A CFS nanozyme and its application in preparing a CFS-ion-MN wound repair microneedle patch

Abstract

The present invention discloses a preparation method of CFS nanozyme. This nanozyme has broad-spectrum bactericidal property, has low toxicity to normal cells, and there is no problem of bacterial drug resistance, and is expected to be used in the field of antibiotic-free sterilization. The CFS-ion-MN wound repair microneedle patch provided by the present invention, as a wound dressing, can transdermally deliver the antibacterial CFS nanozyme to the deep part of the wound, effectively kill bacteria. The CFS nanozyme can also inhibit inflammation in the body through its superoxide dismutase-like activity, promote wound healing, and promote the antibacterial and healing of the wounds of mice infected with Staphylococcus aureus. Among them, Co 2+ and Fe 3+ can inhibit the IL-17 and NF-κB signaling pathways, thereby accelerating wound closure.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and particularly relates to a CFS nanozyme and its application in the preparation of a CFS-ion-MN wound repair microneedle patch. Background Art

[0002] The skin is the body's first line of defense, protecting the human body from external bacteria. Once the skin is damaged, bacteria can invade the body, and bacterial infection is a common problem during the wound healing process. Bacterial invasion can exacerbate inflammation and delay the wound healing process. With the abuse of antibiotics, the drug resistance of bacteria has been continuously increasing, seriously threatening human health. Therefore, developing new antibacterial agents to improve their efficacy in the healing of bacterially infected wounds is an important challenge currently faced. With the penetration and continuous development of nanotechnology in the antibacterial field, nanomaterials are gradually used to replace antibiotics.

[0003] Nanozymes are nanomaterials with enzyme-like properties, having advantages such as low cost, good stability, and mass production. Through the bactericidal mechanism of catalyzing the production of ROS, the generation of drug-resistant bacteria can be avoided when treating bacterially infected wounds. However, excessive ROS can cause damage to normal cells, so it is necessary to balance the generation and clearance of ROS. In addition, some studies have shown that some metal ions (such as zinc, magnesium, cobalt, etc.) can promote cell proliferation and angiogenesis (Wang Jiani, Chen Junyu. Mechanism of metal ions promoting angiogenesis and its application in bone tissue engineering [J]. Chinese Journal of Tissue Engineering Research, 2024, 28(5): 804-812). Co 2+ and Fe 3+ are essential trace elements in the human body, and they participate in cell proliferation and differentiation. It is known that cobalt mimics hypoxia and induces angiogenesis. For example, Nandin et al. synthesized CoNZ with excellent enzyme-like activity and ion release, which can activate HIF-1α to promote blood vessel formation and is of great significance in promoting the healing of diabetic wounds (Nandin et al. Double hits with bioactive nanozyme based on cobalt-doped nanoglass for acute and diabetic wound therapies through anti-inflammatory and pro-angiogenic functions. Bioactive Materials). However, Co 2+ and Fe 3+ The effect of jointly regulating the inflammatory pathway and wound healing is currently unclear.

[0004] Although nanozymes are good potential alternatives to antibiotics, there are some drawbacks to using nanozymes to combat bacterial infections: nanozymes are difficult to penetrate through the stratum corneum, and the lifespan of ROS is short and the diffusion distance is short. Microneedle technology (MN) is used in the field of transdermal drug delivery, which can pierce the stratum corneum and deliver drugs to the dermis, with the advantages of minimally invasive, painless, and efficient. Among them, soluble microneedles can encapsulate drugs / nanoparticles in a polymer matrix and completely dissolve after insertion into the skin. Combining the soluble polymers polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) into a PVA / PVP mixture has become a new tool for preparing biomaterials. There is a literature that designed a PVP / PVA DMNP microneedle (MN) containing lidocaine for local oral anesthesia. The tip of the microneedle consists of the following parts: polyvinylpyrrolidone / polyvinyl alcohol (PVP / PVA), which can quickly dissolve and release lidocaine hydrochloride and achieve rapid anesthesia within 5 minutes. There is no literature publicly available on products using microneedle technology to load CFS nanozymes and metal ions (Co 2+ and Fe 3+ ). Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to solve the above technical problems, and a PVP / PVA microneedle drug delivery system is designed, which is loaded with CFS nanozymes and metal ions (Co 2+ and Fe 3+ ) to promote the wound healing of drug-resistant bacterial infections.

[0006] Technical Solution:

[0007] The present invention provides a CFS nanozyme, and the CFS nanozyme is a (Co3Fe)(S2)4 material, and its preparation method is as follows:

[0008] 1) Dissolve Fe(NO3)3·9H2O, Co(NO3)2·6H2O and dimethylimidazole in ultrapure water, stir evenly and pour into a reaction kettle for reaction. After the reaction is completed, collect the sample and dry it in an oven;

[0009] 2) Place the sample 1 powder obtained in step 1) at the downstream of the tubular furnace, take sodium hypophosphite and place it at the upstream of the tubular furnace, and react under argon gas;

[0010] 3) Place the sample 2 completed in step 2) at the downstream of the tubular furnace, take sublimed sulfur and place it at the upstream of the tubular furnace, and react under argon gas to obtain the final material (Co3Fe)(S2)4 nanozyme.

[0011] Furthermore, the molar ratio of Fe(NO3)3·9H2O and Co(NO3)2·6H2O in step 1) is 1:1 - 1:3.

[0012] Further, the molar ratio of Fe(NO3)3·9H2O and Co(NO3)2·6H2O to dimethylimidazole in step 1) is 1:5 - 1:10.

[0013] Further, the weight ratio of sample 1 to sodium hypophosphite in step 2) is 1:5 - 1:20.

[0014] Further, the mass ratio of sample 2 to sublimed sulfur in step 3) is 1:5 - 1:20.

[0015] Further, the reaction temperature in step 1) is 100 - 180 °C, and the reaction temperatures in steps 2) and 3) are 300 - 400 °C.

[0016] The present invention also provides a CFS-ion-MN wound repair microneedle patch, which is prepared from the above-mentioned CFS nanozyme as a raw material, and the specific preparation method is as follows:

[0017] 1) Prepare a PVP-PVA gel with a mass fraction of 15 - 30 wt%.

[0018] 2) Take 10 - 20 mL of the PVP-PVA gel prepared in step 1), 50 - 100 mg of the CFS nanozyme described in any one of claims 1 - 6, and 0.6 - 1.2 mg of Fe(NO3)3·9H2O and Co(NO3)2·6H2O respectively. Stir and mix evenly in a water bath at 80 - 100 °C for 0.5 - 1 h to form a gel-like substance.

[0019] 3) Pour 0.3 - 0.5 mL of the gel-like substance obtained in step 2) onto a PDMS mold, and use a vacuum pump to evacuate and fill the voids of the needle tips.

[0020] 4) Then add 0.2 - 0.5 mL of the PVP-PVA solution prepared in step 1) as the base, evacuate to remove air bubbles, and then place it in an oven at 30 °C - 60 °C to dry for 3 - 8 hours to obtain CFS-ion-MN.

[0021] 5) Peel the CFS-ion-MN from the mold.

[0022] Further, the mass ratio of PVP to PVA is 9:1 - 9:3.

[0023] Further, the molar ratio of Co 2+ and Fe 3+ in the aqueous solutions of Fe(NO3)3·9H2O and Co(NO3)2·6H2O is 1:1 - 1:3.

[0024] The present invention also provides the application of the CFS-ion-MN wound repair microneedle patch in promoting wound healing.

[0025] Beneficial effects:

[0026] The primary object of the present invention is to provide a method for preparing a CFS nanozyme, which can efficiently catalyze the decomposition of H2O2 to generate ROS, achieve good antibacterial properties against Gram-negative and Gram-positive bacteria in the presence of low concentrations of H2O2, and has broad-spectrum bactericidal properties, has low toxicity to normal cells, and does not have the problem of bacterial drug resistance, and is expected to be used in the field of antibiotic-free sterilization.

[0027] The CFS-ion-MN wound repair microneedle patch provided by the present invention, as a wound dressing, can transdermally deliver the antibacterial CFS nanozyme to the deep part of the wound, effectively kill bacteria, and the CFS nanozyme can also inhibit inflammation in the body through superoxide dismutase-like activity, promote wound healing, and promote the antibacterial and healing of the wounds of mice infected with Staphylococcus aureus. Among them, Co 2+ and Fe 3+ can inhibit the IL-17 and NF-κB signaling pathways, thereby accelerating wound closure. Description of the drawings

[0028] Figure 1 X-ray diffraction spectrogram (a) and scanning electron micrograph (b) of the CFS nanozyme prepared in Example 1.

[0029] Figure 2 Antibacterial performance of the CFS nanozyme prepared in Example 1.

[0030] Figure 3 Synthesis and pictures of CFS-ion-MN.

[0031] Figure 4 Wound healing ability of the CFS-ion-MN prepared in Example 5.

[0032] Figure 5 Transcriptomic analysis and qRT-PCR analysis of the CFS-ion-MN prepared in Example 5 on mouse wounds. Detailed implementation manners

[0033] To deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with the embodiments and the drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0034] Example 1 Preparation of CFS nanozyme

[0035] First, dissolve 1 mmol of Fe(NO3)3·9H2O and 2 mmol of Co(NO3)2·6H2O in 30 mL of ultrapure water (solution A), then dissolve 18 mmol of dimethylimidazole in 20 mL of ultrapure water (solution B). Pour solution B into solution A and stir rapidly for 15 minutes (solution C). Then pour solution C into a 100 mL autoclave and react in an oven at 120 °C for 24 hours. After the reaction, centrifuge, wash three times with an ethanol-water or methanol-water mixture, collect the sample and dry it overnight in an oven at 60 °C. The next day, place 100 mg of the dried powder downstream in a tube furnace, place 1 g of sodium hypophosphite upstream in the tube furnace, and react at 300 °C for 2 hours under an argon atmosphere. After the reaction is complete, place the material downstream in the tube furnace again, place 1 g of sublimed sulfur upstream in the tube furnace, and react at 400 °C for 1 hour under an argon atmosphere to obtain the final material (Co3Fe)(S2)4, which is abbreviated as CFS.

[0036] Figure 1 (a) X-ray diffraction spectrum (XRD) of (Co3Fe)(S2)4 prepared in Example 1, where the peaks of (Co3Fe)(S2)4 are located at 28, 32.5, 36.5, 40.1, 46.6, 55.3, 58, 60.6, 63.1, 77.4, which is consistent with the standard card of (Co3Fe)(S2)4 (PDF#97-062-970). The XRD spectrum demonstrates the successful synthesis of (Co3Fe)(S2)4. Figure 1 (b) SEM shows that CFS presents aggregated irregular spherical nanoparticles.

[0037] Preparation of CFS nanozyme in Example 2

[0038] First, dissolve 1 mmol of Fe(NO3)3·9H2O and 3 mmol of Co(NO3)2·6H2O in 30 mL of ultrapure water (solution A), then dissolve 20 mmol of dimethylimidazole in 20 mL of ultrapure water (solution B). Pour solution B into solution A and stir rapidly for 15 minutes (solution C). Then pour solution C into a 100 mL autoclave and react in an oven at 180 °C for 16 hours. After the reaction, centrifuge, wash three times with an ethanol-water or methanol-water mixture, collect the sample and dry it overnight in an oven at 60 °C. The next day, place 200 mg of the dried powder downstream in a tube furnace, place 1 g of sodium hypophosphite upstream in the tube furnace, and react at 350 °C for 2 hours under an argon atmosphere. After the reaction is complete, place the material downstream in the tube furnace again, place 1 g of sublimed sulfur upstream in the tube furnace, and react at 400 °C for 1 hour under an argon atmosphere to obtain the final material (Co3Fe)(S2)4, which is abbreviated as CFS.

[0039] Preparation of CFS nanozyme in Example 3

[0040] First, dissolve 1 mmol of Fe(NO3)3·9H2O and 1 mmol of Co(NO3)2·6H2O in 30 mL of ultrapure water (solution A), then dissolve 20 mmol of dimethylimidazole in 20 mL of ultrapure water (solution B). Pour solution B into solution A and stir rapidly for 15 minutes (solution C). Then pour solution C into a 100 mL autoclave and react in an oven at 160 °C for 18 hours. After the reaction, centrifuge, wash 3 times with an ethanol-water or methanol-water mixture, collect the sample and dry it overnight in an oven at 60 °C. The next day, place 100 mg of the dried powder downstream of a tubular furnace, place 1 g of sodium hypophosphite upstream of the tubular furnace, and react at 300 °C for 2 hours under argon gas. After the reaction is completed, place the material downstream of the tubular furnace again, place 1 g of sublimed sulfur upstream of the tubular furnace, and react at 400 °C for 1.5 hours under argon gas to obtain the final material (Co3Fe)(S2)4, which is abbreviated as CFS.

[0041] Example 4 Testing the antibacterial property of CFS nanozyme by plate counting method

[0042] Steps of the plate counting method: Activate methicillin-resistant Staphylococcus aureus ( MRSA ) and multidrug-resistant Escherichia coli ( MDR E.coli ) at 37 °C and 150 rmp for 24 hours. Dilute the bacterial solution to 1×10 -5 cfu, then take 50 μL of the bacterial solution and mix it with CFS in the presence or absence of H2O2 (50 μL, 0.5 mM). Incubate in a shaker (37 min, 150 r) for 2 hours, and use the liquid medium instead of the material as the control group. Then, spread the mixed sample on a solid culture dish and perform colony counting after incubating at 37 °C for 24 h.

[0043] Figure 2 Results of the plate counting method for CFS nanozyme against MRSA and MDR E.coli . Among them, compared with the control group and the H2O2 group, the CFS and CFS + H2O2 groups showed excellent antibacterial ability, indicating that CFS has good antibacterial performance, and the colony counting shows that CFS has almost 100% antibacterial ability.

[0044] Example 5 Preparation method of CFS-ion-MN

[0045] Figure 3For the synthesis process of CFS-ion-MN, PVP and PVA were mixed in a ratio of 9:1 and formulated into a 20 wt% gel (PVP-PVA). 20 mL of this gel was taken, and then 100 mg of CFS, 1.2 mg of Fe(NO3)3·9H2O, and 1.2 mg of Co(NO3)2·6H2O were added thereto. Stirring was carried out at 90 °C for 1 hour to form a mixed gel-like substance. Then, 0.5 mL of the solution was cast onto a PDMS mold, and a vacuum pump was used to evacuate the air. Then, 0.1 mL of PVP-PVA gel was added as a base, and after evacuating air to remove bubbles, it was placed in an oven at 40 °C and dried for 4 hours, and finally demolded.

[0046] Example 6 Preparation method of CFS-ion-MN

[0047] PVP and PVA were mixed in a ratio of 9:2 and formulated into a 20 wt% gel (PVP-PVA). 20 mL of this gel was taken, and then 100 mg of CFS, 1.2 mg of Fe(NO3)3·9H2O, and 2.4 mg of Co(NO3)2·6H2O were added thereto. Stirring was carried out at 95 °C for 1 hour to form a mixed gel-like substance. Then, 0.5 mL of the solution was cast onto a PDMS mold, and a vacuum pump was used to evacuate the air. Then, 0.1 mL of PVP-PVA gel was added as a base, and after evacuating air to remove bubbles, it was placed in an oven at 60 °C and dried for 3 hours, and finally demolded.

[0048] Example 7 Wound healing ability of CFS-ion-MN

[0049] Using Staphylococcus aureus as the infection strain, a wound model was created on the back of a mouse. After 5 days of adaptive feeding, a wound with a diameter of 6 mm was created on the back skin of the mouse and infected with Staphylococcus aureus. It was treated with the CFS-ion-MN composite microneedle patch prepared in Example 5. The groups without any treatment, commercial dressing group, CFS group (i.e., CFS nanozyme), MN group (i.e., PVA-PVP gel), and CFS-MN group (i.e., without Co 2+ and Fe 3+ ) were used as control groups. The body weight and food intake of the mice were monitored daily, and the wound healing situation of the mice was observed and photographed.

[0050] Figure 4 It is a treatment picture of the CFS-ion-MN wound repair microneedle patch for antibacterial and promoting wound healing. After 9 days of treatment, the wound healing rates of the control group and the MN group were the lowest, and large scabs could be seen on the wound surface. Followed by the wound dressing group and the CFS group. The healing effects of the CFS-MN and CFS-ion-MN groups were good, and among them, CFS-ion-MN had the best ability to promote wound surface healing.

[0051] Example 8 Transcriptomic analysis and qRT-PCR analysis of mouse wounds

[0052] Mouse wound tissues were taken for transcriptomic analysis and qRT-PCR analysis. Figure 5 For the transcriptomic analysis of mouse wounds, according to the results of KEGG enrichment analysis, the top 10 signaling pathways with the highest enrichment degree were selected for display, and it was found that the pathways related to differentially expressed genes associated with the immune system were NF-κB and IL-17. Genes related to IL-17 and NF-κB were detected by qRT-PCR. The results showed that CFS-MN had a certain inhibitory effect on these genes. In addition, after adding metal ions, CFS-ion-MN could also significantly inhibit the expression of IL-17 and NF-κB and their downstream targets. In summary, these findings indicate that CFS-ion-MN can inhibit inflammation through the IL-17 and NF-κB signaling pathways, thereby further improving the proliferation of keratinocytes and angiogenesis to accelerate wound healing in mice.

[0053] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A CFS nanozyme, characterized in that, The CFS nanozyme is a (Co3Fe)(S2)4 material, and its preparation method is as follows: Dissolve Fe(NO3)3·9H2O, Co(NO3)2·6H2O and dimethylimidazole in ultrapure water, stir evenly and pour into a reaction kettle for reaction. After the reaction, collect the sample and dry it in an oven; Place the sample 1 powder obtained in step 1) downstream of the tube furnace, take sodium hypophosphite and place it upstream of the tube furnace, and react under argon gas; Place the sample 2 after the reaction in step 2) downstream of the tube furnace, take sublimed sulfur and place it upstream of the tube furnace, and react under argon gas to obtain the final material (Co3Fe)(S2)4 nanozyme.

2. The CFS nanozyme according to claim 1, wherein In step 1), the molar ratio of Fe(NO3)3·9H2O to Co(NO3)2·6H2O is 1:1 - 1:

3.

3. The CFS nanozyme according to claim 1, characterized in that, In step 1), the molar ratio of Fe(NO3)3·9H2O and Co(NO3)2·6H2O to dimethylimidazole is 1:5 - 1:

10.

4. The CFS nanozyme according to claim 1, wherein In step 2), the weight ratio of sample 1 to sodium hypophosphite is 1:5 - 1:

20.

5. The CFS nanozyme according to claim 1, wherein In step 3), the mass ratio of sample 2 to sublimed sulfur is 1:5 - 1:

20.

6. The CFS nanozyme according to claim 1, characterized in that, The reaction temperature in step 1) is 100 - 180 °C, and the reaction temperatures in steps 2) and 3) are 300 - 400 °C.

7. A CFS-ion-MN wound repair microneedle patch, characterized in that, Prepared from the CFS nanozyme described in any one of claims 1 - 6 as a raw material, and the specific preparation method is as follows: 1) Prepare a PVP - PVA gel with a mass fraction of 15 - 30 wt%; 2) Take 10 - 20 ml of the PVP - PVA gel in step 1), 50 - 100 mg of the CFS nanozyme described in any one of claims 1 - 6, 0.6 - 1.2 mg of Fe(NO3)3·9H2O and Co(NO3)2·6H2O respectively, and heat and stir in a water bath at 80 - 100 °C for 0.5 - 1 h to mix and stir evenly to form a gel; 3) Pour 0.3 - 0.5 mL of the gel obtained in step 2) onto a PDMS mold, and use a vacuum pump to evacuate and fill the tip gap; 4) Then add 0.2 - 0.5 mL of the PVP - PVA solution as a base, evacuate, remove bubbles, and place it in an oven at 30 °C - 60 °C for drying for 3 - 8 hours to obtain CFS - ion - MN; 5) Peel the CFS - ion - MN from the mold.

8. The CFS-ion-MN wound repair microneedle patch according to claim 7, wherein, The mass ratio of PVP to PVA is 9:1 ~ 9:

4.

9. The CFS-ion-MN wound repair microneedle patch according to claim 7, wherein In the aqueous solutions of Fe(NO3)3·9H2O and Co(NO3)2·6H2O, the molar ratio of Co 2+ and Fe 3+ is 1:1 - 1:

3.

10. Use of the CFS - ion - MN wound - repairing microneedle patch described in any one of claims 6 - 9 in the preparation of a material for promoting wound healing.

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