A Nile Red Derivative with Photodynamic-Photothermal Properties, Its Preparation and Application

Abstract

This invention relates to a Nile Red derivative with photodynamic-photothermal properties, its preparation, and its applications. Two Nile Red derivatives (TPACN and TPAOMCN) were carefully designed and synthesized, with the following structural formulas: Under 660nm laser irradiation, the Nile Red derivatives of this invention can generate a large amount of reactive oxygen species and achieve efficient photothermal conversion, exhibiting powerful photodynamic-photothermal therapeutic effects. They can not only resist multidrug-resistant bacterial infections to promote wound healing but also block the spread of monkeypox virus. The TPAOMCN NFs of this invention can effectively eliminate drug-resistant bacteria in mouse wounds under laser irradiation, regulate inflammatory responses, and promote the healing of infected wounds. In the characteristic pustular lesions of simulated monkeypox, after TPAOMCN NFs combined with laser treatment, the viral titer within the lesions was significantly reduced, the infected lesion tissue was rapidly repaired, and the spread of the virus to healthy mice was effectively blocked, demonstrating great application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Nile Red derivative with photodynamic-photothermal properties and its preparation and application. Background Technology

[0002] Multidrug-resistant bacteria and monkeypox virus pose a significant threat to human health and public health security. On the one hand, bacterial infections cause millions of illnesses and deaths annually. Standard clinical treatments still heavily rely on antibiotics. However, the prevalence and overuse of antibiotics can give rise to "superbugs." The existence of superbugs presents significant challenges to infection treatment and complicates the process, increasing the risk of disease transmission and death. On the other hand, monkeypox is a zoonotic disease caused by monkeypox virus infection. In May 2022, a global monkeypox pandemic affecting more than 100 countries prompted the World Health Organization to declare monkeypox a Public Health Emergency of International Concern. However, current clinical treatment for monkeypox is primarily based on supportive care, supplemented by antiviral drugs. There are currently no specific drugs available for treating monkeypox virus. Based on these considerations, there is an urgent need to develop alternative, novel, and highly effective strategies for treating multidrug-resistant bacterial infections and preventing the spread of monkeypox virus. Summary of the Invention

[0003] The purpose of this invention is to provide a Nile Red derivative with photodynamic-photothermal properties, its preparation and application, in order to solve the problems existing in the current technology.

[0004] A Nile Red derivative with photodynamic-photothermal properties has the following structural formula:

[0005]

[0006] This invention, by altering the electron-donating ability of the donor, not only profoundly affects the structure of the photosensitizer but also plays a significant role in the generation of reactive oxygen species in the photosensitizer. This is mainly reflected in the following aspects: (1) Introducing electron-withdrawing malononitrile and electron-donating triphenylamine groups into the main framework of Nile Red promotes strong intramolecular charge transfer, thereby shifting the absorption wavelength of the molecule to the near-infrared region. Among them, triphenylamine with alkoxy groups has a stronger electron-donating ability, thus enabling TPAOMCN to exhibit the farthest absorption wavelength. (2) The D-π-A molecular structure facilitates the generation of reactive oxygen species through intersystem crossing of photosensitive molecules. The planar structure of Nile Red enhances its light-harvesting ability. Furthermore, triphenylamine can act as a rotor, facilitating the rotation of molecules in an aggregated state, further promoting photothermal conversion while avoiding the quenching of reactive oxygen species. (3) After TPAOMCN is prepared into nanofibers (TPAOMCN NFs), TPAOMCN NFs can effectively kill multidrug-resistant bacteria and monkeypox virus under laser irradiation.

[0007] The preparation method of the above-mentioned Nile Red derivative with photodynamic-photothermal properties includes the following steps:

[0008] (1) Add 2-(9-bromo-5H-benzo[a]phenoxyazine-5-ylidene)malonitrile, (4-(diphenylamino)phenyl)boronic acid or (4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid, tetra(triphenylphosphine)palladium and inorganic base to a mixed solvent;

[0009] (2) Reflux the reaction mixture from step (1) for 12-24 hours; after cooling to room temperature, filter the mixture;

[0010] (3) Pour the filtrate into water, extract, combine the organic layers, wash, dry, filter, concentrate, and purify the resulting mixture.

[0011] Furthermore, the molar ratio of 2-(9-bromo-5H-benzo[a]phenoxyazine-5-ylidene)malonitrile to (4-(diphenylamino)phenyl)boronic acid or (4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid is 1:5-6.

[0012] Furthermore, the mixed solvent is a mixture of degassed toluene, ethanol, and water in a volume ratio of 8:1:1.

[0013] Furthermore, the inorganic base is one or more of potassium carbonate, sodium carbonate, or cesium carbonate.

[0014] The aforementioned nanofibers of Nile Red derivatives with photodynamic-photothermal properties are prepared by blending the aforementioned Nile Red derivatives with polyvinylidene fluoride (PVDF) and then electrospinning. TPAOMCN nanofibers (TPAOMCN NFs) are prepared by blending TPAOMCN with PVDF and then electrospinning. On the one hand, the particle size is difficult to control during nanoparticle preparation; on the other hand, nanoparticles require tail vein injection for in vivo applications, and they will be distributed throughout the body via the bloodstream. Nanofibers are prepared by machine electrospinning, which allows for excellent control of uniformity. Furthermore, nanofibers are applied to wounds, making the process simple and easy to perform.

[0015] The above-mentioned Nile Red derivatives with photodynamic-photothermal properties are used in the preparation of agents to combat drug-resistant bacterial infections. In a mouse model of MRSA infection, TPAOMCN NFs effectively cleared drug-resistant bacteria from mouse wounds under laser irradiation, modulated the inflammatory response, and promoted wound healing.

[0016] The above-mentioned Nile Red derivatives with photodynamic-photothermal properties are used in the preparation of agents to inhibit monkeypox virus. This invention uses a tail-scratching mouse model infected with vaccinia virus as a substitute model for monkeypox to simulate the characteristic pustular lesions of monkeypox. After TPAOMCN NFs combined with laser treatment, the viral titer within the lesions is significantly reduced, the infected lesions are rapidly repaired, and the transmission of the virus to healthy mice is effectively blocked.

[0017] An inhibitory agent for monkeypox virus, comprising the above-mentioned Nile Red derivative having photodynamic-photothermal properties.

[0018] This will provide important insights and reference value for developing new clinical strategies for combating next-generation microbial infections.

[0019] Compared with existing technologies, this invention meticulously designed and synthesized two Nile Red derivatives (TPACN and TPAOMCN), and thoroughly investigated their safe, efficient, and broad-spectrum anti-multidrug-resistant bacteria capabilities and their ability to block the spread of monkeypox virus. Under 660nm laser irradiation, the Nile Red derivatives of this invention can generate a large amount of reactive oxygen species and achieve efficient photothermal conversion, exhibiting powerful photodynamic-photothermal therapeutic effects. They can not only combat multidrug-resistant bacterial infections to promote wound healing, but also block the spread of monkeypox virus. The TPAOMCN NFs of this invention effectively cleared drug-resistant bacteria from mouse wounds under laser irradiation, regulated the inflammatory response, and promoted the healing of infected wounds. In simulated monkeypox pustular lesions, after laser treatment with the TPAOMCN NFs of this invention, the viral titer within the lesions was significantly reduced, the infected lesion tissue was rapidly repaired, and the spread of the virus to healthy mice was effectively blocked, demonstrating great application potential. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of the Nile Red derivative of the present invention;

[0021] Figure 2 This is a synthetic route diagram of the Nile Red derivative of the present invention;

[0022] Figure 3 (a) Fluorescence emission spectrum of photosensitizer; (b) UV absorption spectrum of photosensitizer; (c) Electron cloud arrangement and energy level difference of TPACN and TPAOMCN in HOMO and LUMO orbitals.

[0023] Figure 4 (a) Scanning image of TPAOMCN NFs; (b) Fluorescence intensity change of TPAOMCN NFs co-incubated with DCFH under laser irradiation; (c), (d), and (e) ESR signal intensities of different ROS generated by TPAOMCN NFs under dark or laser irradiation conditions, respectively; (f) Temperature change under 660nm laser irradiation with 0% or 0.5% TPACNON doping; (g) Laser power dependence of TPAOMCN NFs for photothermal conversion; (h) Photothermal stability of TPAOMCN NFs.

[0024] Figure 5 (a) AO / PI fluorescence staining images of MRSA after treatment with PBS, TPAOMCN NFs, and TPAOMCN NFs plus light (SYTO9: green fluorescence; PI: red fluorescence), scale bar: 50 μm; (b) Photographs of the bactericidal effects of different treatments (PBS, TPAOMCN NFs, TPAOMCN NFs plus light) on MRSA; (c) Statistical results of CFU of MRSA after different treatments; (e) GFP staining detection of residual live virus; (f) Comparison of bright-field images of host cells protected by TPAOMCN NFs; (g) Comparison of host cell survival rates after different treatments.

[0025] Figure 6 (a) and (b) are photographs and diagrams of the wounds 12 days after different treatments, respectively; (c) is the statistical result of the changes in wound area of ​​mice receiving different treatments (mean ± standard deviation, n = 3), ****P < 0.0001, t test (and non-parametric test).

[0026] Figure 7 (a) and (b) are photographs of wounds 12 days after different treatments and changes in wound area of ​​mouse tails in different groups during treatment, respectively; (c) viral titer determination; (d) immunohistochemical staining of vaccinia virus antigen; (e) and (f) are determinations of pro-inflammatory factor concentrations. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example 1

[0029] The preparation method of TPACN includes: adding 0.200 g (0.534 mmol) of 2-(9-bromo-5H-benzo[a]phenoxyazine-5-yl)malononitrile, 0.772 g (2.670 mmol) of (4-(diphenylamino)phenyl)boronic acid, 0.058 g (0.050 mmol) of Pd(PPh3)4, and 553 mg (4.00 mmol) of K2CO3 to a degassed solvent mixture of toluene, ethanol, and water (volume ratio = 8:1:1, 50 mL). The reaction mixture is then 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 CH2Cl2. The combined organic layers are washed with brine, dried on anhydrous MgSO4, and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to give 0.200 g (0.371 mmol, 69%) of a black solid 2-(9-(4-(diphenylamino)phenyl)-7a,12a-dihydro-5H-benzo[a]phenoxazine-5-ylidene)malononitrile (TPACN).

[0030] Example 2

[0031] The preparation method of TPAOMCN includes: adding 2-(9-bromo-5H-benzo[a]phenoxyazine-5-yl)malononitrile (0.200 g, 0.534 mmol), (4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid (0.933 g, 2.670 mmol), Pd(PPh3)4 (0.058 g, 0.050 mmol), and K2CO3 (553 mg, 4.00 mmol) to a degassed solvent mixture of toluene, ethanol, and water (volume ratio = 8:1:1, 50 mL). The reaction mixture is then 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 CH2Cl2. The combined organic layers are washed with brine, dried on anhydrous MgSO4, and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to give 0.22 g (0.367 mmol, 69%) of a black solid 2-(9-(4-(bis(4-methoxyphenyl)amino)phenyl)-7a,12a-dihydro-5H-benzo[a]phenoxazine-5-ylidene)malononitrile (TPAOMCN).

[0032] Performance testing:

[0033] 1. Optical properties

[0034] First, the photophysical properties of TPACN and TPAOMCN were investigated using ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy. For example... Figure 3 As shown, neither TPACN nor TPAOMCN exhibits a significant fluorescence emission peak in dimethyl sulfoxide (DMSO) solution. Their main absorption peaks appear at 602 nm and 633 nm, respectively. Theoretical calculations indicate that because TPAOMCN has a greater electron-donating intensity, the difference between its lowest unoccupied orbital (LUMO) and highest occupied orbital (HOMO) is smaller. A smaller LUMO value favors a redshift in the molecular absorption wavelength, which is consistent with the longer absorption wavelength of TPAOMCN in solution.

[0035] 2. Reactive oxygen species generation and photothermal conversion performance

[0036] First, TPAOMCN was blended with polyvinylidene fluoride and then electrospun to prepare TPAOMCN nanofibers (TPAOMCN NFs), such as... Figure 4 (a) The reactive oxygen species (ROS) generation and photothermal conversion of TPAOMCN NFs were then analyzed. Using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as an indicator, the fluorescence intensity of DCFH gradually increased over a 5-minute irradiation period, indicating that TPAOMCN NFs can generate a large amount of ROS, such as... Figure 4 In addition, electron spin resonance (ESR) technology was employed, with 2,2,6,6-tetramethylpiperidine (TEMP) and 5-tert-butoxycarbonyl-5-methyl-1-pyrrolidine N-oxide (BMPO) as the... 1 Spin trappers for O2 and free radicals. For example... Figure 4 As shown in (c), (d), and (e), significant O2 was observed when TPAOMCN NFs coexisted with TEMP or BMPO under laser irradiation. -· ·OH and 1 The ESR signal of O2 indicates that TPAOMCN NFs can simultaneously generate type I and type II reactive oxygen species. This invention further tested the photothermal conversion performance of TPAOMCN NFs. For example... Figure 4 As shown in (f), the temperature of TPAOMCN NFs gradually increased during the 1-minute irradiation period, reaching a maximum of over 75°C. Subsequently, this invention further tested the power dependence and thermal stability of TPAOMCN NFs during photothermal conversion. Figure 4As shown in (g) and (h), the temperature of TPAOMCN NFs gradually increases with increasing laser power. Through three laser on-off cycles, it can be observed that the maximum temperature of TPAOMCN NFs does not decrease significantly, indicating that TPAOMCN NFs possess excellent cyclic stability.

[0037] 3. In vitro antibacterial and antiviral effects

[0038] The antibacterial efficacy of TPAOMCN NFs was evaluated against methicillin-resistant Staphylococcus aureus (MRSA). In short, the antibacterial effects were assessed using AO / PI staining after treatment with PBS, PBS with light, TPAOMCN NFs, and TPAOMCN NFs with light. Figure 5 As shown in (a), only green SYTO9 fluorescence (live bacteria) was observed in the PBS and TPAOMCN NFs groups, while under light conditions, TPAOMCN NFs caused significant red PI fluorescence (dead bacteria) in the bacterial strains. The bactericidal effect of TPAOMCN NFs was further evaluated using the standard colony forming unit (CFU) plate counting method. After 6 hours of incubation with bacteria, the antibacterial effect of TPAOMCN NFs was low under light-free conditions. However, under light conditions, TPAOMCN NFs effectively eliminated most MRSA, achieving a bacterial inhibition rate of 99.9%. Figure 5 (b) and (c). To explore the antiviral activity of laser-induced TPAOMCN NFs, vaccinia virus was co-incubated with PBS and TPAOMCN NFs for 30 minutes, followed by incubation with or without a 660nm laser (0.4W cm⁻¹). -2 Irradiate for 5 minutes. Then, determine the residual live virus after different treatments using GFP staining. Figure 5 As shown in (d), without 660nm laser stimulation, TPAOMCN NFs had almost no killing effect on vaccinia virus. Under 660nm laser irradiation, the PBS group still showed no antiviral effect, while the TPAOMCN NFs group almost completely eradicated the virus, thus enhancing its photothermal antiviral effect. Finally, after infecting host cells (BHK-21) with vaccinia virus of different pretreatments for 12 hours, the "no laser" group and the "PBS + laser" group showed obvious cytopathic effects and decreased cell viability. In contrast, the laser-irradiated TPAOMCN NFs group could completely inhibit host cell death, such as... Figure 5 (e) and (f).

[0039] 4. In vivo antibacterial and wound-healing effects

[0040] Given the excellent antibacterial effects of TPAOMCN NFs exhibited in vitro, this invention further evaluated the in vivo bactericidal effect of TPAOMCN NFs using an MRSA-induced wound infection model and assessed its potential to accelerate wound healing at the in vivo level. Wound size was observed and recorded every two days, such as... Figure 6 As shown in (a), images of mouse wounds after 12 days of treatment with different methods were collected. Mice in the PBS group still had large unhealed wounds. TPAOMCN NFs showed lower therapeutic efficacy in the absence of light and failed to heal wounds. Compared with the un-light-exposed group, the wound area of ​​mice in the TPAOMCN NFs-exposed group was significantly reduced during the observation period, and the external epidermal wounds were almost completely healed after 12 days, indicating that TPAOMCN NFs-mediated photodynamic-photothermal therapy has highly significant antibacterial and wound-healing effects. Figure 6 (b) and (c).

[0041] 5. Live antiviral

[0042] This invention systematically evaluated the antiviral, anti-inflammatory, and wound-healing effects of TPAOMCN NFs. In mice infected with the virus, tail lesions did not improve after intravenous injection of PBS or encapsulation with TPAOMCN NFs without subsequent laser irradiation, and significant scab formation remained. However, in the TPAOMCN NFs-irradiated group, the tail wound area gradually decreased. Figure 7 (a) and (b). Furthermore, detection of vaccinia virus titers in tail lesion tissue showed that after TPAOMCN NFs treatment, 660nm laser irradiation almost eradicated the virus compared to other groups, such as... Figure 7 (c) To assess the virus clearance capabilities of different treatments, tail lesion tissue was collected 7 days later and sections were subjected to immunohistochemical staining (IHC) for vaccinia virus antigens. In the absence of subsequent irradiation, all groups showed extremely high viral antigen-positive immunohistochemical density. In contrast, compared to the PBS group, the TPAOMCN NFs treatment group, which subsequently received 660nm laser irradiation, showed a significantly lower viral antigen-positive immunohistochemical density, such as... Figure 7 (d) Subsequently, the concentrations of pro-inflammatory factors such as IL-1β and IL-6 in tail lesion tissues after different treatments were detected by enzyme-linked immunosorbent assay (ELISA). Figure 7 As shown in (e) and (f), the levels of these pro-inflammatory cytokines in the tail lesion tissue were very high in both the 660 nm laser-irradiated and unirradiated PBS groups, indicating that vaccinia virus infection triggered a strong inflammatory response. Treatment with TPAOMCN NFs resulted in a moderate decrease in the levels of these pro-inflammatory cytokines upon the introduction of a 660 nm laser, demonstrating extremely strong anti-inflammatory activity.

[0043] In a vaccinia virus-infected tail scratch mouse model, used as a surrogate model for monkeypox to mimic the characteristic pustular lesions of monkeypox, TPAOMCN NFs combined with laser treatment significantly reduced viral titers within the lesions, rapidly repaired infected tissue, and effectively blocked viral transmission to healthy mice. This effect of clearing the virus, reducing inflammation, and repairing wounds fully demonstrates the antiviral and skin-beautifying potential of "TPAOMCN NFs + laser" in treating monkeypox.

Claims

1. A Nile Red derivative with photodynamic-photothermal properties, characterized in that, The structure is as follows: 。 2. The method for preparing the Nile Red derivative with photodynamic-photothermal properties according to claim 1, characterized in that, Includes the following steps: (1) Add 2-(9-bromo-5H-benzo[a]phenoxyazine-5-ylidene)malonitrile, (4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid, tetra(triphenylphosphine)palladium and inorganic base to a mixed solvent; (2) Reflux the reaction mixture from step (1) for 12-24 hours; after cooling to room temperature, filter the mixture; (3) Pour the filtrate into water, extract, combine the organic layers, wash, dry, filter, concentrate, and purify the resulting mixture.

3. The preparation method according to claim 2, characterized in that, The molar ratio of 2-(9-bromo-5H-benzo[a]phenoxyazine-5-ylidene)malonitrile to (4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid is 1:5-6.

4. The preparation method according to claim 2, characterized in that, The mixed solvent is a mixture 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, or cesium carbonate.

6. The nanofibers of Nile Red derivatives with photodynamic-photothermal properties according to claim 1, characterized in that, It is prepared by blending the Nile Red derivative with photodynamic and photothermal properties with polyvinylidene fluoride and then electrospinning.

7. The application of the Nile Red derivative nanofibers with photodynamic-photothermal properties as described in claim 1 in the preparation of agents for treating drug-resistant bacterial infections.

8. The application of the Nile Red derivative nanofibers with photodynamic-photothermal properties as described in claim 1 in the preparation of agents to inhibit monkeypox virus.

9. An inhibitory agent for monkeypox virus, characterized in that, Nanofibers containing Nile Red derivatives with photodynamic-photothermal properties as described in claim 1.

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