Acid-base resistant nile blue photosensitizer, preparation method and application thereof

Abstract

The application discloses an acid-alkali resistant Nile blue photosensitizer, a preparation method and application thereof, and a chemical structural general formula of the acid-alkali resistant Nile blue photosensitizer is as follows: wherein R1, R2 and R3 are independently selected from at least one of C1-C6 straight-chain alkane chains. The Nile blue photosensitizer is synthesized through multi-alkylation modification, has acid-alkali resistant characteristics and stable cationic amine salt form, promotes intramolecular charge transfer, promotes the red shift of 20-40 nm of the absorption wavelength and the emission wavelength of the Nile blue photosensitizer (EB series) molecule, and the molar extinction coefficient is increased from about 45000 L.mol ‑1 ·cm ‑1 to about 80000 L.mol ‑1 ·cm ‑1 . The O2 ‑· production efficiency of the EB molecule is increased by 2.2-4.2 times compared with conventional NBS photosensitizers. The improvement of pH stability and the normal existence of cations make the EB molecule exhibit more excellent uptake performance to bacteria, and the fluorescence intensity is 2-3 times that of NBS under the same concentration.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemistry, in particular to an acid and alkali resistant nile blue photosensitizer, a preparation method and application thereof. BACKGROUND

[0002] Biofilm infection is a chronic and long-term bacterial infection disease caused by the massive reproduction of bacteria at the wound site, which has caused serious harm to public health. Because of the shielding effect of bacterial autosecretory polymer in the microenvironment of biofilm infection, conventional antibiotic treatment is difficult to take effect, often requiring excessive use of antibiotics or combined with surgical resection for treatment, but this is easy to bring about antibiotic abuse and additional treatment burden. Recently, photodynamic therapy (PDT) has been developed for antibacterial treatment, which can often achieve rapid and efficient phototherapy effect on the lesion site where the photosensitizer is distributed under the continuous generation of short-lived and strong oxidizing reactive oxygen species (ROS) under light. Because ROS can directly damage proteins, nucleic acids and other life macromolecules, it is often difficult to produce drug resistance, so it is an ideal antibacterial means.

[0003] However, dense bacteria forming biofilm is often accompanied by a low-oxygen microenvironment at the lesion site, which greatly inhibits the therapeutic activity of photosensitizers. For this reason, type I photosensitizers that generate ROS free radicals are developed for anti-hypoxic photodynamic therapy. Among them, nile blue thio (NBS) as a milestone molecule in type I photosensitizers opens up the mechanism exploration and molecular design research of type I PDT. Nile blue thio series molecules exhibit excellent light stability, biocompatibility and ROS generation performance, and are widely concerned in the current domestic and foreign PDT field. The deficiency is that the pH stability of nile blue photosensitizer is insufficient, especially the low alkali tolerance, even in a weak alkaline environment with a pH of 8, cation loss will occur, thereby the photodynamic activity is severely damaged. And, it is also concerned that the molar extinction coefficient of nile blue photosensitizer is insufficient, and the superoxide anion (O2 -· ) generation efficiency needs to be improved. These negative performances are not conducive to its treatment behaviors such as killing bacteria and removing tumor cells in complex physiological environments.

[0004] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0005] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide an acid and alkali resistant nile blue photosensitizer, a preparation method and application thereof, aiming to solve the problems of insufficient molar extinction coefficient and low superoxide anion (O2 -· ) generation efficiency of the existing photosensitizer.

[0006] The technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides an acid-base resistant nile blue photosensitizer, the chemical structure of which is as follows:

[0008]

[0009] wherein R1, R2, R3 are independently selected from C1-C6 linear alkyl chain, at least one of the following.

[0010] In a second aspect, the present application provides a preparation method of the acid-base resistant nile blue photosensitizer, which comprises the following steps:

[0011] dissolving naphthylamine and compound R2I in a first organic solvent, adding a weak alkaline solution under stirring, performing a first reaction, then adding R3I, performing a second reaction, and obtaining a first intermediate;

[0012] dissolving the first intermediate and a Bunte salt containing R1 in a strong acid solution, stirring, then adding an oxidizing agent, performing a third reaction, and obtaining a second intermediate;

[0013] dissolving the second intermediate in a second organic solvent, adding a catalyst and a reducing agent under stirring, performing a fourth reaction, and obtaining the nile blue photosensitizer.

[0014] Preferably, the first organic solvent is at least one of acetonitrile, acetone, tetrahydrofuran, acetic anhydride, DMSO, and DMF;

[0015] The second organic solvent is at least one of acetonitrile, methanol, dichloromethane, ethyl acetate, tetrahydrofuran, and DMF.

[0016] Preferably, the weak alkaline solution is at least one of sodium carbonate, potassium carbonate, sodium bicarbonate, cesium carbonate, sodium acetate, and potassium acetate;

[0017] The strong acid solution is at least one of hydrochloric acid, sulfuric acid, and nitric acid.

[0018] Preferably, the oxidizing agent is at least one of potassium dichromate, potassium permanganate, potassium perchlorate, and potassium superoxide;

[0019] The reducing agent is at least one of copper sulfate, copper chloride, copper acetate, copper oxide, cuprous oxide, and cuprous iodide.

[0020] The catalyst is at least one of manganese dioxide, manganese sulfate, manganese chloride, manganese nitrate, and manganese acetate.

[0021] Preferably, the temperature of the first reaction is 60-120℃, and the time is 1-4 hours.

[0022] The temperature of the second reaction is 60-120 DEG C, and the time is 12-24 hours.

[0023] The temperature of the third reaction is 0-4 DEG C, and the time is 2-10 hours.

[0024] The temperature of the fourth reaction is 60-90 DEG C, and the time is 10-60 minutes.

[0025] In a fourth aspect of the present application, the acid and alkali resistant Nile blue photosensitizer is used for fluorescence imaging of positive bacteria and negative bacteria.

[0026] In a fifth aspect of the present application, the acid and alkali resistant Nile blue photosensitizer is used for preparing a tumor phototherapy product.

[0027] In a sixth aspect of the present application, the acid and alkali resistant Nile blue photosensitizer is used for preparing a broad-spectrum antibacterial phototherapy reagent.

[0028] In a seventh aspect of the present application, the acid and alkali resistant Nile blue photosensitizer is used for preparing a treatment reagent for biofilm infection.

[0029] Beneficial effects: the present application provides an acid and alkali resistant Nile blue photosensitizer, a preparation method and application thereof, a series of Nile blue photosensitizers are synthesized through multi-alkylation modification, have acid and alkali resistant characteristics and stable cationic amine salt form, promote intramolecular charge transfer, promote the red shift of the absorption wavelength and the emission wavelength of the series of Nile blue photosensitizers (EB series) molecules by 20-40 nm, and the molar extinction coefficient is increased from about 45000 L·mol -1 ·cm -1 to about 80000 L·mol -1 ·cm -1 . The O2 -· production efficiency of the EB molecule is increased by 2.2-4.2 times compared with the conventional NBS photosensitizer. The improvement of pH stability and the existence of cationic normal state make the EB molecule exhibit more excellent uptake performance to bacteria, and the fluorescence intensity is 2-3 times that of NBS under the same concentration. And the excellent O2 -· production capacity can help the EB series to realize the bacterial inhibition function excited by red light or sunlight in a low oxygen environment (2% oxygen density), and the required photosensitizer concentration for complete bacterial removal is only 1 / 2 of NBS. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The synthesis route map of the Nile blue photosensitizer provided in the preferred embodiment of the present application.

[0031] Figure 2 The high-resolution mass spectrum of the Nile blue photosensitizer EB-MeP prepared in Example 1.

[0032] Figure 3 NMR spectrum of Nile blue-based photosensitizer EB-MeP prepared for Example 1.

[0033] Figure 4 Mass spectrum of Nile blue-based photosensitizer EB-dEt prepared for Example 2.

[0034] Figure 5 NMR spectrum of Nile blue-based photosensitizer EB-dEt prepared for Example 2.

[0035] Figure 6 High resolution mass spectrum of Nile blue-based photosensitizer EB-dMe prepared for Example 3.

[0036] Figure 7 NMR spectrum of Nile blue-based photosensitizer EB-dMe prepared for Example 3.

[0037] Figure 8 High resolution mass spectrum of Nile blue-based photosensitizer EB-qMe prepared for Example 4.

[0038] Figure 9 NMR spectrum of Nile blue-based photosensitizer EB-qMe prepared for Example 4.

[0039] Figure 10 Absorption spectra of photosensitizers prepared for Comparative Examples 1-2 and Examples 1-4 (in methanol).

[0040] Figure 11 Fluorescence emission spectra of photosensitizers prepared for Comparative Examples and Examples in methanol solution.

[0041] Figure 12 Electrostatic potential distribution of photosensitizers prepared for Comparative Examples and Examples.

[0042] Figure 13 (a) total ROS production; (b) O2 production; and (c) O2 production profile of photosensitizers prepared for Comparative Examples and Examples. 1 -·

[0043] Figure 14 O2 production ability and excitation wavelength dependence of photosensitizers prepared for Comparative Examples and Examples. -·

[0044] Figure 15 pH tolerance test of photosensitizers prepared for Comparative Examples and Examples.

[0045] Figure 16 Imaging results of photosensitizers prepared for Comparative Examples and Examples in SA.

[0046] ​​​Figure 17 Bacterial survival rate of photosensitizers prepared for Comparative Example 1, Comparative Example 2 and Example 2, Example 3 under 660 nm red light excitation (15 minutes) at 2% oxygen concentration.

[0047] Figure 18 Bacterial survival rate of photosensitizers prepared for Comparative Example 1, Comparative Example 2 and Example 2, Example 3 under sunlight excitation (15 minutes) at 2% oxygen concentration.

[0048] Figure 19 Bacterial killing effect of photosensitizers prepared for Example 3 on SA, MRSA and E. coli.

[0049] Figure 20 Bacterial killing effect of photosensitizers prepared for Example 3 on SA, MRSA and E. coli. DETAILED DESCRIPTION

[0050] The present application provides an acid and alkali resistant nile blue photosensitizer, a preparation method and application thereof, in order to make the purpose, technical scheme and effect of the present application more clear and explicit, the present application is further described in detail below. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.

[0051] The present application provides an acid and alkali resistant nile blue photosensitizer, a preparation method and application thereof, in order to make the purpose, technical scheme and effect of the present application more clear and explicit, the present application is further described in detail below. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.

[0052]

[0053] Among them, R1, R2, R3 are independently selected from C1-C6 straight chain alkane, At least one of the above.

[0054] In some embodiments, the acid and alkali resistant nile blue photosensitizer can be used as a type I photodynamic enhanced photosensitizer or as a sunlight activated energy-saving photosensitizer.

[0055] Specifically, the acid and alkali resistant nile blue photosensitizer can have the following structural formula:

[0056]

[0057] The present application also provides a preparation method of the acid and alkali resistant nile blue photosensitizer, as shown in Figure 1 The preparation method comprises the following steps:

[0058] Dissolve naphthylamine and compound R2I in a first organic solvent, add a weak alkaline solution under stirring, carry out a first reaction, then add R3I, and carry out a second reaction to obtain a first intermediate;

[0059] dissolving the first intermediate in a strong acid solution containing a benzyne salt containing R1, stirring, and then adding an oxidizing agent to perform a third reaction to obtain a second intermediate;

[0060] dissolving the second intermediate in a second organic solvent, adding a catalyst and a reducing agent under stirring to perform a fourth reaction to obtain the nile blue photosensitizer.

[0061] In some embodiments, the preparation method comprises the following steps:

[0062] dissolving naphthylamine and a compound R2I in a first organic solvent, adding a weak alkaline solution under stirring, reacting at 60-120℃ for 1-4 hours, and then adding R3I, reacting at 60-120℃ for 12-24 hours to obtain a first intermediate;

[0063] dissolving the first intermediate in a strong acid solution containing a benzyne salt containing R1, stirring, and then adding an oxidizing agent at 0-4℃ to react for 2-10 hours to obtain a second intermediate;

[0064] dissolving the second intermediate in a second organic solvent, adding a catalyst and a reducing agent under stirring, reacting at 60-90℃ for 10-60 minutes to obtain the nile blue photosensitizer.

[0065] In some embodiments, the first organic solvent is selected from at least one of acetonitrile, acetone, tetrahydrofuran, acetic anhydride, DMSO, DMF;

[0066] the second organic solvent is selected from at least one of acetonitrile, methanol, dichloromethane, ethyl acetate, tetrahydrofuran, DMF;

[0067] the weak alkaline solution is at least one of sodium carbonate, potassium carbonate, sodium bicarbonate, cesium carbonate, sodium acetate, potassium acetate;

[0068] the strong acid solution is selected from at least one of hydrochloric acid, sulfuric acid, nitric acid;

[0069] the oxidizing agent is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate, potassium hyperoxide;

[0070] the reducing agent is selected from at least one of copper sulfate, copper chloride, copper acetate, copper oxide, cuprous oxide, cuprous iodide;

[0071] the catalyst is selected from at least one of manganese dioxide, manganese sulfate, manganese chloride, manganese nitrate, manganese acetate.

[0072] The Nile blue photosensitizer is used for fluorescence imaging of positive bacteria and negative bacteria.

[0073] The application further provides application of the Nile blue photosensitizer in preparation of a tumor phototherapy product.

[0074] The application further provides application of the Nile blue photosensitizer in preparation of a broad-spectrum antibacterial phototherapy reagent.

[0075] The application further provides application of the Nile blue photosensitizer in preparation of a treatment reagent for biofilm infection.

[0076] The technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments of the application, and are only used to explain the application, but not limit the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0077] In the embodiments of the application, the chemical reagents are from Anjieji Chemical Reagent Company or Bide Pharmaceutical, the commercial dyes DPBF, DCFH and DHR 123 are purchased from MCE Reagent Company, and the strains used are from Bei Na Biological.

[0078] Embodiment 1

[0079] Preparation of the Nile blue photosensitizer EB-MeP molecule, including the following steps:

[0080] (1) Synthesis of intermediate 1-1: N-methyl-N-pentyl naphthalen-1-amine

[0081]

[0082] Dissolve the raw material naphthalene amine (20 mmol) and methyl iodide (20 mmol) in 30 mL of acetonitrile solvent, add anhydrous potassium carbonate (40 mmol) under stirring, reflux at 80°C for 1 hour, then add excess iodopentane (60 mmol) to the above mixture, terminate the reaction after refluxing at 80°C for 12 hours. After removing potassium carbonate by filtration, remove the organic solvent by reduced pressure distillation, purify the crude product by using a silica gel chromatographic column, and the eluent is petroleum ether: dichloromethane = 50:1 (v / v), to obtain the yellowish oil product of intermediate 1-1, with a yield of 56%.

[0083] (2) Synthesis of EB-MeP molecule

[0084]

[0085] The intermediate 2-1 was obtained by dissolving the bent salt (13 mmol) and intermediate 1-1 (10 mmol) in 10 mL of hydrochloric acid solution (1 M), adding potassium dichromate powder (10 mmol) at 4 °C, and continuously stirring for 1 hour, followed by filtration and washing to obtain a dark blue intermediate 2-1, which was directly subjected to the subsequent reaction.

[0086] The intermediate 2-1 was dissolved in 20 mL of acetonitrile solvent, and then copper sulfate (5 mmol) and manganese dioxide (15 mmol) were sequentially added, and the reaction was refluxed at 80 °C for 1 hour. The organic solvent was removed by reduced pressure distillation, and the crude product was purified using a silica gel chromatographic column with dichloromethane:methanol = 75:1 (v / v) as the eluent to obtain a blue solid EB-MeP molecule with a yield of 32%.

[0087] HRMS (ESI): m / z for C 26 H 32 N3S + ([M] + ): calculated 418.2311; found 418.2306.

[0088] The high resolution mass spectrum of the nile blue photosensitizer EB-MeP molecule prepared in Example 1 is shown in FIG. 1. Figure 2

[0089] 1 H NMR (600 MHz, CDCl3) δ 9.02 (d, J = 8.1 Hz, 1H), 7.98 (d, J = 8.2 Hz, 1H), 7.94 (d, J = 9.4 Hz, 1H), 7.90 (s, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.48 (s, 1H), 7.18 (d, J = 9.3 Hz, 1H), 3.81 (t, J = 7.2 Hz, 2H), 3.62 (q, J = 6.8 Hz, 4H), 3.49 (s, 2H), 1.80-1.85 (m, 2H), 1.31-1.24 (m, 10H), 0.84 (t, J = 6.8 Hz, 3H).

[0090] The nuclear magnetic hydrogen spectrum of the nile blue photosensitizer EB-MeP molecule prepared in Example 1 is shown in FIG. 2. Figure 3

[0091] Example 2

[0092] The preparation of the nile blue photosensitizer EB-dEt molecule includes the following steps:

[0093] (1) Synthesis of intermediate 1-1: N, N-diethyl-1-naphthylamine

[0094] ​​

[0095] The synthesis of intermediate 1-1 of this example was similar to that of intermediate 1-1 of Example 1 to give intermediate 2-1 in 89% yield.

[0096] (2) Synthesis of EB-dEt molecule

[0097]

[0098] Using the same synthetic route as Example 1, the EB-dEt molecule was obtained in 45% yield.

[0099] MS (ESI): m / z for C 24 H 28 N3S + ([M]+): calculated 390.20; found 390.17.

[0100] The mass spectrum of the Nile blue-based photosensitizer EB-dEt molecule prepared in Example 2 is shown in Figure 2. Figure 4

[0101] 1 H NMR (600 MHz, MeOD) δ 9.08 (dd, J = 8.2, 1.0 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 8.04 (d, J = 9.4 Hz, 1H), 7.86 (t, J = 7.3 Hz, 1H), 7.77 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H), 7.52 (s, 1H), 7.45 (dd, J = 9.5, 2.7 Hz, 1H), 7.30 (d, J = 2.7 Hz, 1H), 3.91 (q, J = 7.1 Hz, 4H), 3.72 (q, J = 7.2 Hz, 4H), 1.49 (t, J = 7.1 Hz, 6H), 1.35 (t, J = 7.2 Hz, 6H).

[0102] The mass spectrum of the Nile blue-based photosensitizer EB-dEt molecule prepared in Example 2 is shown in Figure 2. Figure 5

[0103] Example 3

[0104] Preparation of Nile blue-based photosensitizer EB-dMe molecule, comprising the following steps:

[0105] (1) Synthesis of intermediate 1-1: N,N-dimethyl-1-naphthylamine

[0106]

[0107] ​​The synthesis of intermediate 1-1 of this example was similar to that of intermediate 1-1 of Example 1 to give intermediate 3-1 in 92% yield.

[0108] (2) Synthesis of EB-dMe molecule

[0109]

[0110] Using the same synthetic route as Example 1, the EB-dMe molecule was obtained in 41% yield.

[0111] HRMS (ESI): m / z for C 22 H 24 N3S + ([M]+): calculated 362.1685; found 362.1687.

[0112] The mass spectrum of the Nile blue-based photosensitizer EB-dMe molecule prepared in Example 3 is shown in Figure 3. Figure 6

[0113] 1 H NMR (400 MHz, MeOD) δ 8.99 (d, J = 7.4 Hz, 1H), 8.21 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 9.6 Hz, 1H), 7.83 (t, J = 7.3 Hz, 1H), 7.75 (t, J = 7.1 Hz, 1H), 7.39 (q, J = 2.9 Hz, 2H), 7.21 (d, J = 2.7 Hz, 1H), 3.69 (q, J = 7.2 Hz, 4H), 3.54 (s, 6H), 1.34 (t, J = 7.1 Hz, 6H).

[0114] The nuclear magnetic hydrogen spectrum of the Nile blue-based photosensitizer EB-dMe molecule prepared in Example 3 is shown in Figure 4. Figure 7

[0115] Example 4

[0116] Preparation of Nile blue-based photosensitizer EB-qMe molecule, comprising the following steps:

[0117] (1) Synthesis of intermediate 1-1: N,N-dimethyl-1-naphthylamine

[0118]

[0119] (2) Synthesis of EB-qMe molecule

[0120]

[0121] ​​Using the same synthetic route as in Example 1, the methyl-substituted Bent salt was prepared by the ethyl-substituted Bent salt method to obtain the EB-qMe molecule in 29% yield.

[0122] HRMS(ESI):m / z for C 20 H 20 N3S + ([M] + ): Calculated value 334.1372; Measured value 334.1376.

[0123] The mass spectra of the Nile blue photosensitizer EB-qMe molecule prepared in Example 4 are as follows: Figure 8 As shown.

[0124] 1 H NMR(400MHz,MeOD)δ8.70(d,J=7.7Hz,1H),8.05(d,J=7.8Hz,1H),7.73(t,J=7.1H z,1H),7.67(t,J=7.8Hz,2H),7.15(s,1H),6.88(s,1H),3.48(s,6H),3.18(s,6H).

[0125] The 1H NMR spectrum of the Nile blue photosensitizer EB-qMe molecule prepared in Example 4 is shown below. Figure 9 As shown.

[0126] Comparative Example 1 and Comparative Example 2

[0127]

[0128] Comparative Example 1 molecule NBS-NH2 and Comparative Example 2 molecule NBS-Et were prepared by existing methods for synthesizing thio-Nile blue and are used as reference molecules in this invention.

[0129] Absorption spectra of examples and comparative examples

[0130] 3 mM starting stock solutions were prepared for the EB series molecules of Examples 1-4 and the NBS series molecules of Comparative Examples 1-2, for subsequent solution and biological assays. Figure 10 As shown, in methanol solvent, the EB series molecules EB-MeP, EB-dEt, EB-dMe, and EB-qMe exhibit a significant redshift compared to the NBS series comparative molecules NBS-NH2 and NBS-Et. Their maximum absorption wavelength redshifts from approximately 660 nm for NBS to approximately 680 nm, and the EB series also exhibits a wider absorption half-maximum width. Simultaneously, the absorbance and molar extinction coefficient of the EB series are significantly improved, approximately twice that of the NBS series. These beneficial modifications to the absorption spectra enhance the photon absorption capacity of this type of polyalkylated Nile blue structure under red light and sunlight.

[0131] Emission spectra of examples and comparative examples

[0132] The fluorescence emission spectra of the EB series molecules EB-MeP, EB-dEt, EB-dMe, and EB-qMe, and the NBS series comparative molecules NBS-NH2 and NBS-Et, were tested in methanol. The results are as follows: Figure 11 As shown, the maximum fluorescence emission wavelength of the EB series molecules in the examples increased from 680-690 nm to 710-720 nm. Simultaneously, the fluorescence intensity of the EB series molecules in the examples decreased significantly, indicating that after polyalkylation modification, the excited-state energy of these molecules is not consumed by radiative transitions, but rather by other non-radiative transitions, such as ROS generation.

[0133] Electrostatic potential distribution of the examples and comparative examples

[0134] The redshift results from absorption and fluorescence spectra indicate that polyalkylation enhances the intramolecular electronic push-pull effect in Nile Blue-like structures, resulting in increased intramolecular charge transfer efficiency and further charge dispersion. Therefore, this invention uses theoretical calculations to analyze the charge distribution of a series of embodiments and comparative molecules, such as... Figure 12 As shown, comparative examples 1NBS-NH2 and 2NBS-Et exhibited significant charge distribution in the high electrostatic potential region, with concentrated charge distribution at their cation structures. In contrast, the EB series showed no atomic distribution in the high electrostatic potential region, and the intramolecular charge distribution was more uniform. This provides a theoretical basis for explaining the differences in subsequent ROS generation results.

[0135] ROS generation capability of the examples and comparative examples

[0136] To comprehensively analyze the differences in ROS generation capabilities between the examples and comparative examples, a detailed comparison of the total ROS of the six molecules is presented here. 1 O2 and O2 -· The occurrence of this. For example... Figure 13 As shown, the total ROS generation in solution of the EB series of examples and the NBS series of comparative examples was detected using a DCFH probe, and the generation amount was directly proportional to the fluorescence enhancement; the total ROS generation was detected using a DPBF probe. 1 O2 generation was assessed, with absorbance decay representing the generation rate; and O2 was detected using a DHR 123 probe. -· The generation of O2 is directly related to fluorescence enhancement. Results showed that, compared to the comparative NBS series, the EB series in Example 1 generated more O2 via the type I PDT pathway. -·The ROS production is enhanced significantly, about 2.2-4.2 times of the NBS series. Specifically, the total ROS production of Example 1 EB-MeP and Example 2 EB-dEt is less different from that of Comparative Example NBS, but the O2 -· The ROS production capacity can be improved by about 4 times; while the total ROS production of Example 3 EB-dMe and Example 4 EB-qMe is about 1.5 times of the NBS series of Comparative Example, and the O2 -· The ROS production capacity can be improved by about 2.5 times. And as shown in Figure 14 , the O2 -· production capacity and the excitation wavelength are positively correlated, which is regulated by the intramolecular charge transfer effect. Overall, the modification of this type of multi-alkylation enables the EB series molecules to have more excellent ROS production capacity, especially the I-type PDT activity.

[0137] pH tolerance test of examples and comparative examples

[0138] To detect the improvement of pH tolerance of the alkylation substitution of the active secondary amine of benzophenothiazine, the absorption spectrum changes of Example 2 EB-dEt and Example 3 EB-dMe and Comparative Example 1 NBS-NH2 and Comparative Example 2 NBS-Et between pH 5-9 were detected. As shown in Figure 15 , the absorption spectrum changes of examples and comparative examples are not obvious in acidic and neutral solution environment, indicating their acid tolerance. But in alkaline environment, even in alkaline environment of pH 8, the absorption spectrum of the NBS series of comparative examples changes significantly, showing that the absorption spectrum at 660 nm is almost completely quenched, while the absorption spectrum of the basic Nile blue form (at 550 nm) is significantly enhanced. In contrast, the absorption spectrum of the EB series in alkaline environment is minimally affected, with significant alkaline tolerance. Therefore, the EB series of examples constructed here has excellent acid-base tolerance, changing the traditional limitation that Nile blue structures cannot be used in alkaline environment.

[0139] Bacterial imaging analysis of examples and comparative examples

[0140] The bacteria used in the experiment include Staphylococcus aureus (SA), drug-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) cultured in a 37°C incubator, and the culture medium is LB. The bacterial solution was diluted to a density of 10 6 , and transferred to a 35mm culture dish, 6 molecules of examples and comparative examples were added (concentration: 2μM), incubated at 37°C for 30 minutes, and then imaged under a laser confocal microscope (CLSM). As shown in Figure 16As shown, although the EB series of embodiments exhibit lower fluorescence production capacity in solution at the same concentration, the bacterial imaging results show that the intrabacterial fluorescence intensity can be 2-3 times that of the comparative example NBS, due to the attraction of the more stable cationic charge distribution of the EB series to the negatively charged bacterial membrane.

[0141] Antibacterial PDT effect of the embodiments and comparative examples

[0142] To more accurately test the antibacterial phototherapeutic activity of the embodiments and comparative examples, the present application uses the MTT cell / bacterial activity detection method: 10 8 Bacteria at a density of 106cells / mL were transferred to a PBS solution and different groups of photosensitizers were added at gradient concentrations. After incubation for 30 minutes, light treatment was given, and after incubation for 4 hours, MTT solution (1 mg / mL) was added. After the purple solid precipitate was produced, the upper liquid was carefully aspirated, dissolved with DMSO, and the absorbance was tested to calculate the bacterial survival rate. Under red light excitation at 660 nm (Fig. 2), the EB-dEt of Example 2 and the EB-dMe of Example 3 were able to achieve complete elimination of bacteria in a low-oxygen environment (2% oxygen) at a concentration of 5 μM, while the comparative example NBS molecule required 10 μM. Under sunlight excitation (Fig. 3), due to the better total ROS production capacity of the EB-dMe of Example 3, assisted by ultraviolet light in sunlight, the EB-dMe of Example 3 only required 2.5 μM to achieve complete inhibition of bacteria, while at the same concentration, the bacterial killing effect was in the order of the EB-dEt of Example 2, followed by the NBS-Et of Comparative Example 2 and the NBS-NH2 of Comparative Example 1. Figure 17 Figure 18 Antibacterial effect and biofilm destruction effect of the photosensitizer prepared in Example 3

[0143] As an example of Example 3, in addition to exhibiting more excellent anti-SA treatment performance, it also exhibited excellent killing ability against positive MRSA and negative E. coli bacteria. After light treatment with the EB-dMe of Example 3, no bacteria grew on the agarose gel plate and were completely eliminated (Fig. 4). Furthermore, Example 3 was able to significantly destroy the biofilm formed by drug-resistant positive MRSA and negative E. coli bacteria, and the biofilm morphology was destroyed after light treatment, with a significant reduction in bacterial colonies (Fig. 5). Such acid-base tolerant multi-alkylated Nile blue type I photodynamic enhanced photosensitizers have demonstrated more superior broad-spectrum antibacterial performance and low-oxygen environment PDT usability than traditional NBS photosensitizers.

[0144] As an example of Example 3, in addition to exhibiting more excellent anti-SA treatment performance, it also exhibited excellent killing ability against positive MRSA and negative E. coli bacteria. After light treatment with the EB-dMe of Example 3, no bacteria grew on the agarose gel plate and were completely eliminated (Fig. 4). Furthermore, Example 3 was able to significantly destroy the biofilm formed by drug-resistant positive MRSA and negative E. coli bacteria, and the biofilm morphology was destroyed after light treatment, with a significant reduction in bacterial colonies (Fig. 5). Such acid-base tolerant multi-alkylated Nile blue type I photodynamic enhanced photosensitizers have demonstrated more superior broad-spectrum antibacterial performance and low-oxygen environment PDT usability than traditional NBS photosensitizers. Figure 19 Figure 20 As an example of Example 3, in addition to exhibiting more excellent anti-SA treatment performance, it also exhibited excellent killing ability against positive MRSA and negative E. coli bacteria. After light treatment with the EB-dMe of Example 3, no bacteria grew on the agarose gel plate and were completely eliminated (Fig. 4). Furthermore, Example 3 was able to significantly destroy the biofilm formed by drug-resistant positive MRSA and negative E. coli bacteria, and the biofilm morphology was destroyed after light treatment, with a significant reduction in bacterial colonies (Fig. 5). Such acid-base tolerant multi-alkylated Nile blue type I photodynamic enhanced photosensitizers have demonstrated more superior broad-spectrum antibacterial performance and low-oxygen environment PDT usability than traditional NBS photosensitizers.

[0145] ​​It is to be understood that the application is not limited to the examples described above, which can be modified or adapted in several ways by those skilled in the art without departing from the scope of the present application, as defined by the appended claims.

Claims

1. The application of an acid- and alkali-tolerant Nile blue photosensitizer in the preparation of phototherapy reagents against drug-resistant Staphylococcus aureus and Escherichia coli, wherein the chemical structure of the acid- and alkali-tolerant Nile blue photosensitizer is as follows: , or .

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