New application of combined use of chlorogenic acid and copper ions, ROS (reactive oxygen species) response type nano assembly as well as preparation method and application of ROS response type nano assembly

Abstract

The invention provides application of combined use of chlorogenic acid and copper ions in preparation of an external medicine for treating burn and scald wounds. The invention also provides an ROS responsive nano assembly with a network structure, which is characterized in that ROS responsive ketal thiol (TK) is covalently bound with hydrophobic chlorogenic acid (CGA) and a hydrophilic polymer mPEG 2000 to synthesize an amphiphilic polymer mPEG-TK-CGA (PTC), and then the amphiphilic polymer mPEG-TK-CGA (PTC) and Cu < 2 + > are self-assembled to form the PTC-coated Cu NPs. According to the invention, ROS removal, macrophage polarization and regeneration promotion functions are synergistically exerted, and the PTC-coated Cu NPs can effectively relieve oxidative stress, recover mitochondrial functions, reduce cell apoptosis and promote cell migration. The multifunctional linkage treatment strategy can break through pathological circulation of oxidative damage and chronic inflammation, and a comprehensive solution is provided for accelerated healing of infectious deep second-degree burns and scalds.

Description

Technical Field

[0001] The invention relates to a new application of chlorogenic acid and copper ions in combination, and a ROS-responsive nanoassembly and a preparation method and application thereof. Background Art

[0002] The skin plays a vital role in the barrier protection function in the interaction between the body and the external environment, and can effectively resist mechanical damage, microbial invasion and environmental harmful factors from damaging the internal organs. However, severe skin injuries such as deep second-degree and third-degree burns can significantly damage this barrier function, leading to delayed healing, high risk of infection and impaired tissue regeneration. The complexity of burn wound healing stems from multiple factors such as oxidative stress, inflammatory response, immune regulation imbalance and angiogenesis disorders. Therefore, innovative treatment strategies are urgently needed to promote tissue regeneration and prevent infection-related complications.

[0003] A large number of studies have shown that reactive oxygen species (ROS) are the core hub for regulating the skin repair cascade. Under physiological conditions, ROS play a positive role in the host defense mechanism by exerting antibacterial effects and signal transduction functions, and can regulate processes such as cell proliferation, differentiation, and migration. However, excessive production of ROS or impaired antioxidant capacity can lead to redox imbalance and cause significant cell and tissue damage. In infected burn wounds, excessive production of ROS can aggravate the inflammatory response, destroy the function of immune cells, and cause persistent tissue damage through mechanisms such as DNA damage and lipid peroxidation. Therefore, regulating ROS homeostasis has become a key therapeutic target to break through the difficulty of healing infected burns.

[0004] Burn wound healing usually goes through four stages: hemostasis, inflammation, proliferation, and remodeling. Although inflammatory mediators are necessary for the transition from inflammation to proliferation, severe burns often lead to prolonged inflammation due to excessive proinflammatory factors (TNF-α, IL-1β, IL-6), which in turn causes secondary tissue damage and delayed healing. Macrophages play a core role in this stage: although M1 macrophages can clear pathogens by releasing ROS and cytokines, their continuous activation will aggravate the inflammatory response; while M2 macrophages with anti-inflammatory effects can accelerate tissue repair by secreting cytokines such as IL-10 and TGF-β, promoting extracellular matrix deposition and angiogenesis. Studies have found that oxidative stress can enhance M1 activation, while ROS clearance promotes M2 transformation. Therefore, regulating ROS to balance M1 / M2 macrophage polarization has become a potential strategy to improve burn healing.

[0005] Hydrophobic chlorogenic acid (CGA) is a natural polyphenol rich in coffee and various plants, known for its antioxidant and antibacterial properties. By scavenging free radicals, CGA can effectively reduce oxidative stress and regulate inflammatory signaling pathways. In addition, CGA has been shown to promote the polarization of macrophages into the M2 type, thereby enhancing its anti-inflammatory and tissue repair effects.

[0006] Zhang Junwen, Research on the Promotion of Wound Healing in Scald Infections by Calcium Carbonate Encapsulating Chlorogenic Acid, Wenzhou University, 2022. In vitro antibacterial experiments demonstrated that calcium carbonate encapsulating chlorogenic acid particles significantly inhibited the growth of Staphylococcus aureus and Escherichia coli. At the same time, these particles could effectively scavenge hydroxyl radicals and had good antioxidant effects. Finally, we used a scald infection model to evaluate the effect of calcium carbonate encapsulating chlorogenic acid on promoting wound repair. The results showed that the particles could effectively promote cell proliferation and migration, accelerate collagen deposition and epithelial crawling, and thus promote wound healing. Among them, calcium carbonate (CaCO3) itself has pH buffering ability, can neutralize the acidic environment of the wound, and slowly release CGA, with good stability. However, it lacks active targeting, and drug release depends on the degradation of calcium carbonate (pH-sensitive), and the efficiency may be low in non-acidic or low-inflammatory regions. Due to the lack of synergistic antibacterial components, the effect on drug-resistant bacteria or severe infections may be limited. The optimization of drug delivery efficiency (such as permeability enhancement strategies) was not mentioned, which may affect the efficacy of deep wounds. It is more suitable for mild to moderate scalds or chronic wounds (requiring mild antioxidant + collagen repair promotion).

[0007] Application No. 202411072870.X, Invention Title: Synthesis of a Metal-Polyphenol Self-Assembled Nanoparticle Based on Chlorogenic Acid and Its Antitumor Application. By the coordination chelation of the plant polyphenol chlorogenic acid and the transition metal copper ion (Cu 2+ ) under alkaline conditions, a metal-phenolic network nanoparticle with antitumor activity was constructed. This nanoparticle can not only decompose and release drugs in an acidic environment, inducing cuproptosis in tumor cells, but also undergo a Fenton-like reaction with hydrogen peroxide to generate reactive oxygen species and has the ability to consume glutathione, thereby synergistically achieving antitumor effects through chemotherapy and chemodynamic therapy.

[0008] Application No.: 202411551457.1, Invention Title: A Janus Fabric Loaded with Composite Antibacterial Nanoparticles and Its Preparation Method. This application proposes a Janus fabric loaded with composite antibacterial nanoparticles and its preparation method, which relates to the technical field. A Janus fabric loaded with composite antibacterial nanoparticles, one side of the fabric is set as a hydrophobic antibacterial surface, and the other side is set as a hydrophilic wound-healing surface; composite antibacterial nanoparticles containing chlorogenic acid and metal ions are loaded on the fibers on both sides of the fabric. The present invention first prepares antibacterial nanoparticles coupling chlorogenic acid and metal ions, then adsorbs the antibacterial nanoparticles on the surface of the fabric fibers through an impregnation process, and finally, through a unilateral spraying process, hydrophobic anti-fouling and antibacterial modification is carried out on one side of the fabric, and finally a Janus antibacterial fabric is prepared, which is a simple and efficient method for preparing antibacterial fabrics and has great application prospects in the fields of wound anti-infection and wound healing.

[0009] However, for the topical drug treatment of infectious deep second-degree burns and scalds, there are still relatively large defects at present. Summary of the Invention

[0010] The present invention provides a new use of the combined use of chlorogenic acid and copper ions, as well as a ROS-responsive nano-assembly and its preparation method and use.

[0011] The present invention provides a copper nano-assembly rich in thioketal bonds (PTC@Cu NPs), which realizes the targeted delivery of CGA and Cu to the lesion site through the dual effects of ROS scavenging and macrophage M2 polarization, so as to solve the problems of oxidative stress, infection and aplasia in deep burn and scald wounds. 2+

[0012] The present invention provides the use of the combined use of chlorogenic acid and copper ions in the preparation of topical drugs for treating burn and scald wounds.

[0013] Among them, the dosages of the chlorogenic acid and copper ions are as follows: the copper ions corresponding to each mg of chlorogenic acid are 0.256 - 11.52 mg.

[0014] Among them, the dosages of the chlorogenic acid and copper ions are as follows: the copper ions corresponding to each mg of chlorogenic acid are 0.512 mg.

[0015] The present invention provides a network-structured ROS-responsive nano-assembly, which covalently binds ROS-responsive ketothiol (TK) with hydrophobic chlorogenic acid CGA and hydrophilic polymer mPEG 2000 to synthesize amphiphilic polymer mPEG-TK-CGA (PTC), and then self-assembles with Cu 2+ to form PTC@Cu NPs.

[0016] Among them, the structural formula of the PTC is:

[0017]

[0018] where n = 45.

[0019] Among them, the weight ratio of the raw material ROS-responsive ketothiol (TK), chlorogenic acid, and hydrophilic polymer mPEG 2000 is:

[0020] 1 part of raw material ROS-responsive ketothiol (TK), 3.15 parts of chlorogenic acid, and 3.3 parts of hydrophilic polymer mPEG 2000.

[0021] The described PTC and Cu 2+ The dosage ratio is: PTC:Cu 2+ = 1 part: 0.064 part.

[0022] The present invention provides a method for preparing the described ROS-responsive nano-assembly, which includes the following steps:

[0023] a. Dissolve mPEG-COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) in DMSO respectively, and then stir under nitrogen protection in an oil bath at 50 °C for 30 minutes; after the initial activation step, add TK-NH2, and stir magnetically under nitrogen protection, with an oil bath for 24 hours;

[0024] b. Add CGA, NHS, and EDC to the DMSO solution, and stir under nitrogen protection in an oil bath at 50 °C for 30 minutes; then add the stirred CGA solution to the above 24-hour reaction mixture, and stir for another 24 hours under the same conditions (50 °C oil bath, nitrogen protection); after the reaction is completed, obtain the crude product PTC;

[0025] c. Dialyze the crude product PTC in absolute ethanol for 48 hours using a dialysis membrane (MWCO = 2000 kDa); collect the dialysis product with a rotary evaporator, then dissolve it in ethyl acetate, centrifuge at 10000 rpm for 10 minutes to obtain the purified PTC precipitate; dry it in vacuo at room temperature for 2 days;

[0026] d. Dissolve 1 mg / mL PTC and 1 mM Cu 2+ (CuSO4) in methanol, stir at room temperature for 18 hours to prepare PTC@Cu; separate the precipitate by centrifugation at 10000×g for 10 min at 4 °C; wash the collected precipitate with methanol to obtain it.

[0027] The present invention also provides the use of the described network-structured ROS-responsive nano-assembly in the preparation of an external medicine for treating burn and scald wounds.

[0028] Among them, the drug is a drug for treating infected deep second-degree burn and scald wounds.

[0029] Among them, the drug is a drug that promotes cell migration and angiogenesis, and accelerates epithelial regeneration and tissue repair.

[0030] In the raw materials of the present invention, TK can accurately target the high-ROS region of burn and scald wounds by responding to ROS, and under the action of ROS, it breaks and releases chlorogenic acid (CGA) and Cu 2+ ; CGA can effectively scavenge excessive ROS on the wound surface; promote the polarization of macrophages on the wound surface to the M2 type, and accelerate tissue repair; and play a synergistic antibacterial role with the released Cu 2+ mPEG2000 can improve the hydrophobicity of CGA, not only enhancing the biocompatibility of the material, but also enhancing the penetration and absorption efficiency of the drug on the wound surface. By utilizing the hydrophilicity of mPEG 2000, the hydrophobicity and permeability of CGA are improved, making it easier for the burn and scald wounds to absorb CGA; by utilizing the ROS-responsive TK, CGA and Cu 2+ are more precisely targeted to the wound site, and are more suitable for burn / scald wounds with severe oxidative stress.

[0031] The copper ions (Cu 2+ ) of the present invention have a dual role in the biological system: at a controllable concentration, they can exert a strong antibacterial effect by destroying cell membranes, inhibiting metabolic processes, and damaging DNA, and can also promote angiogenesis and tissue regeneration. Ketothiol (TK) is well-known for its selective cleavage characteristics in an ROS (especially hydroxyl radicals and hydrogen peroxide) environment, and can be rapidly cleaved in the wound area with a high ROS concentration to achieve targeted release of therapeutic drugs. This site-specific release can not only enhance the efficacy, but also reduce toxicity by preventing premature leakage of the drug.

[0032] In the present invention, the ROS-responsive TK, the hydrophilic polymer mPEG-COOH, and the hydrophobic CGA are connected through an aminocarboxylic reaction to form an amphiphilic polymer mPEG-TK-CGA (PTC), and then self-assembled with copper ions to form a network-like structure of PTC@Cu NPs. This nanoplatform can accurately release CGA and Cu 2 + . And CGA can neutralize ROS, promote macrophage M2 polarization to inhibit inflammation, and enhance tissue repair; Cu 2+The antibacterial activity can be enhanced through membrane disruption, while angiogenesis is stimulated. Finally, by synergistically exerting ROS scavenging, macrophage polarization, and pro-regenerative functions, PTC@Cu NPs can effectively alleviate oxidative stress, restore mitochondrial function, reduce apoptosis, and promote cell migration. This multifunctional treatment strategy can break the pathological cycle of oxidative damage and chronic inflammation, providing a comprehensive solution for the accelerated healing of infectious deep second-degree burns. Brief Description of the Drawings

[0033] Figure 1 It is a schematic diagram of the preparation process of composite antibacterial nanoparticles;

[0034] Figure 2 It is a characterization experimental diagram of PTC (in the figure, A is the nuclear magnetic resonance hydrogen spectrum ( 1 1H-NMR) of PTC, B is the critical micelle concentration (CMC) of PTC, and C is the negative staining transmission electron microscopy (TEM) image of PTC);

[0035] Figure 3 It is a TEM image of the ROS-responsive nanoassembly of the synthesized PTC@Cu network structure (in the figure, A is the TEM image of PTC@Cu, and B is the TEM mapping image of S, O, N, and Cu in PTC@Cu);

[0036] Figure 4 It is an XPS spectrum of the synthesized ROS-responsive PTC@Cu nanoassembly (in the figure, A is the full spectrum of the elements in the nanoparticles, and B is the X-ray photoelectron spectrum of the Cu element);

[0037] Figure 5 It is a TEM image of the PTC@Cu nanoassembly after the cleavage of TK in response to H2O2 for 4 hours;

[0038] Figure 6 It is the fluorescence image and flow cytometry analysis of the ROS levels in HUVEC and HaCaT cells under the action of PBS, PC (containing 1 μM CGA), PTC (containing 1 μM CGA), and PTC@Cu (containing 1 μM CGA) (in the figure, A is the fluorescence image of HUVEC cells stained with DCFH-DA, B is the flow cytometry analysis diagram of HUVEC cells stained with DCFH-DA, C is the fluorescence image of HaCaT cells stained with DCFH-DA, and D is the flow cytometry analysis diagram of HaCaT cells stained with DCFH-DA);

[0039] Figure 7 It is the synergistic antibacterial effect of CGA and copper ions against Staphylococcus aureus and Escherichia coli in the ROS-responsive nanoassembly;

[0040] Figure 8The synergistic promotion of the migration ability of CGA and copper ions in ROS-responsive nanoassemblies on HUVEC and HaCaT cells (in the figure, A and B are the Transwell migration experiment diagrams and statistical analysis diagrams of HUVEC and HaCaT cells, C is the scratch experiment diagram of HUVEC cells at different time points after H2O2 treatment, and D is the scratch experiment diagram of HaCaT cells at different time points after H2O2 treatment);

[0041] Figure 9 The pro-angiogenic ability of copper ions in ROS-responsive nanoassemblies on HUVEC cells; Figure 10 In the deep second-degree scald-infected wounds of mice, the ability of CGA in ROS-responsive nanoassemblies to promote the transformation of macrophages into the M2 type (in the figure, MB is mixed bacteria, which is a mixture of Staphylococcus aureus and Escherichia coli, and KFX is Kangfuxin Liquid, which serves as a positive control group in animal experiments). Detailed implementation methods

[0042] Example 1 Preparation of the ROS-responsive nanoassemblies of the present invention

[0043] First, 0.66 g of mPEG-COOH, 0.19 g of EDC, and 0.12 g of NHS were respectively dissolved in DMSO. Then, under nitrogen protection, it was stirred in an oil bath at 50 °C for 30 minutes. After the initial activation step, TK-NH2 (0.2 g) was added, and it was magnetically stirred under nitrogen protection for 24 h in an oil bath. CGA (0.63 g), NHS (0.69 g), and EDC (1.15 g) were added to the DMSO solution, and it was stirred in an oil bath at 50 °C under nitrogen protection for 30 minutes. Subsequently, the well-stirred CGA solution was added to the above 24-hour reaction mixture, and it was stirred for another 24 hours under the same conditions (50 °C oil bath, nitrogen protection). After the reaction was completed, the crude product PTC was dialyzed in absolute ethanol using a dialysis membrane (MWCO = 2000 kDa) for 48 hours. The dialyzed product was collected by a rotary evaporator, redissolved in ethyl acetate, and centrifuged at 10000 rpm for 10 minutes to obtain a purified PTC precipitate. It was vacuum dried at room temperature for 2 days. The chemical structure of PTC was analyzed by 1 1H-NMR. 1 mg / mL of PTC and 1 mM Cu 2+ (CuSO4) were dissolved in methanol and stirred at room temperature for 18 hours to prepare PTC@Cu. The precipitate was separated by centrifugation at 10000×g for 10 min at 4 °C using a Dragon Lab D3024R centrifuge. To remove the unreacted components, the collected precipitate was washed three times with methanol. Finally, the purified product was suspended in 1 ml of PBS and sonicated to ensure uniform dispersion in an ice bath. The schematic diagram of the preparation process is shown in Figure 1 .

[0044] Among them, PTC and Cu 2+ This is a parameter selection experiment. 1mg / mL PTC: 1-40mM Cu was used in the experiment. 2+ Because of the toxicity risk of excessive copper ions, 1 mM Cu 2+ .

[0045] The present invention successfully synthesizes PTC@Cu nanoparticles by nanoprecipitation method. First, the amphiphilic polymer PTC is prepared by acylation reaction of hydrophilic polymer polyethylene glycol (mPEG), thioketal (TK) and hydrophobic chlorogenic acid (CGA), and then stirred with copper ions in methanol for 18 hours to self-assemble into PTC@Cu nanoparticles.

[0046] Example 2 Characterization experiment of ROS-responsive nanoassembly of the present invention

[0047] 1. Synthesis and characterization of amphiphilic polymer PTC micelles

[0048] Experimental steps:

[0049] 2 mg of the synthesized PTC was dissolved in 1 mL of deuterated DMSO and placed in a nuclear magnetic resonance tube, and the hydrogen spectrum of PTC was measured using a nuclear magnetic resonance spectrometer.

[0050] PTC powder was dissolved in ultrapure water at a certain concentration ratio, and the critical micelle concentration of PTC was evaluated by surface tension method.

[0051] 100 μL of PTC solution was taken with a pipette and evenly dripped on the ultra-thin copper mesh. After standing for 5 minutes to allow PTC to be fully adsorbed, the PTC liquid was removed and 100 μL of 2% phosphotungstic acid was dripped to cover the copper mesh. After standing for 3 minutes, the dye solution was sucked from the edge of the grid with filter paper and dried naturally at room temperature. After drying, the morphological characteristics of PTC were observed under a transmission electron microscope (TEM) at an accelerating voltage of 80.0 kV.

[0052] Results Figure 2 And analysis:

[0053] H NMR ( 1 H-NMR) analysis confirmed the successful covalent coupling of the intermediate product PTC. The critical micelle concentration (CMC) was determined to be 0.144 mg / mL, which verified its amphiphilic properties of self-assembling into micelles in aqueous solution. Negative staining transmission electron microscopy proved that PTC was a spherical micelle in aqueous solution. The above illustrates the successful synthesis of amphiphilic polymer micelle PTC, and the structural formula of PTC is:

[0054]

[0055] Wherein n=45.

[0056] 2. TEM and Elemental Mapping Analysis of PTC@Cu

[0057] Experimental Procedure:

[0058] Use a pipette to aspirate 100 μL from the PTC@Cu solution and evenly drop it onto a ultra-thin molybdenum grid. After standing for 5 minutes to allow the nanoparticles to fully adsorb, place the molybdenum grid at room temperature and let it dry naturally. After drying, observe the morphological characteristics of PTC@Cu NPs at an accelerating voltage of 80.0 kV under TEM, and simultaneously observe the S, O, N, and Cu element distributions of PTC@Cu under TEM mapping.

[0059] The experimental results are shown in Figure 3 and analysis:

[0060] The TEM image shows that PTC@Cu is a three-dimensional network structure. And the TEM elemental mapping analysis illustrates the coordination relationship between Cu 2+ and the functional groups (-NH2, -SH, -OH) of the PTC micelles. The above indicates that the ROS-responsive network structure PTC@Cu NPs has been successfully prepared by the multi-site coordination of copper ions with PTC.

[0061] 3. XPS Analysis of PTC@Cu NPs

[0062] Experimental Procedure:

[0063] Evenly spread 5 - 10 mg of PTC@Cu powder on indium foil, and gently press it with a clean glass slide to make the sample flat, then load it onto the XPS sample stage. Use Al Kα rays for full-spectrum scanning and high-resolution spectrum analysis to obtain the binding energy information of C, N, O, S, and Cu elements, and determine the surface composition of PTC@Cu and the chemical state of copper ions through peak fitting and database comparison.

[0064] The experimental results are shown in Figure 4 and analysis:

[0065] X-ray photoelectron spectroscopy (XPS) confirms the presence of C, N, O, S, and Cu on the surface of PTC@Cu NPs. The high-resolution Cu 2p spectrum shows characteristic doublets and satellite peaks of the Cu 2p3 / 2 orbital at 933 eV and 934.44 eV, confirming that the copper ions are in the divalent state. The above experimental results illustrate the successful preparation of PTC@Cu and the existence of divalent copper ions.

[0066] 4. TEM Morphological Characterization of PTC@Cu under H2O2 Stimulation

[0067] Experimental Procedure:

[0068] H2O2 was added to 1 mL of the above-prepared PTC@Cu solution to prepare a PTC@Cu solution containing 10 mM H2O2. After standing at room temperature for 4 h, 100 μL was pipetted from this solution and evenly dropped onto a ultra-thin copper mesh. After standing for 5 minutes to allow the nanoparticles to fully adsorb, the copper mesh was left to dry naturally at room temperature. After drying, the morphological characteristics of PTC@Cu containing 10 mM H2O2 were observed under TEM at an acceleration voltage of 80.0 kV.

[0069] The experimental results are shown in Figure 5 and analysis:

[0070] TEM observation showed that after co-incubation of PTC@Cu NPs with 10 mM H2O2 at room temperature for 4 h, the nanoscale network structure was significantly disintegrated. This structural damage was due to the cleavage of the thioacetal (TK) bond in PTC@Cu NPs under oxidative conditions - H2O2 triggered the ROS-responsive degradation of the TK bond. The experimental results of the disintegration of the above network structure illustrate the inherent ROS sensitivity of the thioacetal-based material, indicating its potential for on-demand drug release in a pathological microenvironment with elevated ROS levels (such as infected or inflamed wounds). This result is consistent with previous studies on ROS-responsive nanomaterials, verifying the controlled release mechanism of PTC@Cu NPs in an oxidative environment.

[0071] The beneficial effects of the present invention are demonstrated by the following ergonomics tests.

[0072] Test Example 1 In vitro ROS-responsive consumption experiment of PTC@Cu

[0073] Excessive ROS levels in the wound microenvironment can disrupt the repair process and severely impair wound healing. To investigate the ROS scavenging ability of PTC@Cu NPs in HUVEC and HaCaT cells, intracellular ROS levels were detected by fluorescence imaging and flow cytometry.

[0074] Experimental procedure:

[0075] HUVEC and HaCaT cells were incubated in a 6-well plate for 24 h. Then the cells were stimulated with a 400 μM H2O2 experimental model. Subsequently, HUVEC and HaCaT cells were treated with PBS, PC (containing 1 μM CGA), PTC (containing 1 μM CGA), and PTC@Cu (containing 1 μM CGA). After treatment, the cells were resuspended in serum-free medium and washed twice with PBS. ROS levels were evaluated by incubating the cells with DCFH-DA (10 μM) in serum-free medium at 37 °C for 20 minutes. The stained cells were analyzed by flow cytometry and FlowJo software. For fluorescence imaging, the cells were fixed with 4% paraformaldehyde under standardized conditions.

[0076] The experimental results are shown inFigure 6 And analysis:

[0077] Compared with the H2O2-stimulated model group, PTC@Cu NPs significantly reduced the green fluorescence intensity quantified by DCFH-DA staining and the FITC signal analyzed by flow cytometry under all tested conditions, showing consistent and effective ROS scavenging ability. This may be because the ROS-responsive TK bond-mediated precise release of PTC@Cu NPs releases CGA with antioxidant effects.

[0078] Experimental Example 2 Synergistic antibacterial effect of CGA and Cu in PTC@Cu 2+

[0079] Microbial infections can directly damage the wound site and impede the healing process. Gram-positive bacterium Staphylococcus aureus (S. aureus) often causes early infections in wound healing, while Gram-negative bacterium Escherichia coli (E. coli) is prone to colonize deep wounds and often causes chronic and protracted infections.

[0080] Experimental procedure:

[0081] Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were selected for antibacterial activity evaluation. NaCl, PC, PTC, and PTC@Cu NPs solutions (50 μL, each reagent containing 1 mM CGA) or NaCl and Staphylococcus aureus or Escherichia coli solutions (50 μL, 1×10 7 CFU mL-1) were mixed at 37 °C for 1 hour. After incubation, the bacterial suspension was diluted 50-fold, and 60 μL of the diluted sample was spread on an agar plate. After incubation at 37 °C for 24 hours, the colony-forming units (CFUs) were counted, and the bacterial density was determined by measuring the optical density at 600 nm using a microplate reader. The colony counting was performed using ImageJ software to analyze the bacterial survival rate. The bacterial viability was calculated using the following formula:

[0082] Treatment CFUs represent the colony-forming units observed after experimental treatment; while Control CFUs represent the baseline colony count of the control group.

[0083] The experimental results are shown in Figure 7 And analysis:

[0084] ​The agar plate colony formation experiment showed a gradient enhancement in antibacterial activity: PTC@Cu was the most sensitive to the two bacteria, followed by PTC, and PC had the weakest effect. This stepwise improvement in antibacterial efficacy (PC < PTC < PTC@Cu) stemmed from a triple synergistic mechanism: (1) the "see-saw effect" between hydroxyl groups and copper ions could enhance the bactericidal efficiency; (2) copper ions could enhance the antibacterial activity of chlorogenic acid (CGA); (3) the ROS-responsive TK bond in PTC could rapidly release CGA at the infection site, achieving a high local drug concentration. In contrast, PC lacked ROS responsiveness and relied only on slow diffusion drug release.

[0085] Experimental Example 3 In vitro cell migration and angiogenesis promotion effects of PTC@Cu

[0086] The directed movement of HUVEC cells and HaCaT cells plays a key regulatory role in the processes of neovascularization and re-epithelialization of wounds. To evaluate the effects of nanoparticles on cell migration and angiogenesis, the present invention jointly adopted the Transwell migration experiment, scratch experiment of cells, and angiogenesis experiment of HUVEC.

[0087] Experimental procedure:

[0088] HUVEC (2×10 4 cells / well) and HaCaT (5×10 4 cells / well) cells were evenly cultured on a transwell polycarbonate membrane with 8-μm pores (37 °C, 24 hours). Then the upper chamber was treated with the same experimental model and reagent concentration as in the ROS experiment. After fixation in freshly prepared 4% paraformaldehyde (w / v) at 4 °C for 16 h, the cell specimens were stained with 0.2% (w / v) crystal violet at room temperature for 40 minutes and washed 3 times with PBS for 5 minutes each time. The non-migrated cells on the upper surface of the transwell polycarbonate membrane were gently removed with a cotton swab. Then it was air-dried for 3 days, and images of the migrated HUVEC and HaCaT cells on the lower surface were collected under natural light of a microscope and analyzed with ImageJ software.

[0089] In addition, HUVEC and HaCaT cells were cultured in 6-well plates. For scratch analysis, a sterile pipette tip was introduced into the cell monolayer. At the designated time points, the plates were taken out, and images of the scratched wounds were captured using a fluorescence microscope under natural light to evaluate cell migration and growth.

[0090] The experimental results are shown in Figure 8 and analysis:

[0091] The results of the Transwell assay showed that compared with the control group, more migrated cells were observed on the outer side of the polycarbonate membrane in the PTC@Cu NPs treatment group, indicating that the nanoparticles could significantly promote the migration ability of the two types of cells. The scratch assay further verified this trend and confirmed the promoting effect of PTC@Cu NPs on cell motility. These results suggest that PTC@Cu NPs may promote cell migration by scavenging ROS and the action of Cu 2+ to jointly construct a microenvironment conducive to cell migration, thereby promoting cell migration.

[0092] Experimental procedure: HUVEC cells were first exposed to 400 μM H2O2 in serum-free medium for 24 hours. Next, they were incubated with PC, PTC, PTC@Cu (each containing 1 μM chlorogenic acid, CGA) or PBS for 24 hours. Then, the medium containing the indicator was replaced with serum-free medium and incubated overnight, and the cells were separated and resuspended. Subsequently, HUVEC cells (2×10 4 cells / well) were cultured in a matrigel-coated 96-well plate for 6 hours. Images were taken using a microscope (Olympus, Japan). The experimental results are shown in Figure 9 and analysis:

[0093] After culturing on Matrigel matrix gel for 6 hours, the HUVEC cells treated with PTC@Cu NPs formed more tubular structure connections than the H2O2 treatment group. This pro-angiogenic effect can be attributed to the lower cytotoxicity of PTC@Cu NPs and the continuously released pro-angiogenic factor Cu 2+ .

[0094] Test Example 4 Promoting effect of PTC@Cu on the transformation of M2 macrophages in deep second-degree burn-infected skin wounds

[0095] Anti-inflammatory macrophage M2 phenotype promotes tissue repair by secreting IL-10 and TGF-β cytokines and promoting extracellular matrix deposition and angiogenesis. ROS levels severely affect macrophage polarization because oxidative stress enhances M1 activation while ROS scavenging promotes the M2 phenotype. Therefore, regulating the ROS balance of M1 / M2 macrophage polarization is a promising strategy for improving burn wound healing.

[0096] Experimental procedure:

[0097] To evaluate macrophage polarization, skin tissues of each group after burn infection treatment on the 14th day were collected.

[0098] The collected skin wound tissues were ground, placed on a 70-μm cell strainer for filtration, and the strainer was washed with PBS. After centrifuging the cell suspension, it was resuspended and washed with PBS, centrifuged again, and the cell pellet was collected. The centrifugation conditions were 4°C, 300 g, and 10 minutes. 5 mL of pre-cooled red blood cell lysis buffer was added to each tube, placed on ice, and after 15 minutes, 5 mL of PBS was added and centrifuged under the same conditions. After washing and centrifuging twice with PBS, it was resuspended with 3 mL of PBS. 100 μL of the cell suspension was taken from each skin tissue and aliquoted into 1 EP tube, i.e., 100 μL / tube. Prepare a flow cytometry staining solution for M2 macrophages (CD45 + , F4 / 80 + , CD206 + ) and add it to the EP tube. Incubate at 4°C on a shaker in the dark for 45 minutes. After that, 900 μL of PBS was added to each EP tube, and the cells were filtered through a 200-mesh flow cytometry filter screen into a 5-mL flow tube. The stained cells were detected using a flow cytometer and analyzed with FlowJo software.

[0099] The experimental results are shown in Figure 10 and analysis:

[0100] The experimental results showed that compared with other groups, NPs could significantly promote the polarization of M2 macrophages (CD45 + , F4 / 80 + , CD206 + ). It was illustrated that after PTC@Cu precisely targeted and responded to ROS through TK, the clearance of ROS by CGA could promote the transformation of macrophages into the M2 phenotype to accelerate the healing of infected wounds. The present invention proved that PTC@Cu NPs demonstrated significant potential in promoting the healing of infectious wounds through a synergistic multi-mechanism. The nanoparticles could effectively scavenge reactive oxygen species (ROS), alleviate oxidative stress, protect cells from ROS-induced mitochondrial dysfunction and apoptosis, thereby maintaining cell integrity and function. In addition, PTC@Cu NPs exhibited potent antibacterial activity against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, which was attributed to the combined action of copper ions, chlorogenic acid (CGA), and ROS-responsive drug release. More importantly, the nanoparticles could promote cell migration and angiogenesis, accelerating epithelial regeneration and tissue repair. Experiments on a mouse burn model showed that PTC@Cu NPs significantly accelerated wound healing by alleviating oxidative damage, promoting M2 polarization of macrophages to inhibit inflammatory responses, and enhancing proliferative migration. The nanoparticles could effectively promote the treatment of infectious wounds and chronic wounds, providing an innovative solution for the healing of deep second-degree burn infected wounds.

Claims

1. Use of chlorogenic acid and copper ions in combination in the preparation of an external medicine for treating burn and scald wounds.

2. The use according to claim 1, wherein: The dosages of the chlorogenic acid and copper ions are as follows: the copper ions corresponding to each mg of chlorogenic acid are 0.256 - 11.52 mg.

3. The use according to claim 1, wherein: The dosages of the chlorogenic acid and copper ions are as follows: the copper ions corresponding to each mg of chlorogenic acid are 0.512 mg.

4. A ROS-responsive nanoassembly with a network structure, characterized in that: It covalently binds ROS-responsive ketothiol (TK) with hydrophobic chlorogenic acid CGA and hydrophilic polymer mPEG 2000 to synthesize amphiphilic polymer mPEG-TK-CGA (PTC), and then combines it with Cu 2+ to self-assemble into PTC@Cu NPs.

5. The ROS-responsive nanoassembly according to claim 4, wherein: The structural formula of the PTC is as follows: In the formula, n = 45.

6. The ROS-responsive nano-assembly of the network structure according to claim 4, characterized in that: The weight ratio of the raw materials ROS-responsive ketothiol (TK), chlorogenic acid, and hydrophilic polymer mPEG 2000 is as follows: 1 part of raw material ROS-responsive ketothiol (TK), 3.15 parts of chlorogenic acid, and 3.3 parts of hydrophilic polymer mPEG 2000. The PTC and Cu described above 2+ The dosage ratio is: PTC:Cu 2+ = 1 part: 0.064 part.

7. A method for preparing the ROS-responsive nanoassembly according to any one of claims 4-6, characterized in that: It includes the following steps: a. Dissolve mPEG-COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) in DMSO respectively, and then stir under nitrogen protection in an oil bath at 50 °C for 30 minutes; after the initial activation step is completed, add TK-NH2, and stir magnetically under nitrogen protection, with an oil bath for 24 hours; b. Add CGA, NHS, and EDC to the DMSO solution, and stir under nitrogen protection in an oil bath at 50 °C for 30 minutes; then add the stirred CGA solution to the above 24-hour reaction mixture, and stir for another 24 hours under the same conditions (50 °C oil bath, nitrogen protection); after the reaction is completed, obtain the crude product PTC; c. Dialyze the crude product PTC in absolute ethanol with a dialysis membrane (MWCO = 2000 kDa) for 48 hours; collect the dialysis product with a rotary evaporator, dissolve it in ethyl acetate again, centrifuge at 10000 rpm for 10 minutes to obtain the purified PTC precipitate; dry it in vacuum at room temperature for 2 days; d. Dissolve 1 mg / mL PTC and 1 mM Cu 2+ (CuSO4) in methanol and stir at room temperature for 18 h to prepare PTC@Cu; the precipitate is separated by centrifugation at 10,000×g for 10 min at 4 °C; the collected precipitate is washed with methanol to obtain the product.

8. Use of the ROS-responsive nano-assembly of the network structure according to any one of claims 4 - 6 in the preparation of an external medicine for treating burn and scald wounds.

9. The use according to claim 8, wherein: The medicine is for treating infected deep second-degree burn and scald wounds.

10. The use according to claim 8, characterized in that: The medicine is for promoting cell migration and angiogenesis, and accelerating epithelial regeneration and tissue repair.

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