Anthocyanin-platinum (II) complex Pt-CH2-CY and application thereof

Abstract

The invention relates to the field of biological medicines, in particular to an ultrasonic / near-infrared light dual-driven platinum (II) complex and a preparation method thereof, provides an anthocyanin-platinum (II) complex Pt-CH2-C which can doubly excite response performance under specific ultrasonic and near-infrared light power, overcomes the defect that existing antibiotics easily cause bacterial drug resistance, and particularly provides an anthocyanin-platinum (II) complex Pt-CH2-C which can be used for preparing an antibiotic drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug. The Pt-CH2-CY complex has a relatively strong near-infrared light / ultrasonic-assisted inhibition effect on staphylococcus aureus, and a new molecular basis is provided for development of novel efficient antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an ultrasound / near-infrared light dual-driven anthocyanin-platinum (II) complex Pt-CH2-CY and its uses. Background Art

[0002] The long-term use of antibiotics has led to the emergence of a large number of drug-resistant bacteria, and the emergence of drug-resistant bacteria has caused serious health hazards globally. For example, in 2019, 4.95 million death cases worldwide were directly related to drug-resistant bacteria, of which 1.27 million were clearly attributed to drug resistance. Especially in severe infections, the mortality rate of patients infected with drug-resistant bacteria is more than 30% higher than that of patients infected with sensitive bacteria. In particular, when ICU patients are complicated with bloodstream infections, the mortality rate can reach 50%. The research and development cycle of new antibiotics takes 10 - 15 years, while drug resistance can emerge in just 2 - 3 years, and only 12% of the existing R & D pipelines target Gram-negative drug-resistant bacteria. Therefore, developing new treatment methods for combating drug-resistant bacterial infections has important practical significance.

[0003] Ultrasound technology has been widely used in clinical medicine, specifically including diagnostic imaging, drug delivery, thrombus treatment, gene therapy, site-specific drug activation, and sonodynamic therapy (SDT), etc. Among them, sonodynamic therapy is a relatively new treatment method, and currently, explorations have been carried out in the field of medical anti-bacteria. The principle of SDT is that under the action of ultrasound, acoustic cavitation will occur in the liquid medium. After the acoustic cavitation collapses, shear force on bacteria will be generated, directly causing bacterial death, and at the same time, heat or light will be generated to activate sonosensitizer molecules. When sonosensitizer molecules are activated, they can achieve antibacterial effects through two pathways. One is to sensitize surrounding oxygen molecules and water molecules to generate highly physiologically toxic reactive oxygen species (ROS), causing the inactivation of bioactive molecules of bacteria or cells in the lesion. The other is that the activated sonosensitizer molecules will produce a thermal effect, causing the death of bacteria in the lesion. Compared with traditional chemical drug antibiotics, the advantage of SDT is that it is not easy to cause bacteria to develop drug resistance, because it causes bacterial death through direct physical effects or chemical reactions, rather than the biomolecular binding mechanism of traditional antibiotics. In addition, SDT uses ultrasound as an energy source, which has strong practical significance in the treatment of deep lesions in the body.

[0004] Meanwhile, the Photodynamic Therapy has been used in the treatment of bacterial infections in skin wounds in some countries. Photodynamic therapy requires the presence of oxygen, photosensitizers (PSs), and light. The main principle of antibacterial photodynamic therapy is the absorption of light and the transfer of energy. In antibacterial photodynamic therapy, a photochemical reaction that generates reactive oxygen species occurs; after light irradiation, the PSs transition from the ground state (S0) to the excited singlet state (S1), and then through intersystem crossing (ISC) become the excited triplet state (T1); next, it can return to the ground state (S0) by generating reactive oxygen species (ROS) through two mechanisms (type I and type II). Type I is that the T1 photosensitizer transfers electrons or protons to the organic substances of the cell, generating various free radicals that then react with molecular oxygen to generate reactive oxygen species, mainly superoxide radicals (•O2 - ), hydroxyl radicals (•OH), and hydrogen peroxide radicals (H2O2), etc., so type I is usually called the free radical mechanism. Type II is that the photosensitizer of T1 transfers energy to the triplet oxygen molecule through energy transfer, and then generates singlet oxygen ( 1 O2), and this mechanism is also called the singlet oxygen mechanism. The generated reactive oxygen species are the effective substances that can kill bacteria. Since the antibacterial photodynamic effect is based on the oxidative damage of the bacterial membrane, resulting in cytotoxic effects, it can be used for all types of microorganisms without considering drug resistance barriers. However, most of the currently clinically used anti-infective photosensitizers have absorption in the visible light range, and their defect is that it is difficult to achieve effective treatment of deep tissue infections. Therefore, the current main strategy is to adjust the absorption of the photosensitizer to the near-infrared range to reduce the defects of the material itself. Selecting a suitable molecule with both ultrasonic activity and near-infrared activity as an ultrasonic / light combined treatment reagent is expected to fundamentally improve the treatment effect of drug-resistant bacterial infections without considering drug resistance issues. Summary of the Invention

[0005] Aiming at the problem that the anti-infective photosensitizers in the prior art have ineffective curative effects on deep tissue infections, the present invention provides an anthocyanin-platinum (II) complex with dual excitation responses to ultrasound / near-infrared light, which is expected to provide a new molecular basis for realizing ultrasound-near-infrared photodynamic dual-mode treatment of in vivo bacterial infections.

[0006] The present invention first provides an anthocyanin-platinum (II) complex Pt-CH2-C, and the structural formula of the complex Pt-CH2-CY is as follows: .

[0007] The above complex Pt-CH2-CY has dual excitation response performance under specific ultrasound and near-infrared photodynamic conditions.

[0008] The present invention also provides a method for preparing the above-mentioned anthocyanin-platinum (II) complex Pt-CH2-CY, which comprises the following steps: In the first step, using acetic anhydride as a solvent, 1,2,3,3-tetramethyl-3H-indolium iodide reacts with 3-hydroxymethylene-1-formyl-2-chloro-1-cyclohexene to generate intermediate A: 2-((E)-2-((E)-2-chloro-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide. The reaction formula is as follows: ; In the second step, using N,N-dimethylformamide (DMF) as a solvent and potassium carbonate as an acid-binding agent, intermediate A reacts with 4-mercaptopyridine to obtain intermediate B: 1,3,3-trimethyl-2-((E)-2-((E)-2-(pyridin-4-ylthio)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium iodide. The reaction formula is as follows: ; In the third step, intermediate B reacts with cisplatin salt activated by silver nitrate to obtain the anthocyanin-platinum (II) complex Pt-CH2-CY. The reaction formula is as follows: .

[0009] Further, in the third step, the method for treating cisplatin with silver nitrate is as follows: Dissolve cisplatin in N,N-dimethylformamide (DMF), then add silver nitrate. After the reaction at room temperature is completed, centrifuge to remove the precipitate, and directly use the supernatant for the next step of the reaction. The reaction formula is as follows: .

[0010] Further, in the first step, using acetic anhydride as a solvent, prepare a 3-hydroxymethylene-1-formyl-2-chloro-1-cyclohexene solution with a concentration of 0.010 M. Then, mix 1,2,3,3-tetramethyl-3H-indolium iodide and 3-hydroxymethylene-1-formyl-2-chloro-1-cyclohexene in a molar ratio of 220:100 with the solution, heat up to 125 - 135 °C, and react for 24 - 36 hours. After the reaction is completed, use a rotary evaporator to remove the reaction solvent. The crude product is separated and purified by a silica gel chromatographic column to obtain intermediate A. Among them, the silica gel powder has a specification of 200 - 300 mesh, and the chromatographic eluent is a mixed solution of dichloromethane:methanol with a volume ratio of 10:1.

[0011] Further, in the second step, using N,N-dimethylformamide (DMF) as a solvent, a 4-mercaptopyridine solution with a concentration of 0.010 M is prepared. Then, the intermediate product A and potassium carbonate (K2CO3) are added to the solution for a mixed reaction. Among them, the molar ratio of the intermediate product A, potassium carbonate (K2CO3) to 4-mercaptopyridine is 110:100:100. The reaction temperature is 125 - 135 °C, and the reaction time is 36 - 48 hours. After the reaction, the reaction solvent is removed using a rotary evaporator. The crude product is separated and purified by a silica gel chromatographic column to obtain intermediate product B. Among them, the silica gel powder has a specification of 200 - 300 mesh, and the chromatographic eluent is a mixed solution of chloroform:methanol with a volume ratio of 10:1.

[0012] Further, in the third step, first using N,N-dimethylformamide (DMF) as a solvent, a cisplatin solution with a concentration of 0.01 M is prepared. Then, silver nitrate is added according to the molar ratio of silver nitrate to cisplatin of 220:100 as an activator for cisplatin to remove chloride ions. The reaction temperature is 20 - 30 °C, and the reaction time is 24 - 36 hours. After the reaction, it is set aside.

[0013] Still further, in the third step, first using N,N-dimethylformamide (DMF) as a solvent, a silver nitrate-activated cisplatin salt with a concentration of 0.01 M is prepared. Then, intermediate product B is added according to the molar ratio of intermediate product B to cisplatin salt of 100:50. At 20 - 30 °C, the reaction time is 24 - 36 hours. After the reaction, the solvent DMF is removed by vacuum distillation. The obtained crude product is recrystallized and separated using a methanol-ether mixed solution with a volume ratio of 1:2 - 1:3 to obtain the complex Pt-CH2-CY.

[0014] The anthocyanin platinum (II) complex Pt-CH2-CY of the present invention has the following advantages: (1) Compared with platinum photosensitizer complexes with the same function, the preparation processes of the two complexes involved in this patent are relatively simple, the separation process is simple, and the yield is considerable; (2) Compared with traditional sonosensitizers or photosensitizers, the Pt-CH2-CY molecule of the present invention has dual excitation source responsiveness and has a more efficient antibacterial effect; (3) The antibacterial mechanism through biodynamic or photodynamic antibacterial reagents is that reactive oxygen species (ROS) generated under ultrasonic or light excitation act as actual antibacterial substances, which is different from the biochemical action of traditional antibiotics. Specifically, it is not easy to induce bacteria to produce drug resistance; (4) The dual-responsive Pt-CH2-CY molecule of the present invention can be activated by ultrasound, near-infrared light, or simultaneously to play an antibacterial role. Ultrasound or near-infrared light has strong penetration ability, and it is expected to achieve the treatment of bacteria interference in deep tissues in the body.

[0015] The present invention further provides an antibacterial use of an anthocyanin-platinum (II) complex Pt-CH2-CY under ultrasound and near-infrared photodynamic activation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 1H NMR spectrum of Pt-CH2-CY prepared in Example 1 of the present invention; Figure 2 UV-Vis absorption spectrum of Pt-CH2-CY in Example 2 of the present invention; Figure 3 Fluorescence emission spectrum of Pt-CH2-CY in Example 2 of the present invention; Figure 4 Fluorescence intensity change diagram of DCFH solution at 488 nm under irradiation of a 808 nm light source with an intensity of 40 mW / cm 2 when irradiated, the test time interval is 60 s; Figure 5 Fluorescence intensity change diagram of DCFH solution at 488 nm under the action of ultrasound at 1 MHz and 0.7 W / cm 2 when Pt-CH2-CY in Example 3 of the present invention is used, the test time interval is 60 s; Figure 6 Killing efficiency diagram of Pt-CH2-CY in Example 4 of the present invention under irradiation of a 808 nm light source with an intensity of 40 mW / cm 2 against Staphylococcus aureus; Figure 7 Photograph of the number of colonies on the plate medium of Staphylococcus aureus under irradiation of a 808 nm light source with an intensity of 40 mW / cm 2 when Pt-CH2-CY in Example 4 of the present invention is irradiated; Figure 8 Killing efficiency diagram of Pt-CH2-CY in Example 5 of the present invention under the action of ultrasound at 1 MHz and 0.7 W / cm 2 against Staphylococcus aureus; Figure 9 Photograph of the number of colonies on the plate medium of Staphylococcus aureus under the action of ultrasound at 1 MHz and 0.7 W / cm 2 when Pt-CH2-CY in Example 5 of the present invention is used. DETAILED DESCRIPTION OF THE INVENTION

[0017] The technical solutions of the present invention will be further described below with reference to the accompanying drawings, but the present invention is not limited by the embodiments.

[0018] Example 1: Preparation of the complex Pt-CH2-CY.

[0019] In this embodiment, the chemical structural formula of the complex Pt-CH2-CY is:

[0020] (1) 1,2,3,3-Tetramethyl-3H-indolium iodide (66.22 mg, 0.22 mmol), 3-hydroxymethylene-1-formyl-2-chloro-1-cyclohexene (17.20 mg, 0.10 mmol) and 1 mL of acetic anhydride were placed in a 10 mL single-necked flask. Nitrogen was filled into the single-necked flask to expel oxygen. Under the protection of a nitrogen atmosphere, the reaction mixture was heated to 130 °C and maintained for 36 hours. After the reaction was completed, the reaction mixture was poured into 100 mL of water, and extracted with 20 mL of dichloromethane. The organic phase was collected and dried with anhydrous magnesium sulfate (200 mg). Subsequently, the organic phase was removed using a rotary evaporator. The crude product was purified by column chromatography on silica gel (200 - 300 mesh) (the eluent was dichloromethane:methanol (10:1, volume ratio)). The purified product 2-((E)-2-((E)-2-chloro-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide was obtained as a dark brown solid. The reaction formula is shown as follows.

[0021]

[0022] (2)2-((E)-2-((E)-2-chloro-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (67.12 mg, 0.11 mmol), 4-mercaptopyridine (11.10 mg, 0.10 mmol), and the acid-binding agent potassium carbonate (K2CO3, 10.60 mg, 0.1 mmol) and 2 mL of N,N-dimethylformamide (DMF) were placed in a 10 mL single-necked flask. Nitrogen was introduced into the single-necked flask to expel oxygen, and the reaction mixture was heated to 125 °C under a nitrogen atmosphere and maintained for 24 hours. After the reaction was completed, the reaction mixture was poured into 100 mL of water, and extracted with 20 mL of dichloromethane. The organic phase was collected and dried over anhydrous magnesium sulfate (200 mg), and then the organic phase was removed using a rotary evaporator. The crude product was purified by column chromatography on silica gel (200 - 300 mesh) (the eluent was chloroform:methanol (10:1, v / v)). After purification, the product 1,3,3-trimethyl-2-((E)-2-((E)-2-(pyridin-4-ylthio)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium iodide was obtained as a black solid. The reaction scheme is shown in the following formula.

[0023]

[0024] (3)Cisplatin (30.00 mg, 0.1 mmol) was placed in a 50 ml round-bottom flask, and 10 mL of the solvent N,N-dimethylformamide was added. After cisplatin was dissolved, silver nitrate (37.16 mg, 0.22 mmol) was added, and the mixture was stirred in the dark at room temperature for 24 hours. After the reaction was completed, the silver chloride precipitate was removed by centrifugation, and the supernatant was directly used in the next reaction without further treatment. The reaction scheme is shown in the following formula.

[0025]

[0026] (4) 1,3,3-Trimethyl-2-((E)-2-((E)-2-(pyridin-4-ylthio)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium iodide (137.04 mg, 0.20 mmol) prepared in step (2) was placed in a 5 mL single-necked flask together with the silver nitrate-activated platinum salt (product in step (3)) obtained in step (3) and 1 mL of N,N-dimethylformamide (DMF). Nitrogen was charged into the single-necked flask to expel oxygen, and the reaction mixture was stirred under a nitrogen atmosphere in the dark at 25 °C for 24 hours. After the reaction, the solvent DMF was removed by vacuum distillation, and the crude product was recrystallized and separated using a methanol-ether mixed solution (volume ratio of methanol to ether is 1:2.5) to obtain the product complex Pt-CH2-CY as a dark green solid powder. The reaction equation is shown below. Its 1H NMR spectrum is as Figure 1 shown.

[0027]

[0028] Example 2: Detection of Photophysical / Chemical Properties of Complex In this example, first, the characterization of ultraviolet-visible absorption spectrum, fluorescence emission spectrum was carried out, and the photodynamic ability of Pt-CH2-CY was detected by DCFH reactive oxygen species probe, that is, the ability to generate reactive oxygen species. In the photodynamic detection and bactericidal experiment, the complex Pt-CH2-CY was formulated into a concentrated working solution of 1 mg / mL, and the solvent was DMSO:H2O = 1:9, and corresponding dilution should be carried out during actual use.

[0029] (1) Characterization of ultraviolet-visible absorption spectrum: The concentrated working solution of the complex was diluted with dichloromethane to a final concentration of 10 μM and a volume of 2.5 mL, placed in a 3 mL quartz cuvette, and its ultraviolet-visible absorption spectrum was measured using an ultraviolet spectrophotometer. The results are as Figure 2 shown. The maximum ultraviolet absorption of Pt-CH2-CY is at 790 nm, in the near-infrared range, indicating the possibility of its near-infrared responsiveness.

[0030] (2) Characterization of near-infrared fluorescence emission spectrum: The concentrated working solution of the complex was diluted with water to a final concentration of 10 μM and a volume of 2.5 mL, placed in a 3 mL quartz cuvette, and its near-infrared emission spectrum was measured using a near-infrared fluorescence spectrometer. The results are as Figure 3 shown. The maximum emission of Pt-CH2-CY is at 950 nm, in the near-infrared range.

[0031] (3)DCFH Detection: First, add 0.5 mL of 1 mM DCFH-DA ethanol solution to 2 mL of 0.01 N NaOH, stir at room temperature for 30 min, and then adjust the pH of the solution with 10 mL of 25 mM PBS with pH = 7.4. After adjustment, the pH is 7.0. When testing, take 10 μL of the 1.0 mg / mL concentrated working solution of the complex and add it to 990 μL of the DCFH solution. After mixing, take 100 μL and transfer it to a 96-well plate. Measure the fluorescence intensity at 0 s with a microplate reader, and irradiate it with an LED lamp with near-infrared light of 808 nm (40 mW / cm 2 ), record the value every 300 s, and measure until 15 min ends. The results are as Figure 4 shown. The red curve represents the fluorescence signal of the reactive oxygen species probe DCFH in the presence of Pt-CH2-CY. An increase in its signal value indicates the generation of reactive oxygen species; the black curve is the fluorescence signal of the reactive oxygen species probe without adding Pt-CH2-CY. It can be clearly seen from the figure that the probe signal increases, indicating that Pt-CH2-CY has the ability to promote the generation of reactive oxygen species under near-infrared light.

[0032] Example 3: Ultrasonic dynamic performance test of the complex In this example, after the near-infrared photodynamic ability test was completed in Example 2, the ultrasonic dynamic ability test of the complex was subsequently carried out in this example. In the photodynamic detection and sterilization experiment, the Pt-CH2-CY complex was prepared into a 1 mg / mL concentrated working solution, and the solvent was DMSO:H2O = 1:9. Corresponding dilution should be carried out during actual use.

[0033] DCFH Detection: First, add 0.5 mL of 1 mM DCFH-DA ethanol solution to 2 mL of 0.01 N NaOH, stir at room temperature for 30 min. Then adjust the pH of the solution with 10 mL of 25 mM PBS with pH = 7.4 to pH 7.0. When testing, take 10 μL of the concentrated working solution and add it to 990 μL of the DCFH solution. After mixing, take 100 μL and transfer it to a 96-well plate to make the final concentrations 15, 30, and 60 μg / mL respectively. Measure the fluorescence spectrum at 0 s (range 520 - 600 nm) with a microplate reader, and apply ultrasonic waves with a frequency of 1 MHz and a power of 0.7 W / cm 2 to the 96-well plate, and perform a fluorescence spectrum scan every 300 s for a total time of 15 min. The results are as Figure 5 shown. Under ultrasonic action, Pt-CH2-CY can also significantly increase the fluorescence signal of the reactive oxygen species probe, indicating that Pt-CH2-CY has the ability to promote the generation of reactive oxygen species under ultrasonic waves.

[0034] Example 4: Photodynamic antibacterial of the complex (1) Bacterial culture: Prepare a liquid medium for bacterial growth (100 ml LB: 1 g NaCl, 0.5 g yeast powder, 1 g peptone), and culture the Gram-positive bacterium Staphylococcus aureus (S. aureus, a multi-drug resistant bacterium). Take single colonies of SA and add them to 10 mL of LB respectively, and place them in a shaker at 37 °C for 12 hours. Shake until the OD value measured by a nucleic acid protein detector is about 1 (0.996 - 1.005). At this time, it is estimated that the number of bacteria contained in each 1 mL of bacterial liquid is 10 8 CFU / ml.

[0035] (2) Photocatalysis sterilization Experiment on the killing of Staphylococcus aureus by Pt-CH2-CY: Set the concentrations of the Pt-CH2-CY complex to 0, 0.3, 0.4, 0.5, and 0.6 μg / mL respectively. Add 100 μL of Staphylococcus aureus to make it fully mixed, and add 390 μL of PBS to make the total volume of the solution 500 μL. Incubate for 30 min under dark conditions and then irradiate with a near-infrared lamp. The light intensity is 40 mW / cm 2 for 20 min. Design a control group as the group incubated for 20 min under dark conditions without near-infrared light irradiation.

[0036] (3) Spot plate counting: Take out 5 1.5 mL EP tubes and place them on an EP tube rack, and add 900 μL of PBS buffer solution to each tube. First, use a pipette to transfer 100 μL of the original bacterial liquid in the experimental well plate, and dilute the bacterial liquid in gradients to 10 3 CFU / mL, and plate 10 3 CFU / mL. Place the solid medium after spotting (the composition of 100 ml of solid medium is: 1 g sodium chloride, 0.5 g yeast powder, 1 g peptone, 1.5 g agar) in an incubator at 37 °C for overnight culture to allow the bacteria to grow naturally.

[0037] (4) Calculation of sterilization rate: On the overnight solid medium, the bacteria grow into visible bacterial colonies to the naked eye. Calculate its sterilization efficiency according to the change in the number of colonies in the control group (the complex concentration is 0). Sterilization efficiency = (C0 - C) / C0 × 100%, where C is the number of colonies under light irradiation conditions, and C0 is the number of colonies under dark conditions. As Figure 6 shown, after irradiation with near-infrared light, the sterilization efficiency of Pt-CH2-CY is concentration-dependent. The sterilization efficiency is about 30% at a concentration of 0.3 μg / mL, about 70% at 0.4 μg / mL, about 90% at 0.5 μg / mL, and 100% at 0.6 μg / mL (5) Repeated experiment: Repeat the operation in step (4) three times to verify the sterilization test and make the sterilization rate reach 100% after light irradiation. The experimental results are as Figure 6-7 shown.

[0038] Example 5: Ultrasound - powered antibacterial of the complex (1) Bacterial culture: Prepare a liquid medium for bacterial growth (100 ml LB: 1 g NaCl, 0.5 g yeast extract, 1 g peptone), and culture Gram - positive bacterium Staphylococcus aureus (S. aureus, a multi - drug - resistant bacterium). Take single colonies of SA and add them to 10 ml LB respectively, and place them in a shaker at 37 °C for 12 hours. Shake until the OD value measured by a nucleic acid protein detector is about 1 (0.996 - 1.005). At this time, it is estimated that the number of bacteria contained in each 1 ml of the bacterial solution is 10 8 CFU / mL.

[0039] Ultrasound sterilization: First, dilute Staphylococcus aureus to 10 6 CFU / mL. Set the concentrations of Pt - CH2 - CY complex to 0 μg / mL, 0.6 μg / mL, 1.2 μg / mL, 2.4 μg / mL, and 4.8 μg / mL. Add 100 μL of the diluted Staphylococcus aureus respectively, and dilute it with PBS. Incubate for 30 min under dark conditions. Set the frequency of the ultrasonic instrument to 1 MHz and the power to 0.7 W / cm 2 The ultrasonic time is 3 minutes. The blank group is set to directly plate - count without ultrasound at different concentrations.

[0040] (3) Plate - counting: Take out 3 1.5 - mL EP tubes and place them on an EP tube rack. Add 900 μL of PBS buffer solution to each tube. First, use a pipette to transfer 100 μL of the original bacterial solution in the experimental well - plate, and dilute the bacterial solution in a gradient to 10 3 CFU / ml, and plate 10 3 CFU / ml. Place the solid medium after plating (the composition of 100 ml solid medium: 1 g sodium chloride, 0.5 g yeast extract, 1 g peptone, 1.5 g agar) in an incubator at 37 °C for overnight culture to allow the bacteria to grow naturally.

[0041] (4)Calculation of bactericidal rate: On the solid medium overnight, bacteria grew into visible bacterial colonies with the naked eye. The number of colonies on the culture plates with different material concentrations was counted. The bactericidal rate with a material concentration of 0 μg / mL was used as the control group, and the blank group was set to directly count the electroplates without ultrasonic treatment at different concentrations, and its bactericidal efficiency was calculated. Bactericidal efficiency = (C0 - C) / C0 × 100%, where C is the number of colonies after ultrasonic treatment at different concentrations, and C0 is the number of colonies without ultrasonic treatment at different concentrations. The results showed that the bactericidal rate after ultrasonic treatment of the blank group with a material concentration was below 20%, while when the material concentration was 0.6 μg / mL, the bactericidal efficiency was 90%; when the concentrations were 1.2 μg / mL, 2.4 μg / mL, and 4.8 μg / mL, the bactericidal rates after ultrasonic treatment were all 100% (5)Repeated experiment: Repeat the operation in step (4) above, and perform ultrasonic bactericidal treatment on SA with Pt-CH2-CY at different concentrations respectively, and repeat it three times in total to verify the stability of the ultrasonic bactericidal experiment. The results are as Figure 8-9 shown. Except that the bactericidal rate was 90% when the concentration of the Pt-CH2-CY complex was 0.6 μg / mL, the ultrasonic bactericidal rates of the Pt-CH2-CY complex at other concentrations were all 100%, and the bactericidal effect was very ideal.

Claims

1. An anthocyanin-platinum (II) complex Pt-CH2-CY, characterized in that, The structural formula of the complex Pt-CH2-CY is as follows: 。 2. The anthocyanin-platinum (II) complex according to claim 1, characterized in that, The complex Pt-CH2-CY has dual excitation response performance under specific ultrasound and near-infrared photodynamic conditions.

3. A method for preparing the anthocyanin-platinum (II) complex Pt-CH2-CY according to claim 1 or 2, characterized in that, It includes the following steps: Step 1: Using acetic anhydride as the solvent, reacting 1,2,3,3-tetramethyl-3H-indolium iodide with 3-hydroxymethylene-1-formyl-2-chloro-1-cyclohexene to generate intermediate product A: 2-((E)-2-((E)-2-chloro-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide. The reaction formula is as follows: ； Step 2: Using N,N-dimethylformamide (DMF) as the solvent and potassium carbonate as the acid-binding agent, reacting intermediate product A with 4-mercaptopyridine to obtain intermediate product B: 1,3,3-trimethyl-2-((E)-2-((E)-2-(pyridin-4-ylthio)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium iodide. The reaction formula is as follows: ； Step 3: Reacting intermediate product B with cisplatin salt activated by silver nitrate to obtain the cyanidin-platinum (II) complex Pt-CH2-CY. The reaction formula is as follows: 。 4. The preparation method of the anthocyanin-platinum (II) complex Pt-CH2-CY according to claim 3, characterized in that, In Step 3, the method for treating cisplatin with silver nitrate is as follows: Dissolve cisplatin in N,N-dimethylformamide (DMF), then add silver nitrate. After the reaction at room temperature is completed, centrifuge to remove the precipitate, and directly use the supernatant for the next reaction. The reaction formula is as follows: 。 5. The preparation method of the anthocyanin-platinum (II) complex Pt-CH2-CY according to claim 3, characterized in that, In Step 1, using acetic anhydride as the solvent, prepare a 0.010 M solution of 3-hydroxymethylene-1-formyl-2-chloro-1-cyclohexene. Then mix 1,2,3,3-tetramethyl-3H-indolium iodide and 3-hydroxymethylene-1-formyl-2-chloro-1-cyclohexene in a molar ratio of 220:100 with the solution, heat to 125 - 135 °C, and react for 24 - 36 hours. After the reaction is completed, use a rotary evaporator to remove the reaction solvent. The crude product is separated and purified by a silica gel chromatography column to obtain intermediate product A. Among them, the silica gel powder has a specification of 200 - 300 mesh, and the chromatographic eluent is a mixed solution of dichloromethane:methanol with a volume ratio of 10:

1.

6. The preparation method of the anthocyanin-platinum (II) complex Pt-CH2-CY according to claim 3, characterized in that, Step 2: Using N,N-dimethylformamide (DMF) as the solvent, prepare a 0.010 M solution of 4-mercaptopyridine. Then add intermediate product A and potassium carbonate (K2CO3) to the solution and mix them for reaction. Among them, the feeding molar ratio of intermediate product A, potassium carbonate (K2CO3), and 4-mercaptopyridine is 110:100:

100. The reaction temperature is 125 - 135 °C, and the reaction time is 36 - 48 hours. After the reaction is completed, use a rotary evaporator to remove the reaction solvent. The crude product is separated and purified by a silica gel chromatography column to obtain intermediate product B. Among them, the silica gel powder has a specification of 200 - 300 mesh, and the chromatographic eluent is a mixed solution of chloroform:methanol with a volume ratio of 10:

1.

7. The preparation method of the anthocyanin-platinum (II) complex Pt-CH2-CY according to claim 3, characterized in that, In the third step, first, using N,N-dimethylformamide (DMF) as a solvent, a cisplatin solution with a concentration of 0.01 M is prepared. Then, silver nitrate is added according to the molar ratio of silver nitrate to cisplatin being 220:100 as an activator for cisplatin to remove chloride ions. The reaction temperature is 20 - 30 °C, and the reaction time is 24 - 36 hours. After the reaction is completed, it is set aside for use.

8. The preparation method of the anthocyanin-platinum (II) complex Pt-CH2-CY according to claim 3, characterized in that, In the third step, first, using N,N-dimethylformamide (DMF) as a solvent, a silver nitrate-activated cisplatin salt with a concentration of 0.01 M is prepared. Then, intermediate B is added according to the molar ratio of intermediate B to cisplatin salt being 100:

50. At 20 - 30 °C, the reaction time is 24 - 36 hours. After the reaction is completed, the solvent DMF is removed by vacuum distillation. The obtained crude product is recrystallized and separated using a methanol-ether mixed solution with a volume ratio of 1:2 - 1:3 to obtain the complex Pt-CH2-CY.

9. The use of the anthocyanin-platinum (II) complex Pt-CH2-CY according to any one of claims 1 - 8 for activating antibacterial under ultrasound and near-infrared photodynamic.

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