Nano sound-sensitive agent for treating and preventing chronic osteomyelitis and preparation method of nano sound-sensitive agent

Abstract

The invention discloses a nano sound-sensitive agent for treating and preventing chronic osteomyelitis and a preparation method thereof, and the preparation method comprises the following steps: step 1, dropwise adding a zinc acetate solution into a dimethylimidazole solution, fully reacting, and centrifugally washing to obtain ZIF-8; step 2, adding a manganese-containing compound into the ZIF-8 solution, fully reacting, and centrifugally washing to obtain Mn < 2 + >-doped ZIF-8 nanoparticles; and step 3, adding the Mn < 2 + >-doped ZIF-8 nanoparticles into a trehalose solution, fully stirring and mixing, and centrifugally washing to obtain the required sound-sensitive agent. The sonosensitizer obtained by the invention can gradually degrade and release Mn < 2 + > in an acidic microenvironment of an osteomyelitis infected part. The released Mn < 2 + > further enhances the sensitivity of cGAS to bacterial DNA, effectively activates an STING pathway in DCs, promotes maturation of the DCs, enhances the antigen presentation capability, and then promotes proliferation and differentiation of T cells and induces generation of memory B cells, thereby being beneficial to prevention of hidden survival and infection recurrence of bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a nano sonosensitizer for treating and preventing chronic osteomyelitis and a preparation method thereof. Background Art

[0002] Osteomyelitis is a common orthopedic disease, often caused by bacterial infection following trauma, orthopedic surgery, or joint replacement surgery. Staphylococcus aureus is the most common cause of infection, with 50% of infections caused by Staphylococcus aureus (MRSA). Conventional clinical treatment for osteomyelitis involves long-term, high-dose antibiotic therapy combined with multiple surgical debridements. However, due to the presence of drug-resistant bacteria, this treatment often fails to completely eliminate the infection. The immune system plays a key role in defending against pathogens, but trauma not only delays the body's inflammatory response to bacteria but also suppresses cell-mediated immune responses. Furthermore, MRSA, by expressing multiple virulence factors, undermines the host's innate and adaptive immunity, further hindering its effective clearance. Furthermore, MRSA can exploit a "Trojan Horse" strategy to survive within host cells, further complicating its elimination.

[0003] The effective activation of T cells is crucial in the treatment of MRSA infection, among which CD8 + T cells can recognize and eliminate host cells infected by bacteria, while CD4 + T cells play a key role in the formation of memory B cells. As a bridge between innate and adaptive immunity, dendritic cells (DCs) process and present antigens to T cells, promoting their activation. Therefore, efficiently inducing DC maturation is crucial for enhancing immune responses. Extensive progress has been made in DC research, including strategies such as delivering antigenic proteins and agonists. However, these approaches generally suffer from poor targeting, difficulty achieving effective stimulatory concentrations, and potential toxic side effects due to off-target effects. The cyclic guanosine monophosphate-adenylate synthetase-stimulator of interferon genes (cGAS-STING) signaling pathway has reached maturity in oncology, promoting DC maturation through downstream release of type I interferon (IFN-I). However, the in vivo application of existing STING agonists is limited by poor pharmacokinetic properties, low intracellular uptake efficiency, and enzyme instability due to rapid hydrolysis of the phosphodiester bond, resulting in a short half-life and difficulty accumulating to effective intracellular concentrations. In contrast, endogenous DNA released by bacterial death does not need to be transported through the blood, allowing it to more effectively activate the STING pathway.

[0004] For deep-seated infections like osteomyelitis, sonodynamic therapy is a promising approach. Ultrasound-induced reactive oxygen species (ROS) can disrupt bacterial cell membranes through lipid peroxidation, ultimately leading to bacterial death and the release of DNA and other antigens. Because ROS bactericidal activity does not rely on specific biomolecular targets, it can still kill even drug-resistant bacteria. However, ROS have an extremely short half-life, limiting their diffusion distance and weakening their bacterial killing effect. Consequently, they struggle to release sufficient DNA to generate effective immunogenicity. Current osteomyelitis treatment faces the following challenges: The presence of drug-resistant bacteria results in suboptimal clinical outcomes. While ROS bactericidal therapy can eliminate drug-resistant bacteria, its short half-life limits its ability to kill bacteria. Furthermore, the unique pathological environment of osteomyelitis increases the likelihood of recurrence. While activating adaptive immunity can effectively prevent recurrence, conventional treatments often fail to elicit sufficient bacterial immunogenicity to activate adaptive immunity. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a nano sonosensitizer for treating and preventing chronic osteomyelitis and a preparation method thereof.

[0006] The technical solution adopted by the present invention is: a method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis, comprising the following steps:

[0007] Step 1: Add zinc acetate solution dropwise to dimethylimidazole solution, allow to react fully, and centrifuge and wash to obtain ZIF-8;

[0008] Step 2: Add manganese-containing compounds to the ZIF-8 solution obtained in step 1, react fully, and centrifuge and wash to obtain the doped Mn 2+ ZIF-8 nanoparticles;

[0009] Step 3: Add the doped Mn obtained in step 2 2+ The ZIF-8 nanoparticles are added to the trehalose solution, stirred and mixed thoroughly, and then centrifuged and washed to obtain the desired sonosensitizer.

[0010] Furthermore, in step 1, the molar ratio of zinc acetate to dimethylimidazole is 2-3:1-2.

[0011] Furthermore, the reaction in step 1 is a stirring reaction, and the reaction time is 4 to 6 hours.

[0012] Furthermore, the manganese-containing compound in step 2 is manganese acetylacetonate.

[0013] Furthermore, the mass ratio of manganese acetylacetonate to ZIF-8 is 100-200:2-7.

[0014] Furthermore, in step 2, the reaction temperature is 50-60° C., and the reaction time is 12-36 hours.

[0015] Furthermore, in step 3, Mn is doped 2+ The mass ratio of ZIF-8 nanoparticles and trehalose is 1:10~40.

[0016] A nano sonosensitizer for treating and preventing chronic osteomyelitis. The sonosensitizer has a regular dodecahedral structure and a particle size of 100 nm to 300 nm.

[0017] The invention discloses an application of a nano sonosensitizer for treating and preventing chronic osteomyelitis. The sonosensitizer is used as a bactericide in the preparation of medicine.

[0018] Furthermore, the medicine is a medicine for treating and preventing osteomyelitis.

[0019] The beneficial effects of the present invention are:

[0020] (1) The nanoparticles obtained by the present invention (i.e., sonosensitizers, the nanoparticles hereinafter also refer to the sonosensitizers obtained) are doped with Mn 2+ , the doping of manganese ions reduces the band gap of ZIF-8, causing it to produce more ROS under the same conditions;

[0021] (2) The surface of the sonosensitizer obtained by the present invention is modified with trehalose, which gives the nanoparticles the ability to target and bind to Staphylococcus aureus, thereby enhancing the directional killing effect of ROS;

[0022] (3) Mn doped in the sonosensitizer obtained by the present invention 2+ The synergistic effect of the nanoparticles and the surface-modified trehalose significantly enhances the killing efficiency of the nanoparticles on bacteria, induces strong oxidative damage, prompts the bacteria to release a large amount of DNA and other antigens, and stimulates the body's immune response;

[0023] (4) The sonosensitizer obtained by the present invention has pH response characteristics and can gradually degrade and release Mn in the acidic microenvironment of the osteomyelitis infection site. 2+ , enhances sensitivity to bacterial DNA, effectively activates the STING pathway in DCs, promotes their maturation and enhances antigen presentation ability, and then promotes the proliferation and differentiation of T cells, induces the generation of memory B cells, and helps prevent the hidden survival of bacteria and recurrence of infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a SEM image of the nanoparticles obtained in Example 1 of the present invention.

[0025] Figure 2These are characterization diagrams of the nanoparticles obtained in Example 1 of the present invention, a is the XPS spectrum, and b is the FTIR spectrum.

[0026] Figure 3 The ROS performance of the nanoparticles obtained in Example 1 of the present invention under different ultrasound times, a is the change in fluorescence spectrum, and b is the relative intensity.

[0027] Figure 4 This is an image of a bacterial plate after being sterilized by the nanoparticles obtained in Example 1 of the present invention.

[0028] Figure 5 This is a confirmation that the nanoparticles obtained in Example 1 of the present invention promote DCs maturation, a is the DCs maturation rate, and b is the expression of DCs maturation markers.

[0029] Figure 6 These are the experimental results of the nanoparticles obtained in Example 1 of the present invention in mice, where a is the swelling of the mouse legs and b is the bacterial load in the bone marrow.

[0030] Figure 7 The immunotherapy results of the nanoparticles obtained in Example 1 of the present invention on osteomyelitis in mice, a is CD4 + T cell count, b is CD8 + T cell count.

[0031] Figure 8 : The anti-infection ability results of the nanoparticles obtained in Example 1 of the present invention, a is the area of the back abscess, and b is the number of bacteria in the abscess site. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0033] A method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis comprises the following steps:

[0034] Step 1: Add zinc acetate solution dropwise to dimethylimidazole solution, allow to react fully, and centrifuge and wash to obtain ZIF-8; the molar ratio of zinc acetate to dimethylimidazole is 2-3:1-2. The reaction is stirred for 4-6 hours.

[0035] Step 2: Add manganese-containing compounds to the ZIF-8 solution obtained in step 1, react fully, and centrifuge and wash to obtain the doped Mn 2+ The ZIF-8 nanoparticles are prepared; the manganese-containing compound is manganese acetylacetonate. The mass ratio of manganese acetylacetonate to ZIF-8 is 100-200:2-7. The reaction temperature is 50-60°C, and the reaction time is 12-36 hours.

[0036] Step 3: Add the doped Mn obtained in step 22+ The ZIF-8 nanoparticles were added into the trehalose solution, stirred and mixed thoroughly, and then centrifuged and washed to obtain the desired surface-grafted trehalose-coated Mn 2+ Doped ZIF-8 nanoparticles are sonosensitizers. Doped Mn 2 + The mass ratio of ZIF-8 nanoparticles and trehalose is 1:10~40.

[0037] The obtained nanoparticles have a regular dodecahedral structure and a particle size of 100nm to 300nm.

[0038] Example 1

[0039] A method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis comprises the following steps:

[0040] Step 1: Prepare a 12 mM zinc acetate solution and an 8 mM dimethylimidazole solution in methanol. Add equal volumes of the zinc acetate solution dropwise to the dimethylimidazole solution and stir for 5 hours to obtain ZIF-8. Centrifuge at 12,000 rpm for 5 minutes, wash three times, and dry for later use.

[0041] Step 2: Weigh 5 mg of ZIF-8 powder and dissolve it in methanol to obtain a ZIF-8 solution. Add 150 mg of manganese acetylacetonate and react in an oven at 55°C for 24 hours. Then centrifuge at 12,000 rpm for 5 minutes, wash three times, and dry for use to obtain MZ.

[0042] Step 3: Weigh 30 mg of trehalose and dissolve it in RO water. Add methanol to dilute. Weigh 1 mg of the MZ powder obtained in Step 2 and add it to the trehalose solution. Stir overnight. Centrifuge at 12,000 rpm for 5 minutes, wash three times, and dry to obtain TMZ (sonosensitizer).

[0043] Example 2

[0044] A method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis comprises the following steps:

[0045] Step 1: Prepare a 15 mM zinc acetate solution and a 5 mM dimethylimidazole solution in methanol. Add equal volumes of the zinc acetate solution dropwise to the dimethylimidazole solution and stir for 6 hours to obtain ZIF-8. Centrifuge at 12,000 rpm for 5 minutes, wash three times, and dry for later use.

[0046] Step 2: Weigh 7 mg of ZIF-8 powder and dissolve it in methanol to obtain a ZIF-8 solution. Add 200 mg of manganese acetylacetonate and react in an oven at 60°C for 12 hours. Then centrifuge at 12,000 rpm for 5 minutes, wash three times, and dry to obtain MZ.

[0047] Step 3: Weigh 40 mg of trehalose and dissolve it in RO water. Add methanol to dilute. Weigh 1 mg of the MZ powder obtained in Step 2 and add it to the trehalose solution. Stir overnight. Centrifuge at 12,000 rpm for 5 minutes. Wash three times and dry for use to obtain TMZ.

[0048] Example 3

[0049] A method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis comprises the following steps:

[0050] Step 1: Prepare a 10 mM zinc acetate solution and a 10 mM dimethylimidazole solution in methanol. Add equal volumes of the zinc acetate solution dropwise to the dimethylimidazole solution and stir for 4 hours to obtain ZIF-8. Centrifuge at 12,000 rpm for 5 minutes, wash three times, and dry for later use.

[0051] Step 2: Weigh 2 mg of ZIF-8 powder and dissolve it in methanol to obtain a ZIF-8 solution. Add 100 mg of manganese acetylacetonate and react in an oven at 50°C for 36 hours. Then centrifuge at 12,000 rpm for 5 minutes, wash three times, and dry to obtain MZ.

[0052] Step 3: Weigh 10 mg of trehalose and dissolve it in RO water. Add methanol to dilute. Weigh 1 mg of the MZ powder obtained in Step 2 and add it to the trehalose solution. Stir overnight. Centrifuge at 12,000 rpm for 5 minutes. Wash three times and dry to obtain TMZ.

[0053] Figure 1 This is a SEM image of the nanoparticles obtained in Example 1 of the present invention. It can be seen from the image that the obtained nanoparticles are uniform in size and regular in shape, and their particle diameter is between 100 nm and 300 nm.

[0054] Figure 2 The XPS and FT-IR characterization results of the nanoparticles obtained in Example 1 of the present invention are shown in Figures a and b. The XPS fine spectrum of TMZ and the FT-IR characterization results show that manganese exists in TMZ. 2+ The content is higher than that of Mn 4+ , indicating that the post-synthesis ion exchange method successfully converted Mn 2+ Doped into ZIF-8. Figure 2 As can be seen in b, at 1080 and 800 cm -1The presence of symmetric stretching peaks of the trehalose pyran ring and vibration peaks of the oxygen-containing ring proves the successful modification of trehalose.

[0055] The singlet oxygen probe SOSG was used to detect the nanoparticles obtained in Example 1 of the present invention to quantify the ability of TMZ to generate ROS. The results are as follows: Figure 3 Figure 2 shows the fluorescence spectrum of SOSG under different ultrasound exposure times (a), and the relative intensity (b). As can be seen from the figure, when SOSG is oxidized by singlet oxygen, it produces fluorescence at 525 nm. The experimental results show that the fluorescence intensity gradually increases with increasing ultrasound exposure time, demonstrating the continuous generation of singlet oxygen. Compared with ZIF-8, TMZ produces more ROS under the same ultrasound exposure time.

[0056] The plate count method was used to visually evaluate the bactericidal effect of TMZ against Gram-positive MRSA and Gram-negative E. coli. Figure 4 As shown. In the bacterial plate image, control is the control group (PBS group), US is the ultrasound group, TMZ is the group using the nanoparticles obtained in Example 1 of the present invention alone, and the TMZ+US group is the group using the nanoparticles obtained in Example 1 of the present invention and US (ultrasound). As can be seen from the figure, when TMZ and ultrasound are used alone, a large number of bacteria still remain on the plate, and the antibacterial efficiency is low. However, when the nanoparticles obtained in Example 1 of the present invention and ultrasound act together, the bacteria on the plate almost completely disappear, and the antibacterial efficiency is as high as 99%. This shows that when used as a therapeutic drug, the effect is better when combined with ultrasound. The content and concentration of TMZ in the TMZ+US group and the TMZ group are the same.

[0057] The TMZ nanoparticles constructed by the present invention, wherein Mn 2+ The doping of Mn reduces the energy band gap of ZIF-8, enabling it to generate more ROS under the same ultrasonic conditions; at the same time, the modification of trehalose gives TMZ the ability to target and bind to the surface of Staphylococcus aureus, thereby enhancing the directional killing effect of ROS. The synergistic effect of the two greatly improves the killing efficiency of TMZ on bacteria, induces strong oxidative damage, and prompts bacteria to release a large amount of DNA and other related antigens, thereby stimulating the body's immune response. In addition, TMZ has pH-responsive properties and can gradually degrade and release Mn in the acidic microenvironment of the osteomyelitis infection site. 2+ The released Mn 2+ Further enhancing the sensitivity of cGAS to bacterial DNA effectively activates the STING pathway in DCs, promotes their maturation and enhances antigen presentation ability, thereby promoting the proliferation and differentiation of T cells and inducing the generation of memory B cells, thereby helping to prevent the hidden survival of bacteria and recurrence of infection.

Claims

1. A method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis, comprising the following steps: Step 1: Add zinc acetate solution dropwise to dimethylimidazole solution, allow to react fully, and centrifuge and wash to obtain ZIF-8; Step 2: Add manganese-containing compounds to the ZIF-8 solution obtained in step 1, react fully, and centrifuge and wash to obtain the doped Mn 2+ ZIF-8 nanoparticles; Step 3: Add the doped Mn obtained in step 2 2+ The ZIF-8 nanoparticles are added to the trehalose solution, stirred and mixed thoroughly, and then centrifuged and washed to obtain the desired sonosensitizer.

2. The method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis according to claim 1, wherein the molar ratio of zinc acetate to dimethylimidazole in step 1 is 2-3:1-2.

3. The method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis according to claim 1, wherein the reaction in step 1 is a stirring reaction and the reaction time is 4 to 6 hours.

4. The method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis according to claim 1, wherein the manganese-containing compound in step 2 is manganese acetylacetonate.

5. The method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis according to claim 4, wherein the mass ratio of manganese acetylacetonate to ZIF-8 is 100-200:2-7.

6. The method for preparing a nanosonosensitizer for treating and preventing chronic osteomyelitis according to claim 1, wherein the reaction temperature in step 2 is 50-60°C and the reaction time is 12-36 hours.

7. The Mn-containing compound with trehalose grafted on its surface according to claim 1 2+ The preparation method of doped ZIF-8 nanoparticles is characterized in that the Mn doped in step 3 2+ The mass ratio of ZIF-8 nanoparticles and trehalose is 1:10~40.

8. The nanosonosensitizer for treating and preventing chronic osteomyelitis obtained by the preparation method according to any one of claims 1 to 7, characterized in that: The sonosensitizer has a regular dodecahedron structure and a particle size of 100nm to 300nm.

9. The use of a nanosonosensitizer for treating and preventing chronic osteomyelitis according to claim 8, characterized in that: The sonosensitizer is used as a bactericide in the preparation of medicines.

10. The use of a nanosonosensitizer for treating and preventing chronic osteomyelitis according to claim 9, characterized in that: The medicine is a medicine for treating and preventing osteomyelitis.

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