Porphyrin nanoparticle, porphyrin hydrogel, preparation method and application

Abstract

The invention relates to porphyrin nanoparticles, porphyrin hydrogel, a preparation method and application. The preparation method of the porphyrin nanoparticles comprises the following steps: adding 4-[(trimethylsilyl) ethynyl] benzaldehyde and pyrrole into a solvent, dissolving the obtained product in the solvent, adding a metal salt, dissolving the obtained product in the solvent, adding methanol and potassium carbonate, dissolving the obtained product in the solvent, adding CTAB, adding the obtained product into a reducing agent, adding a metal compound, stirring, filtering, washing, and drying to obtain the porphyrin nanoparticles. Obtaining a first intermediate product; hA is dissolved in water, EDCI, NHS and L-Cys are added, the obtained HA-Cys is added into the first intermediate product, and the porphyrin nanoparticles wrapped by hyaluronic acid are obtained. The invention also provides porphyrin hydrogel and a preparation method thereof, and application of the porphyrin hydrogel and the porphyrin nanoparticles. The invention solves the problem that the existing photosensitizer is insufficient in light, heat and chemical stability, and also solves the problem that the singlet oxygen generated by the existing photosensitizer is easily consumed by GSH, resulting in reduced treatment effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to porphyrin nanoparticles, porphyrin hydrogels, preparation methods and applications. Background Art

[0002] Bacterial infections have long been a major threat to human health. With the widespread use and even overuse of antibiotics, bacteria have gradually developed antimicrobial resistance (AMR) to multiple antibiotics. In particular, the emergence of multidrug-resistant bacteria (MDR) has significantly reduced the effectiveness of many traditional antibiotics, or even completely rendered them ineffective. Therefore, developing novel treatments that effectively address bacterial resistance has become a pressing need in current medical research.

[0003] Photodynamic therapy (PDT), as an emerging antibacterial strategy, has attracted widespread attention in recent years due to its unique bactericidal mechanism and its low risk of drug resistance. The core principle of PDT therapy is to use photosensitizers to generate highly oxidative reactive oxygen species (ROS) through photochemical reactions under light of a specific wavelength, especially singlet oxygen ( 1 PDT is a powerful antimicrobial agent that releases oxygen (O2) into the air, causing toxic effects on target cells (such as bacteria or cancer cells), killing the bacteria and disrupting the biofilm, leading to their death or loss of function. Unlike traditional antibiotics, PDT's bactericidal effect is achieved through physical and chemical mechanisms, making it difficult for bacteria to evade its killing effects through genetic mutations or metabolic changes, and therefore less likely to develop drug resistance.

[0004] In PDT therapy, the selection and performance of photosensitizers are crucial. An ideal photosensitizer should possess the following characteristics: (1) high selectivity for target cells (such as bacteria); (2) efficient production of singlet oxygen under specific wavelength illumination; (3) good biocompatibility and low toxicity; and (4) ease of preparation and modification to optimize its performance. However, existing photosensitizers still face some challenges in practical applications, such as insufficient photostability and the singlet oxygen produced being easily consumed by GSH, which reduces the therapeutic effect and limits their bactericidal and antibacterial effects and clinical applications. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide porphyrin nanoparticles, porphyrin hydrogels, preparation methods and applications to address the problem of insufficient light, heat and chemical stability of existing photosensitizers. It can also address the problem that singlet oxygen generated by existing photosensitizers is easily consumed by GSH, resulting in a decrease in therapeutic effect.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing porphyrin nanoparticles comprises the following steps: S1. Add 4-[(trimethylsilyl)ethynyl]benzaldehyde and pyrrole to a first organic solvent to obtain TMS-TEPP (5,10,15,20-tetrakis(trimethylsilylethynyl)phenylporphyrin) as a purple solid; S2, dissolve TMS-TEPP purple solid in the second organic solvent, add metal salt, collect the organic phase, and rotary evaporate to obtain solid powder TMS-M x TEPP(5,10,15,20-tetrakis(trimethylsilylethynyl)phenylmetalloporphyrin,M x are different metal elements, such as Fe, Mn, Zn, Ir, etc.); S3, solid powder TMS-M x TEPP is dissolved in a third organic solvent, and methanol and potassium carbonate are added to obtain a solid powder M x TEPP (5,10,15,20-tetrakis(4-ethynylphenyl)metalloporphyrin); S4, solid powder M x TEPP is dissolved in a third organic solvent, and cetyltrimethylammonium bromide (CTAB) solution is added to obtain self-assembled metalloporphyrin nanoparticles SAM. x TEPP; S5, metal porphyrin nanoparticles SAM x TEPP is added to a reducing agent solution, and then a silver compound solution or a gold compound solution is added to obtain a first intermediate product; S6. Dissolve hyaluronic acid (HA) in water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and L-cysteine ​​(L-Cys) to obtain HA-Cys, and then add HA-Cys to the first intermediate product solution to obtain hyaluronic acid-coated porphyrin nanoparticles.

[0007] According to the above technical means, first, a porphyrin derivative 4-[(trimethylsilyl)ethynyl]benzaldehyde with an alkynyl functional group is used as a raw material and added to a first organic solvent with pyrrole. The porphyrin ring structure is formed through the condensation reaction of the aldehyde group and the pyrrole. Trimethylsilyl (TMS) is used as a protecting group to effectively prevent the acetylene group from having side reactions in subsequent reactions. Secondly, the affinity of the alkynyl group and gold / silver salts is utilized to achieve the in situ growth of gold / silver nanoparticles on the porphyrin derivative, which effectively enhances the light, heat and chemical stability. At the same time, the complexation of metal ions on the porphyrin ring gives the porphyrin derivative special photophysical and photochemical properties (such as enhanced singlet oxygen production ability), so that it has both PTT / PDT and long-lasting antibacterial effects of silver ions. Thirdly, TMS-MxTEPP removes the TMS protecting group under the action of methanol and potassium carbonate to generate MxTEPP. After deprotection, the acetylene group is exposed, which is suitable for subsequent reactions. The energy-chemical reaction provides active sites, effectively improving the reactivity of porphyrin derivatives; fourthly, CTAB is used as a surfactant to guide the self-assembly of MxTEPP molecules into nanoparticles through hydrophobic interaction, so that the self-assembled nanoparticles have a larger specific surface area and higher singlet oxygen production efficiency. At the same time, the self-assembled structure enhances the stability and bacterial targeting of the photosensitizer; fifthly, silver ions (Ag⁺) or gold ions (Au³⁺) are reduced to nanosilver or nanogold by a reducing agent and deposited on the surface of porphyrin nanoparticles. With the help of the photothermal conversion ability of precious metal nanoparticles, the porphyrin nanoparticles are endowed with photothermal therapy (PTT) function. At the same time, precious metals can scavenge glutathione (GSH) and reduce the consumption of singlet oxygen, thereby effectively improving the photodynamic therapy (PDT) effect; finally, the carboxyl group of HA is activated by EDCI and NHS, and reacts with the amino group of L-Cys to generate HA-Cys. HA-Cys binds to the surface of noble metal nanoparticles via thiol groups, forming porphyrin nanoparticles with enhanced biosafety. Experimental studies have shown that these porphyrin nanoparticles exhibit excellent photo-, thermal-, and chemical-stability. Furthermore, they reduce GSH consumption of reactive oxygen species, enhancing the efficacy of photodynamic / chemodynamic therapy. They also rapidly kill bacteria through photothermal (PTT) / photodynamic (PDT) therapy and exhibit long-lasting antibacterial effects, without significant inhibitory effects on normal tissue cells. This effectively addresses the limited photo-, thermal-, and chemical-stability issues of existing photosensitizers, as well as the issue of singlet oxygen generated by existing photosensitizers being easily consumed by GSH, resulting in reduced therapeutic efficacy. The preparation method is simple, easy to operate, and readily adaptable for industrial implementation.

[0008] Preferably, the metal salt is selected from at least one of ferric chloride, ferrous chloride, manganese acetate and zinc acetate.

[0009] Preferably, the metal salt is selected from manganese acetate.

[0010] Preferably, the reducing agent is selected from at least one of tannic acid, sodium borohydride, trisodium citrate, ascorbic acid and hydroxylamine hydrochloride.

[0011] Preferably, the silver compound solution is selected from silver nitrate solution.

[0012] Preferably, the gold compound solution is selected from chloroauric acid solution.

[0013] Preferably, the molar ratio of the TMS-TEPP purple solid to the metal salt is 1:10-45.

[0014] Preferably, the hexadecyltrimethylammonium bromide (CTAB) and the solid powder M x The molar ratio of TEPP is 2.5~5.5:1.

[0015] Preferably, the molar ratio of the reducing agent to the silver salt is 1:3-8.

[0016] Preferably, the reducing agent solution is alkaline.

[0017] Preferably, the step S1 comprises: adding 4-[(trimethylsilyl)ethynyl]benzaldehyde and pyrrole to propionic acid, reacting for 2 h to 5 h, to obtain TMS-TEPP (tetramethylsilane-tetraethyl pyrophosphate) as a purple solid.

[0018] Preferably, the step S2 comprises: dissolving TMS-TEPP purple solid in N', N-dimethylformamide (DMF), adding metal salt, reacting for 8 h to 15 h, collecting the organic phase and solid dilution, and rotary evaporating to obtain solid powder TMS-M x TEPP.

[0019] Preferably, the step S3 comprises: solid powder TMS-M x TEPP was dissolved in tetrahydrofuran (THF), and methanol and potassium carbonate were added to react for 3-5 h. The solution was concentrated and washed with deionized water to obtain a solid powder M. x TEPP.

[0020] Preferably, the step S4 comprises: x TEPP was dissolved in tetrahydrofuran (THF), and cetyltrimethylammonium bromide (CTAB) solution was quickly added. The reaction was carried out at a temperature of 25°C to 40°C for 24 h to 72 h to obtain self-assembled metalloporphyrin nanoparticles (SAM). x TEPP.

[0021] Preferably, the step S5 comprises: dissolving a reducing agent in water and adjusting the pH value to be alkaline to obtain a reducing agent solution, and adding metalloporphyrin nanoparticles SAM to the reducing agent solution. x TEPP, ultrasonic dispersion for 15 min~30 min, adding a silver compound solution or a gold compound solution at a temperature of 25℃~60℃, then reacting at a temperature of 45℃~65℃ for 30 min, and then switching to room temperature reaction for 22 h~26 h to obtain a first intermediate product.

[0022] Preferably, in S5, the reaction is switched to room temperature for 24 h to obtain the first intermediate product.

[0023] Preferably, S6 comprises: dissolving hyaluronic acid (HA) in water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS), reacting at room temperature for 1 h to 3 h, then adding L-cysteine ​​(L-Cys), stirring and reacting at room temperature for 22 h to 24 h, dialyzing for 70 h to 74 h, and drying to obtain HA-Cys, and then adding HA-Cys to the first intermediate product solution, reacting at room temperature for 20 h, dialyzing for 70 h to 74 h, and drying to obtain porphyrin nanoparticles.

[0024] Preferably, S6 comprises: dissolving hyaluronic acid (HA) in water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS), reacting at room temperature for 2 h, then adding L-cysteine ​​(L-Cys), stirring and reacting at room temperature for 24 h, then dialyzing the product in an aqueous solution for 72 h, and freeze-drying to obtain HA-Cys; centrifuging the first intermediate product, washing it three times with PBS buffer, and redispersing the resulting precipitate in a PBS solution to obtain a first intermediate product solution; ultrasonically dispersing the first intermediate product solution for 15 min to 30 min, then adding HA-Cys, reacting at room temperature for 20 h, dialyzing the reaction solution for 72 h, and freeze-drying to obtain porphyrin nanoparticles.

[0025] Preferably, the pH value of the reducing agent solution is 9-11.

[0026] Preferably, the pH value of the reducing agent solution is 11.

[0027] Preferably, the solvent in the cetyltrimethylammonium bromide (CTAB) solution is a mixed solvent of water and tetrahydrofuran (THF), and the volume ratio of water to tetrahydrofuran (THF) in the mixed solvent is 15-5:1.

[0028] Preferably, the first intermediate product solution is obtained by dispersing the first intermediate product in a PBS solution.

[0029] The present invention also provides porphyrin nanoparticles prepared by the preparation method of the present invention.

[0030] The present invention also provides an application of the porphyrin nanoparticles prepared by the preparation method of the present invention as a photosensitizer.

[0031] The present invention also provides a porphyrin hydrogel, comprising porphyrin nanoparticles prepared by the preparation method of the present invention and hydrogel, wherein the porphyrin nanoparticles are loaded in the hydrogel.

[0032] Preferably, the method for preparing the porphyrin hydrogel comprises the following steps: Step 1), dissolving chitosan in acetic acid solution to obtain a first mixed solution; dissolving lysine in water, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS), and adjusting the pH to acidic to obtain a second mixed solution; mixing the first mixed solution and the second mixed solution to obtain lysine-modified chitosan; Step 2), catechol is dissolved in water and the pH is adjusted to acidic, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) are added, and then lysine-modified chitosan solution is added to obtain a second intermediate product; Step 3) adding the second intermediate product to the porphyrin nanoparticle solution, and then adding polyvinyl alcohol (PVA) to obtain a porphyrin hydrogel.

[0033] The chitosan-based viscous hydrogel provided in this application has good hydrophilicity through catechol-modified chitosan. When mixed with polyvinyl alcohol hydrogel, the viscous hydrogel prepared by the freeze-thaw method has both the toughness and viscosity of the hydrogel. It is loaded with the porphyrin antibacterial drugs (porphyrin nanoparticles) provided in this application to prepare various hydrogel dressings, microneedles, patches, etc. for bacterial infections of wounds.

[0034] Preferably, the mass percentage of chitosan in the first mixed solution is 1% to 5%.

[0035] Preferably, the mass ratio of the chitosan to the lysine is 1:0.5-2.

[0036] Preferably, the pH of the second mixed solution is 5-6.

[0037] Preferably, the catechol is dissolved in water and the pH is adjusted to 5-6.

[0038] Preferably, the catechol is a carboxyl functional group-containing catechol, and the carboxyl functional group-containing catechol is selected from at least one of 3,4-dihydroxyphenylacetic acid, tannic acid, caffeic acid and dopamine.

[0039] Preferably, the mass percentage of the second intermediate product in the porphyrin hydrogel is 2% to 5%.

[0040] Preferably, the mass percentage of polyvinyl alcohol in the porphyrin hydrogel is 2% to 5%.

[0041] Preferably, the step 1) comprises: dissolving chitosan in a 1% acetic acid aqueous solution to obtain a first mixed solution; dissolving lysine in deionized water, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS), adjusting the pH value to 5-6 with 1M dilute hydrochloric acid, and reacting for 2 hours to obtain a second mixed solution; mixing the first mixed solution and the second mixed solution for reaction for 24 hours. After the reaction is completed, dialyzing the product in deionized water for 72 hours, and freeze-drying to obtain lysine-modified chitosan.

[0042] Preferably, the step 2) comprises: dissolving catechol containing a carboxyl functional group in an aqueous solution, adjusting the pH value to 5-6, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) and reacting for 2 h to obtain a third intermediate product; dissolving lysine-modified chitosan in a 1% acetic acid aqueous solution, and then mixing with the third intermediate product for reaction for 24 h. After the reaction is completed, dialyzing the product for 72 h and freeze-drying to obtain a second intermediate product.

[0043] Preferably, the step 3) comprises: dissolving the porphyrin nanoparticles in a PBS solution, then adding the second intermediate product, and then mixing with a polyvinyl alcohol (PVA) aqueous solution in equal proportions to obtain a mixture, and repeatedly freezing and thawing the mixture three times at -20°C, with each freezing time being 6 h to 24 h, to form a gel and enhance the gel toughness, and finally obtain a porphyrin hydrogel.

[0044] Preferably, the step 3) comprises: dissolving the second intermediate product in water, then mixing it with a polyvinyl alcohol (PVA) aqueous solution in equal proportions to obtain a mixture, and repeatedly freezing and thawing the mixture three times at -20°C, with each freezing time being 6 h to 24 h, to obtain a common hydrogel.

[0045] The present invention also provides an application of the porphyrin hydrogel as a hydrogel microneedle, a dressing or a patch.

[0046] Beneficial effects of the present invention: The present invention's method for preparing porphyrin nanoparticles uses porphyrin molecules with alkynyl functional groups as raw materials. The affinity between the alkynyl groups and gold / silver salts is exploited to achieve in-situ growth of gold / silver nanoparticles on the porphyrin rings, resulting in the combined effects of PTT / PDT and the long-lasting antibacterial effects of silver ions. Simultaneously, the method reduces GSH consumption of reactive oxygen species, enhancing the efficacy of photodynamic / chemodynamic therapy. This method effectively addresses the issues of insufficient light, heat, and chemical stability of existing photosensitizers, as well as the problem of singlet oxygen generated by existing photosensitizers being easily consumed by GSH, resulting in reduced therapeutic efficacy.

[0047] The preparation method of porphyrin hydrogel of the present invention, chitosan modified by catechol has good hydrophilicity, is mixed with polyvinyl alcohol hydrogel, and the viscous hydrogel prepared by freeze-thaw method has the toughness and viscosity of hydrogel concurrently.By loading the porphyrin antimicrobial drug provided by the application, porphyrin hydrogel is made to have the dual efficacy of porphyrin nanoparticles and hydrogel concurrently, is applied to various hydrogel dressings, microneedles, patches, etc. for wound bacterial infection, has good antibacterial effect and biocompatibility, also has excellent stability and photodynamic / chemodynamic therapy effect, can effectively kill bacteria, promote wound healing, significantly improve the treatment efficiency of wound, for the treatment of wound bacterial infection provides new solution.And the preparation method of this porphyrin hydrogel is simple, raw material is easy to obtain, cost is relatively low, is easy to large-scale production and application, in the field of biomedical material technology, has application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a flow chart of the method for preparing porphyrin nanoparticles of the present invention; Figure 2 TEM spectrum of the metalloporphyrin nanoparticles SAMnTEPP prepared in Example 1; Figure 3 TEM spectrum of porphyrin nanoparticles SAMnTEPP@TA / Ag prepared in Example 1; Figure 4 This is the EDS spectrum of the porphyrin nanoparticles SAMnTEPP@TA / Ag prepared in Example 1; Figure 5 The UV-visible spectra of porphyrin molecules coordinated with different metals; Figure 6 This is a graph showing the glutathione (GSH) consumption of the porphyrin nanoparticles SAMnTEPP@TA / Ag prepared in Example 1; Figure 7 Figure 2 shows the antibacterial effect test results of different self-assembled porphyrin nanoparticles on Staphylococcus aureus; Figure 8 Figure 2 shows the antibacterial effect test results of different self-assembled porphyrin nanoparticles on Pseudomonas aeruginosa; Figure 9 The figure shows the test results of the antibacterial effect of different self-assembled porphyrin nanoparticles on Escherichia coli; Figure 10 For 808 nm laser (laser power 1.0 w / cm 2 ) Temperature-time curves of SAMnTEPP@TA / Ag-HA with different concentrations under irradiation; Figure 11 This is the stability curve of SAMnTEPP@TA / Ag-HA (50 μg / mL); Figure 12 For 808 nm laser (laser power 1.0 w / cm 2 ) Photothermal imaging of SAMnTEPP@TA / Ag-HA with different concentrations under irradiation; Figure 13 This is the EPR test result diagram of SAMnTEPP@TA / Ag-HA; Figure 14 This is the bactericidal effect of SAMnTEPP@TA / Ag-HA at a concentration of 50 μg / mL on Staphylococcus aureus; Figure 15 This is the bactericidal effect of SAMnTEPP@TA / Ag-HA at a concentration of 25 μg / mL on Staphylococcus aureus; Figure 16 This figure shows the effect of SAMnTEPP@TA / Ag nanoparticles on the survival rate of L929 cells before and after encapsulation of hyaluronic acid (HA) molecules; Figure 17 The fluorescence results of SAMnTEPP@TA / Ag-HA nanoparticles staining live and dead cells at different concentrations; Figure 18 The figure shows the adhesion test results of chitosan adhesive hydrogel to materials such as skin, glass, copper sheets and plastics after adding SAMnTEPP@TA / Ag-HA nanoparticles at a concentration of 50 µg / mL. DETAILED DESCRIPTION

[0049] The following will describe the embodiments of the present invention with reference to preferred embodiments. Those skilled in the art will readily appreciate the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are intended only to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0050] Example 1 like Figure 1 As shown, a method for preparing porphyrin nanoparticles comprises the following steps: S1. Add 4-[(trimethylsilyl)ethynyl]benzaldehyde (2 g) and pyrrole (0.8 mL) to propionic acid (30 mL) and reflux at 150°C for 2 h to obtain TMS-TEPP (5,10,15,20-tetrakis(trimethylsilylethynyl)phenylporphyrin) as a purple solid. S2. Dissolve the purple solid TMS-TEPP (200 mg) obtained in S1 in N', N-dimethylformamide (DMF, 50 mL), add manganese acetate, and reflux at 165°C for 10 h. Collect the organic phase and rotary evaporate to obtain solid powder TMS-MnTEPP, wherein the molar ratio of TMS-TEPP purple solid to manganese acetate is 1:30; S3. Dissolve the solid powder TMS-MnTEPP (106 mg) obtained in S2 in tetrahydrofuran (THF, 15 mL), add methanol (5 mL) and potassium carbonate (55 mg), and react at room temperature for 4 h. Concentrate the solution and wash with deionized water to obtain solid powder MnTEPP. S4. Dissolve the solid powder MnTEPP (20 mg) obtained in S3 in tetrahydrofuran (THF, 5 mL), quickly add it to a mixed solution of water / THF (volume ratio 9:1) containing cetyltrimethylammonium bromide (CTAB), control the molar ratio of CTAB to MnTEPP to be 4:1, and react at 40°C for 24 h to obtain self-assembled manganese porphyrin nanoparticles SAMnTEPP; S5. Dissolve tannic acid in water to prepare a 0.05 mM tannic acid aqueous solution, and adjust the pH to 10 with a 1 M sodium hydroxide aqueous solution to obtain a tannic acid alkaline aqueous solution. Add metalloporphyrin nanoparticles SAMnTEPP (5 mg) to the tannic acid alkaline aqueous solution, and ultrasonically disperse for 20 min. Add silver nitrate solution at 60°C, controlling the molar ratio of tannic acid to silver nitrate to be 1:6, and react at 60°C for 30 min, then switch to room temperature for 24 h to obtain a first intermediate product. S6. Dissolve hyaluronic acid (HA, 2.27 g) in water (100 mL), add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.77 g) and N-hydroxysuccinimide (NHS, 0.46 g), and react at room temperature for 2 h. Then add L-cysteine ​​(L-Cys, 0.2 g) and stir at room temperature for 24 h. Then dialyze the product in aqueous solution for 72 h and freeze-dry to obtain HA-Cys. Centrifuge the first intermediate product, wash it three times with PBS buffer, and redisperse the resulting precipitate in PBS solution to obtain the first intermediate product solution. Ultrasonic disperse the first intermediate product solution for 20 min, then add HA-Cys, react at room temperature for 20 h, dialyze the reaction solution for 72 h, and freeze-dry to obtain hyaluronic acid-coated manganese porphyrin nanoparticles SAMnTEPP@TA / Ag-HA.

[0051] Example 2 like Figure 1 As shown, a method for preparing porphyrin nanoparticles comprises the following steps: S1. Add 4-[(trimethylsilyl)ethynyl]benzaldehyde (2.0 g) and pyrrole (0.8 mL) to propionic acid (30 mL) and reflux at 150°C for 2 h to obtain TMS-TEPP (5,10,15,20-tetrakis(trimethylsilylethynyl)phenylporphyrin) as a purple solid. S2. The purple solid TMS-TEPP obtained in S1 (100 mg) was dissolved in N', N-dimethylformamide (DMF, 50 mL), and ferric chloride (FeCl3, 120 mg) was added. The mixture was reacted at 165°C for 1 h, and then NaCl (0.05 g) was added. The reaction was continued under reflux for 5 h. Most of the DMF was removed by distillation under reduced pressure. After cooling, a large amount of cold water was added to crystallize the iron porphyrin. Concentrated hydrochloric acid (10 mL) was then added to acidify the mixture. The mixture was filtered and dried, and recrystallized from dichloromethane / anhydrous ethanol to obtain a brown solid powder TMS-FeTEPP. S3. Dissolve the solid powder TMS-FeTEPP (106 mg) obtained in S2 in tetrahydrofuran (THF, 15 mL), add methanol (5 mL) and potassium carbonate (55 mg), and react at room temperature for 4 h. Concentrate the solution and wash with deionized water to obtain solid powder FeTEPP. S4. Dissolve the solid powder FeTEPP obtained in S3 in tetrahydrofuran (THF, 5 mL), quickly add it to a mixed solution of water / THF (volume ratio 9:1) containing cetyltrimethylammonium bromide (CTAB), control the molar ratio of CTAB to FeTEPP to be 4:1, and react at 40°C for 24 h to obtain self-assembled iron porphyrin nanoparticles SAFeTEPP; S5. Dissolve tannic acid in water to prepare a 0.05 mM tannic acid aqueous solution, and adjust the pH to 10 with a 1 M sodium hydroxide aqueous solution to obtain a tannic acid alkaline aqueous solution. Add self-assembled iron porphyrin nanoparticles SAFeTEPP to the tannic acid alkaline aqueous solution, and ultrasonically disperse for 20 minutes. Add silver nitrate solution at 60°C, controlling the molar ratio of tannic acid to silver nitrate to be 1:6, and react at 60°C for 30 minutes, then switch to room temperature for 24 hours to obtain a first intermediate product. S6. Dissolve hyaluronic acid (HA, 2.27 g) in water (100 mL), add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.77 g) and N-hydroxysuccinimide (NHS, 0.46 g), and react at room temperature for 2 h. Then add L-cysteine ​​(L-Cys, 0.2 g) and stir at room temperature for 24 h. Then dialyze the product in aqueous solution for 72 h and freeze-dry to obtain HA-Cys. Centrifuge the first intermediate product, wash it three times with PBS buffer, and redisperse the resulting precipitate in PBS solution to obtain the first intermediate product solution. Ultrasonic disperse the first intermediate product solution for 20 min, then add HA-Cys, react at room temperature for 20 h, dialyze the reaction solution for 72 h, and freeze-dry to obtain hyaluronic acid-coated iron porphyrin nanoparticles SAFeTEPP@TA / Ag-HA.

[0052] Example 3 like Figure 1 As shown, a method for preparing porphyrin nanoparticles comprises the following steps: S1. Add 4-[(trimethylsilyl)ethynyl]benzaldehyde (2.0 g) and pyrrole (0.8 mL) to propionic acid (30 mL) and reflux at 150°C for 2 h to obtain TMS-TEPP (5,10,15,20-tetrakis(trimethylsilylethynyl)phenylporphyrin) as a purple solid. S2. Dissolve the solid powder TMS-TEPP (100 mg) obtained in S1 in tetrahydrofuran (THF, 15 mL), add methanol (5 mL) and potassium carbonate (55 mg), and react at room temperature for 4 h. Concentrate the solution and wash with deionized water to obtain solid powder TEPP. S3. The solid powder TEPP obtained in S2 was dissolved in tetrahydrofuran (THF, 5 mL), and the mixture was quickly added to a water / THF mixed solution (volume ratio 9:1) containing cetyltrimethylammonium bromide (CTAB), with the molar ratio of CTAB to TEPP being controlled at 4:1. The mixture was reacted at 40°C for 24 h to obtain self-assembled porphyrin nanoparticles SATEPP. S4. Dissolve tannic acid in water to prepare a 0.05 mM tannic acid aqueous solution, and adjust the pH to 10 with a 1 M sodium hydroxide aqueous solution to obtain a tannic acid alkaline aqueous solution. Add self-assembled porphyrin nanoparticles SATEPP to the tannic acid alkaline aqueous solution, and ultrasonically disperse for 20 minutes. Add silver nitrate solution at 60°C, controlling the molar ratio of tannic acid to silver nitrate to be 1:6, and react at 60°C for 30 minutes, and then switch to room temperature for 24 hours to obtain a first intermediate product. S5. Dissolve hyaluronic acid (HA, 2.27 g) in water (100 mL), add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.77 g) and N-hydroxysuccinimide (NHS, 0.46 g), and react at room temperature for 2 h. Then add L-cysteine ​​(L-Cys, 0.2 g) and stir at room temperature for 24 h. Then dialyze the product in aqueous solution for 72 h and freeze-dry to obtain HA-Cys. Centrifuge the first intermediate product, wash it three times with PBS buffer, and redisperse the resulting precipitate in PBS solution to obtain the first intermediate product solution. Ultrasonic disperse the first intermediate product solution for 20 min, then add HA-Cys, react at room temperature for 20 h, dialyze the reaction solution for 72 h, and freeze-dry to obtain hyaluronic acid-coated self-assembled porphyrin nanoparticles SATEPP@TA / Ag-HA.

[0053] Example 4 like Figure 1 As shown, a method for preparing a porphyrin hydrogel comprises the following steps: Step 1), chitosan (5 g) was dissolved in 1% acetic acid aqueous solution (200 mL) to obtain a first mixed solution; lysine (4.1 g) was dissolved in deionized water (50 mL), the pH value was adjusted to 6.0 with dilute ammonia water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.7 g) and N-hydroxysuccinimide (NHS, 1.6 g) were added and reacted for 2 h to obtain a second mixed solution; the first mixed solution and the second mixed solution were mixed and reacted for 24 h. After the reaction was completed, the product was dialyzed in deionized water for 72 h and freeze-dried to obtain lysine-modified chitosan; Step 2), 3,4-dihydroxyphenylacetic acid (2.8 g) was dissolved in an aqueous solution (100 mL), and the pH value was adjusted to 6.0 with dilute ammonia water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.2 g) and N-hydroxysuccinimide (NHS, 1.9 g) were added and reacted for 2 h to obtain a third intermediate product; lysine-modified chitosan (5.5 g) prepared in step 1) was dissolved in a 1% acetic acid aqueous solution (100 mL), and then mixed with the third intermediate product for reaction for 24 h. After the reaction was completed, the product was dialyzed for 72 h and freeze-dried to obtain a second intermediate product; In step 3, the porphyrin nanoparticles SAMnTEPP@TA / Ag-HA prepared in Example 1 were dissolved in a PBS solution, and then the second intermediate product was added. The mixture was then mixed with a polyvinyl alcohol (PVA, 10%) aqueous solution in equal proportions to obtain a mixture. The mixture was repeatedly frozen and thawed three times at -20°C, with each freezing time being 12 h, to obtain a porphyrin hydrogel.

[0054] Example 5 A method for preparing a common hydrogel comprises the following steps: Step 1), chitosan (5 g) was dissolved in 1% acetic acid aqueous solution (200 mL) to obtain a first mixed solution; lysine (4.1 g) was dissolved in deionized water (50 mL), the pH value was adjusted to 6.0 with dilute ammonia water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.7 g) and N-hydroxysuccinimide (NHS, 1.6 g) were added and reacted for 2 h to obtain a second mixed solution; the first mixed solution and the second mixed solution were mixed and reacted for 24 h. After the reaction was completed, the product was dialyzed in deionized water for 72 h and freeze-dried to obtain lysine-modified chitosan; Step 2), 3,4-dihydroxyphenylacetic acid (2.8 g) was dissolved in an aqueous solution (100 mL), and the pH value was adjusted to 6.0 with dilute ammonia water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.2 g) and N-hydroxysuccinimide (NHS, 1.9 g) were added and reacted for 2 h to obtain a third intermediate product; lysine-modified chitosan (5.5 g) prepared in step 1) was dissolved in a 1% acetic acid aqueous solution (100 mL), and then mixed with the third intermediate product for reaction for 24 h. After the reaction was completed, the product was dialyzed for 72 h and freeze-dried to obtain a second intermediate product; In step 3, the second intermediate product was dissolved in water and then mixed with a polyvinyl alcohol (PVA, 10%) aqueous solution in equal proportions to obtain a mixture, which was repeatedly frozen and thawed three times at -20°C, with each freezing time being 12 h, to obtain a common hydrogel.

[0055] Detection and Analysis 1) Transmission electron microscopy (TEM) and EDS analysis The metal porphyrin nanoparticles SAMnTEPP prepared in S4 of Example 1 and the porphyrin nanoparticles SAMnTEPP@TA / Ag prepared in S6 were subjected to transmission electron microscopy (TEM) and EDS analysis of the porphyrin nanoparticles SAMnTEPP@TA / Ag. The results are as follows: Figures 2 to 4 shown.

[0056] from Figures 2 to 4 As shown in the figure, the size of the prepared SAMnTEPP is about 200~300 nm; the small-sized silver tannate (TA / Ag) nanoparticles in the prepared porphyrin nanoparticles SAMnTEPP@TA / Ag are tightly loaded on the surface of SAMnTEPP.

[0057] 2) Ultraviolet-visible spectroscopy (UV-vis) analysis The solid powder MnTEPP prepared in Example 1, the solid powder FeTEPP prepared in Example 2 and the solid powder TEPP prepared in Comparative Example 1 were analyzed by ultraviolet visible spectroscopy (UV-vis), and the results were as follows: Figure 5 shown.

[0058] from Figure 5 The UV-visible spectrum shows that the metal-coordinated porphyrin molecules can cause the absorption peaks of the porphyrin molecules at 410 nm and 500~600 nm to undergo a significant red shift, while the absorption peaks at 590 nm and 650 nm disappear significantly, indicating that the metal-coordinated porphyrin molecules have been successfully synthesized.

[0059] 3) GSH consumption analysis The specific operation steps are as follows: glutathione aqueous solution (GSH, 50 μL, 1 mM) is added to phosphate buffer (PBS, pH 7.4, 1900 μL) to obtain a first mixed solution; then the SAMnTEPP@TA / Ag-HA nanoparticles (500 μg / mL, 50 μL) prepared in Example 1 are added to obtain a second mixed solution; the second mixed solution is reacted at 37°C for different time intervals, and its UV-vis spectrum between 300 and 500 nm is measured. The results are shown in FIG. Figure 6 shown.

[0060] Figure 6 The figure shows the consumption of glutathione (GSH) by SAMnTEPP@TA / Ag nanoparticles. In photodynamic / chemodynamic therapy, GSH in the bacterial microenvironment can consume reactive oxygen species and other substances produced by photosensitizers, thereby reducing the therapeutic efficacy of photosensitizers. Figure 6 The analysis shows that as the reaction time progresses, SAMnTEPP@TA / Ag-HA nanoparticles themselves can slowly consume GSH in the bacterial microenvironment, thereby reducing the consumption of GSH for reactive oxygen species produced by photosensitizers, effectively enhancing the efficacy of photodynamic / chemodynamic therapy.

[0061] 4) Antibacterial efficacy test The specific operation steps are as follows: Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli strains were cultured at 37°C until the logarithmic phase, the concentration of the bacterial solution was determined by the OD value, and the bacterial solution was diluted with broth medium to a concentration of 10 5 CFU / mL; the diluted bacterial solution (10 5 CFU / mL, 120 μL) and SAMs with different metal coordination x TEPP@TA / Ag nanoparticles (1 mg / mL, 80 μL) were mixed and added to the first well of a 96-well plate. The concentration of nanoparticles in the first well was 400 μg / mL. 100 μL of the mixed bacterial solution in the first well was then transferred to the second well. At the same time, 100 μL of the diluted bacterial solution (10 5 CFU / mL), at this time, the concentration of nanoparticles in the first well was updated to 200 μg / mL; then, 100 μL of the mixed bacterial solution in the second well was transferred to the third well, and 100 μL of the diluted bacterial solution (10 5 CFU / mL), at which point the nanoparticle concentration in the second well was 100 μg / mL; this process was repeated until the final nanoparticle concentration was diluted to 6.25 μg / mL. Finally, the 96-well plate was incubated in a 37°C bacterial incubator for 12 h; the SAMs with different metal coordination groups were determined by turbidimetry. x The minimum inhibitory concentration of TEPP@TA / Ag-HA. Figures 7 to 9 shown.

[0062] Figure 7 The minimum inhibitory concentrations of three different nanomaterials (unmetal-coordinated self-assembled porphyrin / silver tannate nanoparticles SATEPP@TA / Ag, self-assembled manganese porphyrin / silver tannate nanoparticles SAMnTEPP@TA / Ag and self-assembled iron porphyrin / silver tannate nanoparticles SAFeTEPP@TA / Ag) against Pseudomonas aeruginosa were compared and analyzed. Figure 8 The minimum inhibitory concentrations of three different nanomaterials (unmetal-coordinated self-assembled porphyrin / silver tannate nanoparticles SATEPP@TA / Ag, self-assembled manganese porphyrin / silver tannate nanoparticles SAMnTEPP@TA / Ag and self-assembled iron porphyrin / silver tannate nanoparticles SAFeTEPP@TA / Ag) against Staphylococcus aureus were compared and analyzed. Figure 9The minimum inhibitory concentrations of three different nanomaterials (unmetal-coordinated self-assembled porphyrin / silver tannate nanoparticles SATEPP-TA / Ag, self-assembled manganese porphyrin / silver tannate nanoparticles SAMnTEPP-TA / Ag and self-assembled iron porphyrin / silver tannate nanoparticles SAFeTEPP@TA / Ag) against Escherichia coli were compared and analyzed.

[0063] from Figures 7 to 9 The results showed that the minimum inhibitory concentrations of the unmetal-coordinated self-assembled porphyrin / silver tannate nanoparticles SATEPP@TA / Ag against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli were all higher than 200 μg / mL. The minimum inhibitory concentrations of the self-assembled manganese porphyrin / silver tannate nanoparticles SAMnTEPP@TA / Ag against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli were 12.5 μg / mL, 50 μg / mL and 12.5 μg / mL, respectively. The minimum inhibitory concentrations of the self-assembled iron porphyrin / silver tannate nanoparticles SAFeTEPP@TA / Ag against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli were 25 μg / mL, 100 μg / mL and 100 μg / mL, respectively. It can be seen that SAMnTEPP-TA / Ag nanomaterial has the best antibacterial effect, and the non-metal-coordinated SATEPP-TA / Ag loaded with silver tannate nanoparticles has the worst antibacterial effect. This proves that among porphyrin molecules loaded with silver tannate nanoparticles of the same mass, the metal-coordinated porphyrin molecules have an important synergistic effect on their antibacterial efficacy, among which the manganese-coordinated self-assembled porphyrin nanoparticles have the best effect.

[0064] 4) Photothermal and photodynamic characterization The specific operation steps were as follows: 500 μL of SAMnTEPP@TA / Ag-HA nanoparticles with different concentrations (0 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL) were placed in a 1.5 mL centrifuge tube and the 808 nm laser (1 W / cm 2 ) for 10 min, and the solution temperature was measured every minute. The solution temperature was detected by a photothermal imager. To evaluate the stability of SAMnTEPP@TA / Ag-HA nanoparticles under near-infrared light irradiation, a specific concentration (50 µg / mL) was selected and irradiated under 808 nm laser for 10 min. The laser was then turned off until the temperature dropped to room temperature, and then the laser was turned on to measure the solution temperature change. The stability test was repeated 5 times. To evaluate the photodynamic activity of SAMnTEPP@TA / Ag nanoparticles at different synthesis stages, the singlet oxygen ( 1 O2). 2,2,6,6-tetramethylpiperidinyl oxide (TEMP) was used as the singlet oxygen1 As an O2 scavenger, 5 μL of TEMP was added to MnTEPP (methanol solution), SAMnTEPP and SAMnTEPP@TA / Ag aqueous solution, irradiated with 808 nm laser for 10 min, and then EPR measurement was performed. Figures 10 to 13 shown.

[0065] Figure 10 For 808 nm laser (1.0 W / cm 2 ) Temperature-time curves of 500 μL aqueous solutions of SAMnTEPP@TA / Ag-HA nanoparticles with different concentrations under continuous irradiation for 10 min. Figure 10 The analysis shows that in the aqueous solution without SAMnTEPP@TA / Ag-HA nanoparticles, the temperature of the aqueous solution did not increase significantly after 10 minutes of laser irradiation; after adding SAMnTEPP@TA / Ag-HA nanoparticles of different concentrations to the aqueous solution, the temperature of the water increased significantly under laser irradiation; when the concentration of SAMnTEPP@TA / Ag-HA was 50 µg / mL, the temperature of the aqueous solution could rise to 54°C within 10 minutes, which proves that SAMnTEPP@TA / Ag-HA nanoparticles have excellent photothermal conversion performance.

[0066] Figure 11 Figure 2 is the stability curve of SAMnTEPP@TA / Ag-HA (50 μg / mL). Figure 11 The analysis shows that after 5 cycles of illumination, the SAMnTEPP@TA / Ag-HA nanoparticles still maintain excellent photothermal stability.

[0067] Figure 12 Photothermal imaging of SAMnTEPP@TA / Ag-HA nanoparticles at different concentrations. Figure 13 The photodynamic activity of SAMnTEPP@TA / Ag at different synthesis stages was studied using electron paramagnetic resonance (EPR), revealing the singlet oxygen generation of the nanoparticles. Figure 12 and Figure 13 The analysis shows that a single manganese-coordinated porphyrin molecule can generate singlet oxygen active substances under 808 nm laser irradiation. After self-assembly to form manganese porphyrin nanoparticles and loading silver tannate nanoparticles, the generation of singlet oxygen is not inhibited, but significantly enhanced, thus proving that the in situ growth of ultra-small silver tannate nanoparticles (<20 nm) on self-assembled manganese porphyrin nanoparticles is conducive to the enhancement of photodynamic activity.

[0068] 5) Photothermal sterilization effect test The specific operation steps are as follows: Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli strains were cultured at 37°C to the logarithmic phase, and then the bacterial solution was centrifuged at 4000 rpm for 5 min at 4°C, the liquid culture medium was removed, and the bacteria were resuspended in PBS buffer. The operation was repeated three times to wash the bacteria and adjust the bacterial solution concentration to 10 8 CFU / mL for later use; then dilute the bacterial solution with PBS buffer to a concentration of 10 6 CFU / mL; 450 μL of the above bacterial solution was pipetted, and different concentrations of SAMnTEPP@TA / Ag-HA nanomaterials (50 μL) were added. The solution was irradiated with 808 nm near-infrared laser for 5 min and 10 min, and then placed in a 37°C shaker for reaction for 0.5 h, 1.0 h, and 2.0 h. 100 μL of the above bacterial solution was pipetted and added to 20 mL of plate count agar for plating. After plating, the solution was placed in a 37°C bacterial incubator for 12 h. Finally, the number of colonies was observed and counted with the naked eye. The results are as follows. Figure 14 and Figure 15 shown.

[0069] Figure 14 and Figure 15 The rapid bactericidal efficacy of SAMnTEPP@TA / Ag-HA nanoparticles against Staphylococcus aureus at concentrations of 50 µg / mL and 25 µg / mL was demonstrated. Figure 14 and Figure 15 The results show that when the concentration of SAMnTEPP@TA / Ag-HA nanoparticles is 50 μg / mL, they can kill nearly 50% of Staphylococcus aureus after being co-cultured with Staphylococcus aureus for 1 h without laser irradiation, and nearly 80% of Staphylococcus aureus after being co-cultured for 2 h. When SAMnTEPP@TA / Ag-HA nanoparticles are exposed to 808 nm laser (1 W / cm 2 ) irradiation, 5 minutes of irradiation killed all S. aureus within 1 hour, and 10 minutes of irradiation killed all S. aureus within 0.5 hours. When the SAMnTEPP@TA / Ag-HA nanoparticle concentration was 25 µg / mL, its rapid bactericidal effect was significantly reduced. Without laser irradiation, after 2 hours of co-incubation, no significant bactericidal effect was observed. However, when the SAMnTEPP@TA / Ag-HA nanoparticles were irradiated with laser for 5 and 10 minutes and co-incubated for 2 hours, they still rapidly killed S. aureus. These results demonstrate that the prepared SAMnTEPP@TA / Ag-HA nanoparticles can achieve rapid bactericidal effects under 808 nm laser light, which is mainly attributed to the synergistic photothermal / photodynamic bactericidal effects of the SAMnTEPP@TA / Ag-HA nanoparticles.

[0070] 6) Cell viability test The specific operation steps are as follows: add L929 cell suspension to a 96-well plate, so that the number of cells in each well is about 5000 cells / well, and the volume is about 100 μL. Then, the 96-well plate is placed in a cell culture incubator for 24 hours; after removing the supernatant, 100 μL of MEM culture medium containing different concentrations of SAMnTEPP@TA / Ag-HA nanoparticles is added to each well and placed in an incubator for 24 hours; after the incubation is completed, the supernatant in the well is removed and CCK-8 solution (10%, 100 μL) is added. After reacting for 1 hour, the plate is tested by microplate reader. The results are as follows Figure 16 and Figure 17 shown.

[0071] Figure 16 The effects of SAMnTEPP@TA / Ag nanoparticles on L929 cell survival before and after encapsulation of hyaluronic acid (HA) molecules were demonstrated. Figure 16 Analysis revealed that when the SAMnTEPP@TA / Ag-HA concentration exceeded 60 µg / mL, the cell viability showed a significant difference compared to the control group. This suggests that when the nanoparticle concentration exceeds 60 µg / mL, it exhibits a certain degree of cytotoxicity. This is primarily due to the high cytotoxicity of the small-sized silver tannate nanoparticles loaded on SAMnTEPP. To reduce the cytotoxicity of the nanoparticles, we modified the surface of the SAMnTEPP@TA / Ag nanoparticles with a layer of thiolated hyaluronic acid molecules. Cytotoxicity results showed that at a concentration of 80 µg / mL, the hyaluronic acid-coated SAMnTEPP@TA / Ag-HA nanoparticles exhibited significantly different cytotoxicity compared to the uncoated SAMnTEPP@TA / Ag nanoparticles, indicating that hyaluronic acid-coated nanoparticles can enhance their biosafety. Figure 17 The fluorescence results of SAMnTEPP@TA / Ag-HA nanoparticles staining live and dead cells at different concentrations are shown in Figure 2. Figure 17 The analysis showed that SAMnTEPP@TA / Ag-HA nanoparticles had relatively low toxicity to cells when the concentration was below 60 μg / mL.

[0072] 7) Adhesion test The specific operation steps are as follows: the hydrogel in Example 4 is made into a hydrogel patch through a mold, and its adhesion to skin, glass, metal and plastic surfaces is tested and photographed for preservation. Figure 18 shown.

[0073] Figure 18The adhesion of chitosan adhesive hydrogel to materials such as skin, glass, copper and plastic is demonstrated in the figure after adding 50 μg / mL SAMnTEPP@TA / Ag-HA nanoparticles, i.e., the porphyrin hydrogel prepared in Example 4. Figure 18 The analysis showed that the prepared chitosan adhesive hydrogel loaded with antibacterial nanoparticles had very good adhesion to curved skin, smooth glass, rough copper sheets and plastics.

[0074] In summary, the method for preparing porphyrin nanoparticles of the present invention utilizes porphyrin derivatives with alkynyl functional groups as raw materials and leverages the affinity of alkynyl groups with gold / silver salts to achieve in situ growth of gold / silver nanoparticles on the porphyrin derivatives. This method combines the long-lasting antibacterial effects of PTT / PDT with those of silver ions, while simultaneously reducing the consumption of reactive oxygen species by GSH, thereby enhancing the efficacy of photodynamic / chemodynamic therapy. This method effectively addresses the issues of insufficient light, heat, and chemical stability of existing photosensitizers, as well as the problem that singlet oxygen generated by existing photosensitizers is easily consumed by GSH, resulting in reduced therapeutic efficacy.

[0075] The preparation method of porphyrin hydrogel of the present invention, chitosan modified by catechol has good hydrophilicity, is mixed with polyvinyl alcohol hydrogel, and the viscous hydrogel prepared by freeze-thaw method has the toughness and viscosity of hydrogel concurrently.By loading the porphyrin antimicrobial drug provided by the application, porphyrin hydrogel is made to have the dual efficacy of porphyrin nanoparticles and hydrogel concurrently, is applied to various hydrogel dressings, microneedles, patches, etc. for wound bacterial infection, has good antibacterial effect and biocompatibility, also has excellent stability and photodynamic / chemodynamic therapy effect, can effectively kill bacteria, promote wound healing, significantly improve the treatment efficiency of wound, for the treatment of wound bacterial infection provides new solution.And the preparation method of this porphyrin hydrogel is simple, raw material is easy to obtain, cost is relatively low, is easy to large-scale production and application, in the field of biomedical material technology, has application value.

[0076] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or modification made by those skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. A method for preparing porphyrin nanoparticles, characterized in that: The following steps are involved: S1. Add 4-[(trimethylsilyl)ethynyl]benzaldehyde and pyrrole to a first organic solvent to obtain TMS-TEPP (5,10,15,20-tetrakis(trimethylsilylethynyl)phenylporphyrin) as a purple solid; S2, dissolving TMS-TEPP purple solid in a second organic solvent, adding metal salt, washing with deionized water after the reaction, extracting with dichloromethane, combining the organic phases, drying and rotary evaporation to obtain solid powder TMS-M x TEPP (5,10,15,20-tetrakis(trimethylsilylethynyl)phenylmetalloporphyrin); S3, solid powder TMS-M x TEPP is dissolved in a third organic solvent, and methanol and potassium carbonate are added to obtain a solid powder M x TEPP (5,10,15,20-tetrakis(4-ethynylphenyl)metalloporphyrin); S4, solid powder M x TEPP is dissolved in a third organic solvent, and cetyltrimethylammonium bromide (CTAB) solution is added to obtain self-assembled metalloporphyrin nanoparticles SAM. x TEPP; S5, metal porphyrin nanoparticles SAM x TEPP is added to a reducing agent solution, and then a silver compound solution or a gold compound solution is added to obtain a first intermediate product; S6. Dissolve hyaluronic acid (HA) in water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and L-cysteine ​​(L-Cys) to obtain HA-Cys, and then add HA-Cys to the first intermediate product solution to obtain porphyrin nanoparticles.

2. The preparation method of porphyrin nanoparticles according to claim 1, wherein The metal salt is selected from at least one of ferric chloride, ferrous chloride, manganese acetate and zinc acetate; and / or, the reducing agent is selected from at least one of tannic acid, sodium borohydride, trisodium citrate, ascorbic acid and hydroxylamine hydrochloride; and / or, the silver compound solution is selected from silver nitrate solution; and / or, the gold compound solution is selected from chloroauric acid solution; and / or, the molar ratio of the TMS-TEPP purple solid to the metal salt is 1:10-45; And / or, the hexadecyltrimethylammonium bromide (CTAB) and the solid powder M x The molar ratio of TEPP is 2.5~5.5:1; and / or, the molar ratio of the reducing agent to the silver salt is 1:3-8; And / or, the reducing agent solution is alkaline.

3. The preparation method of porphyrin nanoparticles according to claim 1, wherein Said S1 comprises: adding 4-[(trimethylsilyl)ethynyl]benzaldehyde and pyrrole to propionic acid, reacting for 2 h to 5 h, to obtain TMS-TEPP (5,10,15,20-tetrakis(trimethylsilylethynyl)phenylporphyrin) as a purple solid; And / or, the step S2 comprises: dissolving TMS-TEPP purple solid in N', N-dimethylformamide (DMF), adding metal salt, reacting for 8 h to 15 h, collecting the organic phase and the solid dilution, and rotary evaporating to obtain solid powder TMS-M x TEPP; And / or, said S3 includes: solid powder TMS-M x TEPP was dissolved in tetrahydrofuran (THF), and methanol and potassium carbonate were added. The reaction was continued for 3-5 h and concentrated to obtain solid powder M. x TEPP; And / or, said S4 includes: solid powder M x TEPP was dissolved in tetrahydrofuran (THF), and cetyltrimethylammonium bromide (CTAB) solution was added. The reaction was carried out at a temperature of 25°C to 40°C for 24 h to 72 h to obtain self-assembled metalloporphyrin nanoparticles (SAM). x TEPP; And / or, the step S5 includes: dissolving a reducing agent in water and adjusting the pH value to be alkaline to obtain a reducing agent solution, adding metalloporphyrin nanoparticles SAM to the reducing agent solution. x TEPP, ultrasonic dispersion for 15 min to 30 min, adding a silver compound solution or a gold compound solution at a temperature of 25°C to 60°C, then reacting at a temperature of 45°C to 65°C for 30 min, and then switching to room temperature for reaction for 22 h to 26 h to obtain a first intermediate product; And / or, S6 includes: dissolving hyaluronic acid (HA) in water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS), reacting at room temperature for 1 h to 3 h, then adding L-cysteine ​​(L-Cys), stirring and reacting at room temperature for 22 h to 24 h, dialyzing for 70 h to 74 h, and drying to obtain HA-Cys, and then adding HA-Cys to the first intermediate product solution, reacting at room temperature for 20 h, dialyzing for 70 h to 74 h, and drying to obtain porphyrin nanoparticles.

4. The preparation method of porphyrin nanoparticles according to claim 3, wherein The pH value of the reducing agent solution is 9-11; And / or, the solvent in the cetyltrimethylammonium bromide (CTAB) solution is a mixed solvent of water and tetrahydrofuran (THF), and the volume ratio of water to tetrahydrofuran (THF) in the mixed solvent is 15-5:1; And / or, the first intermediate product solution is obtained by dispersing the first intermediate product in a PBS solution.

5. A porphyrin nanoparticle, characterized in that: The method is as described in any one of claims 1 to 4.

6. Use of the porphyrin nanoparticles prepared by the preparation method according to any one of claims 1 to 4 as a photosensitizer.

7. A porphyrin hydrogel, characterized in that: The invention comprises porphyrin nanoparticles prepared by the preparation method according to any one of claims 1 to 4 and a hydrogel, wherein the porphyrin nanoparticles are loaded in the hydrogel.

8. The method for preparing the porphyrin hydrogel according to claim 7, wherein: The following steps are involved: Step 1), dissolving chitosan in acetic acid solution to obtain a first mixed solution; dissolving lysine in water, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS), and adjusting the pH to acidic to obtain a second mixed solution; mixing the first mixed solution and the second mixed solution to obtain lysine-modified chitosan; Step 2), catechol is dissolved in water and the pH is adjusted to weak acidity, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) are added, and then lysine-modified chitosan solution is added to obtain a second intermediate product; Step 3) adding the second intermediate product to the porphyrin nanoparticle solution, and then adding polyvinyl alcohol (PVA) to obtain a porphyrin hydrogel.

9. The method for preparing the porphyrin hydrogel according to claim 8, wherein The mass percentage of chitosan in the first mixed solution is 1% to 5%; And / or, the mass ratio of the chitosan to the lysine is 1:0.5-2; and / or, the pH of the second mixed solution is 5-6; and / or, dissolving catechol in water and adjusting the pH to 5-6; And / or, the catechol is a catechol containing a carboxyl functional group, and the catechol containing a carboxyl functional group is at least one selected from 3,4-dihydroxyphenylacetic acid, tannic acid, caffeic acid and dopamine; And / or, the mass percentage of the second intermediate product in the porphyrin hydrogel is 2% to 5%; And / or, the mass percentage of polyvinyl alcohol in the porphyrin hydrogel is 2% to 5%.

10. Use of the porphyrin hydrogel according to claim 7 as a hydrogel microneedle, dressing or patch.

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