Acid-base tolerant nile blue photosensitizer as well as preparation method and application thereof

Abstract

The invention discloses an acid-base tolerant Nile blue photosensitizer as well as a preparation method and application thereof, the chemical structural general formula of the acid-base tolerant Nile blue photosensitizer is as follows: # imgabs 0 #, and R1, R2 and R3 are respectively and independently selected from at least one of C1-C6 straight-chain alkane chains and # imgabs 1 #. A series of Nile blue photosensitizers are synthesized through modification of polyalkylation, the Nile blue photosensitizers have the acid-base tolerance characteristic and a stable cationic amine salt form, the intramolecular charge transfer effect is promoted, red shift of the absorption wavelength and the emission wavelength of series of Nile blue photosensitizers (EB series) molecules is promoted to be 20-40 nm, and the photosensitizers can be applied to the field of photosensitizers. The molar extinction coefficient is increased from 45000 L * mol <-1 > * cm <-1 > to 80000 L * mol <-1 > * cm <-1 >. Compared with the conventional NBS photosensitizer, the O < 2->. Generation efficiency of EB molecules is improved by 2.2-4.2 times. The improvement of pH stability and the normalized existence of cations enable EB molecules to show more excellent uptake performance on bacteria, and the fluorescence intensity under the same concentration is 2-3 times of that of NBS (N-bromosuccinimide).

Description

Technical Field

[0001] The present invention relates to the field of chemical technology, and in particular to an acid-base tolerant Nile blue photosensitizer, and a preparation method and application thereof. Background Art

[0002] Biofilm infection is a chronic, long-term bacterial infection caused by the massive proliferation of bacteria in wounds, which poses a serious threat to public health. Because of the shielding effect of bacterial autocrine polymers in the biofilm infection microenvironment, conventional antibiotic treatment is difficult to be effective against it, and it is often necessary to overuse antibiotics or combine surgical resection for treatment, but this is prone to antibiotic abuse and additional treatment burden. Recently, photodynamic therapy (PDT) has been developed for antibacterial treatment. The short-lived, strongly oxidizing reactive oxygen species (ROS) produced continuously under light can often achieve rapid and efficient phototherapy effects on the lesions where the photosensitizer is distributed. Because ROS can directly destroy macromolecules of life such as proteins and nucleic acids, it is usually difficult for them to develop drug resistance, so it is an ideal antibacterial method.

[0003] However, the formation of dense bacteria in the biofilm is often accompanied by a hypoxic microenvironment at the lesion site, which greatly inhibits the therapeutic activity of the photosensitizer. For this reason, type I photosensitizers that produce ROS free radicals have been developed for anti-hypoxic photodynamic therapy. Among them, thio-Nile blue (NBS), as a milestone molecule in type I photosensitizers, has opened up the mechanism exploration and molecular design research of type I PDT. The thio-Nile blue series of molecules exhibit excellent photostability, biocompatibility and ROS generation performance, and have attracted widespread attention in the current PDT field at home and abroad. The downside is that Nile blue photosensitizers have insufficient pH stability, especially low alkali tolerance. Even in a weak alkaline environment of pH 8, cation loss will occur, thereby severely impairing the photodynamic activity. In addition, it is also noted that the molar extinction coefficient of thio-Nile blue is insufficient, and the superoxide anion (O2 -· ) production efficiency needs to be improved. These negative properties are not conducive to its therapeutic actions such as sterilization and tumor cell removal in complex physiological environments.

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

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an acid-base tolerant Nile blue photosensitizer, a preparation method and application thereof, in order to solve the problems of insufficient molar extinction coefficient of the existing photosensitizer and the problem of superoxide anion (O2 -· ) results in low efficiency.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect of the present invention, there is provided an acid-base tolerant Nile blue photosensitizer, the chemical structure of the Nile blue photosensitizer is as follows:

[0008]

[0009] Wherein, R1, R2, and R3 are independently selected from C1-C6 straight chain alkane chains, At least one of .

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned acid-base tolerant Nile blue photosensitizer, the preparation method comprising 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, and performing a second reaction to obtain a first intermediate;

[0012] The first intermediate and the Bent salt containing R1 are dissolved in a strong acid solution, stirred, and then an oxidant is added to carry out a third reaction to obtain a second intermediate;

[0013] The second intermediate is dissolved in a second organic solvent, a catalyst and a reducing agent are added under stirring, and a fourth reaction is performed to obtain the Nile blue photosensitizer.

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

[0015] The second organic solvent is selected from 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 selected from at least one of hydrochloric acid, sulfuric acid and nitric acid.

[0018] Preferably, the oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate and potassium superoxide;

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

[0020] The catalyst is selected from 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°C and the time is 1-4 hours;

[0022] The temperature of the second reaction is 60-120°C and the time is 12-24 hours;

[0023] The temperature of the third reaction is 0-4°C and the time is 2-10 hours;

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

[0025] In a fourth aspect of the present invention, the acid-base tolerant Nile blue photosensitizer is provided for use in fluorescence imaging of positive bacteria and negative bacteria.

[0026] The fifth aspect of the present invention provides the use of the above-mentioned acid-base tolerant Nile blue photosensitizer in the preparation of tumor phototherapy products.

[0027] The sixth aspect of the present invention provides the use of the above-mentioned acid-base tolerant Nile blue photosensitizer in the preparation of a broad-spectrum antibacterial phototherapy agent.

[0028] A seventh aspect of the present invention provides use of the acid-base tolerant Nile blue photosensitizer in the preparation of a therapeutic agent for biofilm infection.

[0029] Beneficial effects: The present invention provides an acid-base tolerant Nile blue photosensitizer, a preparation method and application thereof. The present invention synthesizes a series of Nile blue photosensitizers through polyalkylation modification, which have acid-base tolerance and a stable cationic amine salt form, promotes the intramolecular charge transfer effect, and causes the absorption wavelength and emission wavelength of the series of Nile blue photosensitizers (EB series) molecules to red-shift by 20-40nm, and the molar extinction coefficient is from 45000L·mol -1 cm -1 Increased to ~80000L·mol -1 cm -1 EB molecule O2 -· The production efficiency is 2.2-4.2 times higher than that of conventional NBS photosensitizer. The improved pH stability and the normalized presence of cations make EB molecules show better uptake performance for bacteria. The fluorescence intensity is 2-3 times that of NBS at the same concentration. And the excellent O2 -· The production capacity can help the EB series achieve bacterial inhibition function stimulated by red light or sunlight in a low oxygen environment (2% oxygen density), and the photosensitizer concentration required for complete bacterial elimination is only 1 / 2 of NBS. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The synthetic route of the Nile blue photosensitizer preferably provided in the embodiment of the present invention.

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

[0032] Figure 3 This is the H NMR spectrum of the Nile blue photosensitizer EB-MeP prepared in Example 1.

[0033] Figure 4 This is the mass spectrum of the Nile blue photosensitizer EB-dEt prepared in Example 2.

[0034] Figure 5 This is the H NMR spectrum of the Nile blue photosensitizer EB-dEt prepared in Example 2.

[0035] Figure 6 This is a high-resolution mass spectrum of the Nile blue photosensitizer EB-dMe prepared in Example 3.

[0036] Figure 7 This is the H NMR spectrum of the Nile blue photosensitizer EB-dMe prepared in Example 3.

[0037] Figure 8 This is the high-resolution mass spectrum of the Nile blue photosensitizer EB-qMe prepared in Example 4.

[0038] Fig. 9 This is the H NMR spectrum of the Nile blue photosensitizer EB-qMe prepared in Example 4.

[0039] Fig.10 The absorption spectra of the photosensitizers prepared in Comparative Examples 1-2 and Examples 1-4 (in methanol) are shown.

[0040] Fig.11 The fluorescence emission spectra of the photosensitizers prepared in comparative examples and examples in methanol solution.

[0041] Fig.12 The electrostatic potential distribution of the photosensitizer prepared in comparative examples and examples.

[0042] Fig.13 (a) Total ROS generation of the photosensitizers prepared in comparative examples and examples; (b) 1 O2 generation; and (c) O2 -· Produce situation.

[0043] Fig.14 O2 of the photosensitizer prepared in the comparative examples and embodiments -· Correlation between generation capacity and excitation wavelength.

[0044] Fig.15 The pH tolerance of the photosensitizers prepared in the comparative examples and examples was tested.

[0045] Fig.16 The imaging results of the photosensitizers prepared in comparative examples and examples in SA are shown.

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

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

[0048] Fig.19 The photosensitizer prepared in Example 3 has a bacterial killing effect on SA, MRSA and E. coli.

[0049] Fig. 20 The photosensitizer prepared in Example 3 has a destructive effect on the biofilm formed by MRSA and E. coli. DETAILED DESCRIPTION

[0050] The present invention provides an acid-base tolerant Nile blue photosensitizer, a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0051] The embodiment of the present invention provides an acid-base tolerant Nile blue photosensitizer, and the chemical structure of the acid-base tolerant Nile blue photosensitizer is as follows:

[0052]

[0053] Wherein, R1, R2, and R3 are independently selected from C1-C6 straight chain alkane chains, At least one of .

[0054] In some embodiments, the acid-base tolerant Nile blue photosensitizer can be used as a type I photodynamic enhancement photosensitizer or as a sunlight-activated energy-saving photosensitizer.

[0055] Specifically, the acid-base tolerant Nile blue photosensitizer may have a structural formula of:

[0056]

[0057] The present invention also provides a method for preparing the acid-base tolerant Nile blue photosensitizer, such as Figure 1 As shown, the preparation method comprises the following steps:

[0058] Dissolving naphthylamine and compound R2I in a first organic solvent, adding a weak alkaline solution under stirring, performing a first reaction, then adding R3I, and performing a second reaction to obtain a first intermediate;

[0059] The first intermediate and the Bent salt containing R1 are dissolved in a strong acid solution, stirred, and then an oxidant is added to carry out a third reaction to obtain a second intermediate;

[0060] The second intermediate is dissolved in a second organic solvent, a catalyst and a reducing agent are added under stirring, and a fourth reaction is performed to obtain the Nile blue photosensitizer.

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

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

[0063] The first intermediate and the Bent salt containing R1 are dissolved in a strong acid solution, stirred, and then an oxidant is added at 0-4° C. and reacted for 2-10 hours to obtain a second intermediate;

[0064] The second intermediate is dissolved in a second organic solvent, a catalyst and a reducing agent are added under stirring, and the reaction is carried out at 60-90° C. 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, and DMF;

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

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

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

[0069] The oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate and potassium superoxide;

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

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

[0072] The embodiment of the present invention also provides the above-mentioned Nile blue photosensitizer for use in fluorescence imaging of positive bacteria and negative bacteria.

[0073] The embodiments of the present invention also provide the use of the above-mentioned Nile blue photosensitizer in the preparation of tumor phototherapy products.

[0074] The embodiment of the present invention also provides the use of the above-mentioned Nile blue photosensitizer in the preparation of a broad-spectrum antibacterial phototherapy agent.

[0075] The embodiments of the present invention also provide the use of the above-mentioned Nile blue photosensitizer in the preparation of a therapeutic agent for biofilm infection.

[0076] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and are only for illustrating the present invention but not limiting the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0077] In the examples of the present invention, the chemicals were all from Anage Chemical Reagent Company or Bid Pharmaceuticals, the commercial dyes DPBF, DCFH and DHR 123 were purchased from MCE Reagent Company, and the strains used were from Beina Biotechnology.

[0078] Example 1

[0079] The preparation of Nile blue photosensitizer EB-MeP molecule includes the following steps:

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

[0081]

[0082] The raw material naphthylamine (20mmol) and iodomethane (20mmol) were dissolved in 30mL acetonitrile solvent, anhydrous potassium carbonate (40mmol) was added under stirring, and the mixture was refluxed at 80°C for 1 hour, and then an excess of iodine pentane (60mmol) was added to the mixture, and the reaction was terminated after refluxed at 80°C for 12 hours. After potassium carbonate was removed by filtration, the organic solvent was removed by reduced pressure distillation, and the crude product was purified by silica gel chromatography column, and the eluent was petroleum ether: dichloromethane = 50:1 (v / v), to obtain intermediate 1-1 as a light yellow oily product with a yield of 56%.

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

[0084]

[0085] Bent salt (13 mmol) and intermediate 1-1 (10 mmol) were dissolved in 10 mL of hydrochloric acid solution (1 M), potassium dichromate powder (10 mmol) was added at 4°C, and stirring was continued for 1 hour. After filtering and washing, a dark blue intermediate 2-1 was obtained, which was directly used for subsequent reactions.

[0086] Intermediate 2-1 was dissolved in 20 mL of acetonitrile solvent, and then copper sulfate (5 mmol) and manganese dioxide (15 mmol) were added in sequence, and the reaction was refluxed at 80°C for 1 hour to terminate the reaction. The organic solvent was removed by vacuum distillation, and the crude product was purified using a silica gel chromatography column with an eluent of dichloromethane: methanol = 75:1 (v / v) 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 value 418.2311; measured value 418.2306.

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

[0089] 1 H NMR (600MHz, CDCl3) δ9.02(d,J=8.1Hz,1H),7.98(d,J=8.2Hz,1H),7.94(d,J =9.4Hz,1H),7.90(s,1H),7.73(t,J=7.5Hz,1H),7.63(t,J=7.4Hz,1H),7.48 (s,1H),7.18(d,J=9.3Hz,1H),3.81(t,J=7.2Hz,,2H),3.62(q,J=6.8Hz,4H) ,3.49(s,2H),1.80-1.85(m,2H),1.31-1.24(m,10H),0.84(t,J=6.8Hz,3H).

[0090] The NMR hydrogen spectrum of the Nile blue photosensitizer EB-MeP molecule prepared in Example 1 is as follows: Figure 3 shown.

[0091] Example 2

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

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

[0094]

[0095] The synthesis method of intermediate 1-1 in this example is similar to that of intermediate 1-1 in Example 1 to obtain intermediate 2-1 with a yield of 89%.

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

[0097]

[0098] The same synthetic route as in Example 1 was used to obtain EB-dEt molecules with a yield of 45%.

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

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

[0101] 1 H NMR (600MHz, MeOD) δ9.08(dd,J=8.2,1.0Hz,1H),8.10(d,J=8.2Hz,1H),8.04(d,J=9.4Hz,1H),7.86(t,J=7.3Hz,1H),7.77(ddd,J=8.3,7.2,1.3Hz,1H),7 .52(s,1H),7.45(dd,J=9.5,2.7Hz,1H),7.30(d,J=2.7Hz,1H),3.91(q,J=7. 1Hz, 4H), 3.72 (q, J = 7.2Hz, 4H), 1.49 (t, J = 7.1Hz, 6H), 1.35 (t, J = 7.2Hz, 6H).

[0102] The NMR hydrogen spectrum of the Nile blue photosensitizer EB-dEt molecule prepared in Example 2 is as follows: Figure 5 shown.

[0103] Example 3

[0104] The preparation of Nile blue photosensitizer EB-dMe molecules includes the following steps:

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

[0106]

[0107] The synthesis method of intermediate 1-1 in this example is similar to that of intermediate 1-1 in Example 1, to obtain intermediate 3-1 with a yield of 92%.

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

[0109]

[0110] The same synthetic route as in Example 1 was used to obtain EB-dMe molecules with a yield of 41%.

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

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

[0113] 1 H NMR (400MHz, MeOD) δ8.99(d,J=7.4Hz,1H),8.21(d,J=8.2Hz,1H),7.95(d,J=9.6Hz,1H),7.83(t,J=7.3Hz,1H),7.75(t ,J=7.1Hz,1H),7.39(q,J=2.9Hz,2H),7.21(d,J=2.7Hz,1H),3.69(q,J=7.2Hz,4H),3.54(s,6H),1.34(t,J=7.1Hz,6H).

[0114] The NMR hydrogen spectrum of the Nile blue photosensitizer EB-dMe molecule prepared in Example 3 is as follows: Figure 7 shown.

[0115] Example 4

[0116] The preparation of Nile blue photosensitizer EB-qMe molecules includes the following steps:

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

[0118]

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

[0120]

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

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

[0123] The mass spectrum of the Nile blue photosensitizer EB-qMe molecule prepared in Example 4 is as follows: Figure 8 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 NMR hydrogen spectrum of the Nile blue photosensitizer EB-qMe molecule prepared in Example 4 is as follows: Fig. 9 shown.

[0126] Comparative Example 1 and Comparative Example 2

[0127]

[0128] The NBS-NH2 molecule of Comparative Example 1 and the NBS-Et molecule of Comparative Example 2 were prepared by the existing thio-nile blue synthesis method and were used as reference molecules in the present invention.

[0129] Absorption spectra of examples and comparative examples

[0130] 3 mM starting stock solutions of the EB series molecules of Examples 1-4 and the NBS series molecules of Comparative Examples 1-2 were prepared for subsequent solution testing and biological testing. Fig.10 As shown, in methanol solvent, the EB series of example molecules EB-MeP, EB-dEt, EB-dMe and EB-qMe show a significant red shift phenomenon relative to the NBS series of comparative molecules NBS-NH2 and NBS-Et, and their maximum absorption wavelength is red-shifted from about 660nm of NBS to about 680nm, and the absorption half-peak width of the EB series is wider. At the same time, the absorbance and molar extinction coefficient of the EB series are significantly improved, about twice that of the NBS series. These beneficial modifications of the absorption spectrum are conducive to improving the photon absorption capacity of such polyalkylated Nile blue structures under red light and sunlight.

[0131] Emission spectra of embodiments and comparative examples

[0132] The fluorescence emission spectra of the EB series example molecules EB-MeP, EB-dEt, EB-dMe and EB-qMe and the NBS series comparative example molecules NBS-NH2 and NBS-Et were tested in methanol. The results are shown in Figure 2. Fig.11 As shown, the maximum fluorescence emission wavelength of the EB series molecules in the embodiment is increased from 680-690nm to 710-720nm. At the same time, the fluorescence intensity of the EB series embodiment molecules is significantly reduced, which shows that after polyalkylation modification, the excited state energy of this type of molecule is not consumed in the form of radiation transition, but in other non-radiation transition modes, such as ROS generation.

[0133] Electrostatic potential distribution of embodiments and comparative examples

[0134] From the test results of the red shift of the absorption and fluorescence spectra, it can be concluded that the modification of polyalkylation makes the electron push-pull effect in the Nile blue structure more significant, that is, the enhancement of the efficiency of charge transfer in the molecule, which leads to further dispersion of the charge. To this end, the present invention analyzes the charge distribution of the series of examples and comparative examples through theoretical calculations, such as Fig.12 As shown, it is found that Comparative Example 1 NBS-NH2 and Comparative Example 2 NBS-Et have obvious distribution in the high electrostatic potential region, and the charge distribution is concentrated at the cationic structure. In contrast, the EB series has no atomic distribution in the high electrostatic potential region, and the charge distribution within the molecule is more uniform. This provides a theoretical basis for the difference in the subsequent ROS generation results.

[0135] ROS production ability of the embodiments and comparative examples

[0136] In order to comprehensively analyze the differences in the ROS production capacity of the embodiments and comparative examples, the total ROS, 1 O2 and O2 -· The production situation. Fig.13 As shown, the experiment used the DCFH probe to detect the total ROS production in the solution of the EB series of the embodiment and the NBS series of the comparative example. The production amount was proportional to the fluorescence enhancement. The DPBF probe was used to detect the 1 O2 production, absorbance decay represents the production rate; and O2 was detected by DHR 123 probe. -· The production of O2 in the EB series of the example was directly related to the fluorescence enhancement. -·The increase in ROS production is particularly significant, about 2.2-4.2 times that of the NBS series. Specifically, the total ROS production of Example 1EB-MeP and Example 2EB-dEt is relatively small compared with the comparative example NBS, but O2 -· The production capacity can be increased to about 4 times; the total ROS production of Example 3EB-dMe and Example 4EB-qMe is about 1.5 times that of the comparative example NBS series, and O2 -· The production capacity can be increased to about 2.5 times. Fig.14 As shown, the O2 of the series of embodiment molecules and comparative example molecules -· The generation capacity is positively correlated with the excitation wavelength, which is regulated by the charge transfer effect within the molecule. Overall, this type of polyalkylation modification enables the EB series molecules to obtain more excellent ROS generation capacity, especially type I PDT activity.

[0137] pH tolerance test of examples and comparative examples

[0138] In order to detect the improvement of pH tolerance of alkylation substitution of active secondary amines of benzophenothiazine, the absorption spectra of Example 2 EB-dEt and Example 3 EB-dMe as well as Comparative Example 1 NBS-NH2 and Comparative Example 2 NBS-Et were tested between pH 5 and 9. Fig.15 As shown, the absorption spectra of the embodiments and comparative examples do not change significantly in acidic and neutral solution environments, indicating their acid tolerance. However, in an alkaline environment, even in an alkaline environment of pH 8, the absorption spectrum of the NBS series of the comparative example changes significantly, as shown by: the absorption spectrum at 660nm is almost completely quenched, while the absorption spectrum of its alkaline Nile blue form (at 550nm) is significantly enhanced. In contrast, the absorption spectrum of the embodiment EB series is minimally affected in an alkaline environment and has significant alkali tolerance. Therefore, the EB series embodiment molecules constructed here have excellent acid-base tolerance, which changes the limitation that the Nile blue structure is traditionally unavailable in an alkaline environment.

[0139] Bacterial imaging analysis of embodiments and comparative examples

[0140] The bacteria used in the experiment included Staphylococcus aureus (SA), drug-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) cultured in a 37°C incubator with LB as the culture medium. The bacterial solution was diluted to 10 6 The samples were transferred to a 35 mm culture dish, and the six molecules of the examples and comparative examples (concentration: 2 μM) were added respectively. After incubation at 37° C. for 30 minutes, imaging tests were performed under a confocal laser scanning microscope (CLSM). Fig.16As shown, although the Example EB series exhibits lower fluorescence generation ability at the same concentration in solution, the bacterial imaging results show that its fluorescence intensity inside bacteria can be 2-3 times that of the comparative example NBS. This is due to the attraction of its more stable cationic charge distribution to the negatively charged bacterial membrane.

[0141] Antibacterial PDT Effects of Examples and Comparative Examples

[0142] In order to more accurately test the antibacterial phototherapy activity of the embodiments and comparative examples, the present invention uses the MTT cell / bacterial activity detection method for testing: 10 8 The bacteria of different density were transferred to PBS solution, and different groups of photosensitizers with gradient concentrations were added. After incubation for 30 minutes, they were treated with light, and MTT solution (1 mg / mL) was added after incubation for 4 hours. After the purple solid precipitated, the upper liquid was carefully aspirated, dissolved in DMSO, and the absorbance was tested to calculate the bacterial survival rate. Under 660nm red light excitation ( Fig.17 ), Example 2 EB-dEt and Example 3 EB-dMe can completely eliminate bacteria in a low oxygen environment (2% oxygen) at a concentration of 5 μM, while the comparative example NBS molecule requires 10 μM. Under sunlight excitation ( Fig.18 ), due to the better total ROS production ability of Example 3EB-dMe, plus the assistance of ultraviolet rays in sunlight, Example 3EB-dMe only needs 2.5μM to achieve complete inhibition of bacteria, while at the same concentration, the bacterial killing effects are Example 2EB-dEt, Comparative Example 2NBS-Et, and Comparative Example 1NBS-NH2, respectively.

[0143] Spectral antibacterial effect and biofilm destruction effect of the photosensitizer prepared in Example 3

[0144] Taking Example 3 as an example, in addition to showing a more excellent anti-SA therapeutic performance, it also showed excellent killing ability against positive bacteria MRSA and negative bacteria E. coli. After the phototherapy treatment of EB-dMe in Example 3, no bacteria grew on the agarose gel plate and were completely eliminated ( Fig.19 ). In addition, Example 3 can significantly destroy the biofilm formed by drug-resistant positive bacteria MRSA and negative bacteria E. coli. The biofilm morphology is destroyed after phototherapy, and the bacterial community is significantly reduced ( Fig. 20 ). This type of acid-base tolerant polyalkylated Nile blue type I photodynamic enhancement photosensitizer has demonstrated superior broad-spectrum antibacterial properties and PDT usability in hypoxic environments compared to traditional NBS photosensitizers.

[0145] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An acid-base tolerant Nile blue photosensitizer, characterized in that: The chemical structure formula of the acid-base tolerant Nile blue photosensitizer is as follows: Wherein, R1, R2, and R3 are independently selected from C1-C6 straight chain alkane chains, At least one of .

2. A method for preparing the acid-base tolerant Nile blue photosensitizer according to claim 1, characterized in that: The preparation method comprises the following steps: Dissolving naphthylamine and compound R2I in a first organic solvent, adding a weak alkaline solution under stirring, performing a first reaction, then adding R3I, and performing a second reaction to obtain a first intermediate; The first intermediate and the Bent salt containing R1 are dissolved in a strong acid solution, stirred, and then an oxidant is added to carry out a third reaction to obtain a second intermediate; The second intermediate is dissolved in a second organic solvent, a catalyst and a reducing agent are added under stirring, and a fourth reaction is performed to obtain the Nile blue photosensitizer.

3. The method for preparing the acid-base tolerant Nile blue photosensitizer according to claim 2, characterized in that: The first organic solvent is selected from at least one of acetonitrile, acetone, tetrahydrofuran, acetic anhydride, DMSO, and DMF; The second organic solvent is selected from at least one of acetonitrile, methanol, dichloromethane, ethyl acetate, tetrahydrofuran and DMF.

4. The method for preparing the acid-base tolerant Nile blue photosensitizer according to claim 2, characterized in that: The weak alkaline solution is at least one of sodium carbonate, potassium carbonate, sodium bicarbonate, cesium carbonate, sodium acetate, and potassium acetate; The strong acid solution is selected from at least one of hydrochloric acid, sulfuric acid and nitric acid.

5. The method for preparing the acid-base tolerant Nile blue photosensitizer according to claim 2, characterized in that: The oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate and potassium superoxide; The reducing agent is selected from at least one of copper sulfate, copper chloride, copper acetate, copper oxide, cuprous oxide, and cuprous iodide; The catalyst is selected from at least one of manganese dioxide, manganese sulfate, manganese chloride, manganese nitrate and manganese acetate.

6. The method for preparing the acid-base tolerant Nile blue photosensitizer according to claim 2, characterized in that: The temperature of the first reaction is 60-120°C and the time is 1-4 hours; The temperature of the second reaction is 60-120°C and the time is 12-24 hours; The temperature of the third reaction is 0-4°C and the time is 2-10 hours; The temperature of the fourth reaction is 60-90° C. and the time is 10-60 minutes.

7. The acid-base tolerant Nile blue photosensitizer according to claim 1 is used for fluorescence imaging of positive bacteria and negative bacteria.

8. Use of the acid-base tolerant Nile blue photosensitizer according to claim 1 in the preparation of tumor phototherapy products.

9. Use of the acid-base tolerant Nile blue photosensitizer according to claim 1 in the preparation of a broad-spectrum antibacterial phototherapy agent.

10. Use of the acid-base tolerant Nile blue photosensitizer according to claim 1 in preparing a therapeutic agent for biofilm infection.

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