A nano phototherapy reagent for ptt / ferroptosis / cdt synergistic treatment, preparation method and application

Abstract

The present application relates to a kind of PTT / iron death / CDT synergistic therapy nano photodiagnosis and treatment reagent, preparation method and application, specifically, the PTT / iron death / CDT synergistic therapy nano photodiagnosis and treatment reagent of the present application, the large conjugated system of the photothermal material Cro-Jul in it makes molecular structure stable, not easy to degrade under NIR-II light irradiation, and has good photothermal conversion efficiency, extremely low biological toxicity and other superior performance;Quercetin and iron ion complex, not only overcome the heat shock protein up-regulation brought by mild photothermal therapy, promote photothermal effect, and Fe (III) in vivo carries out iron death and chemical power, realizes combined therapy, reaches the combined enhanced apoptosis effect;By nano precipitation technology, Cro-Jul and Fe (III) -Qu complex are assembled into Fe (III) -Qu / CJ nano system, not only overcome the disadvantage that small molecule drug is difficult to dissolve in water, and can realize tumor mild PTT synergistic iron death and chemical power therapy mutually promote the treatment method of cell apoptosis, combined with fluorescence imaging and PAI, also can realize diagnosis and treatment integration.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a nanophototherapy reagent for PTT / ferroptosis / CDT synergistic therapy and its application in tumor diagnosis and treatment; this invention also provides a method for preparing the aforementioned nanophototherapy reagent for PTT / ferroptosis / CDT synergistic therapy. Background Technology

[0002] In exploring 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. PTT can achieve precision treatment in two ways: first, by synthesizing phototherapy drugs with specific structures to target the tumor; second, by controlling the size of the light irradiation area to irradiate only the lesion site, thus avoiding damage to normal tissues. This dual-selectivity phototherapy can effectively reduce the systemic toxicity of traditional chemotherapy and radiotherapy.

[0003] Near-infrared II (NIR-II) photothermal materials have attracted increasing attention due to their deeper tissue penetration capabilities. NIR-II photothermal materials developed in recent years can be divided 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.

[0005] Furthermore, during traditional PTT (photothermal therapy), the high temperature can cause burns to surrounding tissues, leading to non-healing conditions. Therefore, the recommended treatment temperature by relevant certification bodies is 40–45°C. However, this temperature can induce upregulation of heat shock proteins (HSPs) in cells and reduce their ability to be treated with photothermal therapy on tumors. Upregulation of HSP70 expression can inhibit tumor cell apoptosis, and inhibiting HSP70 expression can increase the sensitivity of tumor cells to thermotherapy. Quercetin (Qu) has the effect of inhibiting the synthesis of HSP70 in tumor cells, and it also has anti-proliferative, anti-tumor, and inhibitory effects on the synthesis of biological macromolecules, making it a potential chemotherapy drug for tumors; however, its low solubility in water limits its clinical application.

[0006] Chemodynamic therapy (CDT) is a novel tumor treatment technique based on the iron-based Fenton reaction. It converts H₂O₂ into the more toxic ·OH radical through the iron-based Fenton reaction, inducing oxidative stress in tumor cells and leading to apoptosis. However, the effectiveness of CDT is limited by factors such as the limited H₂O₂ content in the tumor microenvironment (TME) and the abundance of reducing substances. To enhance the therapeutic effect of CDT, researchers have developed numerous multifunctional, multi-therapeutic delivery systems.

[0007] Currently, the problem of drug resistance in tumor cells has attracted widespread attention. Single-method treatments can no longer meet the needs of current cancer treatment. Therefore, it is of great significance to develop a treatment method that combines multiple treatment approaches, not only overcoming the disadvantage of small molecule drugs being poorly soluble in water, but also achieving a combination of tumor-mild PTT synergistic ferroptosis and chemodynamic therapy to promote cell apoptosis. Summary of the Invention

[0008] Therefore, the purpose of this invention is to provide a nanophototherapy reagent for synergistic PTT / ferroptosis / CDT treatment, as well as their preparation method and application. Unlike current single-performance reagents, the nanophototherapy reagent Fe(III)-Qu / CJ NPs in this invention combines excellent PTT, ferroptosis and CDT, and combined with fluorescence imaging and PAI, it can achieve integrated diagnosis and treatment.

[0009] Specifically, the present invention provides a nanophototherapy reagent for synergistic treatment of PTT / ferroptosis / CDT, which comprises nanoparticles Fe(III)-Qu / CJ NPs prepared by compound Cro-Jul and complex Fe(III)-Qu;

[0010] The compound Cro-Jul is

[0011] The complex Fe(III)-Qu is a complex formed by quercetin and iron ions through coordination bonds.

[0012] In some specific embodiments of the present invention, the molar ratio of the compound Cro-Jul to the complex Fe(III)-Qu is 1:1.

[0013] In some specific embodiments of the present invention, the compound Cro-Jul is prepared by reaction according to the following reaction route:

[0014]

[0015] In some specific embodiments of the present invention, the specific preparation steps of the compound Cro-Jul are as follows: the strong electron donor julonidine and the strong electron acceptor keto acid are combined in a system of toluene and n-butanol to synthesize the compound Cro-Jul with a DAD structure;

[0016] The molar ratio of the strong electron donor julonidine to the strong electron acceptor ketoacid is greater than or equal to 2:1.

[0017] In some specific embodiments of the present invention, the molar ratio of the strong electron donor julonidine to the strong electron acceptor ketoacid in the specific preparation steps of the compound Cro-Jul is equal to 3:1.

[0018] In some specific embodiments of the present invention, the specific preparation steps of the compound Cro-Jul are as follows: the strong electron donor julonidine and the 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 the compound Cro-Jul with the structure.

[0019] The molar ratio of the strong electron donor julonidine to the strong electron acceptor ketoacid is greater than or equal to 2:1.

[0020] In some specific embodiments of the present invention, the room temperature stirring time in the preparation step of the compound Cro-Jul is 20 min, the heating temperature is 147℃-153℃, and the reaction time is 2 h.

[0021] In some specific embodiments of the present invention, the solvent washing in the preparation step of the compound Cro-Jul is n-hexane, diethyl ether and ethanol washing.

[0022] In some specific embodiments of the present invention, the preparation steps of the complex Fe(III)-Qu are as follows: FeCl3 ethanol solution is added dropwise to quercetin ethanol solution under continuous stirring, stirred evenly, pH is adjusted to alkaline, reacted for a certain time, cooled and filtered, centrifuged, washed with solvent, and dried to obtain the product.

[0023] In some specific embodiments of the present invention, the preparation steps of the complex Fe(III)-Qu are as follows: FeCl3 ethanol solution is added dropwise to quercetin ethanol solution under continuous stirring, stirred evenly, pH is adjusted to 8.5-9.5, reacted for a certain time, cooled and filtered, centrifuged, washed with solvent, and dried to obtain the product.

[0024] In some specific embodiments of the present invention, the solvent washing in the preparation step of the complex Fe(III)-Qu is anhydrous ethanol washing.

[0025] In some specific embodiments of the present invention, the preparation steps of the complex Fe(III)-Qu are as follows: 0.5 mol / L FeCl3 ethanol solution is added dropwise to 0.5 mol / L quercetin ethanol solution under constant stirring, stirred evenly, the pH is adjusted to alkaline with 5% NaOH ethanol solution, the reaction is allowed to proceed for 70 min, the mixture is allowed to stand and cool, filtered, centrifuged at 3000 r / min, washed twice with anhydrous ethanol, and dried to obtain the product.

[0026] In some specific embodiments of the present invention, the preparation steps of the complex Fe(III)-Qu are as follows: 0.5 mol / L FeCl3 ethanol solution is added dropwise to 0.5 mol / L quercetin ethanol solution under constant stirring, stirred evenly, the pH is adjusted to 8.5-9.5 with 5% NaOH ethanol solution, reacted for 70 min, cooled and filtered, centrifuged at 3000 r / min, washed twice with anhydrous ethanol, and dried to obtain the product.

[0027] In some specific embodiments of the present invention, the preparation steps of the Fe(III)-Qu / CJ NPs nanoparticles are as follows: a certain amount of Cro-Jul, Fe(III)-Qu and PEG are taken respectively, Cro-Jul is dissolved in DMSO, Fe(III)-Qu and PEG are dissolved in PBS, Cro-Jul and Fe(III)-Qu solutions are added dropwise to PEG solution and stirred continuously; the solvent is removed from the obtained solution, and the particle size is reduced by ultrasound to prepare Fe(III)-Qu / CJ NPs nanoparticles.

[0028] In some specific embodiments of the present invention, Cro-Jul, Fe(III)-Qu, and PEG are taken in a molar ratio of 1:1:2. Cro-Jul is then dissolved in DMSO, and Fe(III)-Qu and PEG are dissolved in PBS. Cro-Jul and Fe(III)-Qu solutions are added dropwise to the PEG solution while stirring continuously. The solvent is removed by rotary evaporation, and the particle size is reduced by ultrasound to prepare Fe(III)-Qu / CJ NPs nanoparticles.

[0029] This invention also provides a method for preparing a nanophototherapy reagent for synergistic PTT / ferroptosis / CDT treatment, comprising the following preparation steps:

[0030] Synthesis of Cro-Jul: A certain amount of julonidine and keto acid were added to a system of toluene and n-butanol. After stirring at room temperature for a certain time, the mixture was heated to reflux. After the reaction was completed, the compound Cro-Jul was isolated and extracted.

[0031] Synthesis of Fe(III)-Qu: FeCl3 ethanol solution was added dropwise to quercetin ethanol solution under constant stirring. After stirring until homogeneous, the pH was adjusted to alkaline. The reaction was allowed to proceed for a certain period of time, cooled and then filtered. The mixture was centrifuged, washed with anhydrous ethanol, and dried to obtain Fe(III)-Qu.

[0032] Synthesis of Fe(III)-Qu / CJ NPs:

[0033] A certain amount of Cro-Jul, Fe(III)-Qu and PEG were taken respectively. Cro-Jul was dissolved in DMSO, and Fe(III)-Qu and PEG were dissolved in PBS. Cro-Jul and Fe(III)-Qu solutions were added dropwise to PEG solution while stirring continuously. The solvent was removed from the solution and the particle size was reduced by ultrasound to prepare Fe(III)-Qu / CJ NPs nanoparticles.

[0034] In some specific embodiments of the present invention, the molar ratio of julonidine to ketone acid is greater than or equal to 2:1; the molar ratio of FeCl3 to quercetin is 1:1; and the molar ratio of Cro-Jul, Fe(III)-Qu and PEG is 1:1:2.

[0035] In some specific embodiments of the present invention, the preparation method of the nanophototherapy reagent for PTT / ferroptosis / CDT synergistic therapy includes the following preparation steps:

[0036] Synthesis of Cro-Jul: Julonidine and ketone acid in a molar ratio of 3:1 were added to a system of toluene and n-butanol. After stirring at room temperature for a certain period of time, the mixture was heated to reflux. After the reaction was completed, the compound Cro-Jul was isolated and extracted.

[0037] Synthesis of Fe(III)-Qu: 0.5 mol / L FeCl3 ethanol solution was added dropwise to 0.5 mol / L quercetin ethanol solution at a molar ratio of 1:1 under constant stirring. The mixture was stirred until homogeneous, and the pH was adjusted to alkaline 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 Fe(III)-Qu.

[0038] Synthesis of Fe(III)-Qu / CJ NPs: Cro-Jul, Fe(III)-Qu and PEG were prepared in a molar ratio of 1:1:2. Cro-Jul was dissolved in DMSO, and Fe(III)-Qu and PEG were dissolved in PBS. Cro-Jul and Fe(III)-Qu solutions were added dropwise to the PEG solution while stirring continuously. The solvent was removed by rotary evaporation and the particle size was reduced by sonication to prepare Fe(III)-Qu / CJ NPs nanoparticles.

[0039] The present invention also provides a composition for the integrated application of bioimaging, tumor treatment and tumor diagnosis and treatment, comprising the above-mentioned nanophototherapy reagent or the nanophototherapy reagent prepared by the above preparation method.

[0040] This invention also provides the application of the above-mentioned nanophototherapy reagent or the nanophototherapy reagent prepared by the above preparation method in bioimaging, tumor treatment, and integrated tumor diagnosis and treatment.

[0041] This invention also provides the application of the above-mentioned nanophototherapy reagent in the preparation of tumor therapeutic drugs.

[0042] This invention also provides the application of the above-mentioned nanophototherapy reagent in the preparation of drugs for the treatment of human pharyngeal squamous cell carcinoma.

[0043] The present invention has the following significant advantages and effects compared with the prior art:

[0044] 1. In NIR-II photoresponsive nanomaterials, inorganic nanomaterials, such as gold nanoparticles, plasma metal clusters, and carbon nanotubes, have unclear biocompatibility for long-term use; while some small-molecule photosensitizers, such as indocyanine green (ICG) and dihydroporphyrin e6 (Ce6), have poor stability and show significant degradation after laser irradiation. The photothermal material Cro-Jul in this invention belongs to the DAD conjugated system compound, which has advantages such as easy synthesis and good photostability.

[0045] 2. The complex of quercetin and iron ions not only overcomes the upregulation of heat shock proteins caused by mild photothermal therapy and promotes the efficacy of photothermal therapy, but also enables Fe(III) to carry out ferroptosis and chemokinetics in vivo, achieving a combined therapy and a combined enhanced apoptosis effect.

[0046] 3. Currently, most photothermal materials are limited to excitation in the NIR-I light region. Since the penetration depth of light is related to the wavelength, the longer the wavelength, the deeper the penetration depth and the less interference from scattering by biological tissues. Therefore, NIR-I light excitation often only treats superficial tumors, while it is not effective for some deep tumors. The nanophototherapy reagent Fe(III)-Qu / CJNPs in this invention has a wide absorption in the NIR-II window and can be excited by NIR-II light. Fe(III)-Qu / CJ NPs are excited under 980nm laser, which is beneficial for photothermal treatment of deep tumors. At the same time, it has high biological tissue penetration and can obtain high-resolution and high-contrast photoacoustic imaging of tumor tissue. It shows good biocompatibility in in vivo animal experiments. Therefore, the laser penetrates deeper, and PTT in the NIR-II window can treat deeper tumors in vivo and improve the treatment results.

[0047] 4. Unlike current materials with only a single property, the Fe(III)-Qu / CJ NPs in this invention combine excellent PTT, ferroptosis and CDT, and integrate fluorescence imaging and PAI to achieve integrated diagnosis and treatment. Attached Figure Description

[0048] 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.

[0049] Figure 1 The UV absorption spectra of Cro-Jul and Fe(III)-Qu / CJ NPs are shown.

[0050] Figure 2 Transmission electron microscopy image of Fe(III)-Qu / CJ NPs;

[0051] Figure 3 Photothermal heating capacity test data for Fe(III)-Qu / CJ NPs at different concentrations;

[0052] Figure 4 Photoacoustic imaging test data for Fe(III)-Qu / CJ NPs at different concentrations;

[0053] Figure 5 This is data from a comparative cytotoxicity test;

[0054] Figure 6 Fluorescence imaging images at different time points after Ce6@Fe(III)-Qu / CJ was injected into tumor-forming nude mice via the tail vein;

[0055] Figure 7 Fluorescence imaging of FaDu cells incubated with different reagents and illuminated after being stained with DCFH-DA and Hoechst staining;

[0056] Figure 8 This is a graph showing data from quantitative analysis of FaDu cells incubated with different reagents and light after being stained with DCFH-DA and Hoechst staining by cell flow cytometry.

[0057] Figure 9 Fluorescence imaging of FaDu cells stained with different reagents and incubated under light for C11 Bodipy 581 / 591;

[0058] Figure 10 This is a graph showing data from quantitative analysis of FaDu cells stained with different reagents and incubated under light using a flow cytometer for C11 Bodipy 581 / 591 staining;

[0059] Figure 11 This is a graph showing data from MDA testing of FaDu cells stained with different reagents and then exposed to light using C11 Bodipy 581 / 591 staining agents;

[0060] Figure 12 Figure 1: Western blot data of FaDu cells incubated with different reagents and irradiated with light;

[0061] Figure 13 The images show confocal fluorescence images of FaDu cells incubated with different reagents using the mitochondrial membrane potential detection kit (JC-1).

[0062] Figure 14 The images show confocal fluorescence images of FaDu cells incubated with different reagents using a cell apoptosis and necrosis detection kit.

[0063] Figure 15 Graphs showing data from flow cytometry analysis of FaDu cells incubated with different reagents;

[0064] Figure 16 Images of tumor-forming nude mice injected with Ce6@Fe(III)-Qu / CJ at different time points using a small animal imaging system;

[0065] Figure 17 Photoacoustic images of tumor-forming nude mice injected with Ce6@Fe(III)-Qu / CJ at different time points;

[0066] Figure 18 This is a graph showing the weight change data after different reagents were injected during the treatment cycle;

[0067] Figure 19 This is a graph showing the changes in tumor volume during treatment cycles after injection of different reagents;

[0068] Figure 20 Images showing tumor volume after treatment with different reagents;

[0069] Figure 21 This is a graph showing tumor weight data after treatment with different reagents.

[0070] Figure 22 The images show the HE, Hsp70, TUNEL, SLC7A11, and Ki67 staining test results of tumor sections after treatment with different reagents.

[0071] Figure 23 This image shows a test image of HE staining of the internal organs of nude mice after treatment with different reagents.

[0072] Figure 24 This is a data graph showing the analysis of serum after treatment with different reagents; Detailed Implementation

[0073] 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 only 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.

[0074] Example 1: Preparation of a nanophototherapy reagent for synergistic PTT / ferroptosis / CDT therapy

[0075] The specific steps are as follows:

[0076] (1) Synthesis of Cro-Jul

[0077] 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. The mixture was stirred at room temperature for 20 min, then 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. This solid was washed with n-hexane, diethyl ether, and ethanol, and dried to obtain dark brown crystals. The compound Cro-Jul was synthesized in 43.2% yield. The reaction route is as follows:

[0078]

[0079] (2) Synthesis of Fe(III)-Qu

[0080] 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%.

[0081] (3) Synthesis of Fe(III)-Qu / CJ NPs

[0082] Take 80 mg of Cro-Jul and Fe(III)-Qu, 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.

[0083] Example 2: Ultraviolet absorption spectroscopy of Cro-Jul and Fe(III)-Qu / CJ NPs

[0084] Two 2 mL test systems were prepared using Cro-Jul and Fe(III)-Qu / CJ NPs prepared in Example 1, respectively, and tested using a UV absorption spectrometer.

[0085] The test results are attached. Figure 1 As shown.

[0086] Test results show that Cro-Jul and Fe(III)-Qu / CJ NPs both have broad absorption in the 800-1000 nm range.

[0087] Example 3: Morphological observation of Fe(III)-Qu / CJ NPs under transmission electron microscopy

[0088] First, the prepared Fe(III)-Qu / CJ NPs liquid sample was ultrasonically dispersed into a uniform suspension. The powder solution was then placed on a copper mesh using a dropper method and dried. It was ensured that the powder sample was evenly distributed on the copper mesh and free of contaminants. The copper mesh was gently blown with a syringe to prevent any easily falling powder. A suitable field of view was found under a transmission electron microscope (TEM) for imaging. The TEM images are attached. Figure 2 As shown.

[0089] Transmission electron microscopy results show that the Fe(III)-Qu / CJ NPs nanoparticles are about 110 nm in size, spherical in shape, relatively uniform, and well dispersed.

[0090] Example 4: Photothermal heating capacity test of Fe(III)-Qu / CJ NPs with different concentrations

[0091] Take 1 mL of Fe(III)-Qu / CJ NPs solutions of different concentrations and record the temperature in real time every 30 minutes under irradiation with a 980 nm laser using a thermal imager.

[0092] The test results are attached. Figure 3 As shown.

[0093] The test results show that the temperature rise is positively correlated with the concentration of Fe(III)-Qu / CJ NPs solution, and Fe(III)-Qu / CJ NPs have excellent photothermal conversion efficiency of 48.96%.

[0094] Example 5: Photoacoustic Imaging Tests of Fe(III)-Qu / CJ NPs at Different Concentrations

[0095] Fe(III)-Qu / CJ NPs were injected into tumor-bearing nude mice via the tail vein, and images were taken at different time points using an 808nm photoacoustic imaging system; the test results are attached. Figure 4 As shown.

[0096] The test results show that the signal intensity increases with the increase of Fe(III)-Qu / CJ NPs concentration, and Fe(III)-Qu / CJ NPs have excellent photoacoustic imaging performance.

[0097] Example 6: Cytotoxicity Test

[0098] FaDu cells were selected as the in vitro research subject, and all cells were cultured in a humidified incubator at 37°C and 5% CO2. The experiment was divided into five groups, three of which were light-free groups and two of which were light-exposed groups. The light-free groups were incubated with PBS solution, Fe(III)-Qu solution, and Fe(III)-Qu / CJ NPs nanoparticle solution at concentrations of 0 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml, respectively. The light-exposed groups were incubated with Cro-Jul solution and Fe(III)-Qu / CJ NPs nanoparticle solution at concentrations of 0 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml, respectively. The cells were then irradiated with NIR-II (980 nm) and maintained at a certain temperature. The light-free groups served as controls. Cytotoxicity was measured using CCK-8 assay.

[0099] The test results are attached. Figure 5 As shown.

[0100] Test results show that under laser irradiation, Cro-Jul solution and Fe(III)-Qu / CJ NPs can effectively kill tumor cells, with Fe(III)-Qu / CJ NPs showing a more significant killing effect.

[0101] Example 7: Cellular Uptake Test

[0102] First, 8 mg of Cro-Jul, Ce6, and Fe(III)-Qu, and 20 mg of PEG were taken separately. Cro-Jul was dissolved in 0.2 mL of DMSO, and Fe(III)-Qu and PEG were dissolved in PBS. The Cro-Jul, Ce6, and Fe(III)-Qu solutions were added dropwise to the PEG solution while stirring continuously. The solvent was removed by rotary evaporation to obtain Ce6@Fe(III)-Qu / CJ nanoparticles. The Ce6@Fe(III)-Qu / CJ nanoparticles were then cultured with FaDu cells for different times (0, 2, 4, 6, 8 h). The cell nuclei were stained with 1 μL / mL Hoechst solution, and the images were captured using fluorescence confocal microscopy. The test results are attached. Figure 6 As shown.

[0103] Test results show that Ce6@Fe(III)-Qu / CJ nanoparticles are taken up by tumor cells over time.

[0104] Example 8: Intracellular ROS Assay

[0105] Four experimental groups were set up. One group had no PBS solution added, while the other three groups had PBS solutions with 50 μg / mL Cro-Jul, Fe(III)-Qu, and Fe(III)-Qu / CJ nanoparticles added, respectively. The four groups were then incubated with FaDu cells overnight and illuminated. Afterward, the cells were stained with 1 μL / mL DCFH-DA and Hoechst, and qualitative and quantitative tests were performed by fluorescence confocal microscopy and cell flow cytometry.

[0106] Fe(III)-Qu / CJ nanoparticles were incubated with FaDu cells overnight. Cells were stained with DCFH-DA, and qualitative and quantitative analyses were performed using fluorescence confocal microscopy and flow cytometry. The fluorescence confocal images are attached. Figure 7 As shown in the attached figure, the quantitative test data by cell flow cytometry are as follows. Figure 8 As shown. Figure 7 and Figure 8 The Control and Cro-Jul groups showed almost no green fluorescence, while the Fe(III)-Qu+Laser and Fe(III)-Qu / CJ+Laser showed more green fluorescence, indicating that the generation of ROS was due to the presence of iron, further verifying that ferroptosis occurs in cells.

[0107] Example 9: Intracellular lipid peroxide (LPO) assay

[0108] 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 under light. Cells were then stained with C11 Bodipy 581 / 591, and qualitative and quantitative analyses were performed using fluorescence confocal microscopy and flow cytometry. Simultaneously, the LPO lipids in the cultured cells were quantified according to the MDA assay kit instructions. Fluorescence confocal microscopy images are attached. Figure 9 As shown in the attached figure, the quantitative test data by cell flow cytometry are as follows. Figure 10 As shown in the attached figure, the MDA measurement data are as follows. Figure 11 As shown in the figure, 1)Control+Laser, 2)Cro-Jul+Laser, 3)Fe(III)-Qu+Laser, 4)Fe(III)-Qu / CJ+Laser. Figure 9 and Figure 10 These are qualitative and quantitative test results of lipid peroxide production after co-incubation of cells and nanoparticles using different methods. Figure 9 Compared with the Control+Laser and Cro-Jul+Laser groups, the green fluorescence of the Fe(III)-Qu+Laser and Fe(III)-Qu / CJ+Laser groups was more pronounced. Figure 10 Flow cytometry results confirmed this, and since lipid peroxides are products of ferroptosis, this further illustrates the occurrence of ferroptosis. Subsequently, lipid peroxides were quantified again using a malondialdehyde (MDA) kit, which also verified the occurrence of ferroptosis.

[0109] Example 10: Intracellular Western Blot Validation

[0110] 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 incubated with FaDu cells overnight under light. Cells were collected, digested with trypsin, and the cell pellet was collected in 1.5 mL centrifuge tubes. The cells were washed twice with 1×PBS buffer, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. An appropriate amount of cell lysis buffer was added according to the cell volume, with a protease lysis buffer to protease inhibitor ratio of 100:1. The tubes were placed on ice and shaken for 10-30 min to ensure complete cell lysis. The samples were centrifuged in a centrifuge pre-cooled to 4°C at 12000 rpm for 5 min. The supernatant obtained after centrifugation was the total cellular protein. The supernatant was transferred to a new EP tube and stored at -80°C. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit. SDS-PAGE gels were prepared. After calculating the protein concentration, add 5× protein loading buffer. Mix the loading buffer and total protein solution at a 1:5 ratio, incubate at 100°C for 5 minutes, then immediately remove and cool on ice. It can be stored at -20°C for later use. Perform electrophoresis, transfer, blocking, primary antibody application, secondary antibody application, and exposure. The experiments mainly focused on the photothermal-related protein Hsp70 and the ferroptosis-related proteins GPX4 and SLC7A11. Western blot experimental data are attached. Figure 12 As shown in the figure, 1) Control+Laser, 2) Cro-Jul+Laser, 3) Fe(III)-Qu+Laser, 4) Fe(III)-Qu / CJ+Laser. In Figure 12 In the four groups—Control+Laser, Cro-Jul+Laser, Fe(III)-Qu+Laser, and Fe(III)-Qu / CJ+Laser—GPX4 showed a downregulation trend while SLC7A11 showed an upregulation trend, both of which demonstrate the occurrence of ferroptosis. The upregulation of HSP70 demonstrates that the temperature rise of the photothermal material induced the overexpression of heat shock proteins in cells.

[0111] Example 11: Apoptosis Test

[0112] 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 13 and Figure 14 As shown in the attached figure, the quantitative test data from the flow cytometer are as follows. Figure 15 As shown. Figure 13 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 a better tumor cell killing effect. Figure 15 The streaming cytometry results illustrate this point.

[0113] Example 12: In vivo fluorescence imaging and photoacoustic imaging tests

[0114] Ce6@Fe(III)-Qu / CJ (Fe(III)-Qu / CJ nanoparticles simultaneously loaded with commercially available CE6) was injected intravenously into tumor-forming nude mice. Images were taken using a small animal imaging system at 0, 1, 2, 4, 6, 8, 12, and 24 hours under Cy3 channel imaging. The test results are attached. Figure 16 As shown.

[0115] Similarly, Fe(III)-Qu / CJ nanoparticles were injected into tumor-bearing nude mice via the tail vein, and photoacoustic imaging was performed at 808 nm laser for 1 and 6 hours. The test results are attached. Figure 17 As shown.

[0116] Appendix Figure 16 The test results showed that Ce6@Fe(III)-Qu / CJ nanoparticles were enriched in tumor tissues of tumor-forming nude mice after injection. (See attached image) Figure 17 The study demonstrated that the Fe(III)-Qu / CJ nanoparticles possess excellent photoacoustic imaging capabilities and reached the tumor site within 6 hours, enabling photoacoustic imaging of the tumor tissue.

[0117] Example 13: Photothermal Therapy Effect Test of Fe(III)-Qu / CJ NPs in a Nude Mouse Subcutaneous Tumor Model

[0118] 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 received 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 18 and 19 As shown.

[0119] 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 20 and 21 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 22 As shown; HE staining was performed on the five internal organs to evaluate whether there was any damage during the treatment process. The test results are attached. Figure 23 As shown; serum analysis of liver and kidney function, test results are attached. Figure 24 As shown.

[0120] 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.

Claims

1. A nanophototherapy reagent for synergistic PTT / ferroptosis / CDT treatment, characterized in that: The nanoparticles Fe(III)-Qu / CJ NPs are prepared from the compound Cro-Jul, the complex Fe(III)-Qu, and PEG. The compound Cro-Jul is ; The complex Fe(III)-Qu is a complex formed by quercetin and iron ions through coordination bonds.

2. The nanophototherapy reagent for synergistic PTT / ferroptosis / CDT therapy as described in claim 1, characterized in that, The compound Cro-Jul was prepared by the following reaction route: 。 3. The nanophototherapy reagent for synergistic PTT / ferroptosis / CDT therapy as described in claim 2, characterized in that, The specific preparation steps of the compound Cro-Jul are as follows: the strong electron donor julonidine and the 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 the compound Cro-Jul. The molar ratio of the strong electron donor julonidine to the strong electron acceptor ketoacid is greater than or equal to 2:

1.

4. The nanophototherapy reagent for synergistic PTT / ferroptosis / CDT therapy as described in claim 1, characterized in that, The preparation steps of the complex Fe(III)-Qu are as follows: FeCl3 ethanol solution is added dropwise to quercetin ethanol solution under continuous stirring, stirred evenly, pH is adjusted to alkaline, reaction is allowed for a certain time, filtered after standing and cooling, separated by centrifugation, washed with solvent, and dried to obtain the product.

5. The nanophototherapy reagent for synergistic PTT / ferroptosis / CDT therapy as described in any one of claims 1-4, characterized in that, The preparation steps of the Fe(III)-Qu / CJ NPs nanoparticles are as follows: Take a certain amount of Cro-Jul, Fe(III)-Qu and PEG respectively. Dissolve Cro-Jul in DMSO, and dissolve Fe(III)-Qu and PEG in PBS. Add Cro-Jul and Fe(III)-Qu solutions dropwise to the PEG solution and stir continuously. Remove the solvent from the obtained solution and reduce the particle size by ultrasound to prepare Fe(III)-Qu / CJ NPs nanoparticles.

6. A method for preparing a nanophototherapy reagent for synergistic PTT / ferroptosis / CDT therapy, characterized in that, The preparation steps include the following: Synthesis of Cro-Jul: A certain amount of julonidine and keto acid were added to a system of toluene and n-butanol. After stirring at room temperature for a certain time, the mixture was heated to reflux. After the reaction was completed, the compound Cro-Jul was isolated and extracted. Synthesis of Fe(III)-Qu: FeCl3 ethanol solution was added dropwise to quercetin ethanol solution under constant stirring. After stirring until homogeneous, the pH was adjusted to alkaline. After reacting for a certain period of time, the mixture was allowed to stand and cool, then filtered, centrifuged, washed with solvent, and dried to obtain the product. Synthesis of Fe(III)-Qu / CJ NPs: A certain amount of Cro-Jul, Fe(III)-Qu and PEG were taken respectively. Cro-Jul was dissolved in DMSO, and Fe(III)-Qu and PEG were dissolved in PBS. Cro-Jul and Fe(III)-Qu solutions were added dropwise to PEG solution while stirring continuously. The solvent was removed from the solution and the particle size was reduced by ultrasound to prepare Fe(III)-Qu / CJ NPs nanoparticles.

7. The preparation method of the nanophototherapy reagent for PTT / ferroptosis / CDT synergistic therapy as described in claim 6, characterized in that, The molar ratio of julonidine to ketone acid is greater than or equal to 2:1; the molar ratio of FeCl3 to quercetin is 1:1; and the molar ratio of Cro-Jul, Fe(III)-Qu and PEG is 1:1:

2.

8. A composition for the integrated application of bioimaging, tumor treatment and tumor diagnosis and treatment, comprising the nanophototherapy reagent according to any one of claims 1-5 or the nanophototherapy reagent prepared by the preparation method according to any one of claims 6-7.

9. The application of the nanophototherapy reagent as described in any one of claims 1-5 or the nanophototherapy reagent prepared by the preparation method as described in any one of claims 6-7 in the preparation of bioimaging agents, tumor treatment drugs, and integrated tumor diagnosis and treatment drugs.

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