Nile red-based photosensitizer as well as preparation and application thereof

Abstract

The invention relates to a Nile red-based photosensitizer as well as preparation and application thereof, and develops a series of Nile red-based photosensitizers, the structural formula of which is as follows: # imgabs0. By changing the electron donating ability of a donor, the Nile red-based photosensitizers not only have profound influence on the structure of the photosensitizers, but also have important significance on active oxygen generation of the photosensitizers. The NTPA disclosed by the invention has ultrahigh active oxygen generation capacity, has huge potential of resisting multiple drug-resistant bacteria and promoting healing of subcutaneous abscess wounds, and shows the most excellent photodynamic therapy performance. The yield of active oxygen can exceed 470 times within 5 minutes under the irradiation of white light, the NTPA nanoparticles (NPs) show extremely high killing capacity on various drug-resistant bacteria in vitro, the in-vitro bacteriostasis rate can reach 99.9%, and the NTPA nanoparticles also show excellent sterilization and wound healing promoting capacity on the living body level and have huge application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a Nile red-based photosensitizer and a preparation and application thereof. Background Art

[0002] In the 21st century, with the abuse and improper use of antibiotics, the problem of bacterial resistance is becoming increasingly serious worldwide. The emergence of multidrug-resistant bacteria has brought unprecedented challenges to public health security. These bacteria can resist the effects of multiple antibiotics, making common infections difficult to treat and seriously threatening human life and health. According to statistics, millions of people die from drug-resistant bacterial infections every year around the world, and this number continues to rise. Therefore, the development of new antibacterial strategies and materials, especially new antibacterial materials that can effectively combat multidrug-resistant bacteria, has become a top priority for scientific research.

[0003] There are still many challenges in developing alternative treatments and therapeutic drugs for multidrug-resistant bacterial infections, mainly due to the various causes of bacterial resistance, including the widespread dissemination of resistance genes among strains, bacterial efflux pumps leading to reduced effective drug concentrations, poor antibiotic permeability, and the formation of bacterial biofilms. Photodynamic therapy (PDT) has the advantages of non-invasiveness, specific spatiotemporal selectivity, and low drug resistance, and has been considered an effective strategy for the treatment of bacterial infections. The antibacterial properties of PDT largely rely on photosensitizers (PSs), which can generate reactive oxygen species (ROS) under light irradiation, thereby inactivating bacteria. Photodynamic therapy based on the property of photosensitizers to generate reactive oxygen species is a new antibacterial strategy with excellent potential.

[0004] One of the main problems currently faced by photosensitizers in antibacterial therapy is insufficient ROS generation, which limits their effective killing effect on pathogenic bacteria. In addition, the phenomenon of reactive oxygen quenching is also a challenge, that is, photosensitizers under certain conditions (such as molecular aggregation) will lead to quenching of ROS, thereby weakening the effect of antibacterial therapy. These problems urgently need to be overcome through the development and optimization of new photosensitizers to improve the effectiveness and efficiency of antibacterial therapy. Summary of the invention

[0005] The purpose of the present invention is to provide a Nile red-based photosensitizer and its preparation and application, so as to solve the problems existing in the prior art.

[0006] A Nile red-based photosensitizer, the structural formula is as follows:

[0007]

[0008] Among them, the photosensitizer NTPA with triphenylamine as electron donor has super high reactive oxygen generation ability and has great potential to fight multi-drug resistant bacteria and promote the healing of subcutaneous abscess wounds. This is mainly because NTPA has stronger donor-acceptor (DA) properties, which reduces the energy gap (ΔE) between the singlet state (S1) and triplet state (T1) of the photosensitizer. S-T ); secondly, NTPA has a higher spin-orbit coupling constant (SOC) than the other two photosensitizers. According to perturbation theory, a smaller ΔE S-T and higher SOC can effectively promote the intersystem crossing (ISC) of photosensitizers, thereby efficiently generating reactive oxygen species for photodynamic therapy; in addition, the more distorted molecular configuration of NTPA inhibits the quenching of ROS, so NTPA exhibits the best reactive oxygen production and photodynamic therapy performance.

[0009] The preparation method of the above-mentioned Nile red-based photosensitizer comprises the following steps:

[0010] (1) adding 9-bromo-5H-benzo[a]phenoxazine-5-one, diphenylamine or (4-(diphenylamino)phenyl)boric acid or (4-(bis(4-methoxyphenyl)amino)phenyl)boric acid, tetrakis(triphenylphosphine)palladium and an inorganic base into a mixed solvent;

[0011] (2) reflux the reaction mixture of step (1) for 12-24 hours; after cooling to room temperature, filter the mixture;

[0012] (3) The filtrate is poured into water, extracted, and the organic layers are combined, washed, dried, filtered, concentrated, and the resulting mixture is purified.

[0013] Furthermore, the molar ratio of the 9-bromo-5H-benzo[a]phenoxazine-5-one to diphenylamine or (4-(diphenylamino)phenyl)boric acid or (4-(bis(4-methoxyphenyl)amino)phenyl)boric acid is 1:5-7.

[0014] Furthermore, the mixed solvent is a mixed solvent of degassed toluene, ethanol and water in a volume ratio of 8:1:1.

[0015] Furthermore, the inorganic base is one or more of potassium carbonate, sodium carbonate and cesium carbonate.

[0016] The application of the Nile red-based photosensitizer in the preparation of subcutaneous wound healing preparations is particularly used for the healing of MRSA-induced subcutaneous abscess wounds.

[0017] A subcutaneous wound healing preparation comprises the above-mentioned Nile red-based photosensitizer.

[0018] Compared with the prior art, the present invention has developed a series of Nile red-based photosensitizers, and by changing the electron-donating ability of the donor, it not only has a profound effect on the structure of the photosensitizer, but also has important significance for the generation of active oxygen by the photosensitizer. The photosensitizer NTPA of the present invention, which uses triphenylamine as an electron donor, has an ultra-high active oxygen generation ability, has great potential for resisting multi-drug-resistant bacteria and promoting the healing of subcutaneous abscess wounds, and exhibits the most excellent photodynamic therapy performance. Under white light irradiation, the active oxygen production of NTPA can exceed 470 times within 5 minutes, which is much higher than the commercial photosensitizers Ce6 and Bengal rose red. This ultra-high active oxygen generation ability shows extremely high killing ability against various drug-resistant bacteria in vitro, and the antibacterial rate even reaches 99.9%. At the same time, in the abscess model induced by methicillin-resistant Staphylococcus aureus (MRSA), NTPA nanoparticles (NPs) can achieve complete wound healing within 8 days with only a single administration and irradiation, showing its excellent ability to kill bacteria and promote wound healing, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic structural diagram of the Nile red-based photosensitizer of the present invention;

[0020] Figure 2 It is a synthetic route diagram of the Nile red-based photosensitizer of the present invention;

[0021] Figure 3 (a) and (b) are the non-normalized and normalized absorption spectra of the photosensitizer, respectively; (c) and (d) are the non-normalized and normalized photoluminescence spectra of the photosensitizer, respectively; (e) and (f) are the changes in the fluorescence intensity of NTPA in tetrahydrofuran solution with the proportion of water added; (g) and (h) are the changes in the fluorescence intensity of Nile Red in tetrahydrofuran solution with the proportion of water added.

[0022] Figure 4 (a) is the Huang-Reese (HR) factor of Nile Red, NDPA, NTPA and NTPA-OM; (b) is the reorganization energy of Nile Red, NDPA, NTPA and NTPA-OM; (c) is the contribution of bond length, bond angle and dihedral angle to the total reorganization energy.

[0023] Figure 5 (a) and (b) show the changes in the fluorescence intensity of DCFH with and without NTPA under irradiation conditions, respectively; (c) comparison of the PL intensity of DCFH with the participation of Ce6, RB, Nile Red, NDPA, NTPA and NTPA-OM under illumination conditions; (d) and (f) identification of ROS types by DHR123, HPF and ABDA; (g) and (i) show the ESR signal intensities of different ROS generated by NTPA under dark or light conditions, respectively.

[0024] Figure 6 (a) AO / PI fluorescence staining of four bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus) after treatment with PBS, NTPA and NTPA plus light (SYTO9: green fluorescence; PI: red fluorescence), scale bar: 50 μm; (b) is the average value of red fluorescence in (a) (mean ± standard deviation, n = 3), ****P < 0.0001, t test (and non-parametric test); (c) Photos of the bactericidal effects of the four bacteria under different treatments (PBS, NTPA, NTPA plus light); (d) CFU statistics of the four bacteria after different treatments; (e) Scanning electron microscopy images of the four bacteria after NTPA treatment under light or no light conditions, scale bar: 1 μm; (f) Crystal violet staining of methicillin-resistant Staphylococcus aureus biofilm after different treatments; in the figure, L represents light, -L represents no light, and +L represents light.

[0025] Figure 7 (a) Schematic diagram of the healing of MRSA-induced abscess wounds under NTPA plus light treatment in vivo; (b) and (c) are photographs and schematic diagrams of wounds 8 days after different treatments, respectively; (d) and (e) are photographs of MRSA-induced abscess wounds 8 days after different treatments and CFU (colony forming unit) statistical results (mean ± SD, n = 3), ****P < 0.0001, t test (and non-parametric test); In the figure, L represents light, -L represents injection of NTPA but no light, and +L represents injection of NTPA plus light. DETAILED DESCRIPTION

[0026] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] Example 1

[0028] The preparation method of Nile Red, such as Figure 2As shown, including: 9-bromo-5H-benzo [a] phenoxazine-5-one (0.200 g, 0.613 mmol), diethylamine (0.224 g, 3.070 mmol), cesium carbonate (1.303 g, 4.000 mmol), tris (dibenzylideneacetone) dipalladium (0.058 g, 0.063 mmol) and tri (tert-butyl) phosphine (300 mg, 1.482 mmol) are added to a degassed toluene solvent (20 ml). Then, the reaction mixture is refluxed for 12 hours. After cooling to room temperature, the mixture is filtered to remove inorganic salts. The filtrate is poured into water and extracted three times with dichloromethane. The organic layers are combined, washed with brine, then dried over anhydrous magnesium sulfate, and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.030 g (0.094 mmol, 15%) of Nile Red as a brown-black solid.

[0029] Example 2

[0030] The preparation method of NDPA, such as Figure 2 As shown, including: 9-bromo-5H-benzo [a] phenoxazine-5-one (0.200 g, 0.613 mmol), diphenylamine (0.518 g, 3.070 mmol), cesium carbonate (1.303 g, 4.000 mmol), tris (dibenzylideneacetone) dipalladium (0.058 g, 0.063 mmol) and tri (tert-butyl) phosphine (300 mg, 1.482 mmol) are added to a degassed toluene solvent (20 ml). Then, the reaction mixture is refluxed for 12 hours. After cooling to room temperature, the mixture is filtered to remove inorganic salts. The filtrate is poured into water and extracted three times with dichloromethane. The organic layers are combined, washed with brine, then dried over anhydrous magnesium sulfate, and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.050 g (0.120 mmol, 20%) of 9-(diphenylamino)-5H-benzo[a]phenoxazin-5-one (NDPA) as a dark red solid.

[0031] Example 3

[0032] The preparation method of NTPA, such as Figure 2As shown, it includes: 9-bromo-5H-benzo[a]phenoxazine-5-one (0.200 g, 0.613 mmol), (4-(diphenylamino)phenyl)boric acid (0.886 g, 3.070 mmol), tetrakis(triphenylphosphine)palladium (0.058 g, 0.050 mmol) and potassium carbonate (553 mg, 4.00 mmol) are added to a degassed mixed solvent of toluene, ethanol and water (volume ratio 8:1:1, total 50 ml). Then, the reaction mixture is refluxed for 12 hours. After cooling to room temperature, the mixture is filtered to remove inorganic salts. The filtrate is poured into water and extracted three times with dichloromethane. The organic layers are combined, washed with brine, then dried over anhydrous magnesium sulfate, and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.210 g (0.428 mmol, 70%) of dark red solid 9-(4-(diphenylamino)phenyl)-5H-benzo[a]phenoxazin-5-one (NTPA).

[0033] Example 4

[0034] The preparation method of NTPA-OM is as follows: Figure 2 As shown, 9-bromo-5H-benzo[a]phenoxazine-5-one (0.200 g, 0.613 mmol), (4-(bis(4-methoxyphenyl)amino)phenyl)boric acid (1.070 g, 3.070 mmol), tetrakis(triphenylphosphine)palladium (0.058 g, 0.050 mmol) and potassium carbonate (553 mg, 4.00 mmol) were added to a degassed mixed solvent of toluene, ethanol and water (volume ratio 8:1:1, total 50 ml). Then, the reaction mixture was refluxed for 12 hours. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted three times with dichloromethane. The organic layers were combined, washed with brine, then dried over anhydrous magnesium sulfate, and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.200 g (0.363 mmol, yield 59%) of black solid 9-(4-(bis(4-methoxyphenyl)amino)phenyl)-5H-benzo[a]phenoxazin-5-one (NTPA-OM).

[0035] Performance Test:

[0036] 1. Optical properties

[0037] Firstly, the photophysical properties of organic photosensitizers Nile Red, NDPA, NTPA and NTPA-OM were investigated using UV-visible absorption spectroscopy and fluorescence emission spectroscopy. Figure 3As shown in (a) and (b), the absorption peaks of Nile Red, NDPA, NTPA and NTPA-OM in dimethyl sulfoxide (DMSO) solution are clearly at 551, 528, 500 and 524 nanometers, respectively. The present invention also studies their fluorescence properties in DMSO solution, such as Figure 3 (c) and (d) in the figure. Nile Red shows strong red fluorescence. The photoluminescence (PL) intensity of NDPA is very weak, while the emission intensity of NTPA is stronger than that of NDPA, and NTPA-OM does not emit light. Next, in order to prove that NTPA has aggregation-induced emission (AIE) characteristics, the present invention added a large amount of water to the tetrahydrofuran (THF) solution of NTPA. From Figure 3 As can be seen from (e) and (f) in Figure 1, the emission intensity of NTPA increases significantly with the increase of water ratio, which fully demonstrates its strong AIE characteristics. In sharp contrast, the emission intensity of Nile Red drops sharply by more than 145 times, as shown in Figure 1. Figure 3 (g) and (h) in the figure show its intrinsic aggregation-induced quenching (ACQ) property.

[0038] 2. Theoretical calculation

[0039] In order to reveal the unique photophysical properties of photosensitizers, the present invention further analyzes the Huang-Rhys (HR) factor and reorganization energy, which are closely related to the radiationless decay process. The HR factor characterizes the modification of the vibration quantum (absorption or emission) when transitioning from one electronic state to another; a larger HR factor promotes the radiationless decay rate. Figure 4 (a) compares the HR factors of Nile Red, NDPA, NTPA, and NTPA-OM. It is observed that the HR factor of Nile Red is lower than that of NTPA, and significantly lower than that of NTPA-OM and NDPA. In addition, a larger reorganization energy corresponds to a higher radiationless decay rate. Figure 4 As shown in (b), the results show that the total reorganization energy of Nile Red is 234.19 MeV, NTPA is 600.35 MeV, NDPA is 2624.62 MeV, and NTPA-OM is 1394.41 MeV. In addition, the contribution of dihedral angles to the reorganization energy in Nile Red is only 2.19%, which is significantly lower than NDPA (85.46%) and NTPA-OM (68.68%). Figure 4 (c). Therefore, the fluorescence intensity of Nile Red is higher than that of NTPA, and significantly higher than that of NDPA and NTPA-OM. These calculated results are highly consistent with the spectral measurements in the solution state.

[0040] 3. Generation and identification of reactive oxygen species

[0041] The present invention uses 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as an indicator to detect the photosensitivity of NileRed, NDPA, NTPA, NTPA-OM, Bengal Rose (RB) and Ce6. Figure 5 As shown in (a), DCFH alone emits almost no light under white light irradiation. However, in the presence of all photosensitizers, the fluorescence signal of DCFH at 525 nm gradually increases with the extension of irradiation time. After 5 minutes of irradiation, the DCFH emission intensity increases less in the conditions of NTPA-OM, Ce6 and RB; however, NTPA increases the emission intensity of DCFH by more than 470 times, which is better than NDPA (400 times) and Nile Red (325 times), thus clearly indicating that NTPAs have a strong ROS-generating ability, as shown in Figure 2. Figure 5 In addition, the present invention also uses specific indicators to verify the type of ROS generated by NTPA, including for detecting superoxide anions (O2 -· ), dihydrorhodamine (DHR123) for detecting hydroxyl radicals (·OH), hydroxyphenylfluorescein (HPF) for detecting singlet oxygen ( 1 In short, the fluorescence intensity of DHR 123 and HPF was monitored. In the presence of NTPA, the fluorescence intensity of DHR 123 and HPF increased sharply by 28.6 times and 71.6 times, respectively, indicating that ·OH and O2 -· The large amount of generation, such as Figure 5 (d) and (e). The absorbance of ABDA solution decreased by only 2.79%, indicating that NTPA mainly produces ROS through the type I pathway, such as Figure 5 In addition, the present invention also uses electron spin resonance (ESR) technology, using 2,2,6,6-tetramethylpiperidine (TEMP) and 5-tert-butyloxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) as 1 Spin trap for O2 and free radicals. Figure 5 As shown in (g) and (i), under white light irradiation, when NTPA coexists with TEMP or BMPO, obvious O2 -· , OH and 1 ESR signal of O2. In summary, NTPA can generate a large amount of ROS under white light irradiation.

[0042] 4. In vitro broad-spectrum antibacterial properties

[0043] Four bacterial strains were selected, including Escherichia coli (E. coli), Pseudomonas aeruginosa (PA), Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA), to evaluate their broad-spectrum antibacterial effects. In short, after PBS, NTPA and NTPA plus light treatment, AO / PI staining was used for evaluation. Figure 6 (a) and (b) show that only green SYTO9 fluorescence (live bacteria) was observed in the PBS and NTPA groups, while under light conditions, NTPA NPs caused significant red PI fluorescence (dead bacteria) in all bacterial strains. Subsequently, the bactericidal effect of NTPA was further evaluated using the standard colony forming unit (CFU) plate count method. After incubation with bacteria for 6 hours, the antibacterial effect of NTPA under light-free conditions was low, with an inhibition rate of only 3.15%. However, under light conditions, NTPA NPs were able to effectively eliminate most E. coli, with a bacterial inhibition rate of 99.9%. Similar results were observed against PA, S. aureus, and MRSA, as shown in Figure 2. Figure 6 (c) and (d).

[0044] In order to further study the interaction between NTPANPs and bacteria, the present invention co-incubated bacteria with NTPA and observed them by scanning electron microscopy (SEM). Figure 6 As shown in (e), compared with the bacteria treated with NTPA under dark conditions, the surface of the bacteria treated with NTPA under light conditions showed obvious wrinkles and collapse (orange arrows), while the boundaries of the bacteria under dark conditions were clear and smooth (green arrows), indicating that NTPA had a significant local killing effect on bacteria under light conditions. Considering that bacterial biofilms may aggravate infections, effectively inhibiting the formation of biofilms is crucial for treating bacterial infections. Therefore, the present invention studies the ability of NTPA to destroy biofilms. Figure 6 As shown in (f), the total biomass of MRSA biofilm under different treatment conditions was quantified using crystal violet staining. As expected, the PBS group showed strong biofilm formation, characterized by relatively intact purple staining areas, regardless of whether there was light. Pure NTPANPs also failed to effectively inhibit the formation of bacterial biofilms. In contrast, the group treated with NTPA NPs plus light was unable to form a complete biofilm, with an inhibition rate of approximately 88.35%.

[0045] 5. MRSA-induced subcutaneous abscess wound healing

[0046] In view of the excellent antibacterial effect of NTPANPs in vitro, the present invention further evaluated the in vivo bactericidal effect of NTPA using the MRSA-induced abscess model and evaluated its potential to accelerate wound healing at the in vivo level. The size of the wound was observed and recorded every two days. Figure 7 (a) shows the process of in vivo experiments. Figure 7 As shown in (b), the present invention collected images of wounds of mice infected with MRSA and receiving different treatments. By day 8, mice in the PBS group still had large unhealed wounds. NTPA showed a lower therapeutic effect under no light conditions and failed to heal the wounds. Compared with the non-illuminated group, the wound area of ​​mice in the NTPA-illuminated group gradually decreased during the observation period after white light irradiation. After eight days, the external epidermal wounds were almost completely healed, indicating that NTPA-mediated photodynamic therapy has a highly significant antibacterial therapeutic effect. Semi-quantitative results showed that in mice treated with PBS, the wound area decreased by approximately 53.1% and 59.5% after eight days under non-illuminated and illuminated conditions, respectively. The wound area of ​​the NTPANPs-L group decreased by approximately 60.2%. In the NTPA-illuminated group, the wound area of ​​mice decreased by 92.2%, indicating that the effective removal of bacteria effectively promoted wound healing. In contrast, the other groups still showed signs of partial wound suppuration and skin inflammation, Figure 7 Middle (c).

[0047] After treatment, the infected tissues of each group of wounds were collected and homogenized, and then 10-fold dilutions were prepared. 100 μL of each dilution was evenly spread on the LB plate. After incubation at 37°C for 24 hours, the bacterial CFU was observed and the number of bacteria was calculated. Figure 7 As shown in (d) and (e), the bacterial CFU in the NTPA light-irradiated group was significantly reduced by more than 99.9% compared with the other three groups, indicating that MRSA in the wound infected tissue was indeed effectively eliminated due to the potent PDT effect.

Claims

1. A Nile red-based photosensitizer, characterized in that: The structural formula is as follows:

2. The method for preparing the Nile red-based photosensitizer according to claim 1, characterized in that: The following steps are involved: (1) adding 9-bromo-5H-benzo[a]phenoxazine-5-one, diphenylamine or (4-(diphenylamino)phenyl)boric acid or (4-(bis(4-methoxyphenyl)amino)phenyl)boric acid, tetrakis(triphenylphosphine)palladium and an inorganic base into a mixed solvent; (2) reflux the reaction mixture of step (1) for 12-24 hours; after cooling to room temperature, filter the mixture; (3) The filtrate is poured into water, extracted, and the organic layers are combined, washed, dried, filtered, concentrated, and the resulting mixture is purified.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the 9-bromo-5H-benzo[a]phenoxazine-5-one to diphenylamine or (4-(diphenylamino)phenyl)boric acid or (4-(bis(4-methoxyphenyl)amino)phenyl)boric acid is 1:5-7.

4. The preparation method according to claim 2, characterized in that: The mixed solvent is a mixed solvent of degassed toluene, ethanol and water in a volume ratio of 8:1:

1.

5. The preparation method according to claim 2, characterized in that: The inorganic base is one or more of potassium carbonate, sodium carbonate and cesium carbonate.

6. Use of the Nile red-based photosensitizer according to claim 1 in the preparation of a subcutaneous wound healing preparation.

7. A subcutaneous wound healing preparation, characterized in that: Comprising the Nile red-based photosensitizer according to claim 1.

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