Nile red derivative with photodynamic-photothermal performance and preparation and application thereof

Abstract

The invention relates to a Nile red derivative with photodynamic-photothermal performance as well as preparation and application thereof, two Nile red derivatives (TPACN and TPAOMCN) are elaborately designed and synthesized, and the structural formula is as follows: # imgabs0. The Nile red derivative disclosed by the invention can generate a large amount of active oxygen and efficient photothermal conversion under the irradiation of 660nm laser; a strong photodynamic-photothermal treatment effect is shown, multi-drug-resistant bacterium infection can be resisted to promote wound repair, and propagation of monkey pox viruses can be blocked. The TPAOMCN NFs provided by the invention can effectively remove drug-resistant bacteria in mouse wounds under laser irradiation, regulate inflammatory response and promote healing of infected wounds. In simulation of characteristic impetigo of monkey pox, after TPAOMCN NFs and laser treatment, virus titer in lesion is significantly reduced, infected lesion tissue is rapidly repaired, transmission of viruses to healthy mice is effectively blocked, and the TPAOMCN NFs has a huge application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a Nile red derivative with photodynamic and photothermal properties, and a preparation and application thereof. Background Art

[0002] Multidrug-resistant bacteria and monkeypox virus pose a major threat to human health and public health security. On the one hand, bacterial infections cause millions of illnesses and deaths every year. The standard treatment regimen used in clinical practice still relies heavily on the use of antibiotics. However, the prevalence and abuse of antibiotics may give rise to "super bacteria". The existence of super bacteria not only poses a huge challenge to the treatment of infections, but also complicates the treatment process, which increases 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 pandemic of monkeypox virus involving more than 100 countries broke out, prompting the World Health Organization to declare monkeypox a public health emergency of international concern. However, the current clinical treatment regimen for monkeypox is mainly based on supportive treatment, supplemented by some antiviral drugs. There is currently no specific drug for monkeypox virus on the market. Based on the above considerations, there is an urgent need to develop alternative, new and efficient strategies to treat multidrug-resistant bacterial infections and prevent the spread of monkeypox virus. Summary of the invention

[0003] The purpose of the present invention is to provide a Nile red derivative with photodynamic and photothermal properties and its preparation and application, so as to solve the problems existing in the current technology.

[0004] A Nile red derivative with photodynamic and photothermal properties, the structural formula is as follows:

[0005]

[0006] 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, which is mainly reflected in: (1) the electron-withdrawing group malononitrile and the electron-donating group triphenylamine are introduced into the main skeleton of Nile Red to promote strong intramolecular charge transfer, thereby red-shifting the absorption wavelength of the molecule to the near-infrared region; among them, the triphenylamine with alkoxy group has a stronger electron donating ability, thereby making TPAOMCN show the farthest absorption wavelength; (2) the molecular structure of D-π-A is conducive to the generation of active oxygen by photosensitive molecules through intersystem crossing; the main structure of Nile Red is a planar structure, which is conducive to enhancing the light capture ability. In addition, triphenylamine can also act as a rotor to facilitate the rotation of molecules in the aggregated state, further promote photothermal conversion, and avoid quenching active oxygen. (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 method for preparing the above-mentioned Nile red derivative having photodynamic and photothermal properties comprises the following steps:

[0008] (1) adding 2-(9-bromo-5H-benzo[a]phenoxyazine-5-ylidene)malononitrile, (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;

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

[0010] (3) The filtrate is poured into water, extracted, and the organic layers are combined, washed, dried, filtered, concentrated, and the resulting mixture is purified.

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

[0012] Furthermore, the mixed solvent is a mixed solvent 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 nanofibers of the Nile red derivatives with photodynamic-photothermal properties are prepared by blending the Nile red derivatives with photodynamic-photothermal properties with polyvinylidene fluoride and electrospinning. TPAOMCN is blended with polyvinylidene fluoride and prepared into TPAOMCN nanofibers (TPAOMCN NFs) by electrospinning. On the one hand, the size of the particle size is not well controlled during the preparation of nanoparticles; on the other hand, nanoparticles need to be injected into the tail vein in in vivo applications, and they will be distributed throughout the body through the blood. The preparation of nanofibers is electrospun by machine, and the uniformity is well controlled. In addition, the nanofibers are applied to the wound, which is simple and easy to operate.

[0015] The above-mentioned Nile Red derivatives with photodynamic and photothermal properties are used in the preparation of preparations for anti-resistant bacterial infection. In a MRSA-infected mouse model, TPAOMCN NFs effectively removed drug-resistant bacteria from mouse wounds under laser irradiation, regulated inflammatory responses, and promoted the healing of infected wounds.

[0016] The application of the Nile Red derivatives with photodynamic and photothermal properties in the preparation of preparations for inhibiting monkeypox virus. The present invention uses the tail scratch mouse model infected with cowpox virus as a substitute model for monkeypox to simulate the characteristic pustular lesions of monkeypox. After TPAOMCN NFs plus laser treatment, the virus titer in the lesion is significantly reduced, the infected lesion tissue is quickly repaired, and the spread of the virus to healthy mice is effectively blocked.

[0017] A preparation for inhibiting monkeypox virus comprises the above-mentioned Nile red derivative with photodynamic and photothermal properties.

[0018] This will provide important reference significance and value for the development of the next generation of new clinical strategies for anti-microbial infections.

[0019] Compared with the prior art, the present invention has carefully designed and synthesized two Nile Red derivatives (TPACN and TPAOMCN), and has deeply studied its ability to safely and efficiently broadly resist multi-drug resistant bacteria and block the spread of monkeypox virus. The Nile Red derivatives of the present invention can produce a large amount of reactive oxygen and efficient photothermal conversion under 660nm laser irradiation, showing a strong photodynamic-photothermal therapeutic effect, which can not only resist multi-drug resistant bacteria infection to promote wound repair, but also block the spread of monkeypox virus. The TPAOMCN NFs of the present invention effectively remove drug-resistant bacteria in mouse wounds under laser irradiation, regulate inflammatory response, and promote the healing of infected wounds. In the characteristic pustular lesions simulating monkeypox, after the TPAOMCN NFs of the present invention were treated with laser, the viral titer in the lesion was significantly reduced, the infected lesion tissue was quickly repaired, and the spread of the virus to healthy mice was effectively blocked, which has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0022] Figure 3 (a) Fluorescence emission spectrum of the photosensitizer; (b) UV absorption spectrum of the 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) Change in fluorescence intensity 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 when the TPACNON doping amount is 0% or 0.5%; (g) Laser power dependence of photothermal conversion of TPAOMCN NFs; (h) Photothermal stability of TPAOMCN NFs.

[0024] Figure 5 (a) AO / PI fluorescence staining of MRSA after treatment with PBS, TPAOMCN NFs, and TPAOMCN NFs plus light (SYTO9: green fluorescence; PI: red fluorescence), scale bar: 50 μm; (b) Photos of the bactericidal effects of different treatments (PBS, TPAOMCN NFs, TPAOMCN NFs plus light) on MRSA; (c) CFU statistics of MRSA after different treatments; (e) GFP staining to detect residual live viruses; (f) Comparison of bright field images of TPAOMCN NFs protecting host cells; (g) Comparison of the survival rates of host cells after different treatments.

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

[0026] Figure 7 (a) and (b) are photos of wounds 12 days after different treatments and the changes in wound area of ​​the mouse tails in different groups during the treatment process; (c) virus titer determination; (d) vaccinia virus antigen immunohistochemical staining; (e) and (f) are concentration determinations of pro-inflammatory factors. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1

[0029] The preparation method of TPACN comprises: adding 2-(9-bromo-5H-benzo[a]phenoxyazine-5-ylidene)malononitrile (0.200 g, 0.534 mmol), (4-(diphenylamino)phenyl)boric acid (0.772 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). 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 CH2Cl2. The organic layers are combined, washed with brine, then dried over anhydrous MgSO4, 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 (0.371 mmol, 69%) of 2-(9-(4-(diphenylamino)phenyl)-7a,12a-dihydro-5H-benzo[a]phenoxazin-5-ylidene)malononitrile (TPACN) as a black solid.

[0030] Example 2

[0031] The preparation method of TPAOMCN comprises: adding 2-(9-bromo-5H-benzo[a]phenoxyazine-5-ylidene)malononitrile (0.200 g, 0.534 mmol), (4-(bis(4-methoxyphenyl)amino)phenyl)boric 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). 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 CH2Cl2. The organic layers are combined, washed with brine, then dried over anhydrous MgSO4, and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.22 g (0.367 mmol, 69%) of black solid 2-(9-(4-(bis(4-methoxyphenyl)amino)phenyl)-7a,12a-dihydro-5H-benzo[a]phenoxazine-5-ylidene)malononitrile (TPAOMCN).

[0032] Performance Test:

[0033] 1. Optical properties

[0034] Firstly, the photophysical properties of TPACN and TPAOMCN were investigated using UV-visible absorption spectroscopy and fluorescence emission spectroscopy. Figure 3 As shown in the figure, TPACN and TPAOMCN do not have obvious fluorescence emission peaks in dimethyl sulfoxide (DMSO) solution. The main peaks of their absorption spectra appear at 602 and 633 nanometers, respectively. Theoretical calculations show that since TPAOMCN has a greater electron donation intensity, the difference between its lowest unoccupied orbital (LUMO) and highest occupied orbital (HOMO) is smaller. The smaller this value is, the more conducive it is to the red shift of the absorption wavelength of the molecule, which is consistent with the longer absorption wavelength of TPAOMCN in solution.

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

[0036] First, TPAOMCN and polyvinylidene fluoride were blended and prepared into TPAOMCN nanofibers (TPAOMCN NFs) by electrospinning. Figure 4 (a). The reactive oxygen species generation and photothermal conversion performance of TPAOMCN NFs were then analyzed. Using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as an indicator, the fluorescence intensity of DCFH gradually increased within 5 minutes of irradiation, indicating that TPAOMCN NFs can generate a large amount of reactive oxygen species, such as Figure 4 In addition, electron spin resonance (ESR) technology was used, 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 4 As shown in (c), (d), and (e), when TPAOMCN NFs coexisted with TEMP or BMPO under laser irradiation, obvious O2 -· , OH and 1 The ESR signal of O2 indicates that TPAOMCN NFs can simultaneously produce type I and type II reactive oxygen species. The present invention further tests the photothermal conversion performance of TPAOMCN NFs. Figure 4 As shown in (f), within 1 minute of irradiation, the temperature of TPAOMCN NFs gradually increased, and the highest temperature exceeded 75°C. Subsequently, the present invention tested the power dependence and thermal stability of TPAOMCN NFs for photothermal conversion. Figure 4As shown in (g) and (h), the temperature of TPAOMCN NFs gradually increases with the increase of laser power. After three cycles of laser on and off, it can be seen that the maximum temperature of TPAOMCN NFs has no obvious decrease, which shows that TPAOMCN NFs has excellent cycle stability.

[0037] 3. In vitro antibacterial and antiviral

[0038] Methicillin-resistant Staphylococcus aureus (MRSA) was used as the target to evaluate the antibacterial effect of TPAOMCN NFs. Briefly, after treatment with PBS, PBS plus light, TPAOMCN NFs, and TPAOMCN NFs plus light, AO / PI staining was used for evaluation. Figure 5 (a) shows that 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. Subsequently, the bactericidal effect of TPAOMCN NFs was further evaluated using the standard colony forming unit (CFU) plate count method. After incubation with bacteria for 6 hours, the antibacterial effect of TPAOMCN NFs under no light conditions was low. However, under light conditions, TPAOMCN NFs were able to effectively eliminate most MRSA, with a bacterial inhibition rate of 99.9%. Figure 5 To explore the antiviral ability of laser-induced TPAOMCN NFs, vaccinia virus was co-incubated with PBS and TPAOMCN NFs for 30 min, followed by the presence or absence of 660 nm laser (0.4 W cm -2 ) for 5 minutes. Then, the residual live virus after different treatments was determined by GFP staining. Figure 5 As shown in (d), in the absence of 660nm laser stimulation, TPAOMCN NFs had almost no killing effect on vaccinia virus. Under 660nm laser irradiation, the PBS group still had no antiviral effect, while the TPAOMCN NFs group almost completely eradicated the virus, thereby enhancing its photothermal virus-killing effect. Finally, after the host cells (BHK-21) were infected with vaccinia viruses with different pretreatments for 12 hours, the "no laser" group and the "PBS+laser" group showed obvious cell pathology and decreased cell viability. In contrast, the laser-irradiated TPAOMCN NFs group can completely inhibit host cell death, as shown in Figure 5 (e) and (f).

[0039] 4. Live antibacterial infection and promote wound healing

[0040] In view of the excellent antibacterial effect of TPAOMCN NFs in vitro, the present invention further evaluated the in vivo bactericidal effect of TPAOMCN NFs using the MRSA-induced wound infection 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 6 As shown in (a), images of the wounds of mice receiving different treatments were collected 12 days later. The mice in the PBS group still had large unhealed wounds. TPAOMCN NFs showed a lower therapeutic effect under no light conditions and failed to heal the wounds. Compared with the non-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 treatment and wound healing effects, as shown in Figure 2. Figure 6 (b) and (c).

[0041] 5. Live antiviral

[0042] The present invention systematically evaluated the antiviral, anti-inflammatory and wound repair effects of TPAOMCN NFs. After intravenous injection of PBS or encapsulated TPAOMCN NFs without subsequent laser irradiation, the tail lesions of virus-infected mice did not improve and a large number of scabs remained, but the area of ​​the tail wounds of mice in the TPAOMCN NFs light-irradiated group gradually shrank, as shown in Figure 2. Figure 7 In addition, the detection of vaccinia virus titers in the tail lesion tissue showed that after TPAOMCN NFs treatment, 660 nm laser irradiation almost eradicated the virus compared with the other groups, as shown in Figure 2. Figure 7 (c). To evaluate the ability of different treatments to clear the virus, tail lesion tissues were collected 7 days later and sections were subjected to immunohistochemistry (IHC) staining for vaccinia virus antigens. In the absence of subsequent irradiation, all groups showed extremely high viral antigen-positive IHC densities. In contrast, the viral antigen-positive IHC density of the TPAOMCN NFs-treated group that was subsequently irradiated with 660 nm laser was significantly reduced compared with the PBS group, as shown in Figure 2. Figure 7 (d). Subsequently, the concentrations of pro-inflammatory factors such as IL-1β and IL-6 in the 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 proinflammatory cytokines in the tail lesion tissue were very high in the PBS group with or without 660nm laser irradiation, indicating that vaccinia virus infection triggered a strong inflammatory response. When the 660nm laser was introduced, TPAOMCN NFs treatment led to a moderate decrease in the levels of these proinflammatory cytokines, showing a very strong anti-inflammatory activity.

[0043] In the tail-scratch mouse model infected with vaccinia virus as an alternative model of monkeypox to simulate the characteristic pustular lesions of monkeypox, after TPAOMCN NFs plus laser treatment, the viral titer in the lesions was significantly reduced, the infected lesion tissue was rapidly repaired, and the spread of the virus to healthy mice was effectively blocked. This effect of virus clearance, anti-inflammation and wound repair fully demonstrated the antiviral and skin beautification potential of "TPAOMCN NFs+laser" in the treatment of monkeypox.

Claims

1. A Nile red derivative having photodynamic and photothermal properties, characterized in that: The structural formula is as follows:

2. The method for preparing the Nile red derivative having photodynamic and photothermal properties according to claim 1, characterized in that: The following steps are involved: (1) adding 2-(9-bromo-5H-benzo[a]phenoxyazine-5-ylidene)malononitrile, (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 2-(9-bromo-5H-benzo[a]phenoxyazine-5-ylidene)malononitrile to (4-(diphenylamino)phenyl)boric acid or (4-(bis(4-methoxyphenyl)amino)phenyl)boric acid is 1:5-6.

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 or cesium carbonate.

6. The nanofiber of Nile red derivative having photodynamic and photothermal properties according to claim 1, characterized in that: The Nile red derivative with photodynamic and photothermal properties is blended with polyvinylidene fluoride and prepared by electrostatic spinning.

7. Use of the Nile red derivative with photodynamic and photothermal properties according to claim 1 in the preparation of an anti-drug resistant bacterial infection preparation.

8. Use of the Nile red derivative with photodynamic and photothermal properties according to claim 1 in the preparation of a preparation for inhibiting monkeypox virus.

9. A preparation for inhibiting monkeypox virus, characterized in that: It includes the Nile red derivative with photodynamic and photothermal properties according to claim 1.

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