in nature, such as bacteria, can also have natural drug resistance. When antibiotics are used for a long time, the majority of sensitive strains are continuously killed, and drug-resistant strains multiply in large numbers, replacing sensitive strains, and thus the drug resistance rate of bacteria to the drug increases continuously. In the present application, the inventors have not found any drug resistance characteristics of pathogenic bacteria to the nanocomposite antibacterial agent of the present application after a large number of experiments for more than a month.

[0057] As used herein, "mammal" refers to a mammal that can be infected by a pathogen. Further, the pathogen that infects the mammal is a pathogen that can be killed by the nanocomposite HMSN of the present application. Still further, the pathogen that infects the mammal is MRSA. In some embodiments, the mammal includes but is not limited to human, primate, rodent, rabbit, canine, livestock. Specifically, in some embodiments, the mammal includes but is not limited to human, chimpanzee, monkey, mouse, rat, rabbit, dog, pig, cow, sheep.

[0058] The nanocomposites of the present application have significant antibacterial activity against pathogens, such as MRSA. In some embodiments, the nanocomposites of the present application can be used to prevent or treat pathogen infection in primates, rodents, lagomorphs, canids, livestock, and even humans. Further, according to one embodiment of the present application, the nanocomposites of the present application can be used to treat or prevent MRSA pathogen skin infection caused by animal surgery.

[0059] The present application relates to a nanocomposite comprising a carrier, silver, vancomycin (Van), and hemin. In some embodiments, the carrier is a hollow particle comprising mesopores. Further, in some embodiments, the carrier has a particle size of 91 nm to 400 nm; preferably 95 nm to 300 nm; more preferably 100 nm; and the carrier has a Zeta potential of -13.92 ± 0.47 mV. Still further, the carrier is a hollow silica particle comprising mesopores.

[0060] wherein the silver, vancomycin (Van), and hemin are loaded on the carrier. Further, the nanocomposite releases silver ions, vancomycin, and hemin under near-infrared light irradiation.

[0061] In some embodiments, the silver loading in the nanocomposite is 36% to 50%, preferably 42.1%; the vancomycin loading in the nanocomposite is 20% to 29%, preferably 23.5%; and the hemin loading in the nanocomposite is 20% to 30%, preferably 24.1%.

[0062] In some embodiments, the nanocomposite has a particle size of 91 nm to 300 nm; preferably 100 nm to 200 nm; more preferably 180 nm. Still further, the HAVH has a particle size of about 100 nm to 200 nm and a Zeta potential of -26.13 ± 0.96 mV.

[0063] In some embodiments, the method for preparing the nanocomposite of the present application comprises:

[0064] (1) preparing hollow mesoporous silica nanoparticles (HMSN). In some embodiments, the hollow mesoporous silica nanoparticles are prepared by using a template method. Further, in some embodiments, the template is a polystyrene (PS) organic microsphere.

[0065] (2) loading silver. In this step, Ag is loaded into the HMSN to form HAVH@Ag (HA).

[0066] (3) Loading Van. In this application, Van was loaded into the silver-loaded HMSN to form HAVH@Ag@Van (HAV).

[0067] (4) Loading Hemin. In this step, Van and Hemin were loaded onto the surface of the HMSN which has been loaded with Ag and Van to form HAVH@Ag@Van-Hemin (or Hemin-HAVH@Ag@Van, i.e., the nanocomposite, referred to as HAVH).

[0068] In some embodiments, the nanocomposite of the present application can affect the morphology of the pathogen biofilm. In some embodiments, affecting the morphology of the pathogen biofilm includes collapsing, shrinking, and / or rupturing the biofilm on the surface of the pathogen. In some embodiments, affecting the morphology of the pathogen biofilm includes inhibiting biofilm formation.

[0069] In some embodiments, the nanocomposite of the present application can scavenge multiple active oxygen species in the living organism. In some embodiments, the multiple active oxygen species include, but are not limited to, (H2O2), superoxide radical (O2 - ), and hydroxyl radical (·OH). In some embodiments, HAVH can reduce the content of 14.8% of hydrogen peroxide (H2O2), 70.8% of superoxide anion (O2 .- ), and 54.4% of hydroxyl radical (·OH) after near-infrared laser irradiation for 10 minutes.

[0070] In some embodiments, the nanocomposite of the present application can be used to prepare a nanobactericide for preventing and / or treating diseases related to pathogens. In some embodiments, the nanobactericide includes the nanocomposite as described above. In other embodiments, the nanobactericide further includes other adjuvants.

[0071] In some embodiments, the nanobactericide of the present application is a near-infrared laser responsive antibacterial agent.

[0072] In some embodiments, the pathogen is a pathogen that can be killed by any one of silver, vancomycin (Van), and hemin chloride. Further, in some embodiments, the pathogen is methicillin-resistant Staphylococcus aureus (MRSA).

[0073] In some embodiments, the nanocomposites of the present application have photothermal effect, which can be used for photothermal therapy to treat and / or prevent diseases caused by pathogens. In some embodiments, the photothermal effect of the nanocomposites of the present application has concentration dependence and light intensity dependence, and can exhibit good stability within 5 light cycles. In some embodiments, the photothermal conversion efficiency of HAVH is 42.8%, which is much higher than that of hematin chloride and silver alone. After near-infrared laser irradiation for 10 minutes, Ag + and Van in PBS solution are 37.4% and 57.3%, respectively. In contrast, Ag + and Van in HAVH without near-infrared irradiation are almost not released.

[0074] In some embodiments, under the irradiation of near-infrared light, HAVH (256 μg / mL) of the present application can kill about 99% of MRSA (1 x 10 8 CFU / mL) within 4 minutes; while Van (256 μg / mL) alone needs 16 hours. Without near-infrared light irradiation, HAVH takes 60-120 minutes to completely kill MRSA.

[0075] The present application relates to a method for killing or inhibiting the growth of pathogens in vitro, comprising administering an effective dose of the nanocomposites or nanobactericides of the present application to the pathogens.

[0076] In some embodiments, the nanocomposites or nanobactericides of the present application can be used for preparing a medicament for preventing and / or treating pathogen infection in mammals. In some embodiments, the infection is skin infection. In some embodiments, the mammals include but are not limited to humans, primates, rodents, rabbits, canines, and livestock. Further, in some embodiments, the mammals include but are not limited to humans, chimpanzees, monkeys, mice, rats, rabbits, dogs, pigs, cows, and sheep.

[0077] The technical solutions of the present application will be illustrated by the following examples. Those skilled in the art should understand that the following examples are only for illustrating the technical solutions of the present application, and do not limit the scope of protection of the present application.

[0078] Example 1 Preparation of HMSN and HAVH

[0079] (1) Preparation of polystyrene (PS) organic microspheres

[0080] ① A 250 mL three-necked flask was purged with nitrogen for 5 min to remove air;

[0081] ② 95 mL of ethanol and 3 mL of deionized water were added, and stirred uniformly at a speed of 120 rpm;

[0082] (3) 10 mL styrene (Styrene), 0.1 g 2,2'-azobis-isobutyronitrile (AIBN) and 2.25 g polyvinylpyrrolidone (PVP, Mw = 55000) were added, respectively;

[0083] (4) 24 h of heating at 70 °C under a continuous nitrogen flow at 120 rpm;

[0084] (5) 5 min of centrifugation at 10,000 rpm, 3 times of washing with deionized water and ethanol, and 12 h of drying at 60 °C to obtain PS microspheres.

[0085] (2) Synthesis of hollow mesoporous silica nanoparticles (HMSN) using PS microspheres as templates

[0086] (1) 0.40 g of PS microspheres were dispersed in a mixture of 64 mL of ethanol, 58 mL of water and 0.8 mL of ammonia water;

[0087] (2) 0.48 g of CTAB was dissolved in a mixture of 8 mL of water and 4 mL of ethanol, and then added to solution (1);

[0088] (3) After stirring solution (2) for 30 min, 0.86 mL of TEOS was quickly added and stirred for 3 min, and the mixture was left to stand overnight;

[0089] (4) The product was collected by centrifugation, and the temperature was increased to 550 °C at a rate of 1 °C / min and maintained for 6 h; HMSN was obtained.

[0090] (3) Loading of silver HAVH@Ag (abbreviated as HA)

[0091] In a polypropylene flask, 0.031 g of AgNO3 was dissolved in 60 mL of ethanol under ultrasonic action, and then 0.06 g of HMSN ethanol dispersion (10 mL) was added. The mixture was stirred at 50 °C using a magnetic stirrer for 3 h, so that Ag + was fully introduced into the hollow cavity of HMSN. Subsequently, 1.0 mL of n-butylamine ethanol solution (0.01 g / mL) was added, and Ag + was reduced to silver nanoparticles (abbreviated as AgNPs). After 6 h, Ag in-situ grown HMSNs were obtained by centrifugation at 9000 rpm for 10 min, and finally washed with ethanol three times to remove un-reduced Ag + , and freeze-dried to obtain HMSN@Ag (i.e. HA).

[0092] (4) Loading of Van, Hemin to form Hemin-HAVH@Ag@Van (i.e. nanocomposite, abbreviated as HAVH)

[0093] To 1 mL Van solution with concentration of 80 mg / mL, 10 mg HA was added. The mixed suspension was treated by ultrasonic in ice bath for 10 minutes. Then the suspension after ultrasonic was transferred to a vacuum dryer connected with a vacuum pump. The high pressure pump was opened to vacuum seal it. When the vacuum degree in the vacuum box reached -0.1 MPa, it was maintained for 30 minutes, and then returned to normal pressure (the air inside the lumen was removed during the vacuum process, and Van was loaded into the HMSN by air pressure during the process of returning to normal pressure). The cycle was repeated twice to increase the loading efficiency. Centrifugation was performed at a speed of 5000 r / min for 5 minutes to separate the solid phase in the suspension, and then washed twice with ethanol centrifugation to remove the adsorbed and free Van molecules on the outer surface, to obtain HAVH@Ag@Van (HAV).

[0094] Finally, Hemin (2.5 mg / mL) was connected to the surface of HAV by stirring. After centrifugation to collect the precipitate, vacuum drying was performed for 12 h, and grinding into powder to obtain Hemin-HAVH@Ag@Van (i.e. HAVH).

[0095] wherein, Figure 1 is a characterization diagram of HMSN, HA and HAVH according to an embodiment of the present application; wherein, Figure 1 a is a characterization diagram of HMSN, HA and HAVH under transmission electron microscope according to an embodiment of the present application; Figure 1 b is a characterization diagram of HAVH under transmission electron microscope according to an embodiment of the present application. As shown in Figure 1 a- Figure 1 b, it can be clearly seen under microscope that the nanocomposite HAVH constructed by silver, vancomycin, hemin and silica nanoparticles.

[0096] Figure 2 is a characterization diagram of HMSN, HA, HAV and HAVH according to an embodiment of the present application; wherein Figure 2 a is a particle size histogram according to an embodiment of the present application; Figure 2 b is a potential histogram according to an embodiment of the present application.

[0097] As shown in Figure 2 a, the particle size of HMSN is about 91 nm-400 nm; further, the particle size of HMSN is about 95 nm-300 nm; further, the particle size of HMSN is about 100 nm. The particle size of HAVH is about 40 nm-400 nm; further, the particle size of HAVH is about 91 nm-300 nm; further, the particle size of HAVH is about 100 nm-200 nm; further, the particle size of HAVH is about 180 nm.

[0098] like Figure 2 As shown in b, the Zeta potential of HMSN is approximately -13.92 ± 0.47 mV; the Zeta potential of HAVH is approximately -26.13 ± 0.96 mV.

[0099] Example 2: Calculation of HAVH drug loading and NIR response release curve

[0100] The drug loadings of Van and Hemin in HAVH were calculated using ultraviolet-visible spectrophotometry, and the drug loading of Ag in HAVH was determined using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700). The formula is as follows:

[0101] Drug loading capacity = (Van, Ag, or Hemin content encapsulated in HAVH / HAVH content) × 100%. References were used to evaluate the in vitro drug release performance of HAVH (Tang et al., 2022). In short, the experiment was divided into two groups: one without near-infrared (NIR) irradiation (-NIR group) and one with NIR irradiation (+NIR group). First, HAVH was adjusted to a concentration of 250 μg / mL with PBS. In the -NIR group, after standing in 10 mL centrifuge tubes for 0, 2, 6, 10, 20, and 30 minutes, the separated solutions were obtained by filtration. In the +NIR group, a laser (808 nm, 1.0 W cm⁻¹) was used. -2 Irradiate a 1.5 mL tube (containing 200 μL HAVH solution), then collect the separation solution by filtration through a membrane at the same time points, and add 10 mL of PBS. Ag is determined using ICP-MS (Agilent 720ES). + The concentration of Van was determined by LC-MS (SHIMADZU).

[0102] Figure 3 This is the NIR response release curve of HAVH according to an embodiment of this application; wherein Figure 3 a represents Ag according to an embodiment of this application. + Release curve; Figure 3 b is the Van release curve according to an embodiment of this application; Figure 3 c represents the Hemin release response curve according to an embodiment of this application. Figure 3 As shown in Figure a, HAVH irradiated with near-infrared (NIR) light showed that Ag within 10 minutes... + The release rate in PBS solution was approximately 37.4%; Ag was released within 30 minutes. + The release rate is approximately 40%-55%. For example... Figure 3As shown in FIG. 2b, under NIR irradiation, the release rate of Van in PBS solution was about 57.3% within 10 minutes, and the release amount of Van was about 58%-65% within 30 minutes. Figure 3 As shown in FIG. 2c, under NIR irradiation, the release rate of Hemin in PBS solution was about 79.2% within 10 minutes, and the release amount of Hemin was about 80%-90% within 30 minutes.

[0103] Further, it was calculated that the loading amounts of silver, Van and Hemin in HAVH were 42.1%, 23.5% and 24.1%, respectively.

[0104] Figure 4 is the temperature rise curve of H2O, Hemin, HMSN, HA and HAVH (250 μg / mL) under NIR irradiation according to an embodiment of the present application. As shown in FIG. 3, Figure 4 Under NIR irradiation, the temperatures of Hemin, HA and HAVH all increased to different degrees, and the temperature of HAVH increased faster and maintained at a higher temperature. The photothermal effect of HAVH has concentration dependence and light intensity dependence, and can show good stability within 5 light cycles. Under NIR laser irradiation, HAVH can quickly rise from 29.1°C to 49.3°C. The photothermal conversion efficiency of HAVH is 42.8%, which is much higher than that of hemin and silver alone. In contrast, there is almost no release of Ag + and Van in HAVH without NIR irradiation.

[0105] Example 3 In vitro removal of various active oxygen by HAVH

[0106] (1) H2O2 removal activity

[0107] The residual hydrogen peroxide in the post-reaction solution was detected by potassium titanium oxalate (Kastvig et al., 2021). First, 10 μL of H2O2 was added to 10 mL of 8 mM potassium titanium oxalate solution and mixed well to make the concentration of H2O2 30, 60, 90, 120, 150, 180 μg / mL, respectively. The absorbance at 400 nm was measured, and the hydrogen peroxide standard curve was drawn.

[0108] To 1 mL of PBS, Hemin, HMSN, HA and HAVH (256 μg / mL), 15 μL of H2O2 (5 mg / mL) was added, and after 10 min of reaction with and without NIR, the absorbance value at 400 nm was detected and the content of H2O2 was calculated.

[0109] Figure 5a is the clearance rate curve of HAVH on hydrogen peroxide (H2O2) according to an embodiment of the application. As shown in Figure 5 a, HAVH has a higher clearance rate on H2O2, which can reach 10%-20%; preferably, HAVH can reduce the content of hydrogen peroxide (H2O2) by 14.8% after near-infrared laser irradiation for 10 minutes, which is much higher than that of the PBS negative control group, the Hemin group, the HMSN group and the HA group. Among them, the clearance rate of HAVH on H2O2 after far-infrared light irradiation is much higher than that of HAVH without infrared light irradiation.

[0110] (2) Superoxide anion radical clearance

[0111] The superoxide anion radical clearance ability is detected by using the superoxide anion detection kit according to the instructions. First, a mixed solution (containing reagent I application liquid 1.0 mL, reagents II, III and IV each 0.1 mL) is prepared, and 0.05 mL of chlorinated hemin (Hemin), silicon dioxide nanoparticles (HMSN), silver-loaded (HAVH@Ag, or HA), HAVH solution with a concentration of 256 μg / mL is added to the mixed solution and mixed thoroughly. After incubation for 10 min (with or without NIR), incubate in a 37°C water bath for 40 min. Add 2.0 mL of color developing agent and mix well, stand at room temperature for 10 min, adjust to zero with ddH2O, and detect the absorbance value at 550 nm.

[0112] Figure 5 b is the clearance rate curve of HAVH on superoxide anion (O2 - ) according to an embodiment of the application. As shown in Figure 5 b, the clearance rate of HAVH on superoxide anion (O2 - ) is equivalent to that of HA, which can reach 60-80%; preferably, HAVH can reduce the content of superoxide anion (O2 - ) by 70.8% after near-infrared laser irradiation for 10 minutes, which is much higher than the superoxide ion clearance rate of the PBS group, the Hemin group and the HMSN group. Among them, the clearance rate of HAVH on superoxide ion after far-infrared light irradiation is higher than that of HAVH without infrared light irradiation.

[0113] (3) Hydroxyl radical clearance

[0114] According to the classical Fenton reaction, Fe 2+It can catalyze the generation of hydroxyl radicals from H2O2. These hydroxyl radicals react with methyl violet (MV), causing MV to lighten or even become colorless, resulting in a decrease in the maximum absorbance at 582 nm. PBS, Hemin, HMSN, HA, and HAVH (256 μg / mL, 0.1 mL) were added to the reaction solution: MV (1 mg / mL, 0.4 mL) + Fe2SO4 (0.5 mg / mL, 0.8 mL) + H2O2 (3 mg / mL, 0.2 mL) + H2O (2.7 mL). The absorbance at 582 nm was measured after reacting with and without NIR for 5, 10, 20, 40, and 80 min.

[0115] Figure 5 c represents the scavenging rate curve of HAVH against hydroxyl radicals (·OH) according to an embodiment of this application. Figure 5 As shown in c, HAVH exhibits a high scavenging rate for hydroxyl radicals, reaching 40%-60%. Preferably, after 10 minutes of near-infrared laser irradiation, HAVH can reduce the content of hydroxyl radicals (·OH) by 54.4%, which is significantly higher than that of the PBS negative control group, Hemin group, HMSN group, and HA group. Furthermore, the scavenging rate of hydroxyl radicals by HAVH irradiated with far-infrared light is significantly higher than that of HAVH without infrared light irradiation.

[0116] Example 4: HAVH in vitro antibacterial experiment

[0117] Methicillin-resistant Staphylococcus aureus (MRSA, 330041) was cultured in LB medium and incubated overnight in a shaking incubator at 30°C. It was then transferred to fresh LB medium and cultured until the logarithmic growth phase before use. The minimum inhibitory concentrations (MICs) of Van, Hemin, HMSN, HA, and HAVH against MRSA were determined using the microdilution method (Martin et al., 2020). Simply put, 95 μL of MRSA (1 × 10⁻⁶) was used to determine the minimum inhibitory concentrations (MICs) against MRSA. 5 and 1×10 8 The OD values ​​of 5 μL each of Van, Hemin, HMSN, HA, and HAVH (512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg / mL) were gradually diluted and added to 96-well plates, with three replicates. PBS was used as a control. After incubating the 96-well plates at 30°C for 24 h, the OD values ​​of the 96-well plates were measured using a microplate reader. 600 .

[0118] The MRSA was then diluted to 1×10⁻⁶. 5 CFU / mL and 1×10 8 CFU / mL. For 1×10 5CFU / mL MRSA was used with an 808nm laser (1.0W / cm²) to... 2 Irradiation was performed on groups receiving Van (3.8 μg / mL), Hemin (3.9 μg / mL), HMSN (16 μg / mL), HA (16 μg / mL), and HAVH (16 μg / mL) for 10 min. When the MRSA concentration was 1 × 10⁻⁶... 8 At CFU / mL, Van (60 μg / mL), Hemin (62 μg / mL), HMSN (256 μg / mL), HA (256 μg / mL), and HAVH (256 μg / mL) were administered using an 808 nm laser (1.0 W / cm²). 2 Irradiate for 10 minutes. Power is 1.0 W / cm². 2 MRSA treated with HAVH (16 μg / mL) was irradiated with an 808 nm laser at 1 × 10⁻⁶ nm. 5 CFU / mL and MRSA treated with HAVH (256 μg / mL) 1×10 8 The bactericidal performance of HAVH was evaluated by applying CFU / mL for 10 min. Samples were collected at 10, 30, 60, 120, 240, and 480 min after treatment, and the samples were placed flat on LB agar plates for viable cell counting.

[0119] Figure 6 a represents the survival rate of MRSA after treatment with PBS, Van, Hemin, HMSN, HA, and HAVH according to an embodiment of this application; Figure 6 b represents the survival rate of MRSA under different treatment times in -NIR conditions without near-infrared irradiation or +NIR conditions with near-infrared irradiation according to an embodiment of this application.

[0120] like Figure 6 As shown in Figure a, under infrared light irradiation, HAVH can kill almost all MRSA, while PBS control, and single application of Van, Hemin, or HMSN / HA can hardly kill MRSA. Figure 6 As shown in b, under near-infrared light irradiation, HAVH (256 μg / mL) can kill approximately 99% of MRSA (1×10⁻⁶ μg / mL) within 4 minutes. 8 (CFU / mL); while Van alone (256 μg / mL) requires 16 hours. Without near-infrared light irradiation, HAVH takes 60-120 minutes to completely kill MRSA.

[0121] Example 5: Electron microscopy observation of bacterial morphology after different sample treatments

[0122] The morphology of MRSA after different sample treatments was observed using scanning electron microscopy and transmission electron microscopy. (1×10⁻⁶ samples were used.) 8MRSA at CFU / mL were treated with PBS, Van (60 pg / mL), Hemin (62 pg / mL), HMSN (256 pg / mL), HA (256 pg / mL) and HAVH (256 pg / mL) respectively, and irradiated with 808 nm laser (1.0 W / cm2) for 10 min, then treated at 30 °C for 1 h, and collected, fixed with 2.5% glutaraldehyde at 4 °C overnight. After collecting the bacterial precipitate, the bacteria were washed with PBS three times, and then dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol for 15 min, respectively. After drying at room temperature, the bacteria were observed under SEM (Zeiss Sigma 300, Germany). Under transmission electron microscopy, the bacteria were embedded with Spon 812, and then sliced with Leica EM UC7 to obtain 70 nm thin layer sections. The samples were stained with uranyl acetate and lead citrate, and then detected with TEM (FEI Talos F200X, USA) at 80 kV. 2 ) for 10 min, and then treated at 30 °C for 1 h. The bacteria were collected and fixed with 2.5% glutaraldehyde at 4 °C overnight. After collecting the bacterial precipitate, the bacteria were washed with PBS three times, and then dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol for 15 min, respectively. After drying at room temperature, the bacteria were observed under SEM (Zeiss Sigma 300, Germany). Under transmission electron microscopy, the bacteria were embedded with Spon 812, and then sliced with Leica EM UC7 to obtain 70 nm thin layer sections. The samples were stained with uranyl acetate and lead citrate, and then detected with TEM (FEI Talos F200X, USA) at 80 kV.

[0123] Figure 7 a is a scanning electron microscope (SEM) image of MRSA treated with HAVH NIR and PBS for 24 h according to an embodiment of the present application; Figure 7 b is a transmission electron microscope (TEM) image of MRSA treated with HAVH NIR and PBS for 24 h according to an embodiment of the present application. As shown in Figure 7 a and Figure 7 b, under the irradiation of near-infrared light, HAVH releases silver, Hemin and Van therein; while in HAVH without the irradiation of near-infrared light, silver, Hemin and Van are still loaded therein.

[0124] Example 6: Effect of HAVH on biofilm formation

[0125] Biofilm is a powerful barrier for bacteria to prevent antibiotics from entering and resist environmental pressure, and its formation is a key virulence factor for chronic infection caused by multiple microorganisms. Therefore, the ability to inhibit the formation of biofilm and remove the formed biofilm is necessary for excellent antibacterial substances. (H. Koo, R. Allan, R. Howlin, P. Stoodley, L. J. N. r. M. Hall-Stoodley, Targeting microbial biofilms: current and prospective therapeutic strategies, 15(12) (2017) 740-755. Z. Chen, Z. Wang, J. Ren, X. J. A. o. c. r. Qu, Enzyme Mimicry for Combating Bacteria and Biofilms, 51(3) (2018) 789-799. M. Valdes-Pena, N. Massaro, Y. Lin, J. J. A. o. c. r. Pierce, Leveraging Marine Natural Products as a Platform to Tackle Bacterial Resistance and Persistence, 54(8) (2021) 1866-1877.)

[0126] (1) Inhibition of biofilm formation

[0127] Prepare 1x10 8 CFU / mL of MRSA bacterial solution, add to 96-well plates, and treat with PBS, Van (60 μg / mL), Hemin (62 μg / mL), HMSN (256 μg / mL), HA (256 μg / mL), and HAVH (256 μg / mL). Then incubate the samples at 30°C for 36 h. Aspirate the bacterial solution and wash to clarity with PBS buffer, dry the 96-well plate at room temperature, add 200 μL of 1% crystal violet dye to stain for 10 min at room temperature, wash the plate wells to clarity with sterile water, dry the 96-well plate at 55°C, add 200 μL of 33% glacial acetic acid to stand at 37°C for 30 min to fully dissolve the attached crystal violet dye, and detect the absorbance value at 590 nm with an enzyme-labeled instrument. Set three biological replicates for each sample group and calculate the difference.

[0128] Figure 7 c is the inhibition of MRSA biofilm formation after treatment with PBS, Van, Hemin, HMSN, HA, and HAVH according to an embodiment of the present application. As Figure 7As shown in Figure c, HAVH can significantly inhibit the growth of MRSA by suppressing biofilm formation. Among them, HAVH irradiated with near-infrared light has a stronger inhibitory effect on MRSA than HAVH not irradiated with near-infrared light.

[0129] (2) Removal of biofilm

[0130] Prepare 1×10 8 A CFU / mL MRSA bacterial solution was added to 96-well plates and incubated at 30°C for 24 h. After biofilm formation, the wells were rinsed three times with PBS and treated with PBS, Van (60 μg / mL), Hemin (62 μg / mL), HMSN (256 μg / mL), HA (256 μg / mL), and HAVH (256 μg / mL). The samples were then incubated at 30°C for 12 h. The bacterial culture was aspirated and washed with PBS buffer until clear. The 96-well plates were air-dried at room temperature, and 200 μL of 1% crystal violet dye was added and stained at room temperature for 10 min. The wells were washed with sterile water until clear, and the 96-well plates were dried at 55°C. 200 μL of 33% glacial acetic acid was added and the plates were allowed to stand at 37°C for 30 min to fully dissolve the attached crystal violet dye. The absorbance at 590 nm was measured using a microplate reader. Three biological replicates were set up for each sample group, and statistical analysis was performed to calculate the differences.

[0131] Figure 7 d represents the removal effect of PBS, Van, Hemin, HMSN, HA, and HAVH on an established MRSA biofilm according to one embodiment of this application. Figure 7 As shown in d, HAVH can inhibit the growth of MRSA by removing its biofilm. Specifically, HAVH irradiated with near-infrared light exhibits stronger inhibition of MRSA than HAVH not irradiated with near-infrared light.

[0132] Example 8: HAVH in vivo antibacterial experiment

[0133] All experiments were conducted in accordance with the ethical standards established by the Institutional Animal Protection and Use Committee of Hainan University. ICR mice (30g, male, 6–8 weeks old) were purchased from Hunan SJA Laboratory Co., Ltd. MRSA (1×10⁻⁶) was administered subcutaneously to the right side. 10 A mouse infection model was established using the method of (CFU / mL, 100μL).

[0134] Figure 8 a is a treatment experimental design for MRSA infection according to one embodiment of this application. Figure 8 As shown in a, 24 hours after infection, infected mice were randomly divided into 5 groups. On day 0, mice were subcutaneously injected with PBS, PBS_NIR (808nm, 1W / cm). 2, 10min), HAVH (1mg / kg), HAVH (1mg / kg)_NIR (808nm, 1W / cm 2 Near-infrared laser irradiation (NIRS) was performed on the PBS_NIR and HAVH_NIR groups for 10 min, with 100 μL of Van (40 mg / kg) (n=5). The healing process, wound area, and mouse weight were recorded throughout the treatment period (11 days). After CO2 asphyxiation, infected skin tissue was collected, fixed with 4% paraformaldehyde solution, embedded in paraffin, and dehydrated. Wound tissue sections were stained with HE and Masson's staining and observed under a microscope (Nikon Eclipse E100, Japan). Infected tissue was homogenized with PBS (1 mL) and spread onto LB agar plates. After incubation at 30°C for 24 h, bacterial counting was performed (n=5). Subsequently, the homogenate from the skin wound was spread onto LB agar plates, and the bacterial count was recorded. Serum was collected, with serum from healthy mice as a control. Tissue sections from major organs (liver, kidney, spleen) were collected and stained with HE under a microscope.

[0135] Figure 8 b is a thermograph of mice treated with PBS_NIR and HAVH_NIR according to an embodiment of this application; Figure 8 c is the temperature rise curve of mice treated with PBS_NIR and HAVH_NIR according to an embodiment of this application; Figure 8 d is the body weight of mice after treatment with PBS, PBS_NIR, Van, HAVH, and HAVH_NIR according to one embodiment of this application; Figure 9 a is a photograph of the wound after treatment with PBS, PBS_NIR, Van, HAVH, and HAVH_NIR according to an embodiment of this application; Figure 9 b and Figure 9 c is an LB plate photograph after treatment with PBS, PBS_NIR, Van, HAVH, and HAVH_NIR according to an embodiment of this application, and bacterial counts collected from an infected wound. Figure 9 d is the diameter of the wound after treatment with PBS, PBS_NIR, Van, HAVH, and HAVH_NIR according to one embodiment of this application; Figure 10 a and Figure 10 b is HE and Masson staining after treatment with PBS, PBS_NIR, Van, HAVH, and HAVH_NIR according to one embodiment of this application.

[0136] like Figures 8-10 As shown, HAVH irradiated with near-infrared light can kill all MRSA within 10 days and heal MRSA-infected wounds in mice.

[0137] The above examples are only used to illustrate the present application and are not intended to limit the present application. Those skilled in the art can make various changes and modifications without departing from the scope of the present application. Therefore, all equivalent technical solutions should belong to the scope of the present application.

Claims

1. A nanocomposite comprising: The carrier is a silica particle containing hollow mesopores (HMSN); Silver (Ag); Vancomycin (Van); and Hemin chloride; The silver, vancomycin, and hemin chloride are loaded onto a carrier; the nanocomposite releases silver ions, vancomycin, and hemin chloride under near-infrared light irradiation. Among them, silver is loaded in HMSN, Van is loaded in HMSN that has been loaded with silver, and Hemin is loaded on the surface of HMSN that has been loaded with Ag and Van.

2. The nanocomposite according to claim 1, wherein the support particle size is 91 nm-400 nm; and the Zeta potential of the support is -13.92 ± 0.47 mV.

3. The nanocomposite according to claim 2, wherein the carrier particle size is 95nm-300nm.

4. The nanocomposite according to claim 2, wherein the carrier particle size is 100 nm.

5. The nanocomposite according to claim 1, wherein, The nanocomposite contains 36%-50% silver, 20%-29% vancomycin, and 20%-30% heme chloride.

6. The nanocomposite according to claim 5, wherein, The nanocomposite contains 42.1% silver, 23.5% vancomycin, and 24.1% heme chloride.

7. The nanocomposite according to claim 1, wherein, The nanocomposite has a particle size of 91 nm-300 nm and a potential of -26.13 ± 0.96 mV.

8. The nanocomposite according to claim 1, wherein, The particle size of the nanocomposite is 100nm-200nm.

9. A nano-antibacterial agent comprising the nanocomposite as described in any one of claims 1-8.

10. The use of the nanocomposite as described in any one of claims 1-8 in the preparation of nano-antimicrobial agents for the prevention and / or treatment of diseases caused by pathogens.

11. The application according to claim 10, wherein the nano-antibacterial agent is a near-infrared laser-responsive antibacterial agent.

12. The use of the nanocomposite as described in any one of claims 1-8 or the nano-antibacterial agent as described in claim 9 in the preparation of medicaments for the prevention and / or treatment of mammalian pathogen infections.

13. The application according to claim 12, wherein the infection is a skin infection.

14. The application according to claim 12, wherein the mammal is selected from: primates and rodents.

15. The application according to claim 14, wherein the mammal is selected from: humans, rabbits, canines, and livestock.

16. The application according to claim 14, wherein the mammal is selected from: human, chimpanzee, monkey, mouse, rat, rabbit, dog, pig, cow, sheep.

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