NIR-II photothermal material with D-A-D structure, preparation method and application

Abstract

This invention relates to a D-A-D structured NIR-II photothermal material, its preparation method, and its applications. Specifically, the photothermal material of this invention has a simple structure, is easy to synthesize, is easy to purify, and has a high yield. Furthermore, due to its large conjugated system, the molecular structure is stable and not easily degraded under NIR-II light irradiation. In addition, the photothermal material of this invention has excellent optical properties, good photothermal conversion efficiency, extremely low biotoxicity, and broad ultraviolet absorption in the 800-1000 nm range. Therefore, it has great application prospects in near-infrared photodynamic therapy and tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to the application of a DAD-structured NIR-II photothermal material in bioimaging, fluorescent probes, laser dyes, fluorescent sensors, fluorescent labeling, near-infrared photodynamics, photovoltaic cells, and tumor therapy; this invention also provides a method for preparing the photothermal material. Background Technology

[0002] In the exploration of cancer treatment strategies, non-invasiveness, patient tolerability, and the ability to target and kill tumor cells without damaging other normal cells are ideal goals for treating malignant tumors. Photothermal therapy (PTT) is an emerging treatment method that is generally triggered by light, especially near-infrared light (NIR), which can penetrate deep tissues and selectively kill tumor cells under light irradiation without causing damage to normal tissues. Therefore, it is receiving increasing attention.

[0003] Near-infrared II (NIR-II) photothermal materials have attracted increasing attention due to their deeper tissue penetration capabilities, making their application prospects in photothermal therapy (PTT) increasingly promising. NIR-II photothermal materials developed in recent years can be categorized into inorganic and organic materials. Inorganic materials, such as single-walled carbon nanotubes (SWNTs), quantum dots (QDs), and metallic materials, possess excellent photothermal conversion efficiency and photostability; however, their poor biodegradability and potential cytotoxicity hinder their clinical application. Compared to inorganic materials, organic materials have gained significant attention due to their advantages such as ease of synthesis, controllable structure, good biocompatibility, and easy metabolism.

[0004] Currently, the photothermal material widely used in research institutes is the organic fluorescent probe indocyanine green (ICG), which is the only fluorescent contrast agent approved for clinical use by the U.S. Food and Drug Administration (FDA). However, its poor structural stability and inability to specifically target tumors limit its further clinical application. Therefore, developing novel, stable, and multifunctional NIR-II photothermal materials is currently a key focus and challenge in phototherapy research. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a DAD-structured NIR-II photothermal material, as well as its preparation method and applications, which has the advantages of simple structure, easy synthesis, and excellent optical properties.

[0006] Specifically, the present invention provides a compound having the structure shown in formula (I):

[0007] .

[0008] The present invention also provides a method for preparing the compound of formula (I), characterized in that it is prepared by reacting julonidine and keto acid as raw materials according to the following reaction route:

[0009] .

[0010] In some specific embodiments of the present invention, the preparation steps are as follows: a certain molar ratio of julonidine and ketone acid is added to a system of toluene and n-butanol to prepare a DAD-structured NIR-II photothermal material.

[0011] In some specific embodiments of the present invention, the following steps are included: a strong electron donor, julonidine, and a strong electron acceptor, keto acid, are stirred at room temperature for a certain time in a system of toluene and n-butanol, then heated under reflux for a certain time, the solvent is removed by vacuum distillation, cooled to room temperature, filtered to obtain a black-brown solid, then washed with solvent, and dried to obtain a DAD-structured NIR-II photothermal material.

[0012] The molar ratio of the strong electron donor julonidine to the strong electron acceptor ketoacid is greater than or equal to 2:1.

[0013] In some specific embodiments of the present invention, the molar ratio of julonidine to ketone acid is 3:1.

[0014] In some specific embodiments of the present invention, the stirring time at room temperature is 20 min, the heating temperature is 147 ℃-153 ℃, and the reaction time is 2 h.

[0015] The present invention also provides a composition applicable to bioimaging, fluorescent probes, laser dyes, fluorescent sensors, fluorescent labeling, and tumor therapy, comprising the compounds as described above or the compounds prepared by the preparation method described in any of the preceding claims.

[0016] In some specific embodiments of the present invention, the composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.

[0017] The present invention also provides a kit for use in bioimaging and tumor treatment, comprising the compounds described above.

[0018] This invention also provides applications of the above-mentioned compounds in bioimaging, fluorescent probes, laser dyes, fluorescent sensors, fluorescent labeling, near-infrared photodynamics, and photovoltaic cells.

[0019] The present invention has the following significant advantages and effects compared with the prior art:

[0020] 1. The photothermal material of the present invention has a simple structure, is easy to synthesize, is easy to purify, and has a high yield.

[0021] 2. The large conjugated system of the photothermal material of the present invention makes the molecular structure stable and not easily degraded under NIR-II light irradiation.

[0022] 3. The photothermal material of the present invention has excellent optical properties, good photothermal conversion efficiency, extremely low biotoxicity, and broad ultraviolet absorption in the range of 800-1000 nm. Therefore, it has great application prospects in near-infrared photodynamics and tumor treatment. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 For equation (I) Cro-Jul 1 H-NMR spectrum;

[0025] Figure 2 The ultraviolet absorption spectrum of formula (I) Cro-Jul;

[0026] Figure 3 The light scattering test results for the particle size of Cro-Jul nanoparticles according to formula (I);

[0027] Figure 4 The Cro-Jul photothermal conversion curve is given by equation (I).

[0028] Figure 5 The graph shows the stability test data of ICG at 808nm and 0.5W for formula (I);

[0029] Figure 6 The graph shows the stability test data of Cro-Jul at 980nm and 0.5W for formula (I);

[0030] Figure 7 The diagram shows the cytotoxicity test results for Cro-Jul nanoparticles of formula (I).

[0031] Figure 8 The image shows confocal fluorescence images of FaDu cells incubated with different reagents using a mitochondrial membrane potential detection kit (JC-1).

[0032] Figure 9The images show confocal fluorescence images of FaDu cells incubated with different reagents using a cell apoptosis and necrosis detection kit.

[0033] Figure 10 Graphs showing data from flow cytometry analysis of FaDu cells incubated with different reagents;

[0034] Figure 11 This is a graph showing the weight change data after different reagents were injected during the treatment cycle;

[0035] Figure 12 This is a graph showing the changes in tumor volume during treatment cycles after injection of different reagents;

[0036] Figure 13 Images showing tumor volume after treatment with different reagents;

[0037] Figure 14 This is a graph showing tumor weight data after treatment with different reagents.

[0038] Figure 15 The images show the HE, Hsp70, TUNEL, SLC7A11, and Ki67 staining test results of tumor sections after treatment with different reagents.

[0039] Figure 16 This image shows a test image of HE staining of the internal organs of nude mice after treatment with different reagents.

[0040] Figure 17 This is a data graph showing the analysis of serum after treatment with different reagents. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Protection range.

[0043] Example 1 Synthesis of compound (I)

[0044] The specific steps are as follows:

[0045] First, 0.42 g of the strong electron donor julonidine and 0.137 g of the strong electron acceptor ketone acid were added to a system of 8 mL toluene and 24 mL n-butanol. After stirring at room temperature for 20 min, the mixture was refluxed at 147 ℃-153 ℃ for 2 h. The solvent was then removed by vacuum distillation. After cooling to room temperature, the mixture was filtered to obtain a dark brown solid. The solid was washed with n-hexane, diethyl ether, and ethanol, and dried to obtain dark brown crystals. Compound (I) Cro-Jul was synthesized in a yield of 43.2%. The reaction route is as follows:

[0046]

[0047] The proton and carbon spectra of the prepared compounds are shown in the attached figures. Figure 1 As shown, the structure of compound (I) can be confirmed.

[0048] Example 2: Ultraviolet absorption spectroscopy test of compound (I)

[0049] A 2 mL test system was prepared using the compound of formula (I) prepared in Example 1, and the test was performed using a UV absorption spectrometer.

[0050] The test results are attached. Figure 2 As shown.

[0051] Test results show that the compound of formula (I) has a wide absorption range between 800 and 1000 nm.

[0052] Example 3: Particle size light scattering test of compound (I)

[0053] The synthesized compound of formula (I) was dissolved in 1 mL of DMSO and slowly added dropwise to 10 mL of PBS containing the amphiphilic matrix poly(styrene)-block-poly(ethylene glycol). The mixture was stirred at room temperature for 12 hours to remove the organic solvent, then dissolved in PBS and sonicated for 10 minutes to obtain nanofunctionalized organic nanoparticles of compound (I). (See attached...) Figure 3 The dynamic light scattering test results of the nanofunctionalized organic nanoparticles of formula (I) show that their hydrated particle size is about 100 nm. Nanoparticles of this size can effectively penetrate the cell membrane and enter the cell smoothly.

[0054] Example 4: Photothermal conversion efficiency test of the compound of formula (I) prepared by the present invention

[0055] The nanoparticle solution was irradiated with a 980nm laser, and the temperature change was recorded over 10 minutes. After 10 minutes, the temperature change was recorded again until it cooled to room temperature. The photothermal conversion curve of compound (I) is attached. Figure 4As shown, the photothermal conversion efficiency of compound (I) was calculated to be 48.96%, indicating that the newly synthesized Cro-Jul material has good photothermal properties, providing a good foundation for photothermal therapy.

[0056] Example 5: Stability test of the compound of formula (I) prepared by the present invention

[0057] Take 1 mL of ICG and Cro-Jul solution of compound (I) and place them in different 1 cm... 2 In a quartz dish, the ultraviolet absorption was measured every minute under laser irradiation. See attached figures for details. Figure 5 and Figure 6 As shown, the results indicate that the compound Cro-Jul of formula (I) has better stability than commercial ICG.

[0058] Example 6: In vitro phototoxicity and dark toxicity of formula (I) Cro-Jul nanoparticles to tumor cells

[0059] Cells were first seeded in 96-well plates. After cell adhesion, the prepared Cro-Jul nanoparticle solution was added and incubated overnight. Then, the cells were subjected to laser irradiation and light-protected treatment, respectively. Laser irradiation was performed on each well for 5 minutes. Both groups were then analyzed using a CCK-8 assay kit. The results are shown in the attached figure. Figure 7 As shown, the results indicate that Cro-Jul nanoparticles have low dark toxicity and significantly reduced cell survival rate after laser irradiation.

[0060] Example 7: Preparation of nanophototherapy reagents for synergistic PTT / ferroptosis / CDT therapy

[0061] The specific steps are as follows:

[0062] (1) Synthesis of Fe(III)-Qu

[0063] A 0.5 mol / L FeCl3 ethanol solution was added dropwise to a 0.5 mol / L quercetin ethanol solution under constant stirring. The mixture was stirred until homogeneous, and the pH was adjusted to 9.3 with 5% NaOH ethanol solution. The reaction was allowed to proceed for 70 min, and after cooling, the mixture was filtered, centrifuged at 3000 r / min, washed twice with anhydrous ethanol, and dried to obtain the product Fe(III)-Qu, with a yield of 62.36%.

[0064] (2) Synthesis of Fe(III)-Qu / CJ NPs

[0065] Take 80 mg of Cro-Jul and Fe(III)-Qu prepared in Example 1, and 200 mg of PEG. Dissolve Cro-Jul in 2 mL of DMSO, and dissolve Fe(III)-Qu and PEG in 200 mL of PBS (pH 7.4). Add the Cro-Jul and Fe(III)-Qu solutions dropwise to the PEG solution while stirring continuously. Remove the solvent by rotary evaporation and reduce the particle size by sonication to prepare Fe(III)-Qu / CJ NPs, a nanophototherapy reagent for PTT / ferroptosis / CDT synergistic therapy.

[0066] Example 8: Performance Testing of Nanophototherapy Reagents for PTT / Ferropion / CDT Synergistic Therapy

[0067] Implementation Plan 1: Apoptosis Test

[0068] Four experimental groups were set up. One group had no PBS solution added, while the other three groups had PBS solutions supplemented with 50 μg / mL Cro-Jul, Fe(III)-Qu, and Fe(III)-Qu / CJ nanoparticles, respectively. All four groups were then incubated with FaDu cells overnight and illuminated. Confocal fluorescence imaging was performed using a mitochondrial membrane potential detection kit (JC-1) and a cell apoptosis and necrosis detection kit, and the images were analyzed by flow cytometry. The fluorescence confocal images are attached. Figure 8 and Figure 9 As shown in the attached figure, the quantitative test data from the flow cytometer are as follows. Figure 10 As shown. Figure 8 JC-1 demonstrated that mitochondria were damaged in both the Fe(III)-Qu and Fe(III)-Qu / CJ groups, indicating ferroptosis. Furthermore, fluorescent staining results of apoptosis and necrosis showed that combined therapy exhibited better tumor cell killing effects. Figure 10 The streaming cytometry results illustrate this point.

[0069] Implementation Plan 2: Photothermal Therapy Effect Test of Fe(III)-Qu / CJ NPs in a Nude Mouse Subcutaneous Tumor Model

[0070] Nude mice were divided into four groups: (1) blank + Laser, (2) Cro-Jul + Laser, (3) Fe(III)-Qu + Laser, (4) Fe(III)-Qu / CJ nanoparticles, and (5) Fe(III)-Qu / CJ nanoparticles + Laser. The mice underwent tail vein injection of the nanoparticles, followed by 8 minutes of light irradiation 6 hours later. Weight and tumor volume were measured before each light irradiation treatment. Temperature was monitored using a thermal imager during treatment, maintaining a temperature of 40-45℃. Weight and tumor volume measurements are shown in the attached figures. Figure 11and 12 As shown.

[0071] After treatment, mouse serum, heart, liver, spleen, lung, kidney, and tumor tissue were collected. The volume and weight of the tumor spheroids needed to be measured. The measurement results are attached separately. Figure 13 and 14 As shown in the figure, 1) Control + Laser, 2) Cro-Jul + Laser, 3) Fe(III)-Qu + Laser, 4) Fe(III)-Qu / CJ, 5) Fe(III)-Qu / CJ + Laser. Simultaneously, HE, Hsp70, TUNEL, SLC7A11, Ki67, and GPX4 sections were stained. The test results are attached. Figure 15 As shown; HE staining of the five internal organs was performed to evaluate whether there was any damage during the treatment process. The test results are attached. Figure 16 As shown; serum analysis of liver and kidney function, test results are attached. Figure 17 As shown.

[0072] Test results showed that Cro-Jul + Laser, Fe(III)-Qu, and Fe(III)-Qu / CJ + Laser groups all had significant therapeutic effects. Among them, Fe(III)-Qu / CJ was the most effective under light irradiation. Furthermore, Fe(III)-Qu / CJ nanoparticles combined with light irradiation therapy had good biocompatibility.

[0073] In this embodiment, Cro-Jul, Fe(III)-Qu and Fe(III)-Qu / CJ nanoparticles were all prepared from Examples 1 and 7.

[0074] Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and all such modifications and variations are within the protection scope of the present invention.

Claims

1. Application of the compound shown in structural formula (I) in the preparation of drugs for photothermal therapy of tumors: （I）。 2. A pharmaceutical composition comprising the compound of claim 1; the composition further comprising a solvent, an acid, a base, a buffer solution, or a combination thereof.

3. A kit for use in bioimaging and tumor therapy, comprising the compound of claim 1.

4. The use of the compound as described in claim 1 in the preparation of bioimaging and photothermal therapy reagents.

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