Cationic porphyrin-based covalent organic frameworks, their preparation methods, and targeted bactericidal applications

Abstract

This invention relates to a method for preparing and applying a porphyrin-based covalent organic framework, TAPP-VIO, with photothermal / photodynamic / chemical bactericidal properties, belonging to the fields of advanced functional materials and biomedicine. Using 5,10,15,20-tetra-(4-aminophenyl)porphyrin (TAPP) as the structural unit of the synthesized covalent organic framework not only restricts the distance of the photosensitizer but also endows the covalent organic framework with photosensitizing activity. Simultaneously, another structural unit, 4,4'-dibenzaldehyde-bipyridine (VIO), imparts electrostatic repulsion and bacterial targeting between the material layers, constructing a bacterial reaction-targeting material integrating photothermal / photodynamic / chemical bactericidal capabilities. This photothermal / photodynamic / chemical bactericidal integrated material exhibits good stability, bacterial targeting, photothermal heating effect, and superior ability to generate reactive oxygen species, and also possesses good biosafety, showing great potential in the treatment of bacterial infections.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying a porphyrin-based covalent organic framework TAPP-VIO with photothermal / photodynamic / chemical bactericidal properties, belonging to the fields of advanced functional materials and biomedical technology. Background Technology

[0002] Bacterial infection is an acute systemic infection caused by pathogenic or opportunistic pathogens entering the bloodstream through wounds or infected lesions, multiplying, and producing toxins and other metabolic products. It can affect all tissues and organs throughout the body, from superficial bacterial infections to deep tissue and organ infections. In the elderly, children, and those with chronic diseases or weakened immune systems, it can develop into sepsis or septicemia, seriously threatening human health and social development. The advent of antibiotics has initially alleviated this problem and achieved some success in treating bacterial infections. However, with the widespread use of antibiotics, their shortcomings have gradually become apparent, such as low bioavailability, strong toxicity, and bacterial resistance. Therefore, there is an urgent need to develop a new, highly effective treatment for bacterial infections that does not induce drug resistance.

[0003] Phototherapy is a promising treatment method, attracting significant attention due to its non-invasiveness, ease of operation, high specificity, low invasiveness, minimal side effects, and lack of drug resistance. Phototherapy includes photothermal and photodynamic methods, which convert light energy into heat or reactive oxygen species using photosensitizers, causing irreversible damage to bacteria and achieving a therapeutic effect. However, most photosensitizers lack bacterial targeting and specificity, resulting in poor treatment efficacy and non-specific damage to normal tissues. Furthermore, most photosensitizers suffer from self-aggregation quenching and poor photostability in physiological environments, leading to unsatisfactory treatment results. Additionally, the efficiency of phototherapy is limited by the limited penetration depth of light and oxygen depletion. Therefore, there is an urgent need to develop a novel phototherapy agent that does not exhibit aggregation and self-quenching, can target bacteria, and possesses highly efficient bactericidal properties for the treatment of bacterial infections. Summary of the Invention

[0004] To address the above problems, the present invention aims to provide a novel photothermal bactericidal material, TAPP-VIO, synthesized by a Schiff base reaction solvothermal method, which has specific bacterial targeting and integrates photothermal / photodynamic / chemical bactericidal properties. It overcomes and solves the problems of unsatisfactory therapeutic effects caused by the easy aggregation and quenching of traditional photosensitizers under physiological conditions, lack of targeting, and limited therapeutic efficiency.

[0005] The technical solution of the present invention:

[0006] The first aspect of the present invention is to provide a cationic porphyrin-based covalent organic framework with photothermal / photodynamic / chemical synergistic bactericidal ability, wherein the cationic porphyrin-based covalent organic framework has the structure shown in Formula I;

[0007]

[0008] Furthermore, the cationic porphyrin-based covalent organic framework (TAPP-VIO) is prepared by reacting 5,10,15,20-tetra-(4-aminophenyl)porphyrin monomer (TAPP) with 4,4'-dibenzaldehyde-bipyridine monomer (VIO).

[0009] Furthermore, the 5,10,15,20-tetra-(4-aminophenyl)porphyrin monomer (TAPP) has the structure shown in Formula II;

[0010] Furthermore, the 4,4'-dibenzaldehyde-bipyridine monomer (VIO) has the structure shown in Formula I and II;

[0011]

[0012] The second aspect of this application is to provide a method for preparing the cationic porphyrin-based covalent organic framework (TAPP-VIO) material; comprising the steps of: mixing a solution containing 5,10,15,20-tetra-(4-aminophenyl)porphyrin with a solution containing 4,4'-dibenzaldehyde-bipyridine, then adding an acid or base as a catalyst, mixing evenly, reacting at a certain temperature, and then separating, washing, and drying to obtain the product TAPP-VIO.

[0013] Furthermore, the TAPP-VIO material is prepared using an acid-base assisted solvothermal method, with the specific steps as follows:

[0014] (1) A solution containing 4,4'-dibenzaldehyde-bipyridine (VIO), 5,10,15,20-tetra-(4-aminophenyl)porphyrin (TAPP) and mesitylene / n-butanol was mixed at room temperature, and then a catalyst was added and the mixture was continued to be mixed evenly to obtain a reaction solution.

[0015] (2) React the reaction solution at 60-150℃ for 2-7 days;

[0016] (3) After the reaction is complete, the material is centrifuged, washed, and dried to obtain the porphyrin-based covalent organic framework material (TAPP-VIO).

[0017] In some preferred embodiments, the molar ratio of the 5,10,15,20-tetra-(4-aminophenyl)porphyrin to the 4,4'-dibenzaldehyde-bipyridine is 1:2.

[0018] Furthermore, the reaction is carried out in an organic solvent selected from one or more of dioxane, mesitylene, n-butanol, tetrahydrofuran, and benzyl alcohol.

[0019] In some preferred embodiments, the organic solvent is a mixture of mesitylene and n-butanol in a volume ratio of 9:1 to 1:9.

[0020] In one embodiment of the present invention, the ratio of solvent to TAPP in step (1) is 2 mL: 16.8 mg.

[0021] In one embodiment of the present invention, the mixing in step (1) is ultrasonic mixing, and the ultrasonic time is 5-60 min.

[0022] In some embodiments, the reaction system is mixed by ultrasound for 5-60 minutes, at a temperature of 20-200°C, and for 1-7 days. After the product is naturally cooled to room temperature, the suspension is taken, centrifuged at high speed, and the precipitate is washed with solvent. After washing, the product is dried in a vacuum oven to obtain a purplish-black powder (TAPP-VIO).

[0023] In one embodiment of the present invention, the reaction solution described in step (2) needs to undergo three cycles of freezing-nitrogen purging-vacuuming-thawing before the reaction.

[0024] In one embodiment of the present invention, the drying in step (3) is vacuum drying.

[0025] In some embodiments, the catalyst is an acetic acid solution;

[0026] In one embodiment of the present invention, the catalyst in step (1) is an acetic acid solution with a concentration of 6M;

[0027] Preferably, the amount of catalyst is 10% (V / V) of the total reaction system.

[0028] In some preferred embodiments, the separation step uses centrifugation;

[0029] In some preferred embodiments, the centrifugation speed in the centrifugation separation stage is 8000-12000 rpm;

[0030] In some embodiments, the washing step uses one or more of tetrahydrofuran, N,N-dimethylformamide, and acetonitrile.

[0031] In one embodiment of the present invention, the centrifugation speed in step (3) is 8000-12000 rpm, and the precipitate is washed 3-6 times; the washing solvent is one or more of tetrahydrofuran and N,N-dimethylformamide.

[0032] A third objective of this invention is to provide the application of the aforementioned cationic porphyrin-based covalent organic framework in the preparation of bactericidal agents.

[0033] Furthermore, the bactericidal agent is a bacterial-targeted phototherapy / chemotherapy synergistic bactericidal agent;

[0034] Furthermore, the bactericidal agent is a bactericidal agent that promotes wound healing;

[0035] In some embodiments, the bactericidal agent is a liquid formulation; it can be a drop or a spray formulation.

[0036] The fourth objective of this invention is to provide a photothermal / photodynamic / chemical sterilization method for the diagnosis and treatment of non-disease conditions, wherein the cationic porphyrin-based covalent organic framework described above or the cationic porphyrin-based covalent organic framework prepared by the method described above is used as the sterilization material.

[0037] In one embodiment of the present invention, the photothermal / photodynamic sterilization method for non-disease diagnosis and treatment is to add TAPP-VIO for light irradiation;

[0038] The specific steps include: dissolving porphyrin-based covalent organic framework material (TAPP-VIO) in a solvent, then mixing it with bacterial suspension, irradiating it under a suitable light source for 20 minutes, then serially diluting it and coating it onto a solid culture medium, and counting the colonies after culturing for 12-24 hours to obtain the sterilization rate; wherein the solvent is a liquid culture medium, the bacterial suspension includes Staphylococcus aureus and Escherichia coli, and the light source is a white LED lamp.

[0039] The beneficial effects of this invention are:

[0040] (1) The cationic porphyrin-based covalent organic framework (TAPP-VIO) provided by this invention, due to its special porous structure and the repulsive effect between cations, can limit the distance between photosensitizer monomer molecules, thereby reducing the self-aggregation quenching of photosensitizers, ensuring high-density aggregation of porphyrin molecules, and exhibiting good photothermal conversion performance and high efficiency. 1 O2 generation capability achieves excellent photothermal and photodynamic effects, effectively preventing the self-polymerization quenching of photosensitizers and thermal damage caused by continuous exposure to external light sources;

[0041] (2) The TAPP-VIO prepared by this invention has a large surface area, which enhances light absorption capacity. Its ordered pores also help to store oxygen and release reactive oxygen species, thereby improving the effect of photodynamic therapy.

[0042] (3) The method for preparing cationic porphyrin-based covalent organic framework materials provided by the present invention does not contain metals in the synthesis process, and the reaction that generates imino bonds is reversible, which provides the possibility for subsequent degradation of the material. These characteristics endow TAPP-VIO with good biosafety. Attached Figure Description

[0043] Figure 1 Schematic diagram of TAPP-VIO, which integrates photothermal / photodynamic / chemical sterilization, prepared in Example 1;

[0044] Figure 2 Experimental and simulated X-ray powder diffraction patterns of TAPP-VIO prepared in Example 1;

[0045] Figure 3 Infrared spectra of the monomers TAPP and VIO used in Example 1, and the prepared TAPP-VIO;

[0046] Figure 4 Transmission electron microscopy image of TAPP-VIO prepared in Example 1;

[0047] Figure 5 X-ray powder diffraction pattern of TAPP-VIO prepared in Example 2;

[0048] Figure 6 X-ray powder diffraction pattern of TAPP-VIO prepared in Example 3;

[0049] Figure 7 X-ray powder diffraction pattern of TAPP-VIO prepared in Example 4;

[0050] Figure 8 X-ray powder diffraction pattern of TAPP-VIO prepared in Example 5;

[0051] Figure 9 : Photothermal effect diagram of TAPP-VIO prepared in Example 1;

[0052] Figure 10 : A graph showing the ability of TAPP-VIO prepared in Example 1 to generate singlet oxygen;

[0053] Figure 11 Plate sterilization diagram of TAPP-VIO prepared in Example 1;

[0054] Figure 12Figure: The effect of TAPP-VIO prepared in Example 1 on in vivo wound treatment. Detailed Implementation

[0055] To further enhance understanding of the preparation process and technical characteristics involved in this invention, the invention will be further described below with reference to embodiments. Preferred embodiments of the invention will be described below; it should be understood that these embodiments are for better explanation of the invention and are not intended to limit the invention.

[0056] 5,10,15,20-Tetra-(4-aminophenyl)porphyrin (TAPP): CAS No.: 22112-84-1, purchased from Jilin Institute of Advanced Technology, Chinese Academy of Sciences;

[0057] 4,4'-Diphenylaldehyde-Bipyridine (VIO): CAS No.: 1690360-53-2, purchased from Jilin CAS Research & Development Technology Co., Ltd.

[0058] Example 1: Preparation of TAPP-VIO material

[0059] Preparation of a TAPP-VIO material integrating photothermal / photodynamic / chemical sterilization: The photosensitizer 5,10,15,20-tetra-(4-aminophenyl)porphyrin TAPP is confined within a covalent organic framework as a structural unit. This ensures photosensitizer density while reducing its self-aggregation quenching effect, thus improving its photothermal and photodynamic effects. Simultaneously, the cationic groups in another structural unit, 4,4'-dibenzaldehyde-bipyridine VIO, impart electrostatic repulsion between material layers, reducing the self-aggregation quenching of the photosensitizer. Furthermore, its positive charge in solution can bind to the negatively charged membrane surface of bacteria, immobilizing them and reducing their spread. The material preparation includes the following steps:

[0060] (1) 21.9 mg of 4,4'-dibenzaldehyde-bipyridine (VIO, 0.05 mmol) and 16.8 mg of 5,10,15,20-tetra-(4-aminophenyl)porphyrin (TAPP, 0.025 mmol) were added to a solvent consisting of tricresylbenzene / n-butanol (1:1, v:v, 2.0 mL), and sonicated for 5 minutes. Then 0.2 mL of acetic acid was added, and sonication was performed for another 5 minutes.

[0061] (2) The mixture was degassed three times in a Pyrex tube using a refrigeration pump-thaw cycle, and then sealed. The resulting mixture was then reacted at 120°C for 6 days.

[0062] (3) After cooling to room temperature, the final product (TAPP-VIO) was collected by centrifugation, washed five times with THF, and dried under vacuum at 50°C;

[0063] The performance of the prepared TAPP-VIO was tested. Figure 2 The results show that the characteristic diffraction peaks of the cationic porphyrin-based covalent organic framework material (TAPP-VIO) are similar to those of the simulated AA stacking diffraction peaks, but differ significantly from those of the simulated AB stacking diffraction peaks, indicating that the obtained porphyrin-based covalent organic framework is constructed using the AA stacking mode. Figure 3 The results showed that TAPP-VIO exhibited a characteristic stretching vibration peak of -C=N- at 1595 nm, indicating that TAPP-VIO was successfully synthesized. Figure 4 The TAPP-VIO was shown to be approximately 400nm in size, suitable for in vivo phototherapy sterilization.

[0064] Example 2:

[0065] The mixed solvents in Example 1 were adjusted to be tricresyl / n-butanol (1:1, v:v), tricresyl / dioxane (1:1, v:v), tricresyl / ethanol (1:1, v:v), tricresyl / benzyl alcohol (1:1, v:v), o-dichlorobenzene / n-butanol, tetrahydrofuran / H2O, and otherwise consistent with Example 1, to obtain the porphyrin-based covalent organic framework material (TAPP-VIO).

[0066] The obtained porphyrin-based covalent organic framework material (TAPP-VIO) was subjected to performance testing, and the test results are as follows:

[0067] like Figure 5 As shown in the X-ray powder diffraction pattern, TAPP-VIO obtained with mesitylene / dioxane, mesitylene / ethanol, mesitylene / benzyl alcohol, o-dichlorobenzene / n-butanol, or tetrahydrofuran / H2O as the reaction system has a low characteristic diffraction peak at 3.5°, indicating that TAPP-VIO can be effectively prepared in various solvent systems, and the TAPP-VIO prepared with mesitylene / n-butanol as the reaction system has the highest crystallinity.

[0068] Example 3:

[0069] The ratio of the trimethylbenzene / n-butanol mixed solvent in Example 1 was adjusted to (9:1), (7:3), (1:1), (3:7), (1:9) (v:v), while other aspects remained the same as in Example 1, to obtain the porphyrin-based covalent organic framework material (TAPP-VIO).

[0070] The obtained porphyrin-based covalent organic framework material (TAPP-VIO) was subjected to performance testing, and the test results are as follows:

[0071] like Figure 6The X-ray powder diffraction patterns show that TAPP-VIO obtained with reaction systems of (9:1), (7:3), (3:7), and (1:9) exhibits low characteristic diffraction peaks at 3.5°, indicating that the tricresyl / n-butanol solvent system can effectively prepare TAPP-VIO in the volume ratio range of (9:1) to (1:9), with the tricresyl / n-butanol (1:1, v:v) reaction system yielding the TAPP-VIO with the highest crystallinity.

[0072] Example 4:

[0073] By adjusting the reaction temperature in Example 1 to 60℃, 90℃, or 150℃, while keeping other parameters consistent with Example 1, a porphyrin-based covalent organic framework material (TAPP-VIO) was obtained.

[0074] The obtained porphyrin-based covalent organic framework material (TAPP-VIO) was subjected to performance testing, and the test results are as follows:

[0075] like Figure 7 The X-ray powder diffraction pattern shows that the characteristic diffraction peak of TAPP-VIO obtained by adjusting the reaction temperature to 60℃, 90℃ or 150℃ is lower at 3.5°, indicating that TAPP-VIO can be effectively prepared at reaction temperatures of 60-150℃, and the crystallinity of TAPP-VIO obtained at a reaction temperature of 120℃ is the highest.

[0076] Example 5:

[0077] The reaction time in Example 1 was adjusted to 2, 3, 4, 5, and 7 days, while other aspects remained the same as in Example 1, resulting in a porphyrin-based covalent organic framework material (TAPP-VIO).

[0078] The obtained porphyrin-based covalent organic framework material (TAPP-VIO) was subjected to performance testing, and the test results are as follows:

[0079] like Figure 8 The X-ray powder diffraction patterns show that the characteristic diffraction peaks of TAPP-VIO obtained by adjusting the reaction time to 2, 3, 4, 5, and 7 days are relatively low at 3.5°, indicating that TAPP-VIO can be effectively prepared at reaction times of 2-7 days, and the TAPP-VIO obtained at a reaction time of 6 days has the highest crystallinity.

[0080] Example 6:

[0081] The photothermal and photodynamic properties of TAPP-VIO prepared in Example 1 were investigated.

[0082] Photothermal performance: TAPP-VIO was tested at different concentrations (125 μg / mL). -1 250μg mL-1 500 μg mL -1 and 750 μg / mL -1 The solution was dispersed in PBS, and then 1 mL of the solution was added to a 48-well cell culture plate. The plate was then irradiated with a white LED lamp for 20 minutes (using 1 mL of PBS as a control). A FLIR-50 thermal imager was used to record temperature changes during the irradiation process.

[0083] like Figure 9 As shown, at 100mW·cm -2 After 20 minutes of white light irradiation, TAPP-VIO exhibited good photothermal performance, showing a concentration- and time-dependent effect, with a concentration of 125 μg / mL. -1 250μg mL -1 500 μg mL -1 and 750 μg mL -1 The temperature of TAPP-VIO solution systems of different concentrations increased by 10℃, 13℃, 17℃ and 19℃, respectively, while the temperature of PBS, as the control group, increased by only 3℃.

[0084] Photodynamic properties: Using 9,10-anthracitediacyl-bis(methylene)dimaleic acid (ABDA) as the precursor... 1 O2 indicator, add 100 μL LABDA solution (5 × 10⁻⁶) -3 mol L -1 Add 9.9 mL of TAPP-VIO solution of different concentrations to 50 mL centrifuge tubes (the final concentration of TAPP-VIO was 0 μg / mL). -1 125μg mL -1 250μg mL -1 500 μg mL -1 and 750 μg / mL -1 Then use a white LED light (50, 100, or 150mW cm). -2 The solution was irradiated for different durations (0 min, 2.5 min, 5 min, 7.5 min, 10 min, 12.5 min, 15 min, 17.5 min, and 20 min). Afterward, 1 mL of the irradiated solution was removed and centrifuged at the above time points, and the corresponding UV-vis absorption spectra of the suspension were measured using a UV-vis-NIR spectrophotometer.

[0085] Figure 10 a shows 100mW cm -2 The time-dependent absorption spectra of ABDA at 377 nm for different concentrations of TAPP-VIO under irradiation. Figure 10 b shows the effect of 500 μg mL at different irradiation powers. -1The time-dependent absorption spectra of TAPP-VIO and ABDA at 377 nm (A / A0) (A0 and A are the absorbance values ​​of ABDA before irradiation and after a certain time of irradiation, respectively).

[0086] like Figure 10 a and Figure 10 As shown in b, TAPP-VIO exhibits good photodynamic performance, which is time- and concentration-dependent, with a 100mW cm⁻¹ value. -2 After 20 minutes of exposure to white LED light, 500 μg mL -1 The absorbance of ABDA treated with TAPP-VIO decreased to approximately 20% of its initial value.

[0087] Example 7:

[0088] Using TAPP-VIO prepared in Example 1 as a photosensitizer, its photothermal / photodynamic / chemical bactericidal ability was investigated.

[0089] Plate coating experiment: Bacterial suspensions were mixed with TAPP-VIO solution and then incubated on a shaking table at 37°C and 200 rpm for 30 minutes. Subsequently, the bacterial suspensions containing TAPP-VIO were exposed to a white LED light for 20 minutes. Bacterial suspensions were extracted from each group, serially diluted, and then evenly spread onto solid culture media. After incubation at 37°C for 12 to 24 hours, the colony count was performed.

[0090] like Figure 11 As shown, TAPP-VIO exhibits a significant bactericidal effect under white light irradiation, and the bactericidal effect is positively correlated with irradiation time. At 100mW cm⁻¹ -2 After 20 minutes of white light irradiation, TAPP-VIO achieved bactericidal rates of 98.84% and 99.19% against Escherichia coli and Staphylococcus aureus, respectively.

[0091] Example 8:

[0092] The wound healing ability of TAPP-VIO prepared in Example 1 was investigated.

[0093] The wound healing performance of TAPP-VIO was explored using a mouse model based on Staphylococcus aureus infection. The wound healing properties were assessed by administering 500 μg mL of TAPP-VIO. -1 TAPP-VIO was applied to the mouse wound and treated with 100mW cm -2 In vivo synergistic therapy experiments were conducted by irradiating mice with white LED lights for 20 minutes. Mice infected with Staphylococcus aureus were randomly divided into four groups (control group, TAPP-VIO, control + irradiation, and TAPP-VIO + irradiation), and the wound area was recorded during the treatment.

[0094] like Figure 12 As shown, the wounds in the TAPP-VIO and TAPP-VIO+ irradiation groups were smaller in terms of wound area. The wound morphology also demonstrates the wound healing effect of TAPP-VIO; on the 10th day after irradiation, the wounds in the TAPP-VIO+ irradiation group mice not only healed completely but also grew pink new skin, showing a significant synergistic therapeutic effect.

[0095] It should be understood that the above detailed embodiments of this disclosure are merely illustrative or explanatory of the principles of this disclosure, and are not intended to limit the invention. Therefore, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of this invention.

Claims

1. The application of the cationic porphyrin-based covalent organic framework TAPP-VIO in the preparation of a liquid bactericidal agent for promoting wound healing caused by Staphylococcus aureus infection, characterized in that... The bactericidal agent is a bacterial-targeting photothermal / photodynamic / chemical synergistic bactericidal agent, wherein the concentration of TAPP-VIO in the bactericidal agent is 500 μg / mL, and the bactericidal agent is used to apply to wounds infected with Staphylococcus aureus and at 100 mW·cm -2 The TAPP-VIO promotes wound healing caused by Staphylococcus aureus infection under white LED light irradiation for 20 minutes, and the TAPP-VIO has the structure shown in Formula I. Formula I.

2. The application according to claim 1, characterized in that, The TAPP-VIO was prepared by reacting 5,10,15,20-tetra-(4-aminophenyl)porphyrin monomer with 4,4'-dibenzaldehyde-bipyridine monomer.

3. The application according to claim 1 or 2, characterized in that, The TAPP-VIO is an AA stacking mode.

4. The application according to claim 1, characterized in that, The liquid bactericidal preparation is a drop or spray formulation.

5. The application according to claim 4, characterized in that, The liquid bactericidal preparation is a drop, and is used to apply to wounds infected with Staphylococcus aureus.

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