A FeN-T nanozyme composite material, its preparation method and application

Abstract

This invention discloses a FeN-T nanozyme composite material, its preparation method, and its applications, belonging to the field of pharmaceutical formulation technology. The FeN-T nanozyme composite material is composed of iron-based nanozyme, tacrolimus, and polylactic-co-glycolic acid copolymer. This invention prepares the nanozyme composite material (FeN-T) by encapsulating iron-based nanozyme (FeN) with the anti-inflammatory drug tacrolimus (TAC) using PLGA. Experimental verification shows that FeN-T can promote cell migration and has almost no cytotoxicity at effective therapeutic concentrations; it can also improve the antibacterial rate of H2O2; simultaneously, the combined treatment of FeN-T and H2O2 achieves a wound healing rate of 95% within 9 days, significantly better than single treatment. Therefore, this invention provides a multifunctional composite material with highly efficient antibacterial, good biocompatibility, and anti-inflammatory and repair-promoting functions.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to a FeN-T nanozyme composite material, its preparation method, and its application. Background Technology

[0002] Bacterial infections, especially those caused by drug-resistant bacteria, have become a major threat to global public health. In veterinary and clinical practice, traditional antibiotics are the primary treatment. However, the overuse of antibiotics accelerates the development of bacterial resistance. Simultaneously, bacteria readily form biofilms on wound surfaces, creating physical barriers and a slightly acidic environment, severely weakening the efficacy of traditional antibiotics and leading to treatment failure.

[0003] In recent years, nanozymes, as functional nanomaterials with enzyme-like catalytic activity, have provided new ideas for anti-infective therapy. Among them, iron-based nanozymes (FeN) have shown excellent antibacterial properties. However, single FeN nanozymes still face problems such as poor biocompatibility, uncontrollable properties, and lack of anti-inflammatory synergistic effects, which limit their clinical translation.

[0004] Therefore, developing a multifunctional composite material that combines high-efficiency antibacterial activity, good biocompatibility, and anti-inflammatory and repair-promoting functions is of great significance for breaking through the current bottleneck in the treatment of drug-resistant bacterial infections. Summary of the Invention

[0005] The purpose of this invention is to provide a FeN-T nanozyme composite material, its preparation method, and its applications to solve the problems existing in the prior art. Experimental verification shows that FeN-T can promote cell migration and has almost no cytotoxicity at effective therapeutic concentrations; it can also improve the antibacterial rate of H2O2; furthermore, the combined use of FeN-T and H2O2 can achieve a wound healing rate of 95% within 9 days, significantly better than single treatment. This invention provides a multifunctional composite material with highly efficient antibacterial properties, good biocompatibility, and anti-inflammatory and repair-promoting functions.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a FeN-T nanoenzyme composite material, which is composed of iron-based nanoenzymes, tacrolimus, and polylactic acid-glycolic acid copolymer.

[0007] The present invention also provides a method for preparing the FeN-T nanozyme composite material, comprising the following steps: (1) FeCl3·6H2O was dissolved in water, and 2,6-diaminopyridine was added for polymerization. The polymerized solution was dialyzed, frozen and dried to obtain iron-based nanozyme FeN; (2) Dissolve the polylactic acid-glycolic acid copolymer in an organic solvent to obtain a polylactic acid-glycolic acid copolymer solution, mix the polylactic acid-glycolic acid copolymer solution with a BSA solution, and emulsify by ultrasonication to obtain a suspension; FeN and tacrolimus are added to water to obtain water containing FeN and tacrolimus; The suspension was added to the water containing FeN and tacrolimus, and the solvent was evaporated, centrifuged, and freeze-dried to obtain the FeN-T nanoenzyme composite material.

[0008] Optionally, the molar ratio of FeCl3·6H2O to 2,6-diaminopyridine is 4:1; The polymerization conditions were continuous stirring at 37°C for 24 hours; The cutoff value for dialysis is 12 kDa.

[0009] Optionally, the organic solvent includes dichloromethane; The polylactic acid-glycolic acid copolymer solution has a weight volume fraction of 2%, the BSA solution has a weight volume fraction of 3%, and the volume ratio of the polylactic acid-glycolic acid copolymer solution to the BSA solution is 1:5. The ultrasonic emulsification conditions were 60 W for 1 minute; The ratio of FeN, tacrolimus, and water is 10 mg: 1 mg: 10 mL; The volume ratio of the suspension to the water containing FeN and tacrolimus is 3:10. The centrifugation was performed at 10,000 rpm for 10 minutes using a freeze centrifugation method.

[0010] This invention also provides the application of the FeN-T nanozyme composite material in any of the following: (1) Preparation of antibacterial drugs; (2) Prepare products that enhance the antibacterial effect of H2O2; (3) Preparation of antibacterial drugs by combining with H2O2; (4) Prepare drugs that promote wound healing; (5) Prepare products that enhance the effect of H2O2 in promoting wound healing; (6) Prepare drugs that promote wound healing by combining H2O2.

[0011] Optionally, the antibacterial activity includes anti-Escherichia coli.

[0012] Optionally, the wound includes a bacterial skin infection.

[0013] The present invention also provides an antibacterial drug comprising the FeN-T nanozyme composite material, or comprising the FeN-T nanozyme composite material and H2O2.

[0014] The present invention also provides a drug for promoting wound healing, the drug comprising the FeN-T nanozyme composite material, or the drug comprising the FeN-T nanozyme composite material and H2O2.

[0015] Optionally, the drug may also contain pharmaceutically acceptable excipients.

[0016] The present invention discloses the following technical effects: This invention provides a multifunctional composite material that combines highly efficient antibacterial properties, good biocompatibility, and anti-inflammatory and repair-promoting functions. Specific advantages are as follows: Multifunctional Synergistic Therapy: This invention prepares a nanoenzyme composite material (FeN-T) by encapsulating iron-based nanoenzymes (FeN) with the anti-inflammatory drug tacrolimus (TAC) using PLGA. FeN catalyzes the production of ROS from H₂O₂ to exert a bactericidal effect, TAC provides sustained release to inhibit excessive inflammatory responses, and PLGA improves the material's biocompatibility. The three components synergistically achieve an integrated therapeutic strategy of "antibacterial-anti-inflammatory-promoting repair."

[0017] Strong environmental responsiveness: In vitro enzyme activity experiments show that the FeN-T nanoenzyme composite material reaches its peak catalase-like activity under acidic pH conditions (such as pH=4) that simulate the microenvironment of a biofilm, and can efficiently catalyze the production of sufficient ·OH from low concentration H2O2 to achieve precise and efficient sterilization.

[0018] High biocompatibility: Cytotoxicity experiments and in vivo animal experiments have confirmed that FeN-T is almost non-toxic to human umbilical vein endothelial cells and fibroblasts at effective therapeutic concentrations (≤200 μg / mL), and does not cause significant pathological damage to the major organs (heart, liver, spleen, lung, and kidney) of SD rats, demonstrating excellent biocompatibility.

[0019] Significantly enhanced wound healing: In a full-thickness skin infection model in SD rats, the FeN-T combined with H2O2 treatment group achieved a wound healing rate of 95% within 9 days, significantly superior to the single treatment group and the blank control group. Histopathological analysis showed that this combined treatment effectively promoted granulation tissue growth, collagen deposition, and angiogenesis, while reducing scar tissue. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The image shows a transmission electron microscope (TEM) image of the iron-based nanozyme FeN prepared in Example 1, with a scale bar of 100 nm. Figure 2 Particle size distribution (A), polydispersity index (B), and zeta potential (C) of the iron-based nanozyme FeN prepared in Example 1; Figure 3 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of the FeN-T nanozyme composite material prepared in Example 1. Figure 4 The image shows the enzyme activity of the FeN-T nanozyme composite material in Example 2 at different pH values. Figure 5 This is a diagram showing the cytotoxicity results of the FeN-T nanozyme composite material on HUVEC cells and fibroblasts in Example 3; Figure 6 The image shows the scratch assay results of the FeN-T nanozyme composite material promoting HUVEC cell migration in Example 4. Figure 7 This is a diagram showing the in vitro antibacterial test results of the FeN-T nanozyme composite material against Escherichia coli in Example 5; Figure 8 The image shows the healing process of the FeN-T nanozyme composite material combined with H2O2 in the treatment of infected wounds in SD rats in Example 6. Figure 9 HE-stained pathological section of rat skin wound tissue after treatment in Example 6; Figure 10 This is a graph showing the changes in rat body weight during the treatment process in Example 6. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1: Preparation and Characterization of FeN-T Nanozyme Composite Material 1. Synthesis of iron-based nanozymes (FeN) 80 mmol of FeCl3·6H2O was dissolved in 400 mL of deionized water and stirred at room temperature for 1 hour. Then, 20 mmol of DAP was added, and the mixture was continuously stirred at 37 °C for 24 hours to polymerize. The polymerized solution was transferred to a dialysis bag (cutoff value 12 kDa) and dialyzed in deionized water for 24 hours. The dialyzed solution was placed in a petri dish and frozen at -80 °C overnight. Finally, it was freeze-dried to obtain a yellowish-brown iron-based nanozyme FeN powder. TEM results ( Figure 1 The results show that FeN exhibits a uniformly dispersed spindle-shaped structure with a size ranging from 20 to 60 nm.

[0028] 2. Preparation of FeN-T nanoenzyme composite material Dissolve 40 mg of PLGA in 2 mL of dichloromethane to prepare a 2% PLGA solution. Separately, dissolve 300 mg of BSA (bovine serum albumin) in 10 mL of deionized water to prepare a 3% BSA solution. Mix 2 mL of the 2% PLGA solution and 10 mL of the 3% BSA solution, and sonicate in an ice-water bath for 1 minute (60 W) to obtain a suspension (12 mL).

[0029] Take 40 mg FeN and 4 mg tacrolimus and add them to 40 mL of deionized water to obtain deionized water (40 mL) containing FeN and tacrolimus.

[0030] The suspension was added dropwise to deionized water containing FeN and tacrolimus, and the mixture was magnetically stirred for 4 hours to evaporate dichloromethane. Subsequently, the mixture was centrifuged at 10,000 rpm for 10 minutes and washed 2-3 times. The precipitate was placed in a petri dish and frozen at -80°C overnight. After freeze-drying, the FeN-T nanozyme composite material was obtained.

[0031] 3. Characterization of FeN-T nanozyme composite materials The average particle size of FeN-T measured by dynamic light scattering (DLS) was 505.8 ± 15.23 nm, the polydispersity index (PDI) was 0.286 ± 0.00536, and the zeta potential was -29.5 ± 0.69 mV. Figure 2 This indicates that it has good colloidal stability. XPS analysis ( Figure 3 The results showed that the Fe 2p signal on the FeN-T surface was significantly reduced by 80% compared to the uncoated FeN, but Fe was still retained. 2+ The active site confirms successful PLGA encapsulation.

[0032] Example 2: In vitro catalase-like activity assay of FeN-T nanozyme composite material The catalase-like activity of FeN-T was determined using a TMB colorimetric assay. The reaction system contained FeN-T (100 μg / mL), TMB (2 mM), H2O2 (10 mM), and buffer solutions at different pH values ​​(2, 3, 4, 5, 6, 7, 8), with PBS as a control. After reacting at 37°C for 5 minutes, the absorbance (OD) was measured using a microplate reader. 650 ).

[0033] The results are as follows Figure 4 As shown, FeN-T OD at pH=4 650 The value is the highest, reaching 2.236, indicating that it has the best catalytic activity in an acidic environment.

[0034] Example 3 Cytotoxicity evaluation of FeN-T nanozyme composite material The toxicity of FeN-T to human umbilical vein endothelial cells (HUVECs) and fibroblasts was detected using the CCK-8 assay. Cells were co-cultured with different concentrations (50, 100, 200 μg / mL) of FeN-T for 24 h, while the toxicity of different concentrations of FeN and PLGA to the two cell types was detected as controls.

[0035] The results show that ( Figure 5 Under FeN-T treatment at a concentration of ≤200 μg / mL, the survival rate of both cell types was ≥98%, indicating that FeN-T composite nanoparticles have good biocompatibility.

[0036] Example 4: Effect of FeN-T nanozyme composite material on the migration ability of HUVEC cells HUVEC cells in good growth condition were seeded into 6-well plates and cultured at 37°C in a 5% CO2 incubator until the monolayer cell confluence reached more than 90%. Using a 50 μL sterile pipette tip, constant pressure was applied vertically to the bottom of the well plate to create a uniform scratch. The cells were washed three times with PBS (pH=7.4) to remove detached cells. The medium was replaced with 2% fetal bovine serum to inhibit excessive proliferation. The cells were divided into three groups (blank group, FeN-T group, and FeN group; the blank group was supplemented with serum-free medium, and the FeN-T and FeN groups were supplemented with FeN-T and FeN, respectively). Baseline images were immediately taken under an inverted microscope at 0 h, and images were taken at the same location at 12 h and 24 h. The scratched areas were manually delineated using ImageJ software, and the relative migration rate was calculated by pixel analysis (migration rate = (initial area - terminal area) / initial area × 100%).

[0037] The results showed that ( Figure 6 Compared with the blank group and the FeN group, the FeN-T group had the highest scratch healing rate of 56% within 24 hours, indicating that FeN-T can significantly promote endothelial cell migration.

[0038] Example 5: In vitro antibacterial activity of FeN-T nanozyme composite material Escherichia coli was inoculated into LB liquid medium and cultured at 37°C with shaking at 200 rpm until the logarithmic growth phase (OD200). 600 =1), after centrifugation, resuspend in sterile PBS and adjust the bacterial concentration to 1×10⁻⁶. 6 The bacterial culture was divided into four groups (CFU / mL): blank group, FeN-T group, H2O2 group, and H2O2+FeN-T group. 100 μL of bacterial culture from each group was mixed with an equal volume of FeN-T solution (100 μg / mL), 3% H2O2 solution, a mixture of 3% H2O2+FeN-T (100 μg / mL), and broth (blank control). The mixture was incubated at 37℃ in the dark for 2 h, followed by serial dilution of 50 μL of the mixture (10...).-1 Up to 10 -3 The sample was spread onto LB agar plates and incubated at 37°C for 18 h. The antibacterial effect of FeN-T against Escherichia coli was evaluated by colony counting.

[0039] The results are as follows Figure 7 As shown, the H2O2+FeN-T group exhibited an inhibition rate of over 99.99% against Escherichia coli, with the colony count reduced by two orders of magnitude compared to the single treatment groups (the inhibition rates of the FeN-T group and the H2O2 group were 85.67% and 96.73%, respectively). The survival rate of Escherichia coli in the H2O2+FeN-T group (0.01%) was significantly lower than the product of the survival rates of the FeN group and the H2O2 group (0.47%), demonstrating a significant synergistic antibacterial effect.

[0040] Example 6: Therapeutic effect of FeN-T nanozyme composite material on infected wounds in rats An 8 mm diameter full-thickness skin defect was created on the back of SD rats, and 1×10⁸ m² of skin was injected. 8 An infection model was established using a CFU / mL E. coli suspension. The model rats were randomly divided into an infection control group, a FeN-T treatment group (100 μg / mL), an H2O2 treatment group (3%), and a combined treatment group (100 μg / mL FeN-T + 3% H2O2). Each group received the medication (0.5 mL sprayed onto the wound) every 3 days for a total of 3 treatments (days 3, 6, and 9). Body weight was monitored during the experiment. Skin samples were collected from the wound site on days 3, 6, and 9 for HE staining. The healing rate was calculated on day 9.

[0041] like Figure 8 As shown, the combined treatment group achieved near-complete wound healing by day 9, with a healing rate as high as 95%, significantly superior to other groups (healing rate of 65% in the infection group, 88% in the FeN-T treatment group, and 80% in the H2O2 treatment group). HE staining of the skin on day 9 of treatment (…) Figure 9 The results showed that the combined treatment group exhibited dense collagen deposition in granulation tissue, abundant neovascularization, good regeneration of skin appendages, and minimal scar tissue. Simultaneously, HE staining of the heart, liver, spleen, lungs, and kidneys of rats in each group revealed no significant pathological damage. Weight monitoring showed that the combined treatment effectively alleviated the early weight loss caused by H2O2 alone. Figure 10 ).

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A FeN-T nanozyme composite material, characterized in that, The FeN-T nanozyme composite material is composed of iron-based nanozyme FeN, tacrolimus, and polylactic acid-glycolic acid copolymer.

2. The preparation method of the FeN-T nanozyme composite material as described in claim 1, characterized in that, Includes the following steps: (1) FeCl3·6H2O was dissolved in water, and 2,6-diaminopyridine was added for polymerization. The polymerized solution was dialyzed, frozen and dried to obtain iron-based nanozyme FeN; (2) Dissolve the polylactic acid-glycolic acid copolymer in an organic solvent to obtain a polylactic acid-glycolic acid copolymer solution, mix the polylactic acid-glycolic acid copolymer solution with a BSA solution, and emulsify by ultrasonication to obtain a suspension; FeN and tacrolimus are added to water to obtain water containing FeN and tacrolimus; The suspension was added to the water containing FeN and tacrolimus, and the solvent was evaporated, centrifuged, and freeze-dried to obtain the FeN-T nanoenzyme composite material.

3. The preparation method according to claim 2, characterized in that, The molar ratio of FeCl3·6H2O to 2,6-diaminopyridine is 4:1; The polymerization conditions were continuous stirring at 37°C for 24 hours; The cutoff value for dialysis is 12 kDa.

4. The preparation method according to claim 2, characterized in that, The organic solvent includes dichloromethane; The polylactic acid-glycolic acid copolymer solution has a weight volume fraction of 2%, the BSA solution has a weight volume fraction of 3%, and the volume ratio of the polylactic acid-glycolic acid copolymer solution to the BSA solution is 1:

5. The ultrasonic emulsification conditions were 60 W for 1 minute; The ratio of FeN, tacrolimus, and water is 10 mg: 1 mg: 10 mL; The volume ratio of the suspension to the water containing FeN and tacrolimus is 3:

10. The centrifugation was performed at 10,000 rpm for 10 minutes using a freeze centrifugation method.

5. The application of the FeN-T nanozyme composite material as described in claim 1 in any of the following: (1) Preparation of antibacterial drugs; (2) Prepare products that enhance the antibacterial effect of H2O2; (3) Preparation of antibacterial drugs by combining with H2O2; (4) Prepare drugs that promote wound healing; (5) Prepare products that enhance the effect of H2O2 in promoting wound healing; (6) Prepare drugs that promote wound healing by combining H2O2.

6. The application as described in claim 5, characterized in that, The antibacterial activity includes anti-Escherichia coli.

7. The application as described in claim 5, characterized in that, The wounds include skin wounds infected with bacteria.

8. An antibacterial drug, characterized in that, The drug comprises the FeN-T nanozyme composite material according to claim 1, or the drug comprises the FeN-T nanozyme composite material according to claim 1 and H2O2.

9. A drug for promoting wound healing, characterized in that, The drug comprises the FeN-T nanozyme composite material according to claim 1, or the drug comprises the FeN-T nanozyme composite material according to claim 1 and H2O2.

10. The medicament as described in claim 8 or 9, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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