Nano-antibacterial material based on bisoxalate and hypericin as well as preparation method and application of nano-antibacterial material

Abstract

The invention discloses a nano antibacterial material based on bisoxalate and hypericin as well as a preparation method and application of the nano antibacterial material, and belongs to the technical field of antibacterial agents. A supermolecule self-assembly method is used for organically combining a chemiluminescence agent CPPO and a photosensitizer hypericin, the chemiluminescence agent CPPO and the photosensitizer hypericin are tightly combined through chemical acting force, and the antibacterial nanometer material with the optimal size and dispersity is obtained by adjusting the ratio of the chemiluminescence agent CPPO and the photosensitizer hypericin. The antibacterial nano-material disclosed by the invention has excellent antibacterial biofilm performance generated by triple effects of deep permeation, production of active oxygen to kill bacteria and inhibition of H2S from damaging a self-defense mechanism of the bacteria, and still has a relatively good antibacterial effect on methicillin-resistant staphylococcus aureus at low concentration (2 mu g / mL).

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial agents, and particularly relates to a nano antibacterial material based on bisoxalate and hypericin, and a preparation method and application thereof. Background Art

[0002] The abuse of antibiotics and the intelligent evolution of bacteria have led to the rapid spread of drug resistance. Among them, methicillin-resistant Staphylococcus aureus is one of the most dangerous drug-resistant bacteria, which can cause infection and even death. The widespread presence of bacterial biofilms in wound infections is a key factor in the difficulty of eradicating infections. In this context, it is crucial to explore and develop antibacterial materials that do not cause bacterial resistance and are harmless to the environment.

[0003] Antibiotic-free strategies are important emerging methods to address the challenges of antimicrobial resistance. Among them, antimicrobial photodynamic therapy (APDT) uses the combined action of light, oxygen and photosensitizers to destroy microorganisms, and has great potential in antimicrobial applications. APDT uses the combined action of light, oxygen and photosensitizers to destroy microorganisms, and has great potential in antimicrobial applications. Because biofilms cannot prevent APDT from increasing reactive oxygen species in wounds, APDT has the effects of reactive oxygen-induced oxidative stress, weakening of biofilm matrix, adhesion loss and composition changes on the strength of biofilm matrix and metabolic activity of constituent pathogens. The absorbed light energy is always used for bactericidal or antibacterial effects through two key molecular photosensitizer-mediated mechanisms. For example, a patent application with publication number CNl14949206A discloses a new antibacterial therapy for photodynamic treatment of bacterial endophthalmitis, including: selecting TTPy molecules; dissolving TTPy with DMSO to obtain a 5mM mother solution; diluting the mother solution with PBS solution; injecting the diluted mother solution into rat eyes; and subjecting the injection site to certain conditions of light illumination to achieve effective in vivo antibacterial effects. For example, the patent application with publication number CN117618557A discloses a phage-mediated targeted photosensitizer and its preparation method and application. When the phage is targeted and specifically bound to the bacterial surface, the photosensitive molecules also gather on the bacterial surface with the phage. Compared with the treatment with equal concentration of free photosensitive molecules, the photosensitive molecules bound to the phage can be directed to gather around the bacteria. The ROS generated around the bacteria after light irradiation is higher, and the destructive power to the bacteria is stronger.

[0004] However, bacterial biofilms have the effect of limiting the penetration of drugs and photosensitizers, and are susceptible to the penetration depth of ambient oxygen light in tissues, which is affected by the optical properties of tissues and biofilms, limiting the application of APDT. Summary of the invention

[0005] In order to overcome the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a nano antibacterial material based on bis(oxalate) (CPPO) and hypericin, and its preparation method and application, wherein the antibacterial nanomaterial organically combines the chemiluminescent agent CPPO with the photosensitizer hypericin in the form of supramolecular self-assembly, induces chemiluminescence through the characteristics of the bacterial biofilm microenvironment, and activates antibacterial photodynamic therapy in this form. The antibacterial nanomaterial in the present invention has a significant antibacterial effect on methicillin-resistant Staphylococcus aureus.

[0006] The specific technical solutions are as follows:

[0007] The present invention provides a nano antibacterial material based on bisoxalate and hypericin, wherein the nano antibacterial material comprises bisoxalate and hypericin.

[0008] The nano antibacterial material is a nanoparticle formed by supramolecular self-assembly of bisoxalate and hypericin, wherein CPPO and hypericin in the nanoparticle are closely connected by van der Waals force, hydrogen bond, π-π stacking and other forces.

[0009] Preferably, the particle size of the nano antibacterial material is 50 to 60 nm.

[0010] The loading amounts of bisoxalate and hypericin in the nano antibacterial material are 60% to 70% and 30% to 40% respectively.

[0011] The antibacterial nanomaterial needs to realize photodynamic therapy through chemiluminescence self-activation photosensitizer. The emission wavelength of the required chemiluminescent agent needs to correspond to the excitation wavelength of the photosensitizer. The present invention selects the chemiluminescent agent CPPO with an emission wavelength of about 595nm and the excitation wavelength of hypericin at 592nm. The corresponding wavelengths of the two are consistent, thereby ensuring that the combination of the two can spontaneously activate the antibacterial photodynamic therapy.

[0012] The present invention also provides a method for preparing the nano antibacterial material. Under light-proof conditions, bisoxalate and hypericin are used to form the nano antibacterial material through supramolecular self-assembly.

[0013] Preferably, the molar ratio of the bisoxalate to hypericin is 1-5:2-5. Specific molar ratios include but are not limited to 1:1, 2:1, 3:1, 5:1, 1:5, 1:3, and 1:2.

[0014] Specifically, the supramolecular self-assembly (anti-solvent precipitation method) comprises the following steps:

[0015] The bisoxalate is dissolved in anhydrous ethanol to obtain a mixed solution 1, and the hypericin is dissolved in dimethyl sulfoxide to obtain a mixed solution 2. The mixed solution 1 is uniformly added to the mixed solution 2 to generate supramolecular self-assembly.

[0016] Preferably, the molar concentration of the mixed solution 1 is 3 mM, and the molar concentration of the mixed solution 2 is 1 mM.

[0017] The uniform speed is 0.2 mL / min.

[0018] When the molar ratio of CPPO to hypericin is 3:1, the prepared nano antibacterial material has good dispersibility and regular morphology. In the embodiment of the present invention, the molar ratio of 3:1 is used as an example, but it is not limited thereto.

[0019] Preferably, the supramolecular self-assembly condition is: reaction at 25° C. for 30 min.

[0020] The mixed solution obtained by supramolecular self-assembly is centrifuged and washed with water to disperse to obtain supramolecular self-assembled self-activated nanomaterials. The centrifugal speed of the synthesized mixed solution is 10000 rpm, and the supramolecular self-assembled photodynamic nanoparticles obtained after centrifugation are dialyzed for 24 hours.

[0021] The invention also provides application of the nano antibacterial material in preparing an antibacterial agent.

[0022] Preferably, the bacteria in the antibacterial agent is methicillin-resistant Staphylococcus aureus. The antibacterial nanomaterial has an inhibition rate of 98% on methicillin-resistant Staphylococcus aureus.

[0023] The mechanism of the present invention is as follows:

[0024] The present invention utilizes H in the bacterial biofilm microenvironment 2 O 2 Activate the chemiluminescence of CPPO and produce CO 2 Gas, gas molecules help drugs penetrate deep into biological membranes;

[0025] After CPPO in the supramolecular self-assembled self-activated photodynamic nanoparticles is induced to emit light, the photosensitizer hypericin is activated to produce singlet oxygen, thereby killing bacterial biofilms;

[0026] Hypericin itself has the ability to inhibit H 2 The role of S-producing enzyme CBS in reducing bacterial H 2 The production of S destroys the bacteria's own H 2 S's own defense mechanism against oxidative stress.

[0027] The beneficial effects of the present invention are:

[0028] The present invention uses a supramolecular self-assembly method to organically combine the chemiluminescent agent CPPO and the photosensitizer hypericin. The two are tightly combined by chemical forces, and the antibacterial nanomaterial with the most suitable size and dispersion is obtained by adjusting the ratio of the two. The antibacterial nanomaterial in the present invention has the advantages of deep penetration, production of active oxygen to kill bacteria, inhibition of H 2 S has excellent antibacterial biofilm properties produced by the triple action of destroying the bacteria's own defense mechanism, and it still has a good antibacterial effect on methicillin-resistant Staphylococcus aureus at a low concentration (2μg / mL).

[0029] The preparation method of the present invention has simple preparation steps, simple and convenient administration, safe raw materials, reliable and mild reaction conditions and strong biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a scanning electron microscope image of the antibacterial nanomaterial in Example 1 of the present invention.

[0031] Figure 2 It is a transmission electron microscope image of the antibacterial nanomaterial in Example 1 of the present invention.

[0032] Figure 3 It is a schematic diagram of the binding molecule docking of the antibacterial nanomaterial in Example 1 of the present invention.

[0033] Figure 4 is the H of the antibacterial nanomaterial in Example 2 of the present invention 2 O 2 Transmission electron micrograph showing activated gas production.

[0034] Figure 5 Schematic diagram of HOMO / LUMO energy level transition of CPPO, tetracycline and hypericin in the self-activated photodynamic therapy of antibacterial nanomaterials in Example 2 of the present invention.

[0035] Figure 6 This is a graph showing the antibacterial test of the antibacterial nanomaterial in Example 3 of the present invention against methicillin-resistant Staphylococcus aureus.

[0036] Figure 7 This is a live-death graph of the antibacterial nanomaterial in Example 4 against methicillin-resistant Staphylococcus aureus biofilm. DETAILED DESCRIPTION

[0037] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0038] Example 1

[0039] The antibacterial nanomaterial in this example is prepared by the following method, which specifically includes the following steps:

[0040] (1) Accurately weigh 20.31 mg of CPPO and dissolve it in 10 mL of anhydrous ethanol to obtain an anhydrous ethanol solution of CPPO with a molar concentration of 3 mM.

[0041] (2) Accurately weigh 5.04 mg of hypericin and dissolve it in 10 mL of DMSO to obtain a hypericin DMSO solution with a molar concentration of 1 mM.

[0042] (3) The CPPO anhydrous ethanol solution in (1) was loaded into a 20 mL syringe, and the hypericin DMSO solution in (2) was loaded into a 50 mL round-bottom flask, which was placed in a magnetic thermostatic stirrer and set to 25°C. The CPPO anhydrous ethanol solution was injected into the round-bottom flask containing the hypericin DMSO solution at a rate of 0.2 mL / min using a syringe pump. The magnetic stirring was turned on and the reaction was continued for 30 min after the CPPO solution was completely pushed into the round-bottom flask. This step was protected from light throughout.

[0043] (4) The solution obtained in step (3) was centrifuged at 10,000 rpm for 10 min, the supernatant was removed, distilled water was added, and the solution was centrifuged at 10,000 rpm for washing. This step was repeated three times.

[0044] (5) The antibacterial nanomaterial prepared in step (4) is dispersed in water, placed in a 1000 Da dialysis bag, and dialyzed in anhydrous ethanol in the dark for 24 h to obtain the antibacterial nanomaterial.

[0045] (6) Dispersing the antibacterial nanomaterial obtained in step (5) with water, taking 10 μL and placing it on a copper mesh and a silicon wafer, drying it, and observing its morphology under a transmission electron microscope and a field emission scanning electron microscope.

[0046] The scanning electron microscopy and transmission electron microscopy images of the prepared antibacterial nanoparticles are shown in Figure 1 and Figure 2 As shown by Figures 1-2 It can be seen that the antibacterial nanoparticles are generally round bowl-shaped, uniform in size, and well dispersed, and the particle size of the antibacterial nanoparticles is between 50 and 60 nm.

[0047] Example 2

[0048] The antibacterial nanomaterials in this embodiment are 2 O 2 The activation gas production adopts the following preparation method, which specifically includes the following steps:

[0049] Take the antibacterial nanomaterial aqueous dispersion prepared in step (5) of Example 1, add 20 mM H 2 O2 solution, and 10 μL of the liquid after reaction at different time points (5 min, 10 min, 30 min, 2 h) was placed on a copper grid. After drying, it was placed under a transmission electron microscope to observe the morphological changes.

[0050] The antibacterial nanoparticles in this embodiment are 2 O 2 The transmission electron microscopy images under the action of Figure 4 As shown by Figure 4 It can be seen that antibacterial nanoparticles 2 O 2 After the action, bubbles gradually appear over time and break up after 2 hours.

[0051] The HOMO-LUMO orbitals of CPPO, tetracyclic ketone and hypericin were calculated using the calculation software Gaussian09 and the method b3lyp with the relevant atomic basis set of 6-31g(d).

[0052] The HOMO-LUMO energy level transition of the antibacterial nanoparticle self-activated photodynamic therapy in this embodiment is as follows Figure 5 As shown by Figure 5 It can be seen that the HOMO-LUMO energy level difference of the CPPO chemiluminescent intermediate tetracyclic ketone is greater than that of hypericin, and it can successfully activate hypericin to exert the effect of photodynamic therapy.

[0053] Example 3

[0054] The antibacterial performance of the antibacterial nanomaterial prepared in Example 1 was tested, and the specific testing method was as follows:

[0055] (1) Thaw methicillin-resistant Staphylococcus aureus (MRSA) in a 37°C water bath, and activate and inoculate the bacteria in a biosafety cabinet. Use an inoculation loop to dip the bacterial solution in the space above the flame of an alcohol lamp, and continuously streak lines on a culture dish covered with tryptic soy bean paste (TSB) solid culture medium. The lines should be close but not connected. After streaking, burn the inoculation loop with an alcohol lamp. After cooling, use the end of the previous streak as the starting point to make parallel lines. Repeat 3-4 times, and then place the culture dish in a constant temperature incubator at 37°C for 16-18 hours to form colonies for later use.

[0056] (2) Use an inoculating loop to pick 3-5 single colonies from the TSB solid medium containing MRSA in step (1) and inoculate them into a tube containing 2.5 mL of TSB liquid medium. Loosen the tube cap and place it in a 37°C constant temperature air bath shaker. Cultivate at 200 rpm for 16-18 hours to obtain stable bacteria.

[0057] Take 40 μL of the stable phase bacterial solution and inoculate it into a culture tube containing 4 mL of TSB liquid culture medium, place it at 37°C and 200 rpm for 3 to 4 hours until it reaches the logarithmic phase, centrifuge it at 6000-8000 rpm in a centrifuge for 5 minutes, and white bacterial plaques are seen at the bottom of the tube.

[0058] Discard the supernatant, wash the bacteria with PBS buffer, centrifuge and wash twice, then add PBS buffer to adjust the OD 600 Around 0.5 (this value is fixed each time to facilitate the control of bacterial quantity and ensure repeatability in the later stage). At this time, the bacterial concentration is about 5×10 10-11 CFU / mL, dilute the bacteria 10 times and set aside.

[0059] Take an appropriate amount of MRSA suspension in the stable period and adjust the concentration of MRSA suspension to 1.5×10 6 CFU / mL, take 50 μL of the diluted bacterial solution and add it to a 96-well plate.

[0060] PBS solution and PBS solution of antibacterial nanomaterials (2 μg / mL) were added to the microplate containing bacterial solution and incubated for 3 hours in a 37°C constant temperature incubator. In order to produce visible colonies, the diluted bacterial solution (20 mL) was spread on TSB solid medium and then incubated inverted in a 37°C constant temperature incubator overnight. Each sample was replicated 3 times, and the colonies were photographed and recorded with a camera.

[0061] The antibacterial effect of this embodiment on MRSA is as follows Figure 6 As shown, the results show that the antibacterial nanomaterial has a good antibacterial effect.

[0062] Example 4

[0063] The antibacterial nanomaterial prepared in Example 1 was tested for its anti-MRSA biofilm performance, and the specific testing method was as follows:

[0064] (1) Take an appropriate amount of MRSA bacterial suspension in the logarithmic phase and dilute it with TSB liquid medium to a bacterial concentration of about 1×10 6 CFU / mL, after mixing evenly, add 2mL of bacterial solution to each well of a 6-well microplate and culture in a 37°C constant temperature incubator for 48 hours. During the culture process, replace the TSB medium every 24 hours to maintain the nutritional state of the biofilm. Subsequently, discard the medium and wash with PBS buffer to remove planktonic bacteria. Finally, a MRSA biofilm can be formed at the bottom of the well plate.

[0065] (2) PBS solution and PBS solution of antimicrobial nanomaterials (2 μg / mL) were added to the MRSA biofilm and incubated in a 37°C incubator for 4 h. The supernatant was discarded and the biofilm was washed three times with PBS buffer.

[0066] (3) Then, PBS buffer containing SYTO-9 (2 mM) and PI (1 mM) was added in the dark and stained at room temperature for 15 minutes. After staining, the excess dye solution was washed away and the two fluorescent dyes in the biofilm were observed by laser confocal microscopy to evaluate the anti-MRSA biofilm effect of the antibacterial nanomaterial.

[0067] The killing effect of this embodiment on MRSA biofilm is as follows Figure 7 As shown, the results showed that the antibacterial nanomaterial had a good anti-MRSA biofilm effect.

Claims

1. A nano antibacterial material based on bisoxalate and hypericin, characterized in that: The nano antibacterial material includes bisoxalate and hypericin. The nano antibacterial material is nano particles formed by the supramolecular self-assembly of bisoxalate and hypericin.

2. The nano antibacterial material according to claim 1, characterized in that: The particle size of the nano antibacterial material is 50-60 nm.

3. The nano antibacterial material according to claim 1, characterized in that: The loading amounts of bisoxalate and hypericin in the nano antibacterial material are 60% to 70% and 30% to 40% respectively.

4. The method for preparing the nano antibacterial material according to any one of claims 1 to 3, characterized in that: Under the condition of avoiding light, bisoxalate and hypericin form the nano antibacterial material through supramolecular self-assembly.

5. The method for preparing the nano antibacterial material according to claim 4, characterized in that: The molar ratio of the bisoxalate to hypericin is 1-5:2-5.

6. The method for preparing the nano antibacterial material according to claim 5, characterized in that: The supramolecular self-assembly comprises the following steps: The bisoxalate is dissolved in anhydrous ethanol to obtain a mixed solution 1, and the hypericin is dissolved in dimethyl sulfoxide to obtain a mixed solution 2. The mixed solution 1 is uniformly added to the mixed solution 2 to generate supramolecular self-assembly.

7. The method for preparing the nano antibacterial material according to claim 6, characterized in that: The molar concentration of the mixed solution 1 is 3 mM, and the molar concentration of the mixed solution 2 is 1 mM. The uniform speed is 0.2 mL / min.

8. The method for preparing the nano antibacterial material according to claim 4, characterized in that: The conditions for the supramolecular self-assembly are: reaction at 25° C. for 30 min.

9. Use of the nano antibacterial material according to any one of claims 1 to 3 in the preparation of an antibacterial agent.

10. The use according to claim 9, characterized in that: The bacteria in the antibacterial agent is methicillin-resistant Staphylococcus aureus.

Images