A Nile red-based photosensitizer and its preparation and application

Abstract

The present invention relates to a Nile red-based photosensitizer and its preparation and application. The present invention has developed a series of Nile red-based photosensitizers with the following structural formula: The present invention not only has a profound impact on the structure of the photosensitizer by changing the electron donating ability of the donor, but also has important significance for the generation of active oxygen by the photosensitizer. The NTPA of the present invention has ultra-high active oxygen generation capacity, has great potential to fight multi-drug resistant bacteria and promote the healing of subcutaneous abscess wounds, and exhibits the most excellent photodynamic therapy performance. Under white light irradiation, its active oxygen production can be generated more than 470 times within 5 minutes. NTPA nanoparticles (NPs) show extremely high killing ability against various drug-resistant bacteria in vitro, and its in vitro antibacterial rate can reach 99.9%. It also shows its excellent bactericidal and wound healing ability at the living body level, and has great application prospects.

Description

Technical Field

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

[0002] In the 21st century, with the overuse and inappropriate use of antibiotics, bacterial resistance is becoming an increasingly serious problem worldwide. The emergence of multidrug-resistant bacteria has posed an unprecedented challenge to public health and safety. These bacteria are able to resist the effects of multiple antibiotics, making common infections difficult to treat and posing a serious threat to human life and health. According to statistics, millions of people die worldwide each year from drug-resistant bacterial infections, and this number continues to rise. Therefore, the development of new antimicrobial strategies and materials, especially new antimicrobial materials that can effectively combat multidrug-resistant bacteria, has become a top priority in scientific research.

[0003] The development of alternative treatments and therapeutic agents for multidrug-resistant bacterial infections remains challenging, primarily due to the diverse causes of bacterial resistance, including widespread dissemination of resistance genes between strains, reduced effective drug concentrations due to bacterial efflux pumps, poor antibiotic permeability, and the formation of bacterial biofilms. Photodynamic therapy (PDT) has the advantages of being noninvasive, having specific spatiotemporal selectivity, and exhibiting low drug resistance, and has been recognized as an effective strategy for treating bacterial infections. The antibacterial properties of PDT largely rely on photosensitizers (PSs), which can generate reactive oxygen species (ROS) upon light irradiation, thereby inactivating bacteria. Photodynamic therapy, based on the ROS-generating property of photosensitizers, is a novel and promising antibacterial strategy.

[0004] One of the main challenges currently facing the use of photosensitizers in antimicrobial therapy is insufficient ROS production, which limits their effective killing of pathogens. Furthermore, the phenomenon of reactive oxygen species quenching (ROS) is a challenge. Under certain conditions (such as molecular aggregation), photosensitizers can quench ROS, thereby weakening the effectiveness of antimicrobial therapy. These issues urgently need to be overcome through the development and optimization of new photosensitizers to improve the effectiveness and efficiency of antimicrobial 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, which uses triphenylamine as an electron donor, has an extremely high ability to generate reactive oxygen species and has great potential to fight multi-drug resistant bacteria and promote the healing of subcutaneous abscess wounds. This is mainly because NTPA has a stronger donor-acceptor (DA) property, 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 The high SOC can effectively promote the intersystem crossing (ISC) of the photosensitizer, 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 species 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 to 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 was poured into water for extraction, and the organic layers were combined, washed, dried, filtered, concentrated, and the resulting mixture was 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 above-mentioned Nile red-based photosensitizer is used in the preparation of subcutaneous wound healing preparations, especially 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 impact 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 to fight multi-drug resistant bacteria and promote 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 far higher than that of 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, with an inhibition rate of even 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, demonstrating its excellent bactericidal and wound healing ability, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the structure of the Nile red-based photosensitizer of the present invention;

[0020] Figure 2 This is a synthetic route 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 added water; (g) and (h) are the changes in the fluorescence intensity of Nile Red in tetrahydrofuran solution with the proportion of added water.

[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 without and with 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 light 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 images 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 ± SD, n = 3), ****P < 0.0001, t-test (and non-parametric test); (c) photos of the bactericidal effect 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 images 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 in vivo under NTPA plus light treatment; (b) and (c) are photographs and schematic diagrams of the 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 ± standard deviation, n = 3), ****P < 0.0001, t-test (and non-parametric test); in the figure, L represents light, -L represents NTPA injection but no light, and +L represents NTPA injection 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 described in further detail below with reference to the accompanying drawings.

[0027] Example 1

[0028] The preparation method of Nile Red, such as Figure 2As shown, comprising: 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 brownish-black solid.

[0029] Example 2

[0030] The preparation method of NDPA, such as Figure 2 As shown, comprising: 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, the method comprises: adding 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) 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 0.210 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)boronic 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, 59% yield) of 9-(4-(bis(4-methoxyphenyl)amino)phenyl)-5H-benzo[a]phenoxazin-5-one (NTPA-OM) as a black solid.

[0035] Performance testing:

[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 nm, 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. 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 decreases 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] To reveal the unique photophysical properties of photosensitizers, the present study further analyzed 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 vibrational quantum (absorption or emission) during the transition 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 angle 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 solution.

[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, Rose Bengal (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 emission intensity of DCFH increases less in the presence 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 generation ability, as shown in Figure 5. Figure 5 In addition, the present invention also uses specific indicators to verify the type of ROS produced 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 present invention monitored the fluorescence intensity of DHR 123 and HPF. 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 massive generation of 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 trapping agent 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 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, including Escherichia coli (E. coli), Pseudomonas aeruginosa (PA), Staphylococcus aureus (S. aureus), and methicillin-resistant Staphylococcus aureus (MRSA), were selected for evaluation of their broad-spectrum antimicrobial efficacy. Briefly, the antimicrobial activity was evaluated using AO / PI staining after treatment with PBS, NTPA, and NTPA plus illumination. 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 a standard colony-forming unit (CFU) plate count method. After incubation with bacteria for 6 hours, the antibacterial effect of NTPA under no light 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 5. 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 studied the ability of NTPA to destroy biofilms. Figure 6 As shown in (f), the total biomass of MRSA biofilms under different treatment conditions was quantified using crystal violet staining. As expected, the PBS group showed strong biofilm formation, characterized by relatively complete 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] Given the excellent antibacterial effect of NTPANPs in vitro, the present invention further evaluated the in vivo bactericidal effect of NTPA using a 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 in mice infected with MRSA and receiving different treatments. By day 8, the 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 ​​the 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 ​​the 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, infected wound tissues from each group were collected and homogenized, and then 10-fold dilutions were prepared. 100 μL of each dilution was evenly spread on LB plates. After incubation at 37°C for 24 hours, bacterial CFU were 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 use of a Nile red-based photosensitizer in the preparation of a subcutaneous wound healing preparation, characterized in that: The structural formula of the Nile red-based photosensitizer is as follows: 。

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