A dual free radical photodiagnostic agent and its preparation method and application

Abstract

The present invention provides a dual radical photodiagnostic agent and a preparation method and application thereof, belonging to the field of biochemical material technology. The dual radical photodiagnostic agent provided by the present invention has a structure shown in Formula I, has near-infrared zone II fluorescence imaging and photodynamic / photothermal synergistic killing of cancer cells activity, and can produce strong near-infrared zone II fluorescence in the nanoparticle aggregation state, can be specifically located in the mitochondrial organelles of the cell, and perform real-time tracking imaging of the mitochondria. At the same time, the dual radical photodiagnostic agent of the present invention also has excellent light / thermal stability, a high photothermal conversion efficiency of 66%, and an effective ROS generation ability, all of which are more conducive to in vivo near-infrared zone II fluorescence bioimaging, photodiagnosis and anti-cancer application research.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical materials, and in particular relates to a double free radical photodiagnostic agent and a preparation method and application thereof. Background Art

[0002] Cancer has long been a threat to our health and life due to the rapid proliferation, systemic spread and metastasis of cancer cells. Traditional anti-cancer strategies, including radiotherapy, chemotherapy and surgery, still have problems with long-term toxicity, such as unsatisfactory efficacy and adverse side effects. The development of precision medicine and personalized medicine is an important way to solve these problems, which combines diagnosis and treatment into one platform. This system can integrate collective advantages to achieve the maximum therapeutic effect and make up for the shortcomings of simple treatment and diagnosis. At present, some diagnostic procedures have been developed, including magnetic resonance imaging (MRI), X-ray, positron emission tomography (PET), computed tomography (CT) and ultrasound imaging. However, due to radioactivity, low sensitivity and spatiotemporal resolution, these means are not suitable for real-time guidance of surgery or treatment. In contrast, phototherapy diagnostic strategies involving fluorescence imaging (FLI), photoacoustic imaging (PAI), photodynamic therapy (PDT) and photothermal therapy (PTT) can achieve the requirements of accurate diagnosis of target areas and selective lesion-specific treatment.

[0003] High-sensitivity fluorescence imaging has also been widely explored in tumor phototherapy. To date, many organic phototherapy agents have been reported for applications in bioimaging and therapy, most of which are based on FDA-approved near-infrared region I (NIR-I, 650-1000nm) ICG dyes for surgical navigation and treatment. However, these dye molecules generally have defects such as poor photostability, low efficiency of photothermal conversion and reactive oxygen production, which limit their further biotherapy applications. Therefore, it is crucial to develop multifunctional organic phototherapy agents with near-infrared region II (NIR-II, 1000-1700nm) fluorescence emission, excellent photophysical properties, high photothermal conversion and ROS yield. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a dual-radical photodiagnostic agent and a preparation method and application thereof, wherein the dual-radical photodiagnostic agent has near-infrared second-zone fluorescence imaging and photodynamic / photothermal synergistic cancer cell killing activity.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a dual radical photodiagnostic agent, wherein the dual radical photodiagnostic agent has a structure shown in Formula I:

[0007] Formula I;

[0008] Among them, R1 is independent , R2 is independently H or , R3 is independent ;

[0009] Said Where m is an integer from 1 to 12; Where n is an integer from 0 to 6.

[0010] Preferably, the Where m is 3, where n is 0.

[0011] Preferably, the diradical photodiagnostic agent has the structure shown in Formula I-a1 and I-b1:

[0012] Formula I-a1, Formula I-b1.

[0013] The present invention provides a method for preparing the diradical phototherapy reagent, comprising the following steps: mixing a compound having a structure shown in Formula II with 4-dimethylaminocinnamaldehyde or 3,3-bis(4-(dimethylamino)phenyl)propenal, an organic base and a first organic solvent, respectively, and performing a Novegrin reaction to obtain a compound having a structure shown in Formula I; R1 in Formula II is independently , m is an integer ranging from 1 to 12.

[0014] Formula II

[0015] Preferably, the molar ratio of the compound having the structure shown in Formula II to 4-dimethylaminocinnamaldehyde or 3,3-bis(4-(dimethylamino)phenyl)acrolein is 1:2-4; the molar ratio of the compound having the structure shown in Formula II to the organic base is 1:0.5-1.5.

[0016] Preferably, the temperature of the Novoweng reaction is 55-70°C.

[0017] Preferably, the protective gas for the Novoweng reaction is nitrogen or argon.

[0018] The present invention provides a medicine, comprising the biradical photodiagnostic and therapeutic reagent or the biradical photodiagnostic and therapeutic reagent obtained by the preparation method and a pharmaceutically acceptable carrier thereof.

[0019] Preferably, the dosage form of the drug includes nanoparticles, tablets, capsules, injections or suspensions.

[0020] The present invention provides the use of the biradical photodiagnostic agent or the biradical photodiagnostic agent obtained by the preparation method or the drug in the preparation of anti-tumor products or tumor diagnostic products.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The structure shown in Formula I provided by the present invention is a conjugated polyene photodiagnostic agent with near-infrared second-zone fluorescence imaging (NIR-II FLI) guided diagnosis and efficient PDT and PTT treatment functions. By increasing the π coupling length and introducing donor units at both ends of the central acceptor molecule, a larger conjugated plane and an enhanced intramolecular donor-acceptor (DA) interaction can be formed, respectively, to achieve a narrower band gap and a red shift of the absorption and emission wavelengths. In addition, by increasing the DA intensity, the distribution between the highest occupied molecular orbital and the lowest unoccupied molecular orbital can be further separated to achieve a smaller singlet-triplet energy gap, which is beneficial to the intersystem crossing process, promotes the generation of reactive oxygen species (ROS) and enhances the photothermal conversion performance. On the other hand, by introducing a highly twisted electron donor unit as a molecular rotor, as the number of rotors increases, the intermolecular π-π interaction can be significantly suppressed, thereby achieving a high stable diradical luminescence efficiency, photothermal performance and free radical stability performance.

[0023] The photodiagnostic and therapeutic reagent with near-infrared second-zone fluorescence imaging and photodynamic / photothermal synergistic killing of cancer cells provided by the present invention has simple synthesis steps, simple separation and purification operations, and has strong reactive oxygen generation ability; efficient photothermal conversion performance, has strong killing ability against breast cancer cells, and can be used to construct anticancer drugs with efficient photodynamic / photothermal synergistic therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Chemical reaction flow chart for preparing diradical phototherapy reagents.

[0025] Figure 2 Electron paramagnetic resonance (EPR) characterization of PhNVDPP and 2PhNVDPP. A and C are the temperature-dependent ESR measurement spectra using the same amount of 2PhNVDPP and PhNVDPP solid compounds; B and D are the temperature-dependent ESR signal changes fitted by the Bleaney-Bowers equation.

[0026] Figure 3 The crystal structures of PhNVDPP (A) and 2PhNVDPP (B).

[0027] Figure 4Figure 1 is the particle size and Zeta potential diagram of PhNVDPP and 2PhNVDPP nanoparticles in water phase. A and B are the particle size diagrams of PhNVDPP and 2PhNVDPP, respectively; C is the Zeta potential diagram of PhNVDPP and 2PhNVDPP.

[0028] Figure 5 (A) The ultraviolet absorption spectrum and (B) fluorescence emission spectrum of PhNVDPP and 2PhNVDPP nanoparticles.

[0029] Figure 6 Figure 1 is a test graph of the active oxygen generation capacity of PhNVDPP and 2PhNVDPP nanoparticles. A is the test graph of DCFH; B is the test graph of DCFH+PhNVDPP NPs; C is the test graph of DCFH+2PhNVDPP NPs.

[0030] Figure 7 Figure 1 is a photothermal performance diagram of 2PhNVDPP nanoparticles. A is a photothermal curve of 2PhNVDPP NPs at different concentrations after 808nm laser irradiation; B is a photothermal curve of 2PhNVDPP NPs at 50μM under different 808nm laser power densities; C is a photothermal stability diagram of 2PhNVDPP NPs during 6 heating and cooling cycles; D is a calculation diagram of the photothermal conversion efficiency of 2PhNVDPP NPs.

[0031] Figure 8 This is the photothermal performance diagram of PhNVDPP nanoparticles.

[0032] Fig. 9 This is the cellular co-localization diagram of 2PhNVDPP nanoparticles.

[0033] Fig.10 This is a graph showing the photodynamic / photothermal synergistic killing ability of 2PhNVDPP nanoparticles on human breast cancer cells (HeLa). DETAILED DESCRIPTION

[0034] The present invention provides a dual radical photodiagnostic agent, wherein the dual radical photodiagnostic agent has a structure shown in Formula I:

[0035] Formula I; wherein R1 is independently , R2 is independently H or , R3 is independent The present invention wherein m is an integer of 1 to 12, preferably m is an integer of 2 to 10, and more preferably m is 3; Where n is an integer of 0 to 6, preferably n is an integer of 0 to 3, and more preferably n is 0.

[0036] In the present invention, the diradical photodiagnostic agent has the structure shown in Formula I-a1 and I-b1:

[0037] Formula I-a1, Formula I-b1.

[0038] The present invention also provides a method for preparing the diradical photodiagnostic agent, comprising the following steps: mixing a compound having a structure shown in formula II with 4-dimethylaminocinnamaldehyde or 3,3-bis(4-(dimethylamino)phenyl)propenal, an organic base and a first organic solvent, respectively, and performing a Brainwegg reaction to obtain a compound having a structure shown in formula I.

[0039] In the present invention, R1 in Formula II is independently , m is an integer of 1 to 12, preferably m is an integer of 2 to 10, and more preferably m is 3. The present invention does not specifically limit the source of the compound having the structure of formula II, and can be synthesized using commercially available products or methods well known to those skilled in the art. The compound having the structure shown in formula II of the present invention is simple to prepare, easy to obtain, and easy to modify, and has a rigid and stable planar structure. The present invention further modifies the compound having the structure shown in formula II to obtain the diradical photodiagnostic agent of the present invention.

[0040]

[0041] Formula II

[0042] In the present invention, the molar ratio of the compound having the structure shown in formula II to 4-dimethylaminocinnamaldehyde or 3,3-bis(4-(dimethylamino)phenyl)acrolein is 1:2-4, preferably 1:2.5-3.5; the molar ratio of the compound having the structure shown in formula II to the organic base is 1:0.5-1.5, preferably 1:0.8-1.2. The organic base of the present invention is preferably pyridine.

[0043] In the present invention, the temperature of the Novoweng reaction is 55-70°C, preferably 58-65°C, and more preferably 60°C.

[0044] In the present invention, the protective gas for the Novoweng reaction is nitrogen or argon, and argon is selected.

[0045] In the present invention, there is no particular limitation on the type of the organic solvent, and a conventional solvent is used that can dissolve the raw materials and ensure that the Novovenge reaction proceeds smoothly, and the organic solvent is preferably chloroform. The present invention does not particularly limit the amount of the organic solvent, as long as it can allow the reaction to proceed smoothly.

[0046] In the present invention, the Novoweng reaction is preferably carried out under stirring conditions. The present invention has no particular limitation on the stirring rate, as long as the stirring is uniform.

[0047] In the present invention, there is no particular limitation on the time of the nephelometric reaction. The reaction is preferably monitored by a TLC plate (i.e., thin layer chromatography spot plate) known in the art until the compound having the structure shown in Formula II completely disappears.

[0048] In the present invention, after the Novovenge reaction, the Novovenge reaction product system is further post-processed, and the post-processing includes the following steps: extracting and concentrating the reaction solution obtained by the Novovenge reaction to obtain a concentrate; performing column chromatography on the concentrate to obtain a compound having a structure shown in Formula I. The extraction of the present invention includes the following steps: after the reaction system is cooled to room temperature, adding an NH4Cl aqueous solution to the Novovenge reaction solution (the reaction solution cooled to room temperature), extracting the aqueous phase with dichloromethane for multiple times, and the volume ratio of dichloromethane to water used in each extraction is preferably 1:2~4; the organic phase after dichloromethane extraction is dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a concentrate. The concentrate obtained by the present invention is separated by column chromatography to obtain a column chromatography product; the eluent used for the column chromatography is a mixed solution of dichloromethane and petroleum ether, wherein the volume ratio of dichloromethane to petroleum ether is preferably 1:0.5~3. The present invention removes the solvent in the column chromatography product to obtain a compound having a structure shown in Formula I. The present invention has no particular limitation on the method for removing the solvent, and any conventional method for removing the solvent, such as rotary evaporation, may be used.

[0049] The present invention also provides a drug, comprising the biradical phototherapy agent or the biradical phototherapy agent obtained by the preparation method and a pharmaceutically acceptable carrier thereof. The dosage form of the drug of the present invention includes tablets, nanoparticles, capsules, injections or suspensions, preferably nanoparticles.

[0050] The present invention also provides a biradical photodiagnostic agent nanoparticles, and the preparation method thereof comprises the following steps: dissolving the biradical photodiagnostic agent having the structure shown in Formula I and polyethylene glycol-distearoyl phosphatidylethanolamine in tetrahydrofuran (THF) solution respectively, and ultrasonicating them into water under ultrasonic treatment; then dialyzing through a dialysis bag to obtain a uniform, transparent dark blue solution, and filtering with a polyvinylidene fluoride membrane (PVDF) syringe to obtain a nanoparticle solution (NPs); finally, storing the nanoparticle solution in a refrigerator for standby use. The nanoparticle solution obtained by the present invention can be diluted with PBS for cell imaging. The present invention uses the standard curve of the biradical photodiagnostic agent having the structure shown in Formula I in THF solution as a reference, and calculates the concentration of the final nanoparticles by ultraviolet absorption spectroscopy.

[0051] The present invention provides the use of the biradical phototherapy reagent or the biradical phototherapy reagent obtained by the preparation method or the drug in the preparation of anti-tumor products or tumor diagnosis products. The tumors described in the present invention include but are not limited to breast cancer, gastric cancer, esophageal cancer, liver cancer, uterine cancer and lung cancer.

[0052] In the present invention, the biradical photodiagnostic agent has NIR-II FLI guided diagnosis and efficient PDT and PTT treatment functions, and is a highly stable biradical photodiagnostic agent of conjugated polyenes; by increasing the π coupling length at both ends of the central acceptor molecule and introducing donor units, a larger conjugated plane and enhanced intramolecular DA interaction can be formed respectively, achieving a narrower band gap and red shift of absorption and emission wavelengths; in addition, by increasing the DA intensity, the distribution between the highest occupied molecular orbital and the lowest unoccupied molecular orbital can be further separated to achieve a smaller singlet-triplet energy gap, which is beneficial to the intersystem crossing process, promotes the generation of ROS and enhances the photothermal conversion performance. The present invention introduces a highly twisted electron donor unit as a molecular rotor in the biradical photodiagnostic agent, and as the number of rotors increases, the intermolecular π-π interaction can be significantly suppressed, thereby achieving high stability biradical luminescence efficiency, photothermal performance and biradical stability performance improvement.

[0053] In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.

[0054] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Example 1

[0056] The chemical reaction flow chart of preparing the diradical phototherapy reagent in this embodiment is as follows: Figure 1 shown.

[0057] 1. PhNVDPP (I-a1) (C 38 H 46 The preparation method of N4O2) compound is as follows:

[0058] (1) Under argon atmosphere, compound 1 (0.20 g, 0.72 mmol) and 4-dimethylaminocinnamaldehyde (0.51 g, 2.9 mmol) were dissolved in 5 mL of CHCl3 solution, and piperidine (0.069 g, 0.81 mmol) was slowly added dropwise to the solution. The reaction was carried out at 60°C overnight. The reaction solution was cooled to room temperature to obtain a crude product of the Nouvelle reaction.

[0059] (2) Add NH4Cl aqueous solution (20 mL×3) to the crude product of the naovinger reaction, and extract the aqueous phase three times with dichloromethane (20 mL). Finally, the organic phase after extraction was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product of PhNVDPP. The crude product of PhNVDPP was purified by column chromatography (petroleum ether / dichloromethane 3:1) to obtain a dark blue solid powder (0.23 g, yield 55%).

[0060] The obtained solid was characterized, and the specific data are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.87 (br, 1H),8.73 (br, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 7.07 – 7.04(m, 1H), 6.97 – 6.84 (m, 1H), 6.72 – 6.66 (m, 6H), 6.32 (d, J = 14.8 Hz, 2H), 3.85 – 3.76 (m, 4H), 3.07 (s, 6H), 3.03 (s, 6H), 1.72 – 1.66 (m, 4H), 1.50 –1.42 (m, 4H), 1.01 – 0.98 (m, 6H). 13 C NMR (126 MHz, CDCl3) δ 161.44, 144.76,144.32, 143.53, 140.54, 129.87, 128.78, 124.70, 114.86, 112.17, 112.01,108.94, 40.30, 40.22, 40.14, 32.14, 20.25, 13.85. HRMS:[M+H] + calcd: 591.3699, found: 591.3697.

[0061] According to the above characterization data, the structural formula of the obtained dual radical photodiagnostic agent PhNVDPP is as shown in Formula I-a1.

[0062]

[0063] Formula I-a1

[0064] 2.2PhNVDPP (I-b1) (C 54 H 64 The preparation method of N6O2) compound is as follows:

[0065] (1) Under argon atmosphere, compound 1 (0.19 g, 0.68 mmol) and 3,3-bis(4-(dimethylamino)phenyl)propenal (0.50 g, 1.7 mmol) were dissolved in 5 mL of CHCl3 solution, and piperidine (0.069 g, 0.81 mmol) was slowly added dropwise to the solution. The reaction was carried out at 60°C overnight. The reaction solution was cooled to room temperature to obtain a crude product liquid of the Novovenge reaction.

[0066] (2) Add NH4Cl aqueous solution (20 mL×3) to the crude product of the navongo reaction, and extract the aqueous phase three times with dichloromethane (20 mL). Finally, the organic phase after extraction is dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude 2PhNVDPP product.

[0067] (3) The crude 2PhNVDPP product was separated and purified by column chromatography. The eluent used for column chromatography was a mixture of dichloromethane and petroleum ether in a volume ratio of 1:1, to obtain a dark green solid powder (0.15 g, yield 25.6%).

[0068] The obtained solid was characterized, and the specific data are as follows: 1 H NMR (600 MHz, C2D2Cl2) δ 8.49 (br,2H), 7.19 – 7.15 (m, 8H), 6.68 (d, J = 8.5 Hz, 4H), 6.59 (d, J = 8.5 Hz, 4H),6.37 (d, J = 14.1 Hz, 2H), 5.39 (br, 2H), 3.55 (br, 4H), 2.97 (s, 12H), 2.93(s, 12H), 1.46 – 1.44 (m, 4H), 1.26 – 1.22 (m, 4H), 0.85 (t, J = 7.3 Hz, 6H).HRMS:[M+H] +calcd: 829.5169, found: 829.5162.

[0069] According to the above characterization data, the structural formula of the obtained dual radical photodiagnostic agent 2PhNVDPP is as shown in Formula I-b1.

[0070]

[0071] Formula I-b1

[0072] 3. Performance testing

[0073] (1) Electron paramagnetic resonance (EPR) test of PhNVDPP (I-a1) and 2PhNVDPP (I-b1). The specific steps are as follows: ESR measurement was collected using a Bruker EMXplus spectrometer in continuous wave mode. The same amount (20 mg) of the two compounds was added to the quartz tube, and the temperature was increased from 243K to 348K to obtain the variable temperature ESR spectrum. The results are shown in Figure 2. Figure 2 shown.

[0074] from Figure 2 As can be seen in A and C, the ESR signal g factor of 2PhNVDPP and PhNVDPP is 2.0030, indicating the presence of unpaired carbon-centered radicals. As the temperature increases from 243K to 348K, the ESR signal intensity gradually increases, indicating that there are more thermally activated triplet diradical signals at higher temperatures. At the same time, due to the enhanced DA interaction within the 2PhNVDPP molecule, which promotes spin delocalization, it is observed that 2PhNVDPP shows a stronger diradical ESR signal than PhNVDPP. In addition, Figure 2 B and D in the figure are the temperature-dependent ESR signal changes fitted by the Bleaney-Bowers equation. The fitting results show that the singlet-triplet energy gap (∆E ST ) is -4.21 kcal mol -1 , PhNVDPP is -4.19 kcal mol -1 It can be seen that both 2PhNVDPP and PhNVDPP have the properties of open-shell singlet diradicals.

[0075] (2) The crystal structures of PhNVDPP and 2PhNVDPP were obtained by slow evaporation in a dichloromethane and acetonitrile system. The specific steps are as follows: The crystal diffraction data of PhNVDPP and 2PhNVDPP were measured by a single crystal X-ray diffractometer (SuperNova, Rigaku) ​​under a Cu target microfocus X-ray source.

[0076] The results are as follows Figure 3 shown. Figure 3 A and B are the crystal structures of PhNVDPP and 2PhNVDPP, respectively. Both compounds show a typical J-aggregation stacking mode. For PhNVDPP, the interlayer distance between two adjacent molecules is 3.460Å, and the slip angle is 20.08°. For 2PhNVDPP, the interlayer distance between adjacent molecules is 5.184Å, and the slip angle is 47.26°. The intermolecular distance is much larger than that of PhNVDPP. The introduction of the terminal 4,4-dimethylaminophenyl donor unit gives 2PhNVDPP a more twisted conformation with greater steric hindrance, forming a looser molecular stacking in the aggregated state, which can effectively avoid unfavorable π-π plane stacking and contribute to the generation of highly stable diradical fluorescence. At the same time, this relatively loose molecular stacking will not completely restrict intramolecular rotation, which also helps to improve the photothermal conversion ability in the aggregated state.

[0077] Example 2

[0078] 1. Preparation of PhNVDPP or 2PhNVDPP Nanoparticles

[0079] 2 mg of PhNVDPP or 2PhNVDPP prepared in Example 1 and 10 mg of polyethylene glycol-distearoyl phosphatidylethanolamine were dissolved in 1 mL of THF solution, and then ultrasonicated in 10 mL of ultrapure water for 5 minutes under ultrasonic treatment (150 W). After that, a uniform, transparent dark blue solution was obtained by dialyzing for 12 hours through a dialysis bag with a molecular weight cutoff of 3500. After that, the nanoparticles (NPs) were concentrated using a centrifugal ultrafiltration device. The nanoparticles were filtered with a 0.45 μm polyvinylidene fluoride membrane (PVDF) syringe to obtain a PhNVDPP nanoparticle solution. The PhNVDPP nanoparticle solution can be diluted with PBS for cell imaging. Finally, the prepared PhNVDPP nanoparticle solution was stored in a 4 ° C refrigerator for later use.

[0080] 2. Particle size and Zeta potential test of PhNVDPP and 2PhNVDPP nanoparticles

[0081] The particle size and potential of the prepared PhNVDPP and 2PhNVDPP nanoparticles (NPs) were measured by dynamic light scattering (DLS) and Zeta potential. Figure 4 shown.

[0082] from Figure 4 It can be seen that the hydrodynamic diameters of PhNVDPP and 2PhNVDPP NPs are 50 nm and 117 nm, respectively. In addition, the Zeta potentials of PhNVDPP and 2PhNVDPP NPs are -57.58 and -57.65 mV, respectively.

[0083] 3. Ultraviolet absorption spectrum and fluorescence emission spectrum test of PhNVDPP and 2PhNVDPP nanoparticles

[0084] The aqueous solutions of PhNVDPP and 2PhNVDPP nanoparticles were prepared into a test concentration of 10 μM, and placed in a UV spectrophotometer and a fluorescence spectrometer to collect UV absorption spectra and fluorescence emission spectra. Figure 5 shown.

[0085] from Figure 5 It can be seen that PhNVDPP NPs have a maximum absorption at 623nm and a maximum fluorescence emission at 780nm. In contrast, after the introduction of the second 4,4-dimethylaminophenyl donor unit, 2PhNVDPP NPs have a maximum absorption at 708nm and a maximum NIR fluorescence emission at 1011nm, with a Stokes shift of up to 231nm, which is mainly attributed to its stronger intramolecular DA interaction. The tail NIR-II emission of 2PhNVDPP NPs can reach 1300nm, indicating its potential applicability in NIR-II fluorescence imaging-guided therapy.

[0086] 4. Test of the reactive oxygen species generation capacity of PhNVDPP and 2PhNVDPP nanoparticles

[0087] 2',7'-Dichlorodihydrofluorescein (DCFH) was used to detect reactive oxygen species. 50 μL of DCFH (concentration: 40 μM) was mixed with PhNVDPP or 2PhNVDPP nanoparticle solution (working concentration: 10 μM). A control group was prepared by adding 2 mL of PBS to 50 μL of DCFH. Afterwards, the mixture was exposed to laser irradiation (0.5 W cm -2 ), and the fluorescence spectrum intensity at about 530 nm was recorded every 10 seconds in a fluorescence spectrofluorometer (excitation wavelength: 489 nm). The results are shown in Figure 6 shown.

[0088] Figure 6 The ROS generation of PhNVDPP and 2PhNVDPP nanoparticles was investigated using 2',7'-dichlorodihydrofluorescein (DCFH) as a probe, which emits bright green fluorescence upon detection of ROS. Figure 6 As shown, the signal intensity of DCFH alone showed negligible changes after 180 seconds of laser irradiation. In contrast, the intensity of DCFH gradually increased in the presence of PhNVDPP and 2PhNVDPP nanoparticles. PhNVDPP and 2PhNVDPP nanoparticles exhibited excellent ROS production capabilities, which may be due to the nanoparticles' stronger light absorption effect in the near-infrared region.

[0089] 5. Photothermal performance and photothermal conversion efficiency test of 2PhNVDPP nanoparticles

[0090] Photothermal property test: Nanoparticle (50 μM) solution was exposed to 808 nm (0.1 W cm -2 , 0.2W cm -2 , 0.3W cm -2 , 0.4W cm -2 and 0.5W cm -2 ) laser power density, and the temperature change was monitored by a FLIR E8-XT camera. At the same time, 2PhNVDPP NPs solutions with different concentrations (0 μM, 5 μM, 15 μM, 25 μM, and 50 μM) were prepared and irradiated at 808 nm (0.5 W cm -2 ) The laser was irradiated for 5 minutes and the temperature change during the irradiation process was monitored.

[0091] A 2PhNVDPP NPs (50 μM) solution was heated at 808 nm with 0.5 W cm -2 The laser was irradiated for 5 minutes to allow the temperature to reach equilibrium. At this point, the laser was turned off and the solution was cooled to room temperature. During this process, the temperature of the solution was recorded every 15 seconds, and the temperature change was recorded five times. The results are shown in Figure 1. Figure 7 shown.

[0092] like Figure 7 As shown in A, the temperature changes rapidly during the initial irradiation time, reaches a plateau after 5 minutes of irradiation, and exhibits a concentration-dependent temperature change. Under the same laser power density irradiation conditions, the temperature increment of pure water is negligible. Specifically, even when the concentration of nanoparticles is as low as 25 μM, the temperature still rises to 48°C. In addition, the temperature change of 2PhNVDPP NPs can also be well regulated by adjusting the laser power ( Figure 7 B), which fully proves that the photothermal performance of nanoparticles is closely related to the laser power density or concentration. To evaluate the photothermal stability of 2PhNVDPP NPs in aqueous solution, the 808 nm (0.5 W cm -2 , 5min) laser irradiation 6 heating / cooling cycles, such as Figure 7 As shown in Figure C, 2PhNVDPP NPs exhibit good photothermal stability. The photothermal conversion efficiency of 2PhNVDPP NPs was calculated to be 66% ( Figure 7 D).

[0093] 6. Photothermal performance test of PhNVDPP nanoparticles,

[0094] The specific steps of the photothermal performance test are as follows: irradiate the PhNVDPP nanoparticle solutions with different concentrations (0, 15, 25, 50, 100 μM) with 808 nm laser, and then -2 The laser was irradiated for 5 minutes, and the temperature changes during the illumination were monitored by a FLIR E8-XT camera. Figure 8 shown.

[0095] Figure 8 PhNVDPP nanoparticles were exposed to 808 nm 0.5 W cm -2 Photothermal properties under laser. As the concentration increases from 0 to 100µM, PhNVDPP NPs show a temperature increase, reaching 26°C at concentrations up to 100μM.

[0096] 7. Cell co-localization test of 2PhNVDPP nanoparticles

[0097] The specific steps of the cell co-localization test are as follows: human cervical cancer (HeLa) cells were inoculated in a confocal microscope culture dish and incubated for at least 24 hours. Then 2PhNVDPP NPs (30μM) were added and incubated for 6 hours. The cells were washed 3 times with PBS, and the green fluorescent mitochondrial probe (Mito-Tracker Green, 1μM) was added after 2PhNVDPP NPs treatment and incubated for another 30 minutes. Then, the cells were observed in sequential scanning mode using a confocal microscope (Zeiss). 2PhNVDPP NPs (λex=543nm, λem=long pass 570nm). The results are shown in Fig. 9 shown.

[0098] Fig. 9 The intracellular distribution of 2PhNVDPP NPs. Confocal imaging results confirmed that the red fluorescence of 2PhNVDPP NPs overlapped well with the green fluorescence of the commercial mitochondria-specific probe (Mito-Tracker Green) in the figure, with a Pearson correlation coefficient of 0.801, indicating that 2PhNVDPP NPs can specifically target mitochondria and can be used for cell mitochondrial staining and high-quality fluorescence imaging.

[0099] 8. Testing of the photodynamic / photothermal synergistic killing ability of 2PhNVDPP nanoparticles on human breast cancer cells (HeLa)

[0100] The specific steps of the photodynamic / photothermal synergistic killing ability test are as follows: HeLa cells were seeded on a 96-well plate and cultured in a humidified incubator at 37°C in 5% CO2, 20% O2 in DMEM medium (containing 10% FBS and 1% penicillin / streptomycin). Then, cells in the logarithmic growth phase were collected and cultured at 5×103 The cells were seeded at a density of 10 cells / cm2 in a 96-well plate and incubated for 24 h. Subsequently, the medium was replaced with fresh medium containing different concentrations of 2PhNVDPP nanoparticles. After further incubation for 20 h, the cells were exposed to 808 nm laser irradiation (0.5 W cm -2 ) for 6 minutes. At the same time, dark cytotoxicity studies were performed on cells incubated with 2PhNVDPP nanoparticles without laser irradiation. After further incubation for 4 hours, the culture medium was removed and washed 3 times with PBS. The cells were then incubated with fresh serum-free medium containing 10% CCK-8 in the dark for 1 hour. Finally, the absorbance of the product was measured at a wavelength of 450nm using a microplate reader. The results are expressed as the survival percentage of cells after different treatments relative to control cells without any treatment. The results are shown in Fig.10 shown.

[0101] Fig.10 The results of the phototherapy ability of 2PhNVDPP nanoparticles on cancer cells. When the concentration of 2PhNVDPP nanoparticles increased to 45 μM, the cell viability was still close to 100%, indicating that the dark cytotoxicity of 2PhNVDPP nanoparticles was negligible. -2 , 6 min), obvious concentration-dependent phototoxicity was observed, and the viability of HeLa cells dropped to about 16% when the concentration was 45 μM, confirming that 2PhNVDPP nanoparticles have excellent synergistic phototherapeutic effects in vitro.

[0102] It can be seen from the above embodiments that the dual radical photodiagnostic and therapeutic reagent with near-infrared second-zone fluorescence imaging and photodynamic / photothermal synergistic killing of cancer cells provided by the present invention has simple synthesis steps, simple separation and purification operations, strong reactive oxygen generation ability, efficient photothermal conversion performance, strong killing ability against breast cancer cells, and can be used to construct anticancer drugs with efficient photodynamic / photothermal synergistic therapeutic effects.

[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A dual free radical photodiagnostic agent, characterized in that: The diradical photodiagnostic agent has a structure shown in Formula I: Formula I; Among them, R1 is independent , R2 is independently H or , R3 is independent ; Said Where m is an integer from 1 to 12; Said Where n is an integer from 0 to 6.

2. The dual free radical photodiagnostic agent according to claim 1, characterized in that: Said Where m is 3, where n is 0.

3. The dual free radical photodiagnosis and treatment reagent according to claim 1, characterized in that: The diradical photodiagnostic agent has the structure shown in formula I-a1 and I-b1: Formula I-a1, Formula I-b1.

4. The method for preparing the dual radical photodiagnosis and treatment reagent according to any one of claims 1 to 3, characterized in that: The following steps are involved: The compound having the structure shown in Formula II is mixed with 4-dimethylaminocinnamaldehyde or 3,3-bis(4-(dimethylamino)phenyl)propenal, an organic base and a first organic solvent, respectively, and subjected to a Novegrin reaction to obtain a compound having the structure shown in Formula I; Formula II; R1 in Formula II is independently , m is an integer ranging from 1 to 12.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the compound having the structure shown in formula II to 4-dimethylaminocinnamaldehyde or 3,3-bis(4-(dimethylamino)phenyl)acrolein is 1:2-4; the molar ratio of the compound having the structure shown in formula II to the organic base is 1:0.5-1.

5.

6. The preparation method according to claim 4, characterized in that: The temperature of the brain wenge reaction is 55~70℃.

7. The preparation method according to claim 4, characterized in that: The protective gas for the Novoweng reaction is nitrogen or argon.

8. A drug, characterized in that It comprises the diradical phototherapy reagent as described in any one of claims 1 to 3 or the diradical phototherapy reagent obtained by the preparation method as described in any one of claims 4 to 7 and a pharmaceutically acceptable carrier thereof.

9. The drug according to claim 8, characterized in that The drug is available in the form of nanoparticles, tablets, capsules, injections or suspensions.

10. Use of the diradical photodiagnostic reagent according to any one of claims 1 to 3, the diradical photodiagnostic reagent obtained by the preparation method according to any one of claims 4 to 7, or the drug according to any one of claims 8 to 9 in the preparation of anti-cervical cancer products or cervical cancer diagnostic products.

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