Visual supramolecular photosensitizer capable of being used for photodynamic antibacterial treatment and preparation method of visual supramolecular photosensitizer

Abstract

The invention discloses a visual supramolecular photosensitizer capable of being used for photodynamic antibacterial treatment and preparation, and belongs to the technical field of supramolecules. The supramolecular photosensitizer is prepared from two kinds of molecules, namely diaryl ethylene molecules (DAE) and Aza-BODIPY, and # imgabs0 # F-127, sodium alginate, the synthesized diaryl ethylene molecules (DAE) and the synthesized Aza-BODIPY are subjected to mixed layer assembly in the presence of Ca < 2 + > to form the supramolecular photosensitizer. Structures of diarylethene molecules comprise one or more of the following four: # imgabs1 # Aza-BODIPY structural formulas comprise one or more of the following D0-D2: # imgabs2 #

Description

Technical Field

[0001] The present invention relates to a preparation method of a supramolecular photosensitizer that can be used for photodynamic therapy and can switch fluorescence, and its molecular structure can undergo reversible structural changes through light excitation, thereby causing changes in optical properties. These properties include color change, photochemical reaction, and fluorescence switching, and the visualization of photodynamic therapy can be achieved through changes in color and fluorescence, belonging to the field of functional material technology. Background Art

[0002] Fluorescence imaging technology has been widely used in the measurement of pathophysiological microenvironments due to its advantages such as non-invasive detection, high spatiotemporal resolution, simple operation, and real-time monitoring, and several fluorophores such as indocyanine green and methylene blue have been approved by the US Food and Drug Administration for clinical use. There are many methods for controlling the fluorescence emission of fluorescent materials to switch between "ON" and "OFF", including temperature, mechanical control, force, and acid. Photosensitive fluorescence switching is currently one of the most convenient and precisely controlled fluorescence methods, and unlike irreversibly switching fluorescent probes, reversible fluorescent photoswitches can enhance the spatial resolution and accuracy of the signal through their strictly controlled photoresponse switching between the ON and OFF states - even for images showing autofluorescence signals.

[0003] Fast photoresponsive diarylethenes (DAE) compounds have been widely studied due to their excellent thermal stability and fatigue resistance compared to other photochromic molecules, such as azobenzene, spiropyran, and fulgide. DTE can be converted from an open-ring form to a closed-ring form under ultraviolet (UV) irradiation, with simultaneous color change, and from a closed-ring form to an open-ring form under visible light irradiation, with the color returning to the initial state. Generally, the open and closed ring states of dithienylethylene molecular switches are non-fluorescent, and fluorescence can be obtained by introducing heterocyclic rings such as benzothiophene aromatic rings, pyrazole, pyrrole, and furan. Fluorescent diarylethenes (DAEs) have high thermal stability, reversibility, fatigue resistance, and fast switching rates, making them ideal candidates for super-resolution imaging. In particular, the open-type DAEs have been successfully applied to super-resolution microscopy based on photoactivated localization microscopy (PALM) to reveal soft matter structures with the advantage of high fluorescence on / off contrast.

[0004] Photodynamic therapy (PDT) is a new technology that uses the photodynamic effect to diagnose and treat diseases. Its action is based on the photodynamic effect, which is a photosensitization reaction with biological effects involving oxygen molecules. Its mechanism of action is that under the excitation of external light sources, the photosensitizer absorbs light energy and transitions from the ground state to the singlet excited state, and then to the triplet excited state through intersystem crossing. The photosensitizer in the triplet excited state can transfer electrons or energy with surrounding biological molecules or oxygen, generating free radicals or reactive oxygen species (ROS) that are damaging to biological molecules such as proteins and nucleic acids, thereby exerting therapeutic activity. It has the advantages of few side effects, low possibility of drug resistance, immediate treatment, and the ability to interrupt treatment at any time.

[0005] BODIPY and its derivatives are a class of multifunctional photosensitizing organic dyes with the characteristics of small Stokes shift, strong absorption, narrow absorption and emission, low photobleaching, high fluorescence quantum efficiency, large polarity, pH insensitivity and stable chemical structure, and can be applied in the fields of medicine, energy, environmental protection, etc. In addition, BODIPY has the characteristics of easy modification. The typical fluorescence decay can be converted into a triplet non-radiative intersystem crossing (ISC) by introducing different numbers of heavy atoms and electron-withdrawing / donating groups at different positions of BODIPY, thereby enhancing the ability of BODIPY to generate singlet oxygen for photodynamic therapy (PDT). Therefore, fluoroborate dipyrrole (BODIPY), especially aza-fluoroborate dipyrrole (Aza-BODIPY) derivatives, are revealed as valuable candidates for photodynamic photosensitizers (PS) due to their excellent physical and chemical properties.

[0006] Individual diarylethenes and Aza-BODIPY photosensitizing small molecules are not water-soluble and biocompatible, which hinders their practical application. Some natural or synthetic polymers have special physical and chemical properties and can be used as drug carriers to achieve targeted delivery and controlled release of drugs. By constructing a drug-carrying platform, drugs can better cross biological barriers, reduce side effects, and improve drug efficacy. Therefore, we can select specific polymers and use their hydrophilic and hydrophobic properties to form non-covalent interactions with photosensitive small molecules to form supramolecular photosensitizers, thereby improving the water solubility, biocompatibility and other properties of small molecules, and then achieve precise regulation and treatment of organisms, bringing new possibilities for biomedical research and clinical practice.

[0007] In addition, supramolecular photosensitizers can be applied in the field of biomedical diagnosis. By utilizing the optical properties and response characteristics of supramolecular photosensitizers, new diagnostic tools can be developed for detecting and diagnosing diseases or lesions. For example, by observing the changes in the luminescence properties of supramolecular photosensitizers under different illumination conditions, accurate detection and diagnosis of diseased tissues can be achieved, providing an important basis for early disease screening and treatment. Summary of the invention

[0008] The invention provides a supramolecular photosensitizer for visualized photodynamic antibacterial treatment and a preparation method thereof.

[0009] The present invention discloses a supramolecular photosensitizer for visualized photodynamic antibacterial therapy, which is composed of two molecules, diarylethene molecules (DAE) and Aza-BODIPY. Sodium alginate and the synthesized diarylethene (DAE) and Aza-BODIPY in Ca 2+ In the presence of , supramolecular photosensitizers are formed by mixed layer assembly, such as Fig.19 shown.

[0010] Further, a visual supramolecular photosensitizer that can be used for photodynamic antibacterial therapy of the present invention is to encapsulate diarylethene molecules (DAE) and Aza-BODIPY into a matrix composed of sodium alginate and The nanoparticle shell is composed of a core-shell structure of supramolecular photosensitizer.

[0011] The structures of diarylethenes include one or more of the following four types (DAE-p, A1, A2, A3), which undergo ring-opening and ring-closing structural changes and fluorescence changes under irradiation at 365nm and 808nm:

[0012]

[0013] Among them, DAE-p undergoes open-closed ring structure changes under 365nm and 808nm irradiation:

[0014]

[0015] The dithienylethylene cores of A1, A2, and A3 undergo ring-opening and ring-closing structural changes under irradiation at 365nm and 808nm:

[0016]

[0017] The structural formula of Aza-BODIPY includes one or more of the following D0-D2:

[0018]

[0019] The preparation method of the supramolecular photosensitizer for visualized photodynamic antibacterial therapy described in the present invention is as follows:

[0020] (1) Weigh and sodium alginate, dissolved in deionized water and sonicated to completely dissolve them to form a nanoshell solution;

[0021] (2) preparing a DMSO solution of a mixture of diarylethenes (DAE) and Aza-BODIPY to form a core solution;

[0022] and adding dropwise to the nanoshell solution of step (1) under the action of ultrasound, continuing ultrasound for a period of time, and stirring after the ultrasound to allow for full self-assembly;

[0023] (3) adding CaCl2 solution and continuing ultrasonication to fully cross-link the nanoshell liquid to form supramolecular photosensitizer nanoparticles with a more compact and stable core-shell structure;

[0024] (4) The nanoparticles prepared in step (3) are centrifuged to separate the small molecules not encapsulated therein, and washed with ultrapure water for multiple times. Finally, the washed nanoparticles are placed in a ventilated and dry environment for drying for later use.

[0025] In the further step (1) The mass ratio of sodium alginate to sodium alginate is 4:1.

[0026] In the further step (2), the mass ratio of the diarylethene molecules (DAE) to Aza-BODIPY is 1:10-10:1. The total mass of the diarylethene molecules (DAE) and Aza-BODIPY is The mass ratio is 1:10-1:20.

[0027] In the further step (3), the concentration of CaCl2 added is 15 mmol / L.

[0028] The visualized supramolecular photosensitizer obtained by the present invention can be used for photodynamic antibacterial therapy. Since both molecules have corresponding reactions under 365nm ultraviolet light and 808nm near-infrared light, it can be used to prepare visualized photodynamic therapy materials, fluorescent contrast agents, and photochromic agents. It can be further used to prepare visualized photodynamic therapy materials and visualized antibacterial agents. When used, it is irradiated alternately with 365nm ultraviolet light and 808nm near-infrared light.

[0029] The present invention is achieved by In the presence of Ca2+, the mixed layer assembly of diarylethenes (DAE) and Aza-BODIPY with sodium alginate formed a core-shell structured supramolecular photosensitizer, especially D1-DAE-p NPs, which solved the problem of DAE-p's photochromic performance being inhibited in H2O. Not only did it restore the photochromic performance of DAE-p in H2O solution, but it also made it have excellent water solubility. In addition, the test showed that the presence of D1 did not cause D1-DAE-p NPs to lose the photochromic and fluorescent switching effects. In terms of photodynamic performance, the effect of DAE-p on the singlet generation of D1 was also investigated. Whether it was the UV-visible absorption spectrum test using 1,3-diphenylisobenzofuran (DPBF) as a probe for singlet oxygen detection, or the EPR test using 2,2,6,6-tetramethylpeperidine (TEMP) as a singlet oxygen scavenger, the test results showed that the open-ring and closed-ring forms of DAE-p had almost no effect on the generation of singlet oxygen in D1, indicating that D1-DAE-p NPs have similar photodynamic properties to D1. In terms of fatigue resistance, under alternating irradiation with 365nm ultraviolet light and 808nm near-infrared light, D1-DAE-p NPs have relatively good fatigue resistance, and The introduction of sodium alginate macromolecules has certain positive significance in improving its fatigue resistance, giving it greater advantages in practical applications.

[0030] In short, use The supramolecular photosensitizer D1-DAE-p NPs formed by mixing with sodium alginate in the presence of Ca2+ and encapsulating DAE-p and D1 at the same time not only retains the photochromic and fluorescent switching properties of DAE-p, but also has the photodynamic properties of D1. DAE-p can undergo a closed ring under 365nm ultraviolet light and produce a color change from blue to green, while D1 can also produce singlet oxygen under 365nm ultraviolet light. While the distribution of the supramolecular photosensitizer D1-DAE-p NPs can be viewed with the naked eye, it also has a bactericidal effect and can be applied in surface antibacterial coatings and other directions. In addition, under the irradiation of 808nm near-infrared light, DAE-p changes from a closed ring with color but no fluorescence to an open ring with no color and fluorescence, which is still accompanied by the production of singlet oxygen, and its bactericidal effect is good, with the potential to achieve integrated diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1(a)-(b) are the changes in the absorption spectrum of DAE-p in DMSO solution under 365nm and 808nm illumination, respectively; (c)-(d) are the changes in the absorption spectrum of DAE-p in H2O solution under 365nm and 808nm illumination, respectively; (e)-(f) are the changes in the absorption spectrum of DAE-p@F127 under 365nm and 808nm illumination, respectively; (g)-(h) are the changes in the absorption spectrum of DAE-p NPs under 365nm and 808nm illumination, respectively; (i) Color change diagram under 365nm and 808nm illumination;

[0032] Figure 2 Absorption spectra of D1-DAE-p NPs under (a) 365nm and (b) 808nm illumination; (c) color changes of D1-DAE-pNPs under 365nm illumination and different illumination time

[0033] Figure 3 In mixed DMSO solutions with DAE-p and D1 concentration ratios of 1:1, 1:2, 1:3, 1:4, and 1:5, the color changes of the solutions before and after irradiation with 365 nm light;

[0034] Figure 4 The fluorescence spectrum of DAE-p in DMSO solution changes with time under (a) 365nm and (b) 808nm illumination; The fluorescence spectrum of DAE-p in H2O solution changes with time under (c) 365nm and (d) 808nm illumination;

[0035] Figure 5 Fluorescence emission spectra of DAE-p@F127 under (a) 365nm, (b) 808nm illumination, and laser confocal imaging under (c) 365nm, (d) 808nm illumination; fluorescence emission spectra of D1-DAE-p NPs under (e) 365nm, (f) 808nm illumination, and laser confocal imaging under (g) 365nm, (h) 808nm illumination;

[0036] Figure 6 Changes in the absorption spectra of D1 containing DPBF over time under (a) DMSO solution at 808 nm illumination, (b) H2O solution at 808 nm (1 W·cm-2) illumination, and (c) H2O solution at 365 nm illumination; (d) normalized summary of the absorption spectra.

[0037] Figure 7 At 808nm (1W·cm -2) in H2O solution containing DPBF, the absorption spectra of (a) D1 and (b) D1@F127 change with time and their (c) normalized summary; in H2O solution under 365nm light, the absorption spectra of (d) D1 and (b) D1@F127 change with time and their (f) normalized summary.

[0038] Figure 8 D2 containing DPBF (a) DMSO solution under 808 nm light, (b) H2O solution under 808 nm (1 W·cm -2 ) under light of 365 nm; (c) the change of absorption spectrum of H2O solution under light of 365 nm; (d) normalized summary of absorption spectrum.

[0039] Fig. 9 At 808nm (1W·cm -2 ) in H2O solution containing DPBF, (a) D2 and (b) D2@F127 absorption spectra change with time and (c) normalized summary; in H2O solution containing DPBF under 365nm light, (d) D2 and (b) D2@F127 absorption spectra change with time and (f) normalized summary;

[0040] Fig.10 In DMSO solution, 808nm (1W·cm -2 ) under the illumination of (a) D0, (b) D1, (d)

[0041] D2, (e) Changes of ICG absorption spectrum over time and its (c) color difference and (f) normalized processing diagram;

[0042] Fig.11 In H2O solution, 808nm (1W·cm -2 ) under the illumination of (a) D0, (b) D1, (d)

[0043] D2, (e) Changes of ICG absorption spectrum over time and its (c) color difference and (f) normalized processing diagram;

[0044] Fig.12 Changes of absorption spectra of (a) D0, (b) D1, (d) D2, and (e) ICG containing DPBF over time in H2O solution and under 365 nm light, as well as their (c) color differences and (f) normalized processing diagrams;

[0045] Fig.13Absorption spectra of (a) D1, (b) D1@F127, and (c) D1-DAE-p(O)NPs solutions containing DPBF change with irradiation time under 808 nm light in H2O solution; (d) normalized absorption spectra

[0046] Fig.14 Singlet oxygen generated by D1@F127 and D1-DAE-p(O)NPs (a) before and (b) after 808 nm irradiation. 1 O2) changes; D1@F127 and D1-DAE-p(C)NPs, singlet oxygen (O2) produced before (c) 365nm irradiation and after (d) 365nm irradiation. 1 O2) changes;

[0047] Fig.15 (a) DAE-p, (b) DAE-p@F127, (c) D1-DAE-p NPs in H2O solution, changes in the maximum absorption peak at 600-800 nm under alternating illumination at 365 nm and 808 nm; (d) Linear fitting of the absorption values ​​of the closed-loop state after 365 nm illumination in each cycle;

[0048] Fig.16 (a) Changes in the maximum emission peaks of DAE-p, (b) DAE-p@F127, and (c) D1-DAE-p NPs in H2O solutions under alternating illumination at 365 nm and 808 nm; (d) Linear fitting of the emission values ​​of the closed-loop state after 365 nm illumination in each cycle;

[0049] Fig.17 D1 is (a) protected from light, (b) 365nm (35mW·cm -2 , 5min) of ultraviolet light, (c) simulated fluorescent light (100

[0050] mW cm -2 , 30min), (d) 808nm (1W·cm -2 , 10min) antibacterial effect under near-infrared light

[0051] Fig.18 (a) Particle size (b) morphology (c) Zeta potential characterization of D1-DAE-p NPs.

[0052] Fig.19 Schematic diagram of the synthesis process of supramolecular photosensitizers for visualizing photodynamic antibacterial therapy.

[0053] Fig. 20 , Fig.21 Corresponding to the NMR mass spectrum of DAE-p;

[0054] Fig. 22 , Fig.23 Corresponding to the NMR mass spectrum of A1;

[0055] Fig.24 , Fig.25 Corresponding to the NMR mass spectrum of A2;

[0056] Fig.26 , Fig. 27 Corresponding to the NMR mass spectrum of A3.

[0057] Fig.28 , Fig.29 NMR mass spectrum corresponding to D0;

[0058] Fig.30 , Fig.31 Corresponding to the NMR mass spectrum of D1;

[0059] Fig.32 , Fig.33 Corresponding to the NMR mass spectrum of D2. DETAILED DESCRIPTION

[0060] The present invention will be further described below in conjunction with examples, but the present invention is not limited to the following examples.

[0061] Example 1

[0062] The method for preparing the diarylethene molecule of the present invention comprises the following steps:

[0063] (1) Synthesis of 4-bromo-5-methylthiophene-2-carboxaldehyde

[0064]

[0065] Specifically, the method includes: taking a clean magnetic particle and blowing it dry, placing it in a 250mL single-mouth flask, first adding 5-methyl-2-thiophene aldehyde (1eq, 10.0g), then adding 40mL glacial acetic acid, and finally adding N-bromosuccinimide (NBS, 1.2eq, 16.95g), and the solution is orange-yellow. Stirring at room temperature for 48h, and detecting the intermediate point plate. After the reaction is completed, extracting with saturated sodium thiosulfate, water and ethyl acetate, taking the upper organic solution, adding silica gel to spin-dry and preparing a sample, and using petroleum ether: ethyl acetate = 50:1 eluent to pass through the column, an orange-yellow powder 1 (6.8g, yield of about 68%) is obtained.

[0066] (2) Synthesis of 3-bromo-5-(diethoxymethyl)-2-methylthiophene

[0067]

[0068] Weigh 4-bromo-5-methylthiophene-2-carboxaldehyde (1eq, 2g), triethyl orthoformate (1.2eq, 1.744g, 1.96mL), ammonium chloride (1eq, 0.554g), put into a 100mL single-mouth flask, and then add 40mL of ethanol. Put in a clean magnetic bar, stir at room temperature for 8h, and detect the reaction during the reaction. After the reaction is completed, the ethanol is dried under reduced pressure, and then extracted with saturated sodium bicarbonate, water, saturated sodium chloride and dichloromethane. Take the lower organic solution and put it into anhydrous magnesium sulfate in a conical flask to absorb water for 1h, then filter, dry under reduced pressure and pump with a water pump for 10min, and pump with an oil pump for 10min to ensure that the water has been completely removed, and obtain a yellow oily liquid 2 (3.69g, yield is about 85.85%), without purification, and immediately proceed to the next step.

[0069] (3) Synthesis of 4,4'-(perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-2-carboxaldehyde)

[0070]

[0071] Take a 250mL two-necked bottle, put in a clean magnet and dry 3-bromo-5-(diethoxymethyl)-2-methylthiophene (1eq, 3.69g), and dissolve it in pure steamed tetrahydrofuran (60mL), evacuate, and pass argon. Put the reaction bottle into a cold well at -78℃ and let it stand for 30min, then add n-butyl lithium (1.1eq, 0.934g, 6.1mL) dropwise and react for 30min, then add octafluorocyclopentene (0.7eq, 1.97g, 1.25mL) and the solution gradually turns yellow-brown, continue to react in the cold well for 2h, then take it out and stir it at room temperature in the dark for 12h. Add hydrochloric acid and a few drops of water, and stop the reaction if there is no purple spot on the plate after a few minutes. After the reaction is completed, add a little saturated ammonium chloride to quench the unreacted n-butyl lithium, and decompress and spin dry. Then it was extracted with saturated sodium chloride, water and dichloromethane, the lower organic solution was taken, silica gel was added and spin-dried to prepare a sample, and dichloromethane: petroleum ether = 2:1 was used as the eluent to pass through a column to obtain a yellow powder 3 (1.3 g, yield of about 23%).

[0072] (4) Synthesis of 2-(4-(pyridin-4-yl)phenyl)acetonitrile

[0073]

[0074] Weigh 4-bromobenzeneacetonitrile (1.2eq, 2g), 4-pyridine boronic acid (1eq, 1.25g) and add them to a 100mL two-necked bottle containing a 2M potassium carbonate aqueous solution, and add 40mL isopropanol. Vacuum and pass argon gas, add tetrakis(triphenylphosphine)palladium (0.03eq, 0.353g) solution under the protection of argon gas and avoid light, and the solution is dark green. Reflux and stir at 70℃ and react for 7h in the dark. Extract with saturated sodium chloride and ethyl acetate, take the upper organic solution, dry with anhydrous magnesium sulfate, evaporate under reduced pressure to prepare a sample, and purify it by column with an eluent of petroleum ether: ethyl acetate = 1:2 to obtain white powder 4 (1.3g, yield is about 40%).

[0075] (5) Synthesis of A1:

[0076] Dissolve 4,4'-(perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-2-carboxaldehyde) (1eq, 0.2g) and 2-(4-(pyridin-4-yl)phenyl)acetonitrile (2eq, 0.183g) in tert-butanol and stir at 50°C for 1h. Evacuate, pass argon, heat to 80°C, and then add a methanol solution of sodium methoxide (1.7eq, 0.04g) dropwise. The solution changes from light yellow to red and then to black. Stir at 80°C in the dark for 3h before stopping the reaction. During this period, the reaction is monitored by a plate. After the solvent is dried under reduced pressure, extract with saturated sodium chloride, water and ethyl acetate. Take the upper organic solution and pass it through a column with petroleum ether:ethyl acetate = 1:2 as the eluent to obtain a light yellow powder A1 (0.183g, yield of about 48%).

[0077] (6) Synthesis of A2

[0078] Take a 100mL two-necked bottle and put in a magnetic particle of suitable size. Weigh 4,4'-(perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-2-carboxaldehyde) (1eq, 0.4g) and 2-(4-(pyridin-4-yl)phenyl)acetonitrile (4eq, 0.72g) into a two-necked bottle, add tert-butyl alcohol, evacuate, and pass argon. After heating the temperature to 80°C, add the methanol solution in which sodium methoxide (1.7eq, 0.085g) is completely dissolved to the reaction bottle dropwise with a needle, and stir at 80°C for 5h. During this period, observe the reaction by tapping the plate, and ensure that the reaction is protected from light throughout the process. After the solvent is dried under reduced pressure, extract it with saturated sodium chloride, water and ethyl acetate, remove the upper organic solution, and use pure ethyl acetate as the eluent to pass through the column to obtain a yellow-green powder A2 (0.47g, yield is about 42%).

[0079] (7) Synthesis of A3

[0080] Dissolve cisplatin (4eq, 0.15g) and silver nitrate (4eq, 0.087g) in 20mL of DMF, stir at 55°C for 24h, and white solid is found to precipitate. Centrifuge the white silver chloride to obtain a light yellow solution. Weigh A2 (1eq, 0.1g) and add it to the light yellow solution obtained above, and continue to stir at 55°C in the dark for 24h. Cool the above reaction solution to room temperature, centrifuge it with ether as a poor solvent, pour off the suspension, rinse it repeatedly with ethanol, evaporate it under reduced pressure, and then vacuum dry it to obtain black solid A3 (0.22g, yield is about 89%).

[0081] Among them, DAE-p has been reported before and will not be described in detail here.

[0082] The structure of Aza-BODIPY is as follows:

[0083] The present invention relates to a class of Aza-BODIPY molecules that can generate singlet oxygen under both 365nm ultraviolet light and 808nm near-infrared light, and the structure is as follows:

[0084]

[0085] The preparation method of the Aza-BODIPY molecule involved in the present invention, which can generate singlet oxygen under 365nm ultraviolet light and 808nm near-infrared light, is as follows:

[0086] (1) Synthesis of (E)-3-(5-methylthiophen-2-yl)-1-phenylpropyl-2-en-1-one

[0087]

[0088] Take a 500mL round-bottom flask, put (6g, 5.13mL, 47.6mmol, 1eq.) 5-methyl-2-thiophenecarboxaldehyde and 100mL methanol. Then, after the mixture solution is cooled to 0°C, sodium hydroxide aqueous solution (50mL, 50%wt) is added dropwise under the action of a magnetic stirrer. After 20 minutes, (5.71g, 5.57mL, 47.5mmol, 1eq.) acetophenone is added dropwise, and then the solution is heated to room temperature and stirred overnight. The methanol is spin-dried and extracted with water, ethyl acetate and saturated NaCl to obtain a yellow solid 1 (11.12g, 95%).

[0089] (2) Synthesis of 3-(5-methylthiophen-2-yl)-4-nitro-1-phenylbutan-1-one

[0090]

[0091] Take a 500mL round-bottom flask, add nitromethane (14.87g, 13.04mL, 0.24mol, 5eq.), (E)-3-(5-methylthiophen-2-yl)-1-phenylpropyl-2-en-1-one (11.12g, 48mmol, 1eq.) and diethylamine (17.83g, 25.11mL, 0.24mol, 5eq.) into the 500mL round-bottom flask, then add 300mL methanol, heat to 66°C in an oil bath, reflux overnight, and the solution changes from orange to blood red in about 30 minutes. Evaporate the solvent methanol by vacuum distillation, and dissolve the crude product with 150mL dichloromethane. Wash the organic layer with brine and water, let it stand for stratification, dry the organic layer with anhydrous sodium sulfate, and evaporate the solvent. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate (15 / 1 V / V)) to give dark yellow oily solid 2 (8.48 g, 32.6%).

[0092] (3) Synthesis of (Z)-3-(5-methylthiophen-2-yl)-N-(3-(5-methylthiophen-2-yl)-5-phenyl-1H-pyrrol-2-yl)-5-phenyl-2H-pyrrol-2-imine

[0093]

[0094] Take a 200mL two-necked bottle, add ammonium acetate (11.16g, 0.14mol, 35eq.), evacuate and pass argon, then dissolve 3-(5-methylthiophene-2-yl)-4-nitro-1-phenylbutane-1-one (1.19g, 4mmol, 1eq.) in 80mL n-butanol and pour into a Schlenk container, and heat the solution under reflux for 48 hours. The solution is cooled to room temperature and dried by rotation, extracted with dichloromethane, water and saturated sodium chloride to obtain a black solid 3 (0.75g, 60%).

[0095] (4) Synthesis of 5,5-difluoro-1,9-bis(5-methylthiophene-2-yl)-3,7-diphenyl-5H-4-14,5-dipyrrolo[1,2-c:2',1'-f][1,3,5,2]triazacytidine D0

[0096]

[0097] Take a 200mL two-necked bottle, add (Z)-3-(5-methylthiophene-2-yl)-N-(3-(5-methylthiophene-2-yl)-5-phenyl-1H-pyrrol-2-yl)-5-phenyl-2H-pyrrol-2-imine (0.75g, 1.5mmol, 1eq.) and 100mL of dichloromethane, and evacuate and pass argon. Stir the mixture at 0°C for 20 minutes, then add N,N-diisopropylethylamine (1.98g, 2.53mL, 15mmol, 10eq.) dropwise in an ice-water bath, stir the mixture solution for 30 minutes, and then add boron trifluoride etherate (6.52g, 5.83mL, 46mmol, 30eq.). Stir at room temperature under argon atmosphere for 50 hours. The filter residue obtained by suction filtration is a dark blue solid that turns red under sunlight (0.3g, 40%).

[0098] (5) Synthesis of 5,5-difluoro-1,9-di(5-methylthiophene-2-yl)-3,7-diphenyl-5H-414,514-dipyrrolo[1,2-c:2',1'-f][1,3,5,2]triazaboropurine-2-carboxaldehyde D1

[0099]

[0100] Take a 50mL round-bottom flask, add D0 (0.05g, 0.093mmol, 1eq.) and dichloromethane (5mL), and place it in an ice bath to cool to 0°C. Add dimethylformamide (0.68g, 0.7mL, 9mmol, 100eq.) dropwise under magnetic stirring, and then slowly add trichlorophosphine (1.4g, 0.9mL, 9mmol, 100eq.) in 6mL dichloromethane to the above solution through a constant pressure funnel. After the solution is heated to room temperature and refluxed at 35°C overnight, 10mL of saturated sodium bicarbonate solution is added to the mixed solution and stirred for 6 hours. Wash the organic layer with brine and water, dry the organic layer with anhydrous sodium sulfate after standing and stratifying, and evaporate the solvent. The crude product is purified by column chromatography on silica gel (petroleum ether / ethyl acetate / (5 / 1V / V)) to obtain a blue solid (0.04g, 27.5%).

[0101] (6) Synthesis of 5,5-difluoro-2,8-diiodo-1,9-di(5-methylthiophen-2-yl)-3,7-diphenyl-5H-4l4,5-dipyrrolo[1,2-c:2',1'-f][1,3,5,2]triazacytidine D2

[0102]

[0103] Take a 100 mL two-necked bottle, add DO (0.22 g, 0.4 mmol, 1 eq.), 10 mL AcOH and 30 mL CHCl3, evacuate, pass argon, stir at room temperature for 20 min, then add iodosuccinimide (NIS), and continue to stir the mixture in the dark overnight at room temperature under argon protection.

[0104] Example 2 Synthesis of a visual supramolecular photosensitizer for photodynamic antibacterial therapy

[0105] The diarylethenes (DAE-p\A1\A2\A3) and Aza-BODIPY (D0\D1\D2) molecules synthesized above were paired and encapsulated in a solution of sodium alginate and In the composed nanoparticles, a supramolecular photosensitizer is formed according to the method described above.

[0106] Through testing, it was found that when DAE-p and D1 (where the mass ratio of DAE-p to D1 is 5.5:1) were selected, the total mass of diarylethene molecules (DAE) and Aza-BODIPY was The supramolecular photosensitizer formed by the method has excellent photochromic, fluorescent switching and photodynamic properties, and can be used to prepare photodynamic therapy materials, fluorescent contrast agents and photochromic agents.

[0107] Its advantages mainly include the following aspects:

[0108] Photochromism:

[0109] 1. The ring opening and closing forms of the diarylethene molecule DAE-p will be significantly inhibited in water, thereby greatly reducing its color change. The DAE-p@F127 formed after encapsulation restores its open and closed ring forms, and the color change is more obvious than in water. Figure 1 ;

[0110] 2. The ring opening and closing forms of the diarylethene molecule DAE-p will be significantly inhibited in water, reducing the change in fluorescence before and after the switch. After encapsulation, supramolecular photosensitizer DAE-p NPs was formed, and its color change reached the same level as that of single use. Package, similar effect. For specific test results, see Figure 1 ; 3. The supramolecular photosensitizer D1-DAE-p NPs can still change color under 365nm ultraviolet light, but the color

[0111] The color change efficiency is slower than that without Aza-BODIPY (D1), and it takes 15 minutes from the original 30 seconds to reach the closed-loop equilibrium state. Under 808nm near-infrared light, the efficiency of DAE-p to restore the open loop decreases relatively little, extending from the original 10 seconds to 70 seconds. For specific test results, see Figure 2 ;

[0112] Fluorescent switch performance:

[0113] 4. The diarylethene molecule DAE-P itself has the performance of fluorescence switch. After encapsulation, the formed assembly DAE-p@F127, as well as the The DAE-p NPs formed after encapsulation still have the performance of fluorescence switching. Figure 4 , 5 ;

[0114] 5. Addition of Aza-BODIPY (D1) and DAE-p in a Ca2+ environment with sodium alginate and

[0115] The supramolecular photosensitive gold D1-DAE-p NPs formed after encapsulation still have the performance of fluorescence switching, that is, the addition of D1 does not cause DAE-p to lose its fluorescence switching performance. Figure 5 ;

[0116] Photodynamic performance:

[0117] 6. Aza-BODIPY (D1) can generate singlet oxygen under 365nm ultraviolet light and 808nm near-infrared light. The test results show that the efficiency of D1 in generating singlet oxygen is the highest under 365nm light in H2O, followed by D1 in organic solvent DMSO under 808nm light, and finally in H2O under 808nm light. For specific results, see Figure 6 ;

[0118] 7. Under 808nm light, The packaging of D1 had almost no effect on the production of singlet oxygen, but under 365nm light, The packaging of D1 has a certain degree of strengthening effect on the ability of D1 to produce singlet oxygen. The specific results are shown in Figure 7 ;

[0119] 8. Aza-BODIPY (D2) can generate singlet oxygen under 365nm ultraviolet light and 808nm near-infrared light, and the highest efficiency of singlet oxygen generation is under DMSO and 808nm light, followed by D2 in H2O, 365nm light, and finally D2 in H2O, 808nm light.

[0120] The order of the rates is different, indicating that the solvent is the main factor affecting the efficiency of D2 in producing singlet oxygen. Figure 8 ;

[0121] 9. Whether under 808nm near-infrared light or 365nm ultraviolet light, Bag

[0122] When other conditions are the same, the efficiency and amount of singlet oxygen produced by D2 are improved to a certain extent.

[0123] Therefore, we can get The conclusion is that the packaging has a positive effect on the production of D2 singlet oxygen. Fig. 9 ;

[0124] 10. Under the near-infrared light of 808nm in the same DMSO solution, the order of ability to produce singlet oxygen is: D2>ICG>D1>

[0125] D0. Thus, we found that D2 has the strongest ability to produce singlet oxygen under 808nm near-infrared light in DMSO solution. Moreover, the introduction of aldehyde group and heavy metal iodine can enhance the ability of Aza-BODIPY to produce singlet oxygen. See the specific results for details. Fig.10 ;

[0126] 11. In the same H2O solution, under 808nm near-infrared light irradiation, the order of ability to produce singlet oxygen is: D2>D0>D1>

[0127] ICG. The same as in DMSO and 808nm light is that D2 still has the strongest ability to produce singlet oxygen, but the ability of ICG and D1 to produce singlet oxygen is relatively weaker. It can be found that the type of solvent has a great influence on the ability of the same substance to produce singlet oxygen. For example, the introduction of aldehyde groups can make D1's ability to produce singlet oxygen in DMSO relatively stronger, but in H2O it can make its ability to produce singlet oxygen relatively weaker. See the specific results. Fig.11 ;

[0128] 12. Under the same H2O solution and 365nm ultraviolet light, the order of singlet oxygen generation capacity is: D0>D1>

[0129] D2>ICG. ICG produces almost no singlet oxygen. Compared with D2, which has the strongest ability to produce singlet oxygen under DMSO or 808nm near-infrared light, it is relatively insensitive to 365nm ultraviolet light, resulting in a significant decrease in its ability to produce singlet oxygen under 365nm light. See the specific results for details. Fig.12 ;

[0130] 13. Under 808 nm light, DAE-p always maintains a stable open-ring form. The presence of open-ring DAE-p makes the photodynamic effect of D1-DAE-p NPs similar to that of D1 alone in H2O or in combination with The D1@F127 assembly formed by the encapsulation is similar, which to some extent shows that the existence of the open-ring form of DAE-p does not affect the photodynamic performance of D1. Fig.13 ;

[0131] Under 14.365nm light, DAE-p always maintains a closed ring form, and DAE-p that always maintains a closed ring form has no effect on the production of singlet oxygen in D1. Therefore, we can conclude that D1-DAE-p NPs have a photodynamic force similar to that of D1.

[0132] Performance. See the specific results Fig.14 ;

[0133] Anti-fatigue performance:

[0134] 15. The fatigue resistance of the cyclic switch is shown in Fig.15 and 16 .

[0135] Antibacterial activity: Staphylococcus aureus (S.aureus) was cultured in LB medium to 0.8×108CFU·mL-1, and then diluted to 2×105CFU·mL-1 using 5% DMSO / PBS. 50uL of D1 dispersion of different concentrations (1024ug·mL-1, 512ug·mL-1, 256ug·mL-1, 128ug·mL-1, 64ug·mL-1, 0ug·mL-1) was added to a 96-well plate, and then 50uL of bacterial suspension was added to each well. The 1W·cm-2 808nm light source, 100mW·cm-2 simulated sunlight light source, and 35mW·cm-2 365nm light source were used for 10min, 30min, and 5min, respectively. 3.5uL of suspension was added to the solid culture plate from each well, and the plate was cultured in a 37°C incubator for 24h before taking photos to evaluate the antibacterial properties of the molecule.

[0136] 16.D1 can produce singlet oxygen to destroy bacterial cell membranes under 35mW·cm-2 365nm ultraviolet light, 100mW·cm-2 simulated fluorescent light, or 1W·cm-2 808nm near-infrared light, showing certain antibacterial properties. See the specific results for details. Fig.17 ;

[0137] 17. The visualization of the present invention can be used for photodynamic antibacterial therapy of supramolecular photosensitizer, using sodium alginate and

[0138] Through the host-guest supramolecular force wrapping, spherical nanoparticles within the range of 332nm can be formed. The specific test results are shown in Fig.18 .

Claims

1. A supramolecular photosensitizer for visualized photodynamic antibacterial therapy, characterized in that: It is composed of two molecules, diarylethene (DAE) and Aza-BODIPY. F-127, sodium alginate, diarylethene (DAE) and Aza-BODIPY synthesized in Ca 2+ Under the presence of , supramolecular photosensitizers are formed by mixed-layer assembly.

2. A supramolecular photosensitizer for visualized photodynamic antibacterial therapy according to claim 1, characterized in that: The diarylethene molecules (DAE) and Aza-BODIPY are encapsulated in a mixture of sodium alginate and In the nanoparticle shell composed of F-127, a supramolecular photosensitizer with a core-shell structure is formed.

3. A supramolecular photosensitizer for visualized photodynamic antibacterial therapy according to claim 1, characterized in that: The structures of diarylethenes include one or more of the following four types (DAE-p, A1, A2, A3), which undergo ring-opening and ring-closing structural changes and fluorescence changes under irradiation at 365nm and 808nm: The structural formula of Aza-BODIPY includes one or more of the following D0-D2:

4. A method for preparing a supramolecular photosensitizer for visualized photodynamic antibacterial therapy according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Weigh F-127 and sodium alginate were dissolved in deionized water and sonicated to completely dissolve them to form a nanoshell solution; (2) preparing a DMSO solution of a mixture of diarylethenes (DAE) and Aza-BODIPY to form a core solution; and adding dropwise to the nanoshell solution of step (1) under the action of ultrasound, continuing ultrasound for a period of time, and stirring after the ultrasound to allow for full self-assembly; (3) adding CaCl2 solution and continuing ultrasonication to fully cross-link the nanoshell liquid to form supramolecular photosensitizer nanoparticles with a more compact and stable core-shell structure; (4) separating the small molecules not encapsulated in the nanoparticles prepared in step (3) by centrifugation; The nanoparticles are washed with ultrapure water for multiple times, and finally the clean nanoparticles are placed in a ventilated and dry environment for drying for later use.

5. The preparation method according to claim 4, characterized in that: In step (1) The mass ratio of F-127 to sodium alginate is 4:1; In step (2), the mass ratio of diarylethene molecules (DAE) to Aza-BODIPY is 1:10-10:1; the total mass of diarylethene molecules (DAE) and Aza-BODIPY is The F-127 has a mass ratio of 1:10-1:

20.

6. The preparation method according to claim 4, characterized in that: The added concentration of CaCl2 in step (3) is 15mmol / L.

7. The preparation method according to claim 4, characterized in that: The preparation method of diarylethene molecules comprises the following steps: (1) Synthesis of 4-bromo-5-methylthiophene-2-carboxaldehyde First add 5-methyl-2-thiophene aldehyde, then add glacial acetic acid, and finally add N-bromosuccinimide solution to turn orange-yellow; react at room temperature, and separate from the system after the reaction; (2) Synthesis of 3-bromo-5-(diethoxymethyl)-2-methylthiophene Weigh 4-bromo-5-methylthiophene-2-carboxaldehyde, triethyl orthoformate, and ammonium chloride, put them into a reactor, then add ethanol, react at room temperature, separate from the system after the reaction, and immediately proceed to the next step of reaction; (3) Synthesis of 4,4'-(perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-2-carboxaldehyde) Put in dry 3-bromo-5-(diethoxymethyl)-2-methylthiophene and dissolve it in tetrahydrofuran, evacuate, pass argon, put in a cold well at -78°C, then add n-butyl lithium dropwise, add octafluorocyclopentene solution and gradually turn into yellow-brown, continue to react in the cold well for 2 hours, then take out, stir and react at room temperature in the dark; then add hydrochloric acid and a few drops of water, stop the reaction if there is no purple spot on the plate after a few minutes, add a little saturated ammonium chloride after the reaction is completed to quench the unreacted n-butyl lithium, and then separate; (4) Synthesis of 2-(4-(pyridin-4-yl)phenyl)acetonitrile Weigh 4-bromobenzeneacetonitrile and 4-pyridineboronic acid, add them to a bottle containing an aqueous solution of potassium carbonate, add isopropanol, evacuate and pass argon, add tetrakis(triphenylphosphine)palladium solution under the protection of argon to form a dark green color, reflux with stirring and avoid light for reaction, separate after the reaction to obtain a white powder; (5) Synthesis of A1: Dissolve 4,4'-(perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-2-carboxaldehyde) and 2-(4-(pyridin-4-yl)phenyl)acetonitrile in tert-butanol, stir, evacuate, pass argon, heat, add a methanol solution of sodium methoxide dropwise, and continue stirring in the dark for a period of time, then stop the reaction, separate, and obtain a light yellow powder A1; 2 eq of 2-(4-(pyridin-4-yl)phenyl)acetonitrile for every 1 eq of 4,4'-(perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-2-carboxaldehyde); (6) Synthesis of A2 Weigh 4,4'-(perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-2-carboxaldehyde) and 2-(4-(pyridin-4-yl)phenyl)acetonitrile, add tert-butyl alcohol, evacuate, and pass argon; heat, add a methanol solution in which sodium methoxide is completely dissolved dropwise into the reaction system, continue stirring, and protect from light throughout the reaction; separate, and a yellow-green powder A2 can be obtained; For every 1 eq of 4,4'-(perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-2-carbaldehyde), there is 4 eq of 2-(4-(pyridin-4-yl)phenyl)acetonitrile; (7) Synthesis of A3 Dissolve cisplatin and silver nitrate in DMF, heat and stir, a white solid precipitates, centrifuge the white silver chloride to obtain a light yellow solution, weigh A2 and add it to the light yellow solution obtained above, continue to stir and react at 55°C in the dark, after the reaction is completed, cool and separate to obtain a black solid A3; The structure of Aza-BODIPY is as follows: (1) Synthesis of (E)-3-(5-methylthiophen-2-yl)-1-phenylpropyl-2-en-1-one 5-Methyl-2-thiophenecarboxaldehyde and methanol were added, and then the mixture solution was cooled to 0°C, and then sodium hydroxide aqueous solution was added dropwise under stirring, and then acetophenone was added dropwise, and the solution was heated to room temperature, stirred overnight, and separated to obtain a yellow solid 1; (2) Synthesis of 3-(5-methylthiophen-2-yl)-4-nitro-1-phenylbutan-1-one Nitromethane, (E)-3-(5-methylthiophen-2-yl)-1-phenylpropyl-2-en-1-one and diethylamine were added to the reactor, and methanol was added, and heated to reflux overnight; separated to obtain a dark yellow oily solid 2; (3) Synthesis of (Z)-3-(5-methylthiophen-2-yl)-N-(3-(5-methylthiophen-2-yl)-5-phenyl-1H-pyrrol-2-yl)-5-phenyl-2H-pyrrol-2-imine Take the reactor, add ammonium acetate, evacuate and pass argon, then dissolve 3-(5-methylthiophen-2-yl)-4-nitro-1-phenylbutan-1-one in n-butanol and inject it into a Schlenk container, and heat the solution under reflux for 48 hours; cool the solution to room temperature, separate and obtain a black solid 3; (4) Synthesis of 5,5-difluoro-1,9-bis(5-methylthiophene-2-yl)-3,7-diphenyl-5H-4-14,5-dipyrrolo[1,2-c:2',1'-f][1,3,5,2]triazacytidine D0 Take a reactor, add (Z)-3-(5-methylthiophene-2-yl)-N-(3-(5-methylthiophene-2-yl)-5-phenyl-1H-pyrrol-2-yl)-5-phenyl-2H-pyrrol-2-imine and dichloromethane, evacuate and pass argon, stir the mixture at 0°C for reaction, then add N,N-diisopropylethylamine dropwise in an ice-water bath, continue to stir the mixture solution, then add boron trifluoride etherate, stir and react at room temperature under argon atmosphere, and after the reaction, filter to obtain a dark blue solid residue that turns red under sunlight; (5) Synthesis of 5,5-difluoro-1,9-di(5-methylthiophene-2-yl)-3,7-diphenyl-5H-414,514-dipyrrolo[1,2-c:2',1'-f][1,3,5,2]triazaboropurine-2-carboxaldehyde D1 Take a reactor bottle, add D0 and dichloromethane, and place it in an ice bath to cool to 0°C. Add dimethylformamide dropwise under stirring, and then add dichloromethane containing trichlorophosphine to the above solution; heat the solution to room temperature and reflux overnight, separate and purify to obtain a blue solid; (6) Synthesis of 5,5-difluoro-2,8-diiodo-1,9-di(5-methylthiophen-2-yl)-3,7-diphenyl-5H-4l4,5-dipyrrolo[1,2-c:2',1'-f][1,3,5,2]triazacytidine D2 Take the reactor, add DO, AcOH and CHCl3, evacuate, pass argon, stir at room temperature for a while, add iodosuccinimide (NIS), and continue to stir the mixture in the dark overnight at room temperature under argon protection.

8. Use of the supramolecular photosensitizer for visualized photodynamic antibacterial therapy according to any one of claims 1 to 3 for preparing visualized photodynamic therapy materials, fluorescent contrast agents, and photochromic agents.

9. Use of the supramolecular photosensitizer for visualized photodynamic antibacterial therapy according to any one of claims 1 to 3 to prepare a visualized antibacterial agent.

10. Different types of molecules that react simultaneously under 365nm and 808nm irradiation, characterized in that, The diarylethene photochromic molecule undergoing ring-opening and ring-closing structure changes under irradiation at 365nm and 808nm has the following structure: Aza-BODIPY molecules can generate singlet oxygen under 365nm ultraviolet light and 808nm near-infrared light. The structure is as follows:

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