Zwitterionic photosensitizer material, nano particle, and preparation method and application of zwitterionic photosensitizer material and nano particle

Abstract

The invention belongs to the technical field of antibacterial materials, and particularly relates to a zwitterionic photosensitizer material, nanoparticles and a preparation method and application of the zwitterionic photosensitizer material and the nanoparticles. The structural formula of the zwitterionic photosensitizer material is shown in the specification, wherein x is any integer between 3 and 5; r1 and R2 groups are shown in the specification. The zwitterionic photosensitizer material based on sulfonation has excellent hydrophilicity and biological activity, can achieve an efficient sterilization effect at a low dosage, and has efficient curability on mouse living body wound infection, so that the zwitterionic photosensitizer material can be used for preparing a medicine for treating wound infection, and the problems that in the prior art, aggregation causes a fluorescence quenching effect, and the toxicity is poor are solved. And the problem of poor antibacterial treatment effect caused by reduction of ROS yield due to aggregation is solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial materials, and particularly relates to a zwitterionic photosensitizer material, nanoparticles and a preparation method and application thereof. BACKGROUND

[0002] At present, bacterial infection is still an important challenge in the medical field, although antibiotics have always been the cornerstone of infection management, but the harm of superbugs caused by antibiotic abuse far exceeds the therapeutic effect. The essence of bacterial biofilm is extracellular polymer, including polysaccharide, protein, extracellular DNA and lipid. One of the bacterial drug resistance mechanisms is the physical barrier produced by bacterial biofilm, which degrades or resists antibiotics through chelation and enzymes. In order to effectively solve the drug-resistant bacterial infection, the development of new antibacterial agents, such as antibacterial peptides, nanoparticles, cationic polymers and photosensitizers (PS), provides innovative strategies for bacterial treatment.

[0003] PS-mediated phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), is a new type of antibacterial treatment method, which has the characteristics of high antibacterial efficiency, non-invasive and low toxicity. They can destroy or penetrate bacterial biofilm, and then destroy bacterial biomolecules in the presence of heat and reactive oxygen species (ROS). Therefore, bacteria are almost impossible to develop resistance to PS, because their mechanism of action is different from traditional antibiotics. When excited by light, PS transitions from the ground state to the singlet excited state, and part of the energy is generated by non-radiative decay. They can also reach the triplet excited state through intersystem crossing, and transfer electrons to the surrounding substrates (water and oxygen) to form type I and type II ROS. PTT produces a high-temperature environment that directly destroys bacteria and the stubborn structure of the biofilm, but there is still a problem of high PS dose side effects. While PDT kills bacteria by combining with bacteria at a lower PS dose and generating intracellular ROS, which effectively makes up for the shortcomings of PTT. Therefore, the complementary mechanism of the two treatment methods, i.e. the synergistic therapy of PDT and PTT, shows great potential.

[0004] In recent years, compared with traditional aggregation-induced fluorescence quenching PS, aggregation-induced emission (AIE) PS shows higher ROS generation efficiency and photothermal performance in the aggregated state, becoming an ideal candidate material for phototherapy. Some AIE PSs have poor water solubility, and need carriers to realize biological application, such as polyethylene glycol (PEG) and liposomes, which makes the process of biomaterial manufacturing complex.

[0005] Therefore, it is of great significance to provide a photosensitizer material with good hydrophilicity and biological activity, and further good antibacterial effect. SUMMARY

[0006] The present application aims to solve one or more of the technical problems existing in the prior art, and at least provide a beneficial alternative. Specifically, the present application provides a photosensitizer material with good hydrophilicity and biological activity, which can be used for preparing a drug for treating wound infection, and has good antibacterial effect.

[0007] Therefore, the first aspect of the present application provides a zwitterionic photosensitizer material.

[0008] Specifically, the structure formula of the zwitterionic photosensitizer material is as follows: ; Among them, x takes any integer between 3-5; R1, R2 groups are both: .

[0009] Specifically, x represents the number of carbon atoms on the alkyl chain, and when it takes any integer between 3-5, the endpoints are included, i.e. x can take 3, 4, 5.

[0010] Specifically, the sulfonic acid group is a strong polar group with hydrophilic group characteristics, which can form hydrogen bonds with water molecules, significantly improving the water solubility of organic matter, facilitating subsequent biological experimental operation. In addition, the photosensitizer carries triphenylamine, which has the property of aggregation-induced emission. The active oxygen generation capacity and photothermal properties of the photosensitizer material will be enhanced in the aggregation state. In a 99% aqueous solution, the molecules aggregate to form small molecular nanoparticles, and the molecules can effectively enter the bacterial intracellular, and generate ROS and heat to kill bacteria under light irradiation.

[0011] The second aspect of the present application provides a preparation method of the photosensitizer material of the first aspect of the present application.

[0012] Specifically, the preparation method of the photosensitizer material comprises the following steps: (1) mixing 3, 4-disubstituted maleic anhydride derivative and 4-aminopyridine, and reacting to obtain an intermediate product; (2) mixing the intermediate product and alkylsultone, and reacting to obtain the photosensitizer material; In step (1), the structure formula of the 3, 4-disubstituted maleic anhydride derivative is as follows: .

[0013] Preferably, in step (1), the molar ratio of the 3,4-disubstituted maleic anhydride derivative and 4-aminopyridine is 1:(0.8-2.2).

[0014] Further preferably, the molar ratio of the 3,4-disubstituted maleic anhydride derivative and 4-aminopyridine is 1:(1-2).

[0015] Preferably, in step (1), the temperature of the reaction is 130-190°C; further preferably, the temperature of the reaction is 140-180°C.

[0016] Preferably, in step (1), the time of the reaction is 7-17h; further preferably, the time of the reaction is 8-16h.

[0017] Preferably, in step (1), the reaction is carried out in an organic solvent; further preferably, the organic solvent comprises N,N-dimethylformamide (DMF).

[0018] Preferably, in step (1), the reaction is carried out under the condition of a basic catalyst; further preferably, the basic catalyst comprises triethylamine.

[0019] Preferably, in step (1), the volume-molar ratio of the organic solvent to the 3,4-disubstituted maleic anhydride derivative is (18-45)mL:1mmol; further preferably, the volume-molar ratio of the organic solvent to the 3,4-disubstituted maleic anhydride derivative is (20-40)mL:1mmol.

[0020] Preferably, in step (1), the volume-molar ratio of the basic catalyst to the 3,4-disubstituted maleic anhydride derivative is (0.05-0.22)mL:1mmol; further preferably, the volume-molar ratio of the basic catalyst to the 3,4-disubstituted maleic anhydride derivative is (0.05-0.2)mL:1mmol.

[0021] Preferably, in step (2), the molar ratio of the intermediate product and alkylsultone is in the range of 1:(0.9-2.2); further preferably, in step (2), the molar ratio of the intermediate product and alkylsultone is in the range of 1:(1-2).

[0022] Preferably, the alkylsultone comprises any one of 1,3-propane sultone, 1,4-butane sultone, 1,5-pentane sultone.

[0023] Specifically, when the alkyl sulfonyl lactone is 1,3-propane sulfonyl lactone, a photosensitizer material with 3 carbon alkyl chains can be obtained, i.e., x=3; when the alkyl sulfonyl lactone is 1,4-butane sulfonyl lactone, a photosensitizer material with 4 carbon alkyl chains can be obtained, i.e., x=4; and when the alkyl sulfonyl lactone is 1,5-pentane sulfonyl lactone, a photosensitizer material with 5 carbon alkyl chains can be obtained, i.e., x=5.

[0024] Preferably, in step (2), the reaction temperature is 65-110℃; more preferably, the reaction temperature is 70-100℃.

[0025] Preferably, in step (2), the reaction time is 5.5-11 hours; more preferably, the reaction time is 6-10 hours.

[0026] Preferably, in step (2), the reaction is carried out in an organic solvent; more preferably, the organic solvent includes ethanol (EtOH).

[0027] Preferably, in step (2), the reaction is carried out under alkaline catalytic conditions; more preferably, the alkaline catalyst includes piperidine.

[0028] Preferably, in step (2), the volume molar ratio of the organic solvent to the intermediate product is (18-45) mL:1 mmol; more preferably, the volume molar ratio of the organic solvent to the intermediate product is (20-40) mL:1 mmol.

[0029] Preferably, in step (2), the volume molar ratio of the alkaline catalyst to the intermediate product is (0.9-3.3) mL:1 mmol; more preferably, the volume molar ratio of the alkaline catalyst to the intermediate product is (1-3) mL:1 mmol.

[0030] A third aspect of the present invention provides a nanoparticle.

[0031] Specifically, the raw materials for preparing the nanoparticles include the photosensitizer material described in the first aspect of this invention.

[0032] Preferably, the raw materials for preparing the nanoparticles also include organic solvents and unsuitable solvents.

[0033] Preferably, the organic solvent includes dimethyl sulfoxide (DMSO).

[0034] Preferably, the unsuitable solvent includes at least one of water and phosphate buffered saline (PBS buffer); more preferably, the unsuitable solvent includes ultrapure water.

[0035] A fourth aspect of the present invention provides a method for preparing the nanoparticles described in the third aspect of the present invention.

[0036] Specifically, the preparation method of the nanoparticles includes the following steps: The photosensitizer material and organic solvent are mixed, and then a poor solvent is added to obtain the nanoparticles.

[0037] Preferably, in the system, the volume ratio of the undesirable solvent to the organic solvent is ≥99%.

[0038] The fifth aspect of the present invention provides the use of the photosensitizer material described in the first aspect of the present invention and / or the nanoparticles described in the third aspect of the present invention in the preparation of antibacterial reagents.

[0039] Specifically, the photosensitizer material at 660 nm has a strength of 0.3 W / cm². 2 Laser irradiation can efficiently generate reactive oxygen species and heat, thereby achieving low-dose antibacterial effects. This characteristic gives it excellent potential for antibacterial and wound-healing applications, making it a promising candidate for clinical wound infection treatment. Nanoparticles, in solution conditions, exhibit highly effective killing effects against Staphylococcus aureus and drug-resistant bacteria. This application utilizes special light irradiation to generate a large amount of heat and reactive oxygen species for efficient sterilization, achieving synergistic PDT / PTT treatment.

[0040] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows: (1) The sulfonated zwitterionic photosensitizer material of the present invention is a new photosensitizer material with excellent hydrophilicity and bioactivity. This material can overcome the problems of fluorescence quenching effect caused by aggregation in the prior art, as well as the decrease in ROS production caused by aggregation, resulting in poor antibacterial treatment effect.

[0041] (2) The preparation process of this invention is simple. It synthesizes antibacterial materials with high hydrophilicity and bioactivity through a simple sulfonation reaction. The photosensitizer material and nanoparticles have a highly efficient bactericidal effect at low doses. After special light irradiation, a large amount of heat and active oxygen are generated for highly efficient bactericidal effect, realizing PDT / PTT synergistic treatment. At the same time, the intermediate and photosensitizer materials prepared by this invention have high yields, both reaching 99%.

[0042] (3) The sulfonated zwitterionic photosensitizer material of the present invention has good killing effect on Staphylococcus aureus and has high curative effect on live wound infection in mice. Therefore, it can be used to prepare drugs for treating wound infection. Attached Figure Description

[0043] Figure 1 This is a synthetic route diagram of the zwitterionic photosensitizer material of Example 1 of the present invention; Figure 2The ultraviolet absorption and fluorescence spectra of PY and PYSO3 nanoparticles of this invention are shown. Figure 3 This is a particle size diagram of the PY and PYSO3 nanoparticles of the present invention; Figure 4 This is a photothermal cycle diagram of PY and PYSO3 nanoparticles of the present invention; Figure 5 The graph shows the reactive oxygen species yield and superoxide anion yield of PY and PYSO3 nanoparticles of this invention. Figure 6 The graph shows the cytotoxicity evaluation results of PY and PYSO3 in this invention. Figure 7 This is a diagram showing the in vitro antibacterial activity of PY and PYSO3 nanoparticles of the present invention. Figure 8 This is a scanning electron microscope image of the in vitro antibacterial properties of PYSO3 nanoparticles from this invention. Figure 9 This is an in vitro imaging image of the PYSO3 nanoparticles of this invention against biofilm-dead bacteria. Figure 10 This is a graph showing the change in mouse body weight during the in vivo antibacterial test of the PYSO3 nanoparticles of this invention. Figure 11 This is a graph showing the results of in vivo antibacterial testing of PYSO3 nanoparticles in mice to assess wound growth. Detailed Implementation

[0044] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0045] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0046] Example 1 This embodiment provides a method for preparing a zwitterionic photosensitizer material, the specific steps of which are as follows: (1) Synthesis of 3,4-bis(4-methoxyphenyl)cyclobut-3-ene-1,2-dione (PSQ): Aluminum trichloride (2.57 g, 19.29 mmol) was added to two reaction flasks, followed by the addition of 3,4-dichlorocyclobut-3-ene-1,2-dione (1.32 g, 8.80 mmol). Anisole (2.1 g, 19.29 mmol) was slowly added dropwise for Friedel-Crafts acylation. The mixture was stirred at 50 °C for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted with dichloromethane, and the solvent was removed by vacuum rotary evaporation. The crude product was then purified by silica gel column chromatography to obtain a yellow solid product (50% yield). The characterization results of the product's 1H NMR spectrum are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.11 (d, J =9.0 Hz, 4H), 7.04 (d, J = 9.0 Hz, 4H), 3.92 (s, 6H); The characterization results of the product's carbon NMR spectrum are as follows: 13 C NMR (126 MHz, CDCl3) δ 196.46,184.60, 163.64, 130.59, 121.43, 114.89, 55.76; (2) Synthesis of 3,4-bis(5-(4-(diphenylamino)phenyl)thiophen-2-yl)furan-2,5-dione (MAHTHTPA): 500 mg of PSQ obtained in step (1) was dissolved in 100 mL of tetrahydrofuran. After irradiation with a UV lamp (410 nm, 25 W) for 6 hours, the crude product was purified by silica gel column chromatography to obtain an indigo blue solid with a yield of 50%. The characterization results of the product's 1H NMR spectrum are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.02 (d, J =4.1 Hz, 2H), 7.50 (d, J = 8.7 Hz, 4H), 7.29 (dd, J = 8.8, 6.9 Hz, 10H), 7.13 (d, J = 7.6 Hz, 8H), 7.09 (d, J = 7.4 Hz, 3H), 7.07 (d, J = 3.4 Hz, 3H), 7.05(s, 2H); The characterization results of the product's carbon NMR spectrum are as follows: 13C NMR (126 MHz, CDCl3) δ 164.81,151.51, 148.90, 147.17, 134.49, 129.61, 127.23, 126.49, 125.22, 123.92,123.09, 122.72; (3) Synthesis of 3,4-bis[5-(4-(diphenylamino)phenyl)thiophen-2-yl]-1-(pyridin-4-yl)-1H-pyrrole-2,5-dione (PY): MAHTHTPA (0.75 g, 1.0 mmol) obtained in step (2), pyridin-4-amine (0.14 g, 1.5 mmol) and triethylamine (0.1 mL) were dissolved in 20 mL of N,N-dimethylformamide and reacted at 160 °C for 12 hours. After the reaction was completed, the mixture was extracted with dichloromethane and the solvent was removed by vacuum distillation to obtain a deep purple-blue powder (0.82 g) with a yield of 99%. The characterization results of the product's 1H NMR spectrum are as follows: 1 H NMR (400 MHz, DMSO-d6) delta (TMS, ppm):8.71 (d, J = 6.1 Hz, 2H), 7.91 (d, J = 4.0 Hz, 2H), 7.67-7.50 (m, 8H), 7.34(t, 8H), 7.10 (m, 12H), 6.97 (d, J = 8.7 Hz, 6H); The characterization results of the product's carbon NMR spectrum are as follows: 13 C NMR (101 MHz, DMSO-d6) delta (TMS, ppm):168.91, 167.79, 150.48, 149.07, 147.84, 146.57, 129.73, 127.45, 126.94,126.20, 124.72, 123.86, 123.52, 122.29, 119.94. HRMS (C 53 H 36 N4O2S2): m / z824.2280 (M+, calcd 825.2355); (4) Synthesis of 4-[4-(3,4-bis{5-[4-(diphenylamino)phenyl]thiophen-2-yl}-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pyridin-1-onthiol-1-yl]butane-1-sulfonate (PYSO3): PY (0.82 g, 1.0 mmol) obtained in step (3), 1,2-oxothiacyclohexane-2,2-dioxide (1,4-butanesulfonyl lactone) (0.21 g, 1.5 mmol) and piperidine (2.0 mL) were dissolved in 20 mL of ethanol and reacted at 85 °C for 8 hours; after the reaction was completed, the solvent was removed by vacuum distillation to obtain a deep purple-blue powder (0.82 g), with a yield of 99%; The characterization results of the product's 1H NMR spectrum are as follows: 1 H NMR (500 MHz, DMSO-d6) delta (TMS, ppm):9.15 (d, J = 7.3 Hz, 2H), 8.38 (d, J = 7.3 Hz, 2H), 7.96 (d, J = 4.1 Hz, 2H), 7.65 (d, J = 8.8 Hz, 4H), 7.59 (d, J = 4.1 Hz, 2H), 7.35 (m, 7.5 Hz, 8H),7.14-7.06 (m, 12H), 6.97 (d, J = 8.8 Hz, 4H), 4.61 (t, 2H), 2.44 (m, 11.8 Hz,2H), 2.07-1.99 (m, 2H), 1.62-1.57 (m, 2H); The characterization results of the product's carbon NMR spectrum are as follows: 13 C NMR (126 MHz, DMSO-d6) delta (TMS, ppm):146.52, 145.79, 129.76, 126.95, 124.81, 123.96, 122.13, 50.36, 29.74, 21.71.HRMS (C 57 H 44 N4O5S2):m / z 961.2507 (M, calcd 961.2573).

[0047] Example 1: Synthesis route diagram of zwitterionic photosensitizer material as shown below Figure 1 As shown.

[0048] Experimental Example 1: Ultraviolet Absorption Spectroscopy and Fluorescence Spectroscopy Test The specific steps for preparing PY and PYSO3 nanoparticles are as follows: 4.12 mg of PY powder was dissolved in 5 mL of DMSO to obtain a 1 mM PY stock solution; 4.73 mg of PYSO3 powder was dissolved in 5 mL of DMSO to obtain a 1 mM PYSO3 stock solution; 10 μL of PY stock solution, 10 μL of PY stock solution, 10 μL of PY SO3 stock solution and 990 μL of water were mixed to prepare 10 μM PY and PYSO3 nanoparticle test solutions.

[0049] The ultraviolet absorption spectra of PY and PYSO3 nanoparticles were detected using ultraviolet absorption spectroscopy; the fluorescence emission spectra of PY and PYSO3 nanoparticles (excitation light 560 nm) were detected using fluorescence spectroscopy. The ultraviolet absorption and fluorescence spectra of PY and PYSO3 nanoparticles are as follows: Figure 2 Wherein, PY Abs and PYSO3Abs represent the absorption spectra of PY and PYSO3, respectively; PY PL and PYSO3PL represent the fluorescence emission spectra of PY and PYSO3, respectively.

[0050] Depend on Figure 2 It can be seen that the two molecules exhibit similar maximum absorption peaks at 560 nm and have maximum emission near the 775 nm near-infrared emission region.

[0051] Experimental Example 2: Particle Size Test Using the same method as in Example 1, 1 mM PY stock solution and PYSO3 stock solution were prepared; 10 μL each of PY stock solution and PYSO3 stock solution were added to 990 μL of PBS solution to prepare PY and PYSO3 nanoparticle test solutions.

[0052] The particle size of PY and PYSO3 nanoparticles was measured using a dynamic scattering light spectrometer, and the results are as follows: Figure 3 As shown in the figure. Here, PDI represents the particle size distribution index.

[0053] Depend on Figure 3 It can be seen that the average particle size of PY nanoparticles is 149.7 nm, and the average particle size of PYSO3 nanoparticles is 84.23 nm. PY exhibits aggregation in the 3000-5000 nm range, indicating its poor hydrophilicity; while PYSO3 nanoparticles do not exhibit aggregation, indicating that the photosensitizer material prepared in this invention has good hydrophilicity.

[0054] Experimental Example 3: Photothermal Cycling Test Using the same method as in Experimental Example 1, 1 mM PY stock solution and PYSO3 stock solution were prepared; 1 μL each of PY stock solution and PYSO3 stock solution were added to 99 μL of PBS solution to prepare PY and PYSO3 nanoparticle test solutions.

[0055] The temperature of the PY and PYSO3 nanoparticle test solution was monitored using a temperature monitor at 660 nm and 0.3 W / cm². 2 The test was started under laser irradiation. The photothermal cycling test results for PY and PYSO3 nanoparticles are as follows: Figure 4 As shown, where, Figure 4 Figure (A) in the diagram is the photothermal cycle diagram of PY. Figure 4 Figure (B) shows the photothermal cycle of PYSO3 nanoparticles.

[0056] Depend on Figure 4 It can be seen that, in the five-cycle thermal simulation, both PY and PYSO3 nanoparticles exhibit excellent photothermal conversion performance.

[0057] Experimental Example 4: Reactive Oxygen Species Test and Superoxide Anion Test Using the same preparation method as in Example 1, 10 μM test solutions of PY and PYSO3 nanoparticles were prepared. The total ROS generation efficiency of PY and PYSO3 nanoparticles was detected using a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, and the superoxide anion yield generated by PY and PYSO3 nanoparticles was detected using a dihydrorhodamine 123 (DHR123) fluorescent probe. The reactive oxygen species generation efficiency and superoxide anion generation efficiency of PY and PYSO3 nanoparticles are shown in the figure below. Figure 5 As shown. Among them. Figure 5 Figure (A) shows the reactive oxygen species generation efficiency of PY and PYSO3 nanoparticles. Figure 5 Figure (B) shows the superoxide anion generation efficiency of PY and PYSO3 nanoparticles; fw represents the volume fraction of the poor solvent, I in the vertical axis represents the fluorescence intensity of the nanoparticles at different water volume fractions, and I0 represents the initial fluorescence intensity of the nanoparticles in pure DMSO.

[0058] Depend on Figure 5 It can be seen that under 660nm laser irradiation, the fluorescence intensity of DCFH-DA in PY rapidly increased to nearly 110 times, and the fluorescence intensity of DCFH-DA in PYSO3 nanoparticles rapidly increased to nearly 113 times, significantly higher than that of the commercial photosensitizer Ce6, indicating that both PY and PYSO3 molecules have strong reactive oxygen species (ROS) generation efficiency. Simultaneously, the fluorescence intensity of DHR123 in PY rapidly increased to nearly 66 times, and the fluorescence intensity of DHR123 in PYSO3 rapidly increased to nearly 94 times, indicating that PYSO3 nanoparticles have a higher superoxide anion generation efficiency.

[0059] Experimental Example 5: Cytotoxicity Assessment Using cervical cancer cells (HeLa cells) as a model, the cytotoxicity of photosensitizer materials was evaluated.

[0060] After HeLa cells were seeded in 96-well plates and cultured for 24 hours, they were incubated with PBS solutions of different concentrations of PY and PYSO3 (0 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively) for 20 minutes. Then, 100 μL of DMEM (10% fetal bovine serum) medium was added and the cells were cultured for 24 hours. Finally, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) was added and the cells were cultured for 4 hours. The UV absorption intensity at 570 nm was measured to study its biotoxicity.

[0061] The preparation process of PBS solutions of different concentrations of PY and PYSO3 was as follows: First, 1 mM PY stock solution and 1 mM PYSO3 stock solution were prepared according to the same method as in Experimental Example 1; then they were mixed with PBS to obtain the solution.

[0062] The cytotoxicity assessment results of PY and PYSO3 are as follows: Figure 6 As shown. Among them, Figure 6 Figure (A) shows the cytotoxicity assessment results of PY; Figure 6 Figure (B) shows the cytotoxicity assessment results of PYSO3 nanoparticles.

[0063] Depend on Figure 6 It can be seen that at concentrations of 100 μM and below, the cells still maintain an 80% survival rate. Under the same conditions, the PYSO3 treatment group also showed similar experimental results, indicating that PY and PYSO3 nanoparticles have good biocompatibility at 100 μM.

[0064] Experimental Example 6: In vitro antibacterial activity test The in vitro antibacterial activity of PY and PYSO3 nanoparticles was evaluated using the plate count method, specifically: Bacterial solutions of Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA) (bacterial numbers ATCC 25923 (S. aureus) and ATCC 43300 (MRSA), purchased as lyophilized powder, revived for 2 generations in LB broth) were centrifuged at 7200 rpm for 90 seconds and washed three times with sterile PBS. The washed bacteria were then resuspended in PBS to obtain a bacterial solution with OD600=1 using a visible spectrophotometer. The bacterial solution was diluted 1000 times with PBS and then mixed with equal volumes of different concentrations (0 μM, 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM) of PY and PYSO3 for testing. In this method, PY and PYSO3 were dissolved in DMSO to obtain a 1 mM solution, which was then diluted with PBS to obtain test solutions of PY and PYSO3 nanoparticles at different concentrations.

[0065] For the laser (+) group, the samples were incubated at 170 rpm and 37°C for 30 minutes, using a 660 nm (0.3 W / cm²) laser. 2 The samples were irradiated with laser for 10 minutes, followed by incubation for 80 minutes. For the dark group (Laser (-)), the samples were incubated at 170 rpm and 37°C for 2 hours. Subsequently, the samples from the light and dark groups were diluted 25-fold with PBS, and 100 μL of the sample was evenly spread on a plate. After incubation at 37°C for 24 hours, the number of colony-forming units in each plate was observed.

[0066] The in vitro antibacterial activity of PY and PYSO3 nanoparticles, such as Figure 7 As shown. Among them, Figure 7 Figure (A) shows the in vitro antibacterial activity of PY; Figure 7 Figure (B) shows the in vitro antibacterial activity of PYSO3 nanoparticles.

[0067] Depend on Figure 7 It can be seen that PY and PYSO3 exhibit bactericidal effects against Staphylococcus aureus and MRSA under dark conditions, while Figure 7 In Figure (A), PY showed a weak antibacterial effect against Staphylococcus aureus after being exposed to light. Figure 7 In Figure (B), low concentrations of PYSO3 showed excellent antibacterial activity against Staphylococcus aureus and MRSA under light conditions, indicating that the sulfonated photosensitizer PYSO3 has excellent antibacterial activity.

[0068] Experimental Example 7: Scanning Electron Micrographs of In Vitro Antibacterial Properties The prepared bacteria (same as those in Example 6) were washed three times with PBS and then resuspended in 1 mL of PBS to obtain S. aureus and MRSA bacterial PBS suspensions. Then, 10 μL of PYSO3 (1 mM, prepared as in Example 1) was added to 1 mL of S. aureus and MRSA bacterial PBS suspensions, respectively. For the PYSO3+L group, a 660 nm laser (0.3 W / cm²) was used. 2 Irradiation was performed for 10 min; for the PYSO3 group and the PBS group, the samples were kept in the dark at 37°C for 40 min; subsequently, the bacterial samples were fixed with 2.5% glutaraldehyde for 12 h, and dehydrated continuously with gradient ethanol solutions (20%, 40%, 60%, 80%, 90% and 100%) for 15 min at each concentration; finally, gold sputtering was performed and the samples were observed by scanning electron microscopy (SEM).

[0069] Scanning electron microscopy image of PYSO3 nanoparticles' in vitro antibacterial properties, as shown below. Figure 8 As shown. Figure 8The scale bars for all figures are 1 μm.

[0070] Depend on Figure 8 As can be seen, the bacteria in the PYSO3 dark group still maintained a smooth and intact bacterial surface. After being exposed to light, a large area of ​​the bacterial surface collapsed, shrank, and died, indicating that the PYSO3 nanoparticles of this invention have excellent antibacterial properties.

[0071] Experiment Example 8: In vitro imaging test for bacteria that die from biofilm damage Staphylococcus aureus and MRSA biofilms were pre-incubated in confocal imaging dishes for 24 hours and 48 hours, respectively, to obtain mature biofilm structures. After maturation, 30 μM PYSO3 was added to the mature biofilms, and the mature biofilm structures were treated for 30 min each, followed by treatment with a 660 nm laser (0.3 W / cm²). 2 Irradiation for 10 min was recorded as PYSO3+L, while the group that did not undergo laser irradiation was recorded as PYSO3; then SYTOX Blue Dead Cell Stain was applied to the biofilm for 30 min, and the dead bacteria were imaged by confocal laser scanning microscopy (CLSM) (for SYTOX Blue Dead Cell Stain, λex=405nm, λem=430-490nm).

[0072] The preparation process of 30μM PYSO3 is as follows: First, PYSO3 powder is dissolved in DMSO to prepare a 5mM PYSO3 stock solution, which is then diluted with DMSO to a 3mM solution; then 10μL of the 3mM solution is taken and added to 990μL of PBS solution to obtain the final product.

[0073] In vitro imaging of PYSO3 nanoparticles against biofilm-dead bacteria, such as... Figure 9 As shown. By Figure 9 It can be seen that, compared with the dark group, the mature biofilms of Staphylococcus aureus and MRSA at 24 / 48h showed bright blue fluorescence in the laser irradiation group, indicating that their biofilms were destroyed after laser irradiation.

[0074] Test Example 9: In vivo antibacterial test BALB / c mice (n=5 per group) were randomly divided into five groups: a control group without laser treatment (Control), a control group treated with laser treatment (Control+L), a group treated with 50 μM vancomycin, a group treated with 50 μM PYSO3 without laser treatment (PYSO3), and a group treated with 50 μM PYSO3 treated with laser treatment (PYSO3+L). Mice were anesthetized with 1.5% sodium pentobarbital (50 mg / kg), and a 10 mm full-thickness dorsal wound was surgically created. 100 μL of MRSA suspension (1.0 × 10⁻⁶) was then administered. 9 CFU mL -1 Inoculation of the wound bed was performed to establish infection; 24 hours after infection, 50 μL of PBS (control group), PYSO3, or vancomycin were applied topically; 30 minutes later, the laser irradiation group received 660 nm laser (0.3 W / cm²). 2 The mice were irradiated for 10 minutes, while the unirradiated group was placed in the dark; the same treatment was repeated 72 hours after infection. The wound healing process was dynamically monitored every two days by measuring the weight of the mice and taking photographs.

[0075] The graph shows the changes in mouse body weight during the in vivo antibacterial test of PYSO3 nanoparticles. Figure 10 As shown in the figure; the results of the in vivo antibacterial test of PYSO3 nanoparticles in mice wound growth are shown in the figure. Figure 11 As shown.

[0076] Depend on Figure 10 As can be seen, the mice's body weight gradually increased, indicating that PYSO3 nanoparticles have good biocompatibility. Figure 11 It can be seen that, compared with the control group, the PYSO3+L group showed good healing on day 11, indicating that the sulfonated zwitterionic photosensitizer material has a good promoting effect on the healing of bacterial infection wounds in mice.

[0077] In summary, the sulfonated zwitterionic photosensitizer material of this invention has excellent hydrophilicity and bioactivity, and can achieve efficient bactericidal effect at low doses. It has high curative effect on live wound infections in mice, and therefore can be used to prepare drugs for treating wound infections, solving the problems of poor antibacterial treatment effect caused by aggregation leading to fluorescence quenching effect and aggregation leading to a decrease in ROS production in the prior art.

[0078] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A photosensitizer material, characterized in that, The structural formula of the photosensitizer material is as follows: ； Where x takes any integer between 3 and 5; Both R1 and R2 groups are: 。 2. The method for preparing the photosensitizer material according to claim 1, characterized in that, The preparation method includes the following steps: (1) Mix the 3,4-disubstituted maleic anhydride derivative and 4-aminopyridine, react them, and obtain the intermediate product; (2) The intermediate product and alkyl sulfonyl lactone are mixed and reacted to obtain the photosensitizer material; In step (1), the structural formula of the 3,4-disubstituted maleic anhydride derivative is as follows: 。 3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of the 3,4-disubstituted maleic anhydride derivative and 4-aminopyridine is 1:(0.8-2.2); and / or, in step (1), the reaction temperature is 130-190℃; and / or, in step (1), the reaction time is 7-17h.

4. The preparation method according to claim 2, characterized in that, In step (1), the reaction is carried out in an organic solvent; and / or, in step (1), the reaction is carried out under alkaline catalytic conditions.

5. The preparation method according to claim 4, characterized in that, The organic solvent includes N,N-dimethylformamide; And / or, the basic catalyst includes triethylamine; And / or, the volume molar ratio of the organic solvent to the 3,4-disubstituted maleic anhydride derivative is (18-45) mL:1 mmol; And / or, the volume molar ratio of the alkaline catalyst to the 3,4-disubstituted maleic anhydride derivative is (0.05-0.22) mL:1 mmol.

6. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of the intermediate product to the alkyl sulfonyl lactone is 1:(0.9-2.2). And / or, in step (2), the alkyl sulfonyl lactone includes any one of 1,3-propane sulfonyl lactone, 1,4-butane sulfonyl lactone, and 1,5-pentane sulfonyl lactone; And / or, in step (2), the temperature of the reaction is 65-110°C; and / or, in step (2), the reaction time is 5.5-11h; And / or, in step (2), the reaction is carried out in an organic solvent; and / or, in step (2), the reaction is carried out under alkaline catalytic conditions.

7. The preparation method according to claim 6, characterized in that, In step (2), the organic solvent includes ethanol; and / or, in step (2), the alkaline catalyst includes piperidine; And / or, in step (2), the volume molar ratio of the organic solvent to the intermediate product is (18-45) mL:1 mmol; And / or, in step (2), the volume molar ratio of the alkaline catalyst to the intermediate product is (0.9-3.3) mL:1 mmol.

8. A nanoparticle, characterized in that, The raw materials for preparing the nanoparticles include the photosensitizer material as described in claim 1.

9. The method for preparing nanoparticles according to claim 8, characterized in that, The preparation method includes the following steps: The photosensitizer material and organic solvent are mixed, and then a poor solvent is added to obtain the nanoparticles.

10. The use of the photosensitizer material of claim 1 and / or the nanoparticles of claim 8 in the preparation of antibacterial reagents.

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