Copper porphyrin-containing porous polymer with cavity inclusion and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WEIFANG MEDICAL UNIV
- Filing Date
- 2026-02-09
- Publication Date
- 2026-05-12
Smart Images
Figure CN121673505B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a copper porphyrin porous polymer with cavity encapsulation and its applications. Background Technology
[0002] In recent years, reactive oxygen species (ROS)-mediated catalytic therapy strategies have attracted much attention due to their high selectivity, low likelihood of inducing drug resistance, and effective penetration of biomembranes. Among them, the combined application of photodynamic therapy (PDT), photothermal therapy (PTT), and chemodynamic therapy (CDT) has shown great potential. However, these therapies still face many bottlenecks in practical applications, and single-modality treatments often fail to completely eliminate refractory biomembranes.
[0003] Porphyrins and their derivatives possess both photothermal and photodynamic effects, making them valuable for phototherapy, particularly in antibacterial and antitumor treatments. Porphyrins are planar macrocyclic heterocyclic compounds. When porphyrins polymerize, they form stacked structures where the large π-bond planes overlap, creating a π-π stacking effect. π-π stacking enhances nonradiative relaxation and reduces fluorescence radiation, thus affecting photothermal conversion efficiency; close stacking shields the central metal ion, hindering oxygen contact and impacting photodynamic efficiency. Therefore, rational polymer structure design is needed to suppress π-π stacking, enabling porphyrins to exhibit both efficient photothermal and photodynamic effects under specific conditions, achieving synergistic therapy. Cyclodextrins have excellent biocompatibility and antibacterial activity, leading to reports on polymerizing cyclodextrins with porphyrins to enhance antibacterial activity. For example, patent application CN117327210A discloses a porphyrin microporous composite material based on β-cyclodextrin-terephthalaldehyde inclusion complex, its preparation method, and its application. While this patent uses cyclodextrin and porphyrin to form an antibacterial polymer, it does not solve the problem of porphyrin π-π stacking. Compounds with a certain cavity structure, such as cyclodextrin, can avoid π-π stacking by designing to increase the porphyrin interlayer spacing. Patent application CN120173193A discloses a polyrotaxylated covalent organic framework material and its application using cyclodextrin to regulate the interlayer spacing. However, this patent uses γ-cyclodextrin, which has a larger cavity than β-cyclodextrin, to increase the interlayer spacing. Patent application CN121270877A discloses a macrocyclic covalent organic polymer material and its application that suppresses the π-π stacking effect, using a larger macrocyclic polymer to increase the interlayer spacing. In the polymers of the two patents mentioned above, the porphyrins are still located on the same plane, and both use compounds with large cavity structures inserted into planar structures such as porphyrins to avoid π-π stacking. However, for β-cyclodextrin, its cavity structure is smaller, making it difficult to inhibit π-π stacking by increasing the interlayer spacing. Therefore, for β-cyclodextrin and porphyrin, a new structure needs to be designed to effectively inhibit π-π stacking and significantly improve antibacterial efficacy through multimodal antibacterial activity. Summary of the Invention
[0004] To address the aforementioned limitations of existing technologies, the present invention aims to provide a porous copper porphyrin polymer with cavity inclusion and its applications. This invention is based on a conjugated polymer (Cu-CD-Por-COP) constructed from β-cyclodextrin and pyrrole to form porphyrin structural units. During polymerization, a cyclodextrin derivative containing seven acetal groups and pyrrole simultaneously achieve pyrrole cyclization and the combination of macrocyclic porphyrin structures, endowing the material with unique multifunctional synergistic properties: the cyclodextrin skeleton provides abundant cavity inclusion capacity and a high specific surface area, which is beneficial for the adsorption and transport of active species; while the copper-coordinated porphyrin units formed by metal insertion modification contribute excellent light absorption performance, efficient photothermal conversion capability, stable singlet oxygen generation efficiency, and Fenton-like catalytic activity.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] In a first aspect, the present invention provides a copper porphyrin porous polymer with cavity encapsulation, wherein the copper porphyrin porous polymer uses β-cyclodextrin and porphyrin copper complexes as structural units, wherein the porphyrin copper complexes are covalently bonded to β-cyclodextrin and surround the cyclodextrin, and the porphyrin copper complexes surrounding a cyclodextrin are not on the same plane.
[0007] Preferably, the β-cyclodextrin is 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin; and the porphyrin copper complex is porphyrin copper.
[0008] Preferably, the structural formula of the copper porphyrin porous polymer is:
[0009] .
[0010] Preferably, the copper porphyrin porous polymer is prepared by the following method:
[0011] (1) Dissolve 4-hydroxybenzaldehyde and potassium carbonate in DMF and react at room temperature under a protective atmosphere. Then add a DMF solution of iodine-β-cyclodextrin dropwise, heat the reaction, remove DMF under reduced pressure, cool and filter the precipitate, recrystallize to obtain a white precipitate, wash and dry to obtain 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin.
[0012] (2) 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin was added to propionic acid, and pyrrole was added dropwise under a protective atmosphere and stirring to carry out a solvothermal reaction to obtain cyclodextrin porphyrin polymer;
[0013] (3) Add the cyclodextrin porphyrin polymer and copper salt to anhydrous methanol and reflux under a protective atmosphere. After the reaction is completed, filter, wash and dry to obtain the copper coordination polymer.
[0014] Preferably, in step (1), the molar ratio of 4-hydroxybenzaldehyde, potassium carbonate and iodine-β-cyclodextrin is 10:10:1; the heating reaction temperature is 80°C and the time is 24h.
[0015] Preferably, in step (2), the molar ratio of 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin to pyrrole is ≤1:28; the temperature of the solvothermal reaction is 140℃ and the time is 2d.
[0016] Preferably, in step (3), the copper salt is copper acetate; the mass ratio of the cyclodextrin porphyrin polymer to copper acetate is 2.17:1; the reflux reaction temperature is 50°C and the time is 48h.
[0017] A second aspect of the invention provides the use of copper porphyrin porous polymers in the preparation of antibacterial drugs.
[0018] Preferably, the copper porphyrin porous polymer exhibits antibacterial activity by simulating photothermal, photodynamic, and Fenton effects.
[0019] Preferably, the photodynamic therapy is a type I or type II photodynamic therapy.
[0020] The beneficial effects of this invention are:
[0021] (1) This invention designs a novel conjugated polymer (Cu-CD-Por-COP) based on β-cyclodextrin and pyrrole to construct porphyrin structural units, which possesses multiple antibacterial functions that intelligently respond to the infection microenvironment. This material achieves precise fusion of the cyclodextrin porous framework and the porphyrin active center at the molecular scale. The insertion modification of metallic copper further endows the material with excellent Fenton-like catalytic activity, which can effectively utilize the endogenous H2O2 of the infection microenvironment to achieve highly efficient sterilization without relying on exogenous antibiotics.
[0022] (2) The Cu-CD-Por-COP of this invention combines the cavity inclusion capacity of cyclodextrin, the high specific surface area of the porous structure, and the optical and catalytic properties of the porphyrin unit, exhibiting significant photothermal conversion, singlet oxygen generation, and Fenton-like catalytic performance. This material can serve as a synergistic therapeutic platform integrating photothermal / photodynamic / chemical kinetics, achieving simultaneous activation and mutual enhancement of multiple antibacterial mechanisms under near-infrared light irradiation. It can not only effectively destroy bacterial membrane structures but also deeply penetrate and disintegrate biofilms, significantly accelerating the healing of infected wounds and providing a new material solution for the treatment of clinically refractory bacterial infections.
[0023] (3) Cu-CD-Por-COP, as a novel pH-responsive conjugated polymer-based therapeutic platform, integrates multiple functions such as cavity inclusion, porous adsorption, photothermal conversion, photodynamic generation and endogenous catalysis, providing a promising solution for the efficient treatment of drug-resistant bacterial infections and related refractory wounds, and demonstrating broad application potential in the fields of biomedical materials and catalytic therapy. Attached Figure Description
[0024] Figure 1 (a) Fourier transform infrared spectra of 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin, CD-Por-COP, and Cu-CD-Por-COP; (b) Fourier transform infrared spectra of Cu-CD-Por-COP. 13 (c) Powder XRD of Cu-CD-Por-COP; (d) Thermogravimetric analysis curve of Cu-CD-Por-COP; (e) XPS spectrum of Cu-CD-Por-COP; (f) XPS spectrum of C1s; (g) XPS spectrum of N1s; (h) XPS spectrum of O2p; (i) High-resolution XPS spectrum of Cu2p;
[0025] Figure 2 (a) Scanning electron microscope (SEM) image of Cu-CD-Por-COP at a scale of 500 nm; (b) Scanning electron microscope (SEM) image of Cu-CD-Por-COP at a scale of 200 nm; (c) Scanning electron microscope (SEM) image of Cu-CD-Por-COP at a scale of 100 nm; (d) TEM image of Cu-CD-Por-COP at a scale of 200 nm; (e) TEM image of Cu-CD-Por-COP at a scale of 200 nm; (f) TEM image of Cu-CD-Por-COP at a scale of 5 nm; (g) Elemental distribution map of Cu-CD-Por-COP.
[0026] Figure 3(a) Temperature curves of Cu-CD-Por-COP and CD-Por-COP at different concentrations; (b) Photothermal curves of Cu-CD-Por-COP and CD-Por-COP at different power densities; (c) Infrared thermographic images of Cu-CD-Por-COP at different concentrations; (d) Concentration-temperature curves of Cu-CD-Por-COP and CD-Por-COP in the concentration range of 50~200 µg / mL; (e) Laser power-temperature curves of Cu-CD-Por-COP and CD-Por-COP at a concentration of 150 µg / mL; (f) Temperature change curves of Cu-CD-Por-COP aqueous solution (150 μg / mL) during four laser irradiation on / off cycles; (g) Relationship between cooling time and the negative natural logarithm of temperature for Cu-CD-Por-COP aqueous solution; (h) CD-Por-COP aqueous solution (150 µg / mL) (i) Temperature change curves of CD-Por-COP aqueous solution during four laser irradiation on / off cycles; (ii) Relationship curve between cooling time and the negative natural logarithm of temperature.
[0027] Figure 4 (a) UV-Vis spectrum of DPBF under laser irradiation; (b) UV-Vis spectrum of Cu-CD-Por-COP + DPBF under laser irradiation; (c) Comparison of DPBF attenuation rates induced by Cu-CD-Por-COP under laser irradiation; (d) UV-Vis spectrum of MB under laser irradiation; (e) UV-Vis spectrum of Cu-CD-Por-COP + MB under laser irradiation; (f) Comparison of MB attenuation rates induced by Cu-CD-Por-COP under laser irradiation; (g) Fluorescence spectrum of DHR123 under laser irradiation; (h) Fluorescence spectrum of Cu-CD-Por-COP + DHR123 under laser irradiation; (i) Comparison of DHR123 attenuation rates induced by Cu-CD-Por-COP under laser irradiation; (j) 1 Electron paramagnetic resonance (EPR) spectrum of O2; Electron paramagnetic resonance (EPR) spectrum of (k)•OH; (l)O2 •- Electron paramagnetic resonance (EPR) spectrum;
[0028] Figure 5 (a) UV absorption of each group near 650 nm; (b) UV absorption of Cu-CD-Por-COP at 650 nm under illumination or without illumination; (c) UV absorption peaks of Cu-CD-Por-COP solutions of different concentrations at 650 nm (pH 5.5); (d) UV absorption of •OH generated by Cu-CD-Por-COP solution with a concentration of 150 μg / mL in PBS at different pH values.
[0029] Figure 6 (a) Photographs of Staphylococcus aureus colonies formed after treatment in each group; (b) Photographs of Escherichia coli colonies formed after treatment in each group; (c) Survival rate of Staphylococcus aureus after treatment in each group; (d) Survival rate of Escherichia coli after treatment in each group.
[0030] Figure 7 (a) Fluorescent images of Staphylococcus aureus after co-staining various treatments; (b) Live-to-dead bacteria ratio of Staphylococcus aureus after various treatments; (c) Fluorescent images of Escherichia coli after co-staining various treatments; (d) Live-to-dead bacteria ratio of Escherichia coli after various treatments.
[0031] Figure 8 (a) TEM images of Staphylococcus aureus after co-staining with various treatments; (b) TEM images of Escherichia coli after co-staining with various treatments;
[0032] Figure 9 (a) Hemolysis of different concentrations of Cu-CD-Por-COP; (b) MTT assay of NIH / 3T3 cells after 24 h of treatment with different concentrations; (c) Staphylococcus aureus biofilm assay; (d) Escherichia coli biofilm assay; (e) Cell migration after 24 h of treatment with different concentrations of Cu-CD-Por-COP; (f) Statistical graph of cell migration rate after 24 h of treatment with different concentrations of Cu-CD-Por-COP.
[0033] Figure 10 (a) Photographs of the back wounds of mice infected with Staphylococcus aureus after treatment in each group; (b) Changes in mouse body weight during treatment; (c) Curves showing changes in wound area over time;
[0034] Figure 11 H&E and Masson trichrome staining images of wound tissues in each group on day 9 of wound healing;
[0035] Figure 12 (a) White blood cell count of mice on day 9; (b) Mean hemoglobin concentration of mice on day 9; (c) Platelet count of mice on day 9; (d) Hemoglobin concentration of mice on day 9; (e) Red blood cell volume of mice on day 9; (f) Mean hemoglobin content of mice on day 9; (j) Hematocrit of mice on day 9; (h) Mean red blood cell volume of mice on day 9;
[0036] Figure 13 H&E staining of major organs of mice: heart, liver, spleen, lungs and kidneys;
[0037] Figure 14 : A three-dimensional model diagram of Cu-CD-Por-COP. Detailed Implementation
[0038] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0039] As introduced in the background section, the current combination of cyclodextrin and porphyrin is achieved by expanding the distance between porphyrin layers through the cavity of cyclodextrin, thereby overcoming the π-π stacking between different porphyrin layers. However, the cavity of β-cyclodextrin is small and can only accommodate one benzene ring, which makes the extent to which the distance between porphyrin layers is expanded very limited.
[0040] Based on this, the object of the present invention is to provide a porous copper porphyrin polymer with cavity encapsulation and its application. The present invention introduces acetal groups into cyclodextrin to form derivatives. One cyclodextrin needs to be linked to seven porphyrins, creating steric hindrance, preventing the porphyrins from aligning in the same plane. The porphyrin plane becomes tilted or twisted, thus preventing the porphyrins from aligning parallel to each other and thus preventing the formation of π-π stacking. Figure 14 Thanks to this structure, Cu-CD-Por-COP exhibits a significant photothermal effect under near-infrared light irradiation, while simultaneously generating a large amount of singlet oxygen, achieving synergistic photothermal / photodynamic sterilization. Furthermore, this material can trigger a Fenton-like reaction in acidic infection microenvironments. These three mechanisms reinforce each other, forming a powerful multimodal synergistic antibacterial system that not only effectively disrupts bacterial membrane structures but also deeply penetrates and disintegrates the biofilm matrix.
[0041] This synthetic strategy not only effectively inhibits the intermolecular aggregation of porphyrin units, avoiding photosensitive activity quenching, but also endows the material with unique multi-mode synergistic functions: the cyclodextrin backbone provides excellent cavity inclusion capacity and high specific surface area, promoting the transport of active substances; while the in-situ constructed copper-porphyrin center contributes strong near-infrared absorption, efficient photothermal conversion, stable singlet oxygen generation, and Fenton-like catalytic activity. Specifically, Cu-CD-Por-COP can simultaneously exert photothermal therapy (PTT) and photodynamic therapy (PDT) effects under near-infrared light excitation; in acidic IME, it can further initiate a Fenton-like reaction, utilizing endogenous H2O2 to generate highly toxic •OH, achieving chemokinetic therapy (CDT). These three mechanisms reinforce each other, forming a synergistic antibacterial system that can not only effectively destroy bacterial membrane structures but also deeply penetrate and disintegrate biofilms. The material's pH-responsive characteristics enable it to intelligently release active components at the site of infection, thereby significantly accelerating wound healing while reducing damage to normal tissues.
[0042] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.
[0043] Note: Unless otherwise stated, the pH of the PBS used in this invention is 7.0.
[0044] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.
[0045] Example 1: Preparation of Cu-CD-Por-COP
[0046] (1) Preparation of 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin:
[0047] Using a dry 100 mL graduated cylinder, measure 50 mL of dry dimethylformamide (DM(F)) and add it to a 250 mL three-necked flask (wrapped in aluminum foil). At this point, purge the solution with nitrogen to remove any residual oxygen. Then, add triphenylphosphine (22 g, 83.94 mmol) to the reaction system and stir thoroughly at room temperature for about 10 minutes. Weigh I2 (22.5 g, 87.81 mmol) and dissolve it in 30 mL of DMF solution, stirring continuously until completely dissolved. Using a titration funnel (wrapped in aluminum foil), slowly add 30 mL of the I2 N,N-dimethylformamide solution dropwise to the 250 mL three-necked flask at room temperature, taking about 30 minutes. After the addition is complete, start stirring and heat to 50 °C. Add weighed dry β-cyclodextrin (5.795 g, 5.096 mmol) to the black solution, then slowly heat to 70 °C. Terminate the reaction after stirring for 19 hours under nitrogen protection. Evaporate approximately 50 mL under reduced pressure. The DMF system contained approximately 30 mL of black solution. The solution was slowly stirred at 0°C for about 20 minutes, followed by the addition of 150 mL (3 mol / L) sodium methoxide solution. A yellow precipitate formed. After standing for 10-20 minutes, 400 mL of ice-cold anhydrous methanol was added, producing a large amount of yellow precipitate. After standing for 30 minutes, the precipitate was filtered under vacuum, yielding a deep yellow precipitate. The precipitate was washed with methanol until the yellow color disappeared, then washed three times each with acetone and water. After surface drying, the solid was extracted with anhydrous methanol in a Soxhlet extractor for 22 hours until the extract was colorless. The solid was then removed and dried under vacuum at 65°C for 12 hours to obtain total iodine-β-cyclodextrin.
[0048] 1.22 g of 4-hydroxybenzaldehyde (10 mmol) and 1.38 g of potassium carbonate (10 mmol) were dissolved in 60 mL of DMF. The reaction was carried out at room temperature for 2 hours under nitrogen protection. Then, a solution of 1.904 g of total iodine-β-cyclodextrin (1 mmol) in DMF (20 mL) was slowly added dropwise over 30 minutes. The reaction temperature was raised to 80 °C, and the reaction was completed after 24 hours. After evaporating 60 mL of DMF under reduced pressure, the solution was poured into 200 mL of cold water (0 °C). The precipitate was filtered and recrystallized from 100 mL of DMF-H2O (volume ratio 1:4) to obtain a white precipitate, which was then filtered and washed under vacuum to obtain 7,6-deoxy-6-(4-formylphenyl)-β-cyclodextrin.
[0049] The synthesis route is as follows:
[0050] .
[0051] (2) Preparation of CD-Por-COP
[0052] 0.187 g of 7,6-deoxy-6-(4-formylphenyl)-β-cyclodextrin (0.1 mmol) was added to a 100 mL round-bottom flask containing 20 mL of propionic acid. Under an argon atmosphere, 277 μL (4 mmol) of freshly distilled pyrrole was slowly added dropwise over a total time of 3 min. The system was heated to 140 °C and maintained for 2 days. The reaction mixture was then filtered under reduced pressure to obtain a black powder, which was washed successively with deionized water, DMSO, DMF, methanol, THF, and DCM. The final product was dried under vacuum at 60 °C for 12 h to obtain the cyclodextrin porphyrin polymer CD-Por-COP.
[0053] The synthesis route is as follows:
[0054] .
[0055] (3) Cu-CD-Por-COP
[0056] In anhydrous methanol, 0.217 g of CD-Por-COP was mixed with 0.1 g of copper acetate and refluxed at 50 °C for 48 hours under argon protection. After the reaction was complete, the mixture was cooled to room temperature, and deionized water was poured in to precipitate the reactants, followed by filtration. The mixture was then thoroughly washed with water and methanol successively to remove excess copper salt and byproducts. Finally, the polymer was vacuum dried to obtain the copper coordination polymer Cu-CD-Por-COP.
[0057] The synthesis route is as follows:
[0058] .
[0059] Example 2: Characterization
[0060] To verify the successful synthesis of Cu-CD-Por-COP, Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy were first performed. 1 Determination by H NMR.
[0061] (1) The structure of Cu-CD-Por-COP was analyzed using infrared spectroscopy, such as... Figure 1 As shown in (a), the FT-IR spectrum of Cu-CD-Por-COP simultaneously displays the characteristics of porphyrin (Por) and 7,6-deoxy-6-(4-formylphenyl)-β-cyclodextrin, while also showing the characteristics of 7,6-deoxy-6-(4-formylphenyl)-β-cyclodextrin (V). O-H Peak 3360 cm -1 V C-O-C Peak 1028 cm -1 ) and porphyrin (V N-H Peak 3510 cm -1 The characteristic absorption peak of 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin is also observed at approximately 1700 cm⁻¹. -1 The presence of the polymer almost disappeared, confirming the successful synthesis of the polymer.
[0062] (2) Through solid state 13 C-NMR further confirmed the carbon skeleton structure of the Cu-CD-Por-COP complex. Figure 1 (b) The characteristic signals of saturated carbon (-CH and -CH2, chemical shifts 52.9 ppm and 72.9 ppm) and unsaturated carbon (81.8 ppm and 103 ppm) derived from cyclodextrin (CD) are clearly visible. Characteristic signals of substituted carbon (-OC, 159 ppm) in the benzene ring structure and carbon atoms in the porphyrin macrocyclic structure (118.9 ppm, 130.5 ppm, 147.9 ppm) are also observed. These results further confirm the successful polymerization of Cu-CD-Por-COP.
[0063] (3) The X-ray diffraction (XRD) pattern of Cu-CD-Por-COP is as follows: Figure 1 As shown in (c), only a broad peak is observed between 20 and 30, indicating that Cu-CDPor has an amorphous structure. Figure 1(d) Thermal stability assessment by thermogravimetric analysis (TG(A)) under a nitrogen atmosphere revealed a phased decomposition pattern. Thermogravimetric curves showed that Cu-CD-Por-COP exhibited a mass loss of less than 10% in the initial stage (<100℃), followed by a major mass loss stage (>200℃). The former is attributed to the evaporation of absorbed moisture within the highly polar porous framework, while the latter is related to the decomposition of the porous network structure. Notably, the material retained 42% of its residual mass at 800℃, indicating its excellent thermal stability required for photothermal applications.
[0064] (4) The surface elemental composition of Cu-CD-Por-COP was studied using X-ray photoelectron spectroscopy (XPS). Figure 1 (e) shows the measured spectrum of Cu-CD-Por-COP, revealing clear peaks for Cu, N, C, and S elements. The composition of Cu-CD-Por-COP was characterized by X-ray photoelectron spectroscopy (XPS), confirming the presence of C, N, S, and Fe elements. Figure 1 (f) The C1s spectrum is divided into three peaks, corresponding to the CC / C=C bond (284.80 eV), CO bond (286.38 eV) and C=NC bond (288.73 eV), respectively. Figure 1 The N1s spectrum in (g) shows two clear peaks at 399.29 eV (C=N-(C)) and 400.00 eV (CN-(H), confirming the successful introduction of nitrogen-containing groups. Figure 1 (h) Oxygen content analysis showed that there were three oxidation states in the polymer, corresponding to hydroxyl (-OH, 533.20 eV) and carbonyl (CO, 531.90 eV). Figure 1 In (i), the Cu2p spectrum shows two peaks at 933.23 eV and 954.31 eV, which are attributed to Cu2p, respectively. 3 / 2 and Cu2p 1 / 2 track.
[0065] (5) The morphology and internal structure of Cu-CD-Por-COP were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 2 (a) ~ Figure 2 As shown in Figure 2(c), Cu-CD-Por-COP is an irregular cluster formed by the aggregation of primary nanoparticles, constituting a rough and porous three-dimensional structure. This morphology can provide abundant adhesion sites for bacteria. This indicates that Cu-CD-Por-COP is not obtained through layer stacking; the porphyrins on the cyclodextrin are not on the same plane, forming a three-dimensional structure. Figure 2(d) and Figure 2(e) Low-magnification TEM image shows Cu-CD-Por-COP existing as discrete clusters. Figure 2(f) High-resolution TEM further shows the distinct light and dark contrasts in these structures, indicating the presence of macropores. Figure 2 (g) Elemental surface distribution analysis confirmed that the four characteristic elements C, O, N and Cu are highly uniformly dispersed in the material framework.
[0066] Example 3: Performance Testing
[0067] (1) Photothermal conversion performance test
[0068] Different concentrations of CD-Por-COP and Cu-CD-Por-COP aqueous solutions (0, 50, 100, 150, 200 μg / mL) were prepared and added to 1.5 mL centrifuge tubes, with a total volume of 1 mL for both solutions in each tube. Each centrifuge tube was then placed in an environment with a wavelength of 638 nm and a power density of 1.5 W / cm². -2 The sample was irradiated with an infrared laser for 10 min. Temperature values were displayed on a thermal imaging device, and the temperature rise within 10 min was plotted every 20 s. Additionally, 150 μg / mL aqueous solutions of CD-Por-COP and Cu-CD-Por-COP were prepared and subjected to power densities of 0.5, 1.0, 1.5, and 2.0 W / cm², respectively. -2 The CD-Por-COP and Cu-CD-Por-COP were irradiated with a 638 nm laser for 10 min, with data collected every 20 s for plotting. Subsequently, a cyclic irradiation experiment with the laser on and off was conducted to evaluate the thermal stability of CD-Por-COP and Cu-CD-Por-COP. Prepared aqueous solutions of CD-Por-COP and Cu-CD-Por-COP (150 μg / mL) were irradiated with an infrared laser (power density 1.5 W / cm²). -2 After irradiation for 10 minutes, the laser was turned off, and the Cu-CD-Por-COP was allowed to cool naturally to room temperature. This process was repeated three more times, and the temperature changes throughout the cycle were recorded. One cycle was plotted separately, and the photothermal conversion efficiency was calculated using the temperature change during the descent.
[0069] from Figure 3 (a) and Figure 3 (d) It can be seen that the heating behavior of CD-Por-COP and Cu-CD-Por-COP is related to their concentration. At 1.5 W cm⁻¹ -2After 10 minutes of laser irradiation, as the concentrations of CD-Por-COP and Cu-CD-Por-COP aqueous solutions increased from 50 μg / mL to 200 μg / mL, the temperature range of the CD-Por-COP suspension increased from 41.9℃ to 51.8℃, and the temperature range of Cu-CD-Por-COP increased from 40.8℃ to 52.2℃. In contrast, the temperature of pure water remained almost constant regardless of whether laser irradiation was present. Figure 3 (c) It can be seen that Cu-CD-Por-COP exhibits a wide temperature change range from light blue to bright red within 10 min, indicating that Cu-CD-Por-COP has good photothermal conversion performance. Meanwhile, as... Figure 3 (b) and Figure 3 As shown in (e), the temperature increase of CD-Por-COP and Cu-CD-Por-COP is also positively correlated with the laser density power. When the concentration is 150 μg / mL, the laser power is 0.5, 1.0, 1.5, and 2.0 W / cm². -2 At those times, the temperature increments for CD-Por-COP were 7.9℃, 12.4℃, 20.9℃, and 26.7℃, respectively. The temperature increments for Cu-CD-Por-COP were 6.8℃, 11.8℃, 20.2℃, and 26.8℃, respectively. Figure 3 (f) and Figure 3 As shown in (h), Cu-CD-Por-COP and CD-Por-COP exhibit good durability after four consecutive laser irradiation cycles, with no significant degradation. These results indicate that CD-Por-COP and Cu-CD-Por-COP are durable and efficient photothermal agents for repeated photothermal treatment. The photothermal conversion efficiency of Cu-CD-Por-COP is η (%) = 58.30%; the photothermal conversion efficiency of CD-Por-COP is η (%) = 56.30%. The photothermal conversion efficiency was calculated using the formula published in application CN115845086A, entitled "A Photothermal-Fenton-like Artificial Nanoenzyme and Its Preparation Method and Application". Figure 3 (g) and Figure 3 (i) shows the values of τS and θ in the formula for calculating the photothermal conversion efficiency of Cu-CD-Por-COP and CD-Por-COP.
[0070] (2) Testing of Type I and Type II photodynamic properties
[0071] The photodynamic properties of Cu-CD-Por-COP were investigated. Since the DPBF probe is easily oxidized and further decomposed into 1,2-diphenylmethylbenzene upon contact with reactive oxygen species (ROS), leading to a decrease in absorbance, the photodynamic properties of Cu-CD-Por-COP were studied. A 1 mg / mL stock solution of 1,3-diphenylisobenzofuran (DPBF) was prepared using N,N-dimethylformamide (DMF) as the solvent. 30 μL of this stock solution was serially diluted with 3 mL of DMF, and the absorbance of the solution was calibrated to approximately 1.0. The diluted DPBF solution was then subjected to a 638 nm laser (power density 1.5 W / cm²). 2 Irradiation was performed for 0, 2, 4, 6, 8, and 10 min, with UV absorption immediately after each irradiation to verify the stability of the DPBF probe under the experimental illumination conditions. A 150 μg / mL Cu-CD-Por-COP dispersion was prepared using DMF as the dispersion medium. 30 μL of the DPBF probe was added to this dispersion and thoroughly mixed. The mixture was then transferred to a cuvette and subjected to the same laser parameters (638 nm, 1.5 W / cm²). 2 The samples were irradiated for different durations, and their absorption spectra were measured immediately after each irradiation using a UV spectrophotometer. All experiments were performed in triplicate, and the UV absorption spectra of Cu-CD-Por-COP were plotted based on the UV absorption detection results at each time point.
[0072] according to Figure 4 (a) It can be seen that pure DPBF, after 10 min of laser irradiation (638 nm, 1.5 W / cm), 2 After adding Cu-CD-Por-COP, the absorbance decreased only slightly. With prolonged laser irradiation time, the absorbance decreased uniformly after adding Cu-CD-Por-COP. Figure 4 (b) and Figure 4 In (c), after 10 min of laser irradiation, the absorbance of Cu-CD-Por-COP decreased to 73.66% of its initial value. The results indicate that Cu-CD-Por-COP can be induced by laser irradiation. 1 The generation of O2 leads to a decrease in the absorbance of DPBF.
[0073] Methylene blue (MB) was used as an indicator to assess the hydroxyl radical (•OH) generation capacity of Cu-CD-Por-COP by detecting its UV absorption changes. First, a 1 mg / mL MB stock solution and a Cu-CD-Por-COP aqueous solution were prepared using deionized water. In the control experiment, 30 μL of MB aqueous solution was added to an EP tube, diluted with 3 mL of deionized water, and thoroughly mixed. The mixture was then transferred to a 3 mL cuvette and purified using a 1.5 W / cm² cuvette. 2 Irradiation with a 638 nm laser for 0, 2, 4, 6, 8, and 10 minutes was performed. The UV absorbance was measured immediately after each irradiation to observe the intrinsic changes of MB under illumination. In the experimental group, a solution containing Cu-CD-Por-COP (150 μg / mL) was first prepared, and 30 μL of MB indicator was added to adjust the total volume of the mixture to 3 mL. Subsequently, the same parameters (638 nm, 1.5 W / cm²) were used at the aforementioned time points. 2 The laser was used for irradiation. The absorption spectrum was measured immediately after each irradiation using a UV spectrophotometer. Finally, the UV absorption spectrum of Cu-CD-Por-COP was plotted based on the UV absorption detection results after irradiation, and the amount of hydroxyl radicals (•OH) generated was assessed by the degree of absorbance reduction using an MB indicator.
[0074] As can be seen from Figure 4(d), pure MB under laser irradiation (638 nm, 1.5 W / cm²) 2 After 10 minutes, the absorbance only decreased slightly. However, according to... Figure 4 (e) After adding Cu-CD-Por-COP, the absorbance decreased more significantly with the extension of irradiation time. Figure 4 The quantitative results in (f) show that the addition of Cu-CD-Por-COP to MB resulted in a significant decrease of 77.62% within 10 minutes compared to pure MB (56.15%).
[0075] The dihydrorhodamine 123 (DHR123) fluorescent probe method was used, with 638 nm laser irradiation (1.5 W / cm²). 2 The quantitative evaluation of superoxide anion (O2) in different material systems was carried out. •- Generation capacity. Dihydrorhodamine 123 (DHR123) was used as a fluorescent probe to detect superoxide anion radicals (O2). •-The specific operating steps are as follows: Accurately transfer a PBS solution containing Cu-CD-Por-COP (pH 5.5, material concentration 150 μg / mL), add 3 μL of DHR123 solution (solvent: dimethyl sulfoxide, concentration 5 mM), and then use PBS buffer of the same specification (pH 5.5) to bring the total volume of the mixture to 3 mL to ensure uniform dispersion. Place the prepared mixture under 638 nm laser irradiation, setting the laser power density to 1.5 W / cm². 2 The system was irradiated for 0, 2, 4, 6, 8, and 10 minutes, respectively. Immediately after each irradiation, a fluorescence spectrometer was used to detect the fluorescence signal in the 500–600 nm wavelength range. Changes in fluorescence intensity reflected the O2 levels. •- The formation and dynamic evolution of the substance were investigated. To eliminate interference from probe autofluorescence and laser irradiation, a blank control group experiment was set up: pure DHR123 solution without Cu-CD-Por-COP was detected under exactly the same experimental conditions.
[0076] In Figures 4(g) to 4(i), compared with the small change in ΔI value of pure DHR123, the absorbance increased rapidly after the introduction of Cu-CD-Por-COP. Specifically, as shown in Figure 4(i), after 10 minutes of laser irradiation, the ΔI value of Cu-CD-Por-COP was 6.21, which was higher than that of pure DHR123 (ΔI value 3.8).
[0077] In summary, these results demonstrate that the Cu-CD-Por-COP hybrid type I / II photodynamic activity has great potential for advancements in photodynamic therapy and related applications.
[0078] Electron paramagnetic resonance (EPR) spectroscopy, combined with a specific spin trapping agent, was used to further characterize the reactive oxygen species (ROS) generation spectrum of Cu-CD-Por-COP under laser irradiation, clarifying the types and generation characteristics of ROS during photodynamic processes. The effects of ROS generation under 638 nm laser irradiation (1.5 W / cm²) were systematically investigated. 2 The reactive oxygen species (ROS) generation spectrum was obtained at 10 min. EPR analysis clearly confirmed the simultaneous generation of multiple ROS. Figure 4 (j) TEMP- 1 The unique triplet signal presented in the O2 spectrum, with its three peaks exhibiting equal intensity (1:1:1), confirms the formation of singlet oxygen through energy transfer from the triplet state to molecular oxygen via photosensitizer excitation (type II pathway). 1 O2). Meanwhile... Figure 4(k) The typical peak shape with a 1:2:2:1 quadtet ratio in the DMPO-•OH spectrum confirms the formation of hydroxyl radicals (•OH), which is likely generated through further reduction of O2. •– It generates, or through the strong oxidation of water by photogenerated holes in the valence band. Figure 4 (l) DMPO-O2 •– The characteristic quartet signal (intensity ratio of 1:1:1:1) presented in the spectrum confirms the presence of superoxide anion (O2). •– As a major type I reactive oxygen species (ROS), O2 originates from a process of efficient photoinduced electron transfer from Cu-CD-Por-COP to ambient oxygen molecules. •– / •OH / 1 The O2 ternary reactive oxygen system generates a powerful synergistic antibacterial effect by simultaneously attacking key targets of multiple microorganisms.
[0079] (3) Hydroxyl radical generation capacity test
[0080] Two solutions were prepared: H₂O₂ + TMB and TMB + H₂O₂ + Cu-CD-Por-COP. In both solutions, the concentrations of TMB and H₂O₂ were 100 μl / mL, and the concentration of Cu-CD-Por-COP was 150 μg / mL. The total volume of each solution was 1 mL, and the volume was brought to the nearest whole number with PBS at pH 5.5. The UV absorption of each solution was then measured. Subsequently, Cu-CD-Por-COP solutions (prepared with PBS) with concentrations of 25, 50, 100, 150, and 200 μg / mL were prepared, all with a total volume of 1 mL and a pH of 5.5. The UV absorption of each solution at different concentrations under the same pH conditions was measured. A Cu-CD-Por-COP solution with a concentration of 150 μg / mL was prepared, and the volume was brought to 1 mL with PBS at pH values of 1.5, 2.5, 3.5, 4.5, and 5.5, respectively. The UV absorption of each solution was measured using a UV spectrophotometer. To verify whether light affects the enzyme activity of Cu-CD-Por-COP, an aqueous solution of Cu-CD-Por-COP with a concentration of 150 μg / mL was prepared, and its ultraviolet absorption was measured and plotted before and after irradiation.
[0081] like Figure 5 As shown in (a), Cu-CD-Por-COP only induces TMB to turn the solution blue and exhibits a striking UV adsorption peak at 650 nm in the presence of H2O2. However, Cu-CD-Por-COP alone or H2O2 alone does not induce oxidation. This result indicates that Cu-CD-Por-COP possesses excellent chemokinetic activity for treating CDT. Figure 5(d) shows that the enzyme activity of Cu-CD-Por-COP gradually increases from pH 5.5 to 2.5, indicating that the enzyme activity of Cu-CD-Por-COP reaches its optimum at pH 2.5. Therefore, even in an infectious environment (pH 5.5), Cu-CD-Por-COP exhibits significant catalytic activity, capable of converting low concentrations of H2O2 into toxic •OH, thereby effectively killing bacteria. Figure 5 (c) It was revealed that the activity of the Cu-CD-Por-COP mimic enzyme was related to its concentration, and the activity of the mimic enzyme was significantly increased with the increase of Cu-CD-Por-COP concentration (pH 5.5). Figure 5 (b) The signal intensity was further enhanced after laser irradiation, indicating that photothermal therapy can work synergistically with enzyme therapy to cause severe oxidative damage to various bacteria.
[0082] Test Example 1: In vitro antibacterial performance test
[0083] Staphylococcus aureus and Escherichia coli were used as representatives of Gram-positive and Gram-negative bacteria, respectively, to test the antibacterial properties of Cu-CD-Por-COP.
[0084] (1) The antibacterial test of Cu-CD-Por-COP was divided into 10 groups: I: control (PBS); II: H2O2; III: CD-Por-COP; IV: Cu-CD-Por-COP; V: Cu-CD-Por-COP + H2O2; VI: PBS + laser; VII: H2O2 + laser; VIII: CD-Por-COP + laser; IX: Cu-CD-Por-COP + laser; X: Cu-CD-Por-COP + H2O2 + laser. In the above groups, the total volume of the mixed solution in each group was 1 mL, and the bacterial concentration was 100 μL / mL (colony count was 10). 8 CFU mL -1 The concentrations of CD-Por-COP and Cu-CD-Por-COP were 150 μg / mL, and the concentration of H2O2 was 10 μL / mL. The laser-equipped group was irradiated with a 638 nm infrared laser at a power density of 1.5 W / cm². -2 The irradiation time was 10 minutes. Finally, the mixture from each group was incubated in a shaker at 37°C for 24 hours. 100 μL of the mixture from each group was then transferred to a solid culture medium, and morphology was observed and colony counts were calculated. The results were compared with the control group (PBS group) to evaluate the antibacterial effect.
[0085] As shown in Figure 6, regardless of whether laser irradiation was applied, the number of colony-forming units (CFU) in the Cu-CD-Por-COP H2O2 groups (II and VI) was similar to that in groups I and II, indicating that the low concentration of H2O2 (10 μL / mL) had a negligible effect on bacterial reduction. As for the CD-Por-COP group (III) and the Cu-CD-Por-COP group (IV), the number of CFUs was only slightly lower than that in the PBS group, indicating that the antibacterial effect of using CD-Por-COP (150 μg / mL) and Cu-CD-Por-COP (150 μg / mL) alone was weak. After laser irradiation, the bacterial colony counts in the CD-Por-COP+laser group (VIII) and the Cu-CD-Por-COP+laser group (IX) were significantly reduced, with Staphylococcus aureus survival rates of 11.12 ± 0.191% and 10.98 ± 0.779%, and Escherichia coli survival rates of 14.12 ± 0.336% and 12.53 ± 0.788%, respectively. In contrast, when H2O2 and laser irradiation were used simultaneously, the survival rates of Staphylococcus aureus and Escherichia coli in the Cu-CD-Por-COP + H2O2 + laser group (VIII) dropped sharply to 4.89 ± 0.318% and 6.26 ± 0.253%, respectively, demonstrating the significant advantage of PTT / PDT / CDT cascade synergistic therapy in effectively killing bacteria.
[0086] Experimental Example 2: Bacterial Staining Test
[0087] (1) Staining of live / dead bacteria
[0088] The mixture was divided into 10 groups according to the grouping method in Experiment Example 1 (the total volume of the mixed solution in each group was 1 mL). 400 μL of solution was taken from each group, and 20 μL of SYTO-9 (1.0 × 10⁻⁶) was added to each group. -3 M) and 20 μL PI (1.5 × 10 -3 The solution was incubated at 37°C in the dark for 15 min. Afterwards, excess SYTO-9 and PI were removed by centrifugation. The bacteria in each solution were then resuspended in 50 μL of PBS, and images were observed using an inverted fluorescence microscope.
[0089] As shown in Figure 7(a) and Figure 7As shown in (c), Staphylococcus aureus and Escherichia coli in the control group, PBS+laser group, H2O2 group, H2O2+laser group, CD-Por-COP group, and Cu-CD-Por-COP group all survived, and the red fluorescence disappeared. Significant red fluorescence was detected after treatment with CD-Por-COP+laser, Cu-CD-Por-COP+laser, and Cu-CD-Por-COP+H2O2+laser groups. Consistent with the results obtained by the plate count method, bacteria treated with Cu-CD-Por-COP+H2O2+laser showed the strongest red fluorescence, indicating a significant increase in the number of dead bacteria after multimodal treatment. Furthermore, to more clearly compare the bactericidal effect of Cu-CD-Por-COP, quantitative analysis of bacterial viability and mortality was performed. Figure 7 (b) and Figure 7 (d) It can be clearly seen that the survival rates of Staphylococcus aureus in groups VIII and IX were 14.27 ± 1.748% and 13.25 ± 1.663%, respectively, while the survival rate in group X was 4.47 ± 0.283%. The survival rates of Escherichia coli in groups VIII and IX were 13.05 ± 1.240% and 12.45 ± 0.668%, respectively, while the survival rate in group X was 6.090 ± 0.413%.
[0090] (2) Morphology of bacteria under projection
[0091] The bacteria were divided into 10 groups according to the grouping in Experiment 1. Each group's solution was added to 2.5 wt% glutaraldehyde solution and fixed at 4°C for 24 h. Afterward, the bacteria were washed three times with PBS, embedded in agar, and blocked. The bacteria were then treated sequentially with ethanol solutions of 30 wt%, 50 wt%, 70 wt%, 90 wt%, 95 wt%, and 100 wt% concentrations for 10 min to dehydrate them, followed by treatment with acetone for 3 h, using a gradient permeation embedding method. Finally, the bacteria were negatively stained, fixed on a nickel grid, and observed under a transmission electron microscope.
[0092] like Figure 8As shown, regardless of the laser irradiation intensity, the bacterial surfaces treated with PBS and H2O2 remained smooth and intact; Staphylococcus aureus appeared spherical, and Escherichia coli appeared rod-shaped. After exposure to the Cu-CD-Por-COP group (Group IV), damage to the bacterial cell surface was observed only for one week, indicating that the antibacterial effect of the Cu-CD-Por-COP group was negligible. The cell walls of the Cu-CD-Por-COP+H2O2 group (Group V) also showed damage, indicating that the Fenton effect of this material also has a certain bactericidal effect. After treatment with CD-Por-COP + laser (Group VIII) and Cu-CD-Por-COP + laser (Group IX), the cell membranes were damaged, resulting in cytoplasmic leakage. In the Cu-CD-Por-COP + laser + H2O2 group (Group X), the cell membranes were almost completely destroyed, with a large amount of cytoplasm leaking out, which is consistent with the antibacterial results.
[0093] Experimental Example 3: In vitro biocompatibility experiment
[0094] (1) Cell hemolysis experiment
[0095] Blood was collected from 5-week-old female KM mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). Fresh blood was centrifuged (10000 rpm, 20 min), and the supernatant was discarded to collect red blood cells. The cells were then washed with PBS buffer at a ratio of approximately 1:1 (PBS buffer to red blood cells). After centrifugation, the supernatant was discarded, and this process was repeated four times. Red blood cells were then mixed with PBS buffer at a volume ratio of 3:11. The resulting mixture was then combined with Cu-CD-Por-COP (concentrations of 50, 100, 150, 200, and 250 μg / mL) at a ratio of 1:9 (v / v) and incubated at 37°C for 4 h. 100 μL of the supernatant from each tube was added to a 96-well plate, and the absorbance was measured using a microplate reader. Distilled water served as a positive control, and the PBS group served as a negative control. The calculation formula is as follows:
[0096] Hemolysis volume (%) = (A-An) / (Ap-An) × 100%;
[0097] A: The absorbance obtained by taking the supernatant after adding Cu-CD-Por-COP to red blood cells;
[0098] An: Absorbance obtained by taking the supernatant after adding PBS to red blood cells (negative control).
[0099] Ap: Absorbance obtained by taking the supernatant after adding distilled water to red blood cells (positive control).
[0100] like Figure 9As shown in (a), after co-culturing with blood, Cu-CD-Por-COP exhibited concentration-dependent hemolytic behavior, with the hemolysis rate increasing with increasing concentration. Within the experimental concentration range (50, 100, 150, 200, 250 μg / mL), the hemolysis rate remained below the standard value (4.0%), indicating that Cu-CD-Por-COP has good blood compatibility.
[0101] (2) Cytotoxicity test
[0102] In 96-well plates, NIH / 3T3 mouse embryonic fibroblasts (from the Chinese Academy of Sciences Cell Bank) were stored at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL per well, and the edges of the plate were sealed with 100 μL of PBS to prevent excessive evaporation. After 24 h of incubation, the control group was treated with PBS, while the experimental groups were treated with different concentrations of Cu-CD-Por-COP (50, 100, 150, 200, and 250 μg / mL). After standing for 24 h, the supernatant was discarded, and 10 μL of MTT (tetramethylazolyl blue, 5 mg / mL) was added to each well. After incubation for 4 h, the supernatant was aspirated, and 100 μL of dimethyl sulfoxide was added. After standing for approximately 10 min, the absorbance was measured at 540 nm using a microplate reader. Each experiment was repeated three times.
[0103] like Figure 9 As shown in (b), the cytotoxicity assessment results indicate that Cu-CD-Por-COP has good cell compatibility, with cell viability exceeding 80% at given doses (50-250 μg / mL).
[0104] (3) Cell scratch test
[0105] Prepare three 6-well plates, with three wells per group. The concentrations were 0 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL. Group 0 received the same treatment as the other groups except for no drug administration. NIH / 3T3 cells (from the Chinese Academy of Sciences Cell Bank) were seeded into the 6-well plates and cultured for 24 h. Cells were then scraped from the tip of a sterile pipette and cultured for 12 h, followed by laser irradiation for 10 min. Cells were then washed with PBS, and phase-contrast images were taken using a microscope at 0 h, 12 h, and 24 h. ImageJ software was used to measure cell migration distance and calculate the percentage of wound healing.
[0106] like Figure 9 (e) and Figure 9As shown in (f), the scratch area in all three experimental groups decreased significantly with prolonged culture time. The scratch healing rate of the 50 μg / mL to 200 μg / mL groups was essentially consistent with that of the 0 μg / mL group, indicating that Cu-CD-Por-COP has negligible effect on normal cell migration function. All these results demonstrate that Cu-CD-Por-COP has good biocompatibility and can be used as a therapeutic agent for in vivo infections.
[0107] (4) Bacterial biofilm experiment
[0108] Staphylococcus aureus and Escherichia coli were used for modeling: 150 μL of liquid culture medium and 50 μL of bacterial suspension were added to each well of a 96-well plate, and the plates were incubated for 72 hours. The experiment was divided into four groups: control group (PBS group I), Cu-CD-Por-COP group (II), Cu-CD-Por-COP+H2O2 group (III), Cu-CD-Por-COP+laser group (IV), and Cu-CD-Por-COP+laser+H2O2 group (V). 50 μL of Cu-CD-Por-COP was added to the Cu-CD-Por-COP group; 10 μL of H2O2 was added to the H2O2 group; and the laser group used a laser at 638 nm and 1.5 W / cm². 2 Laser irradiation for 10 min. The above groups were added to the incubated bacterial culture and incubated for another 5 hours. After incubation, the cultures were stained with crystal violet (CV): washed with PBS, fixed with 100 μL of methanol for 30 min, washed with PBS, and finally incubated with 50 μL of 0.1% crystal violet for 20 min. After incubation, washed with PBS until clear, added 100 μL of glacial acetic acid, and incubated for 30 min. The supernatant was then transferred to a blank plate to measure the OD value (490 nm). Biofilm ratio = OD value of groups II to V / OD value of group I.
[0109] like Figure 9 (c) and Figure 9As shown in (d), the color of the Cu-CD-Por-COP group (II) showed no significant change compared to the control group (I), indicating that Cu-CD-Por-COP alone has little effect on biofilms. The Cu-CD-Por-COP+H2O2 group (III) was lighter in color than the Cu-CD-Por-COP group (II), indicating that the peroxidase (CDT)-like activity of Cu-CD-Por-COP can disrupt biofilms. The Cu-CD-Por-COP+laser group (IV) and the Cu-CD-Por-COP+laser+H2O2 group (V) were significantly lighter in color than the other groups, with the Cu-CD-Por-COP+laser+H2O2 group (V) being the lightest. The biofilm elimination effect of Cu-CD-Por-COP was further evaluated by measuring the CV absorbance. It can be clearly seen that the biofilm elimination rates of Staphylococcus aureus in the Cu-CD-Por-COP+laser group (IV) and Cu-CD-Por-COP+laser+H2O2 group (V) were 75.99 ± 1.150% and 79.44 ± 0.800%, respectively, and the biofilm elimination rates of Escherichia coli were 74.58 ± 5.614% and 78.95 ± 2.956%, respectively. This indicates that Cu-CD-Por-COP, as a photosensitizer, has a significant effect on destroying bacterial biofilms, and its effect on destroying Staphylococcus aureus biofilms is more significant.
[0110] Experiment Example 4: Wound Healing Experiment
[0111] Wound trauma models were established using 5-week-old female KM mice weighing approximately 25g (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). Mice were randomly divided into 6 groups: I: Blank group; II: Control group (PBS); III: Cu-CD-Por-COP group; IV: Cu-CD-Por-COP+H2O2 group; V: Cu-CD-Por-COP+laser group; VI: Cu-CD-Por-COP+laser+H2O2 group, with 6 mice in each group. The blank group consisted of normally fed mice used for comparison with the other 5 groups. Before formal infection, the weight of each group of mice was measured. Except for the blank group, all other groups of mice were anesthetized, their back hair was shaved, and the wounds were disinfected with 75wt% ethanol to create a wound with a radius of approximately 5mm. Staphylococcus aureus (1×10⁻⁶) was added to the wound. 6 Mice were infected with Cu-CD-Por-COP at a concentration of 150 μg / mL and H2O2 at a concentration of 10 μL / mL 24 h later. The laser irradiation power was 1.5 W cm⁻¹. -2The irradiation time was 10 min. Changes in the back wounds of mice were recorded on days 1, 3, 5, 7, and 9, and the mice's weight was monitored daily. The weight changes and back wound area were plotted. On day 9, mice were anesthetized, and blood was collected from the eyeballs. After blood collection, the mice were euthanized, and the back wounds and major organ tissues (heart, liver, spleen, lungs, and kidneys) were fixed with 10% formalin. Then, H&E staining and Masson's trichrome staining were performed. Two mL of blood was collected from each group for complete blood count analysis.
[0112] Figure 10 (a) Shows the wound area of mice under different treatments, photographed on days 1, 3, 5, 7, and 9. On day 1, significant wound exudate was observed in all groups, indicating successful establishment of the animal model. With prolonged treatment, wounds in each group gradually healed, but the healing rate varied considerably among groups. It can be seen that the wound repair process in the Cu-CD-Por-COP+H2O2 group was slightly faster than that in the control group and the Cu-CD-Por-COP group, while the Cu-CD-Por-COP+laser and Cu-CD-Por-COP+laser+H2O2 groups showed the fastest wound repair. By day 9, significant wound healing was observed in all groups, with the Cu-CD-Por-COP+laser and Cu-CD-Por-COP+laser+H2O2 groups showing near-complete healing.
[0113] like Figure 10 (c) shows that after 9 days of treatment, the wound area of mice in the Cu-CD-Por-COP+laser+H2O2 group was almost completely healed, with the remaining wound area being 9.83% of the initial wound area, much smaller than that of other groups. Figure 10 (b) As can be seen, similar to the control group, the body weight of mice in other groups did not change significantly throughout the treatment process, indicating that Cu-CD-Por-COP has excellent biocompatibility. To directly estimate the healing efficiency of infected wounds, histological analysis was performed on the newly formed skin at the wound sites of mice after different treatments.
[0114] Figure 11 Histological analysis and Massen staining results showed that new skin layers and capillary formation were observed in all treatment groups. However, compared to groups I-IV, the epidermal layer of the wound treated in groups V and VI was clearer, and the wound area was smaller. The skin in the Cu-CD-Por-COP+laser+H2O2 group almost completely healed, with the appearance of hair follicles and connective tissue. These results indicate that Cu-CD-Por-COP+laser+H2O2 is crucial for accelerating wound healing.
[0115] As shown in Figure 12, similar to the control group mice, there were almost no significant differences in any of the blood routine indicators. Figure 13 As can be seen, no obvious organ damage or abnormalities were observed in the mouse tissues after different treatments. All these results confirm that Cu-CD-Por-COP has good in vivo biocompatibility and can be used as a safe and efficient material-based antibacterial agent.
[0116] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A porous copper porphyrin polymer with cavity encapsulation, characterized in that, The copper porphyrin porous polymer uses β-cyclodextrin and porphyrin copper complex as structural units. The porphyrin copper complex is covalently bonded to β-cyclodextrin and surrounds the cyclodextrin. The porphyrin copper complexes surrounding a cyclodextrin are not on the same plane. The β-cyclodextrin is 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin; the porphyrin copper complex is porphyrin copper; The structural formula of the copper porphyrin porous polymer is: 。 2. The copper porphyrin porous polymer according to claim 1, characterized in that, The copper porphyrin porous polymer is prepared by the following method: (1) Dissolve 4-hydroxybenzaldehyde and potassium carbonate in DMF and react at room temperature under a protective atmosphere. Then add a DMF solution of iodine-β-cyclodextrin dropwise, heat the reaction, remove DMF under reduced pressure, cool and filter the precipitate, recrystallize to obtain a white precipitate, wash and dry to obtain 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin. (2) 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin was added to propionic acid, and pyrrole was added dropwise under a protective atmosphere and stirring to carry out a solvothermal reaction to obtain cyclodextrin porphyrin polymer; (3) Add the cyclodextrin porphyrin polymer and copper salt to anhydrous methanol and reflux under a protective atmosphere. After the reaction is completed, filter, wash and dry to obtain the copper coordination polymer.
3. The copper porphyrin porous polymer according to claim 2, characterized in that, In step (1), the molar ratio of 4-hydroxybenzaldehyde, potassium carbonate and iodine-β-cyclodextrin is 10:10:1; the heating reaction temperature is 80℃ and the time is 24h.
4. The copper porphyrin porous polymer according to claim 2, characterized in that, In step (2), the molar ratio of 7-6-deoxy-6-(4-formylphenyl)-β-cyclodextrin to pyrrole is ≤1:28; the temperature of the solvothermal reaction is 140℃ and the time is 2d.
5. The copper porphyrin porous polymer according to claim 2, characterized in that, In step (3), the copper salt is copper acetate; the mass ratio of the cyclodextrin porphyrin polymer to copper acetate is 2.17:1; the reflux reaction temperature is 50°C and the time is 48h.
6. The use of the copper porphyrin porous polymer according to any one of claims 1 to 5 in the preparation of antibacterial drugs.
7. The application according to claim 6, characterized in that, The copper porphyrin porous polymer exhibits antibacterial activity by simulating photothermal, photodynamic, and Fenton effects.
8. The application according to claim 7, characterized in that, The photodynamics are type I and type II.