CN117679509A - Nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment, preparation method and application - Google Patents
Nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment, preparation method and application Download PDFInfo
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Abstract
The invention relates to a PTT/iron death/CDT cooperative therapy nanometer photodiagnosis and treatment reagent, a preparation method and application thereof, in particular to the PTT/iron death/CDT cooperative therapy nanometer photodiagnosis and treatment reagent, wherein a large conjugated system of a photo-thermal material Cro-Jul ensures that a molecular structure is stable, is not easy to degrade under NIR-II light irradiation, and has excellent performances such as good photo-thermal conversion efficiency, extremely low biotoxicity and the like; the complex of quercetin and iron ions not only overcomes the up-regulation of heat shock proteins caused by mild photothermal treatment and promotes the photothermal effect, but also ensures that Fe (III) carries out iron death and chemical power in vivo, thus realizing combined therapy and achieving the combined enhanced apoptosis effect; the Cro-Jul and Fe (III) -Qu complex are assembled into a Fe (III) -Qu/CJ nano system by a nano precipitation technology, so that the defect that a small molecular medicine is indissolvable in water is overcome, a treatment method for promoting apoptosis by combining moderate tumor PTT with iron death and chemodynamic therapy can be realized, and diagnosis and treatment integration can be realized by combining fluorescence imaging and PAI.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a nanometer light diagnosis and treatment test for PTT/iron death/CDT cooperative treatment and application thereof in tumor diagnosis and treatment; the invention also provides a method for preparing the nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment.
Background
In the search for cancer treatment strategies, having non-invasive and patient tolerability, and being able to target killer tumor cells without damaging other normal cells is an ideal target for the treatment of malignant tumors. Photothermal therapy (Photothermal therapy, PTT) is an emerging treatment method that is generally triggered by light, especially Near-infrared (NIR) capable of penetrating deep tissues, selectively killing tumor cells under light conditions, and not causing normal tissue damage. PTT can be treated precisely in two ways: firstly, synthesizing a light therapeutic drug with a specific structure, so that the light therapeutic drug can target and position tumors; on the other hand, the size of the illumination range is controlled, and only the lesion part is irradiated, so that the damage of normal tissues is not caused. The dual selective light therapy can effectively reduce systemic toxicity caused by traditional chemotherapy and radiotherapy methods.
Near infrared two-region (NIR-II) photothermal materials are of increasing interest due to their deeper tissue penetration capabilities. NIR-II photothermal materials developed in recent years can be classified into inorganic materials and organic materials. Inorganic materials such as single-walled carbon nanotubes (SWNTs), quantum Dots (QDs), metallic materials, etc. have good photo-thermal conversion efficiency and photo-stability, etc., but potential cytotoxicity caused by poor biodegradability has prevented their clinical application. Compared with inorganic materials, organic materials have the advantages of easy synthesis, controllable structure, good biocompatibility, easy metabolism and the like, and are paid attention to.
The photothermal material adopted by more researches at present is organic fluorescent probe indocyanine green (Indocyanine green, ICG), which is the only fluorescent contrast agent approved by the United states food and drug administration (Food and drug administration, FDA) at present and can be used for clinic, but the further clinical application of the photothermal material is limited due to the obvious defects of poor structural stability, inability of specifically targeting tumors and the like. The development of new stable, multifunctional NIR-II photothermal materials is therefore an important and difficult point of current optical therapeutic research.
In addition, when conventional PTT is performed, high temperatures can cause burns in surrounding tissues, causing non-healable phenomena. Therefore, the recommended treatment temperature according to the relevant certification authorities is 40-45 ℃, but this in turn promotes up-regulation of the heat shock proteins (Heat shock protein, HSP) of the cells and reduces the ability of photothermal treatment of tumors. Up-regulation of HSP70 expression can inhibit apoptosis of tumor cells, and inhibition of HSP70 expression can increase sensitivity of tumor cells to hyperthermia. Quercetin (Qu) has the effect of inhibiting the synthesis of tumor cells HSP70, and can resist hyperplasia, resist tumor and inhibit the synthesis of biological macromolecules, thereby being a potential tumor chemotherapeutic medicine; however, the low solubility of quercetin in water limits its clinical application.
Chemotherapy (Chemodynamic Therapy, CDT), a novel class of tumor treatment techniques based on the iron-based Fenton reaction, which treats H through the iron-based Fenton reaction 2 O 2 The conversion into OH with higher toxicity causes oxidative stress reaction in tumor cells to cause apoptosis; however, CDT action is also exerted by H in TME 2 O 2 Limited content, more reducing substances, and the like. To enhance the therapeutic effects of CDT, researchers have developed a number of multi-functional, multi-therapy delivery systems.
At present, the problem of drug resistance of tumor cells is widely concerned, and a single treatment system can not meet the requirements of the current cancer treatment, so that the development of a treatment method for overcoming the defect that small molecular drugs are indissolvable in water and realizing the mutual combination of tumor mildness, PTT and iron death and chemodynamic therapy to promote apoptosis is significant by combining multiple treatment means.
Disclosure of Invention
In view of the above, the present invention aims to provide a nano-photodiagnosis and treatment reagent for PTT/iron death/CDT co-therapy, and a preparation method and application thereof, wherein the nano-photodiagnosis and treatment reagent Fe (III) -Qu/CJ NPs of the present invention integrates excellent PTT, iron death and CDT, and combines fluorescence imaging and PAI, so that diagnosis and treatment integration can be realized, unlike the current single-performance reagent.
Specifically, the invention provides a nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment, which comprises nanometer particles Fe (III) -Qu/CJ NPs prepared from a compound Cro-Jul and a complex Fe (III) -Qu;
the compound Cro-Jul is
The complex Fe (III) -Qu is a complex formed by quercetin and iron ions through coordination bonds.
In some embodiments of the invention, the molar ratio of the compound Cro-Jul to the complex Fe (III) -Qu is 1:1.
In some embodiments of the invention, the compound Cro-Jul is prepared by reacting according to the following reaction scheme:
in some embodiments of the invention, the compound Cro-Jul is prepared by the following steps: synthesizing a compound Cro-Jul with a D-A-D structure by using a strong electron donor julolidine and a strong electron acceptor croconic acid in a system of toluene and n-butanol;
the mass ratio of the material of the strong electron donor julolidine and the material of the strong electron acceptor croconic acid is more than or equal to 2:1.
In some embodiments of the invention, the compound Cro-Jul is prepared in a mass ratio of the strong electron donor julolidine to the strong electron acceptor croconic acid equal to 3:1.
In some embodiments of the invention, the compound Cro-Jul is prepared by the following steps: stirring strong electron donor julolidine and strong electron acceptor croconic acid in a toluene and n-butanol system at room temperature for a certain time, then heating and refluxing for a certain time, distilling under reduced pressure to remove solvent, cooling to room temperature, filtering to obtain a black brown solid, washing with the solvent, and drying to obtain a compound Cro-Jul with a structure;
the mass ratio of the material of the strong electron donor julolidine and the material of the strong electron acceptor croconic acid is more than or equal to 2:1.
In some embodiments of the invention, the compound Cro-Jul is prepared in a step of stirring at room temperature for 20min, heating at 147-153 ℃ and reacting for 2h.
In some embodiments of the invention, the solvent wash in the step of preparing the compound Cro-Jul is an n-hexane, diethyl ether and ethanol wash.
In some embodiments of the invention, the complex Fe (III) -Qu is prepared as follows: feCl is added under continuous stirring 3 Dripping ethanol solution into quercetin ethanol solution, stirring, adjusting pH to alkaline, reacting for a certain time, standing, cooling, filtering, centrifuging, washing with solvent, and oven drying to obtain the final product.
In some embodiments of the invention, the complex Fe (III) -Qu is prepared as follows: feCl is added under continuous stirring 3 Dripping ethanol solution into quercetin ethanol solution, stirring, adjusting pH to 8.5-9.5, reacting for a certain time, standing, cooling, filtering, centrifuging, washing with solvent, and oven drying to obtain the final product.
In some embodiments of the invention, the solvent wash in the complex Fe (III) -Qu preparation step is an absolute ethanol wash.
In some embodiments of the invention, the complex Fe (III) -Qu is prepared as follows: stirring continuously to make 0.5mol/L FeCl 3 Dripping ethanol solution into 0.5mol/L quercetin ethanol solution, stirring, and adding 5% NaOH ethanolAdjusting the pH of the alcohol solution to be alkaline, reacting for 70min, standing, cooling, filtering, centrifugally separating at 3000r/min, washing with absolute ethyl alcohol for 2 times, and drying to obtain the product.
In some embodiments of the invention, the complex Fe (III) -Qu is prepared as follows: stirring continuously to make 0.5mol/L FeCl 3 Dripping the ethanol solution into 0.5mol/L quercetin ethanol solution, stirring uniformly, regulating the pH to 8.5-9.5 with 5% NaOH ethanol solution, reacting for 70min, standing, cooling, filtering, centrifuging at 3000r/min, washing with absolute ethanol for 2 times, and drying to obtain the product.
In some embodiments of the invention, the nanoparticle Fe (III) -Qu/CJ NPs are prepared as follows: taking a certain amount of Cro-Jul, fe (III) -Qu and PEG respectively, dissolving the Cro-Jul in DMSO, dissolving the Fe (III) -Qu and the PEG in PBS, dropwise adding the Cro-Jul and the Fe (III) -Qu into the PEG solution respectively, and continuously stirring; the solvent is removed from the prepared solution, and the particle size is reduced by ultrasonic treatment, so that the nano-particle Fe (III) -Qu/CJ NPs are prepared.
In some specific embodiments of the present invention, cro-Jul, fe (III) -Qu, PEG were taken in a mass ratio of 1:1:2, respectively, then Cro-Jul was dissolved with DMSO, fe (III) -Qu and PEG were dissolved with PBS, cro-Jul and Fe (III) -Qu solutions were added dropwise to PEG solutions, respectively, and the prepared solutions were stirred continuously, the solvent was removed by spin evaporation, and the particle size was reduced by sonication to prepare nanoparticle Fe (III) -Qu/CJ NPs.
The invention also provides a preparation method of the nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment, which comprises the following preparation steps:
synthesis of Cro-Jul: adding a certain amount of julolidine and croconic acid into a system of toluene and n-butanol, stirring at room temperature for a certain time, heating and refluxing for reaction, and separating and extracting to obtain a compound Cro-Jul after the reaction is finished;
synthesis of Fe (III) -Qu: feCl is added under continuous stirring 3 Dripping ethanol solution into quercetin ethanol solution, stirring, adjusting pH to alkaline, reacting for a certain time, standing, cooling, filtering, centrifuging, washing with anhydrous ethanol, and oven drying to obtain Fe (III) -Qu.
Synthesis of Fe (III) -Qu/CJ NPs:
taking a certain amount of Cro-Jul, fe (III) -Qu and PEG respectively, dissolving the Cro-Jul in DMSO, dissolving the Fe (III) -Qu and the PEG in PBS, dropwise adding the Cro-Jul and the Fe (III) -Qu into the PEG solution respectively, and continuously stirring; the solvent is removed from the prepared solution, and the particle size is reduced by ultrasonic treatment, so that the nano-particle Fe (III) -Qu/CJ NPs are prepared.
In some embodiments of the invention, the mass ratio of julolidine to croconic acid is 2:1 or more; the FeCl 3 And quercetin in a mass ratio of 1:1; the mass ratio of Cro-Jul, fe (III) -Qu and PEG is 1:1:2.
In some embodiments of the invention, the preparation method of the nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment comprises the following preparation steps:
synthesis of Cro-Jul: adding julolidine and croconic acid with the mass ratio of 3:1 into a system of toluene and n-butanol, stirring at room temperature for a certain time, heating and refluxing for reaction, and separating and extracting to obtain a compound Cro-Jul after the reaction is finished;
synthesis of Fe (III) -Qu: 0.5mol/L FeCl is added in a mass ratio of 1:1 under continuous stirring 3 Dripping ethanol solution into 0.5mol/L quercetin ethanol solution, stirring uniformly, regulating pH to be alkaline with 5% NaOH ethanol solution, reacting for 70min, standing, cooling, filtering, centrifuging at 3000r/min, washing with anhydrous ethanol for 2 times, and oven drying to obtain Fe (III) -Qu;
synthesis of Fe (III) -Qu/CJ NPs: taking Cro-Jul, fe (III) -Qu and PEG with a molar ratio of 1:1:2 respectively, dissolving the Cro-Jul in DMSO, dissolving the Fe (III) -Qu and the PEG in PBS, dropwise adding the Cro-Jul and the Fe (III) -Qu into the PEG solution respectively, and continuously stirring; the solvent of the prepared solution is removed by rotary evaporation, and the particle size is reduced by ultrasonic, so that the nano-particle Fe (III) -Qu/CJ NPs are prepared.
The invention also provides a composition applied to integration of biological imaging, tumor treatment and tumor diagnosis and treatment, which comprises the nano-photodiagnosis and treatment reagent or the nano-photodiagnosis and treatment reagent prepared by the preparation method.
The invention also provides application of the nano-photodiagnosis and treatment reagent or the nano-photodiagnosis and treatment reagent prepared by the preparation method in biological imaging, tumor treatment and tumor diagnosis and treatment integration.
The invention also provides application of the nano-photodiagnosis and treatment reagent in preparing tumor treatment medicines.
The invention also provides application of the nano-photodiagnosis and treatment reagent in preparing medicaments for resisting human pharyngeal squamous cancer tumor.
Compared with the prior art, the invention has the following remarkable advantages and effects:
1. in the NIR-II light response nanomaterial, the biocompatibility of inorganic nanomaterial such as gold nanoparticle, plasma metal cluster, carbon nanotube, etc. used for a long time is not clear; and some small molecule photosensitizers, such as indocyanine green (ICG), chlorin e6 (Ce 6) and the like, have poor stability and obvious attenuation after laser irradiation. The photothermal material Cro-Jul belongs to a D-A-D conjugated system compound and has the advantages of easy synthesis and preparation, good light stability and the like.
2. The complex of quercetin and iron ions not only overcomes the upregulation of heat shock proteins caused by mild photothermal treatment by using the quercetin, promotes the photothermal curative effect, but also realizes the combined therapy by Fe (III) performing iron death and chemical power in vivo, thereby achieving the combined enhanced apoptosis effect.
3. Most of the photo-thermal materials are only excited in an NIR-I light interval at present, and as the penetration depth of light is related to the wavelength, the longer the wavelength is, the deeper the penetration depth is, and the less scattering interference is caused by biological tissues, so that the light excitation in the NIR-I light can only treat superficial tumors, but the effect is poor for some deep tumors; the nanometer photodiagnosis and treatment reagent Fe (III) -Qu/CJ NPs in the invention has wide absorption in an NIR-II window, can be excited by NIR-II light, is favorable for photothermal treatment of deep tumors by exciting Fe (III) -Qu/CJ NPs under 980nm laser, has high-efficiency biological tissue penetrating power, can obtain high-resolution and high-contrast photoacoustic imaging of tumor tissues, and has better biocompatibility in vivo animal experiments, so that the laser penetration is deeper, PTT can treat deeper tumors in vivo in the NIR-II window, and the treatment result is improved.
4. Unlike available material with single performance, the nanometer particle Fe (III) -Qu/CJ NPs of the present invention has excellent PTT, iron death and CDT integrated, and combines fluorescent imaging and PAI to realize diagnosis and treatment.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the ultraviolet absorption spectra of Cro-Jul and Fe (III) -Qu/CJ NPs;
FIG. 2 is a transmission electron microscope image of Fe (III) -Qu/CJ NPs;
FIG. 3 is a graph showing the photo-thermal heating capacity test data of Fe (III) -Qu/CJ NPs with different concentrations;
FIG. 4 is photoacoustic imaging test data for different concentrations of Fe (III) -Qu/CJ NPs;
FIG. 5 is cytotoxicity comparative test data;
FIG. 6 is a graph of fluorescence imaging at various times after injection of Ce6@Fe (III) -Qu/CJ tail vein into a tumor-bearing nude mouse;
FIG. 7 is a fluorescence imaging of FaDu cells incubated and illuminated with DCFH-DA and Hoechst staining with different reagents;
FIG. 8 is a data diagram of quantitative tests performed by a cell flow cytometer on FaDu cells incubated and illuminated with different reagents for DCFH-DA and Hoechst staining;
FIG. 9 is a fluorescence imaging of FaDu cells incubated and illuminated with different reagents stained with C11 Bodipy 581/591;
FIG. 10 is a data diagram of a quantitative assay of FaDu cells incubated with different reagents for C11 Bodipy 581/591 staining using a cell flow cytometer;
FIG. 11 is a data diagram of MDA testing of FaDu cells incubated with different reagents for C11 Bodipy 581/591 staining and illuminated;
FIG. 12 is a graph of Western blot experimental data of FaDu cells incubated with different reagents and illuminated;
FIG. 13 is a fluorescence confocal image of FaDu cells incubated with different reagents using mitochondrial membrane potential detection kit (JC-1);
FIG. 14 is a fluorescence confocal image of FaDu cells incubated with different reagents using apoptosis and necrosis detection kit;
FIG. 15 is a graph of data for flow-through quantitative testing of FaDu cells incubated with different reagents;
FIG. 16 is an image of a small animal imager for various times of injection of Ce6@Fe (III) -Qu/CJ in an oncological nude mouse;
FIG. 17 is a photograph of photoacoustic imaging of Ce6@Fe (III) -Qu/CJ injected tumor-forming nude mice at different times;
FIG. 18 is a graph of treatment cycle weight change data following injection of different agents;
FIG. 19 is a graph showing tumor volume change data for treatment cycles following injection of different agents;
FIG. 20 is a photograph of tumor volumes after treatment with different agents;
FIG. 21 is a graph of tumor weight data after treatment with different agents;
FIG. 22 is a graph of staining of HE, hsp70, TUNEL, SLC7A11, ki67 sections of tumors treated with different agents;
FIG. 23 is a graph showing HE staining of five viscera of nude mice treated with different agents;
FIG. 24 is a data graph of analytical tests performed on serum following treatment with different agents;
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it should be apparent that the described embodiments are only some of the embodiments of the present invention and should not be used to limit the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
Example 1: preparation of nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment
The specific operation steps are as follows:
(1) Synthesis of Cro-Jul
Adding 0.42g of strong electron donor julolidine and 0.137g of strong electron acceptor croconic acid into a system of 8mL of toluene and 24mL of n-butanol, stirring at room temperature for 20min, stirring and refluxing for 2h at 147-153 ℃, removing the solvent by reduced pressure distillation, cooling to room temperature, filtering to obtain a black brown solid, washing with n-hexane, diethyl ether and ethanol, and drying to obtain a black brown crystal, wherein the yield of the synthesized compound Cro-Jul is 43.2%; the reaction route is as follows:
(2) Synthesis of Fe (III) -Qu
Stirring continuously to make 0.5mol/L FeCl 3 Dripping the ethanol solution into 0.5mol/L quercetin ethanol solution, stirring uniformly, regulating the pH to 9.3 with 5% NaOH ethanol solution, reacting for 70min, standing, cooling, filtering, centrifuging at 3000r/min, washing with absolute ethanol for 2 times, and drying to obtain the product Fe (III) -Qu with the yield of 62.36%.
(3) Synthesis of Fe (III) -Qu/CJ NPs
80mg of Cro-Jul and Fe (III) -Qu and 200mg of PEG were taken, the Cro-Jul was dissolved in 2mL of DMSO, the Fe (III) -Qu and PEG were dissolved in 200mL of PBS with pH=7.4, and the Cro-Jul and Fe (III) -Qu solutions were added dropwise to the PEG solution, respectively, and stirring was continued. The solvent is removed by rotary evaporation of the prepared solution, and the particle size is reduced by ultrasonic treatment, so that the nanometer photodiagnosis and treatment reagent Fe (III) -Qu/CJ NPs for PTT/iron death/CDT cooperative treatment is prepared.
Example 2: cro-Jul and Fe (III) -Qu/CJ NPs ultraviolet absorption Spectrometry test
Two 2mL test systems were prepared using Cro-Jul and Fe (III) -Qu/CJ NPs prepared in example 1, respectively, and tested using an ultraviolet absorption spectrometer.
The test results are shown in figure 1.
The test results show that Cro-Jul and Fe (III) -Qu/CJ NPs have wide absorption between 800 nm and 1000 nm.
Example 3: morphological observation of Fe (III) -Qu/CJ NPs under Transmission Electron microscopy
Firstly, dispersing a prepared Fe (III) -Qu/CJ NPs liquid sample into a uniform suspension by ultrasonic, placing a powder solution on a copper mesh by adopting a sample dripping method, and drying; ensuring that the powder sample is uniformly distributed on the copper mesh and has no pollutant; the copper net is lightly blown by the ear washing ball, so that no powder easy to fall is ensured, a proper visual field is found under a transmission electron microscope instrument for shooting, and a transmission electron microscope image result is shown in figure 2.
The transmission electron microscope result shows that: the Fe (III) -Qu/CJ NPs nano particles have the size of about 110nm, are spherical in shape, are relatively uniform and are well dispersed.
Example 4: photo-thermal heating capability test of Fe (III) -Qu/CJ NPs with different concentrations
1mL of a solution of Fe (III) -Qu/CJ NPs of different concentrations was taken and the temperature was recorded in real time every 30 using a thermal imager under 980nm laser irradiation.
The test results are shown in FIG. 3.
The test results showed that the temperature rise was positively correlated with the concentration of the Fe (III) -Qu/CJ NPs solution, and that the Fe (III) -Qu/CJ NPs had excellent photo-thermal conversion efficiency of 48.96%.
Example 5: photoacoustic imaging test of Fe (III) -Qu/CJ NPs with different concentrations
Fe (III) -Qu/CJ NPs were injected into tumor-bearing nude mice via tail vein and photographed with 808nm photoacoustic imager at different time points; the test results are shown in FIG. 4.
The test results show that the signal intensity is enhanced with the increase of the concentration of Fe (III) -Qu/CJ NPs, and the Fe (III) -Qu/CJ NPs have excellent photoacoustic imaging performance.
Example 6: cytotoxicity test
FaDu cells were selected as in vitro subjects, all cells at 37℃with 5% CO 2 Is cultured in a humid atmosphere incubator. The experiments are divided into five groups ofThree of the groups were no-light groups, two were light groups, and no-light groups were incubated FaDu cells with PBS solution, fe (III) -Qu solution, and Fe (III) -Qu/CJ NPs nanoparticle solution at concentrations of 0. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, 200. Mu.g/ml, 400. Mu.g/ml, respectively; the light groups were incubated with FaDu cells using 0. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, 200. Mu.g/ml, 400. Mu.g/ml Cro-Jul solution and Fe (III) -Qu/CJ NPs nanoparticle solution, respectively, and then the cells were irradiated with NIR-II (980 nm) and maintained at a temperature, and cytotoxicity was measured with CCK-8 using the light-free groups as a control.
The test results are shown in FIG. 5.
Test results show that under the irradiation of laser, cro-Jul solution and Fe (III) -Qu/CJ NPs can effectively kill tumor cells, wherein the killing effect of Fe (III) -Qu/CJ NPs is more obvious.
Example 7: cell uptake test
8mg of Cro-Jul, ce6 and Fe (III) -Qu,20mg of PEG, respectively, were first taken. Cro-Jul was dissolved in 0.2mL DMSO, fe (III) -Qu and PEG were dissolved in PBS, and Cro-Jul, ce6 and Fe (III) -Qu solutions were added drop-wise to the PEG solution, respectively, and stirring was continued. The prepared solution was subjected to spin evaporation to remove the solvent and obtain Ce6@Fe (III) -Qu/CJ nanoparticles, and then Ce6@Fe (III) -Qu/CJ nanoparticles were incubated with FaDu cells for different times (0, 2,4,6,8 h), and then nuclei were stained with 1. Mu.L/mL Hoechst and photographed by fluorescence confocal imaging. The test results are shown in FIG. 6.
The test results show that Ce6@Fe (III) -Qu/CJ nanoparticles are taken up by tumor cells over time.
Example 8: intracellular ROS assay
Four experiments were set up, one of which was not added with PBS solution, the other three were added with 50. Mu.g/mL Cro-Jul, fe (III) -Qu, and Fe (III) -Qu/CJ nanoparticles, respectively, and then the four groups were incubated overnight with FaDu cells, respectively, and were illuminated, after which the cells were stained with 1. Mu.L/mL DCFH-DA and Hoechst, respectively, and fluorescence confocal and cell-flow cytometry were performed for qualitative and quantitative tests.
The Fe (III) -Qu/CJ nanoparticles were incubated with FaDu cells overnight, the cells were stained with DCFH-DA, fluorescence confocal and cytometry were performed qualitatively and quantitatively, fluorescence confocal images were shown in FIG. 7, and quantitative measurement data of the cytometry were shown in FIG. 8. The Control and Cro-Jul groups in fig. 7 and 8 showed little green fluorescence, while more green fluorescence compared to Fe (III) -qu+laser and Fe (III) -Qu/cj+laser, indicating that ROS production was due to the presence of iron, further verifying that iron death occurred in the cells.
Example 9: intracellular Lipid Peroxide (LPO) assay
Four experiments were set up, one without addition of PBS solution, the other three with 50. Mu.g/mL of Cro-Jul, fe (III) -Qu, and Fe (III) -Qu/CJ nanoparticles, respectively, and then the four were incubated overnight with FaDu cells, respectively, and illuminated, after which the cells were stained with C11 Bodipy 581/591, fluorescence confocal, and cell-flow cytometry for characterization and quantification. Meanwhile, the cultured cells were subjected to lipid LPO quantification according to the MDA assay kit instructions. The fluorescence confocal images are shown in fig. 9, the quantitative test data of the cell flow meter are shown in fig. 10, and the MDA measurement data are shown in fig. 11, wherein 1) control+laser, 2) Cro-Jul+laser, 3) Fe (III) -Qu+laser, and 4) Fe (III) -Qu/CJ+laser. Fig. 9 and 10 are qualitative and quantitative test results of lipid peroxide production after co-incubation of cells with nanoparticles using different methods, and fig. 9 shows that the green fluorescence of both the Fe (III) -qu+laser and Fe (III) -Qu/cj+laser groups is more pronounced than that of the control+laser and Cro-jul+laser groups, and the flow results of fig. 10 demonstrate this point, whereas lipid peroxide is the product of iron death, further illustrating the occurrence of iron death. Lipid peroxides were then re-quantified using malondialdehyde MDA kit, again verifying the onset of iron death.
Example 10: intracellular Western Blot validation
Four experiments were set up, one of the three PBS solutions was not added, and the other three PBS solutions were added separately with 50. Mu.g/mL Cro-Jul, fe (III) -Qu, and Fe (III) -Qu/CJ nanoparticles, respectively, and then the four groups were incubated overnight with FaDu cells, respectively, and irradiated with light. After cells were collected and digested with pancreatin, the cells were collected as pellet in 1.5ml centrifuge tubes, washed twice with 1 XPBS buffer, centrifuged at 1000rpm for 5min and the supernatant discarded. Adding appropriate amount of cell lysate according to cell amount, placing the protease lysate and protease inhibitor at a ratio of 100:1 on ice, and shaking for 10-30min to allow the cells to be fully lysed. The sample was placed in a centrifuge pre-chilled to 4℃at 12000rpm for 5min. And obtaining the supernatant after centrifugation, namely the total cell protein. The supernatant was aspirated and placed in a fresh EP tube and stored at-80 ℃. Protein concentration determination was performed using the PierceTM BCA Protein Assay Kit kit. SDS-PAGE gels were prepared. After calculating the protein concentration, adding 5 Xprotein loading buffer solution, uniformly mixing the loading buffer solution and the total protein solution according to the ratio of 1:5, boiling in water bath at 100 ℃ for 5min, immediately taking out and cooling on ice, and storing at-20 ℃ for standby. Performing electrophoresis, transferring film, sealing, primary antibody, secondary antibody, exposure and the like. Experiments were mainly focused on the photothermal related protein Hsp70 and the iron death related proteins GPX4 and SLC7a11. The western blot data is shown in FIG. 12, where 1) control+laser, 2) Cro-jul+laser, 3) Fe (III) -Qu+laser, 4) Fe (III) -Qu/CJ+laser. In the four groups of FIG. 12control+Laser, cro-Jul+Laser, fe (III) -Qu+Laser and Fe (III) -Qu/CJ+Laser, GPX4 showed a down-regulation trend and SLC7A11 showed an up-regulation trend, both of which demonstrated the occurrence of iron death. Up-regulated expression of HSP70 demonstrated that warming of the photothermal material caused overexpression of heat shock proteins by the cells.
Example 11: apoptosis test
Four experiments were set up, one of the three PBS solutions was not added, and the other three PBS solutions were added separately with 50. Mu.g/mL Cro-Jul, fe (III) -Qu, and Fe (III) -Qu/CJ nanoparticles, respectively, and then the four groups were incubated overnight with FaDu cells, respectively, and irradiated with light. Confocal photographing is carried out by using a mitochondrial membrane potential detection kit (JC-1) and an apoptosis and necrosis detection kit respectively, and detection is carried out by using a cell flow meter. The fluorescence confocal images are shown in fig. 13 and 14 respectively, and the quantitative measurement data of the flow meter are shown in fig. 15. JC-1 in FIG. 13 demonstrates that cell mitochondria are damaged in both the groups Fe (III) -Qu and Fe (III) -Qu/CJ, demonstrating the onset of iron death, and that additional apoptosis and necrotic fluorescent staining results indicate that the combination therapy exhibits better tumor cell killing, as demonstrated by the flow results of FIG. 15.
Example 12: in vivo fluorescence imaging and photoacoustic imaging testing
Ce6@Fe (III) -Qu/CJ (Fe (III) -Qu/CJ nanoparticles loaded with commercial CE 6) tail vein were injected into the tumor-forming nude mice, and the test results were shown in FIG. 16 by photographing 0,1,2,4,6,8, 12 and 24 hours under the Cy3 channel with a small animal imager.
Likewise, fe (III) -Qu/CJ nanoparticles were injected into the tumor-forming nude mice via the tail vein, photo-acoustic imaging was performed at the laser 808nm for 1,6 hours, and the test results are shown in FIG. 17.
The test results of FIG. 16 show that the tumor-bearing nude mice are enriched in tumor tissue after injecting Ce6@Fe (III) -Qu/CJ nanoparticles. Fig. 17 demonstrates that the nanoparticle Fe (III) -Qu/CJ has good photoacoustic imaging capability and has reached the tumor site at 6h, enabling photoacoustic imaging of tumor tissue.
Example 13: photothermal treatment efficacy test of Fe (III) -Qu/CJ NPs in nude mice subcutaneous tumor model
Nude mice were divided into (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 groups, tail intravenous injection of nanomaterials was performed, and after 6h treatment with light was performed for 8 minutes. Weighing and tumor volume measurement were carried out before each phototherapy, temperature detection was carried out with a thermal imager, and the temperature was maintained at 40-45 ℃, and the weighing and tumor volume measurement were shown in fig. 18 and 19, respectively.
After the treatment, the serum, heart, liver, spleen, lung and kidney of the mice and tumor tissues are taken, the volume and weight of the tumor balls are measured, and the measurement results are shown in figures 20 and 21, respectively, wherein 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, and the test results are shown in FIG. 22; HE staining the five internal organs, evaluating whether the treatment process is damaged, and the test result is shown in fig. 23; serum was analyzed for liver and kidney function, and the test results are shown in FIG. 24.
The test results show that all of Cro-Jul+Laser, fe (III) -Qu/CJ+Laser groups have obvious therapeutic effects, wherein under illumination, the Fe (III) -Qu/CJ therapeutic effects are most effective, and the Fe (III) -Qu/CJ nanoparticles have good biological safety in combination with illumination therapy.
Claims (10)
1. A nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment, which is characterized in that:
comprising nanoparticles Fe (III) -Qu/CJ NPs prepared from the compound Cro-Jul and the complex Fe (III) -Qu;
the compound Cro-Jul is
The complex Fe (III) -Qu is a complex formed by quercetin and iron ions through coordination bonds.
2. The nano-photodiagnosis and treat-ment agent for PTT/iron death/CDT co-treatment according to claim 1, wherein the compound Cro-Ju is prepared by reacting according to the following reaction scheme:
3. the nano-photodiagnosis and treat agent for PTT/iron death/CDT co-treatment according to claim 2, wherein the compound Cro-Jul is prepared by the following steps: stirring strong electron donor julolidine and strong electron acceptor croconic acid in a toluene and n-butanol system at room temperature for a certain time, then heating and refluxing for a certain time, distilling under reduced pressure to remove solvent, cooling to room temperature, filtering to obtain a black brown solid, washing with the solvent, and drying to obtain a compound Cro-Jul;
the mass ratio of the material of the strong electron donor julolidine and the material of the strong electron acceptor croconic acid is more than or equal to 2:1.
4. The PTT/iron death/CDT co-catalyst of claim 1The co-therapeutic nanometer photodiagnosis and treatment reagent is characterized in that the preparation steps of the complex Fe (III) -Qu are as follows: feCl is added under continuous stirring 3 Dripping ethanol solution into quercetin ethanol solution, stirring, adjusting pH to alkaline, reacting for a certain time, standing, cooling, filtering, centrifuging, washing with solvent, and oven drying to obtain the final product.
5. The PTT/iron death/CDT co-therapeutic nanophotodiagnostic agent according to any one of claims 1 to 4, wherein said nanoparticle Fe (III) -Qu/CJ NPs is prepared by the steps of: taking a certain amount of Cro-Jul, fe (III) -Qu and PEG respectively, dissolving the Cro-Jul in DMSO, dissolving the Fe (III) -Qu and the PEG in PBS, dropwise adding the Cro-Jul and the Fe (III) -Qu into the PEG solution respectively, and continuously stirring; the solvent is removed from the prepared solution, and the particle size is reduced by ultrasonic treatment, so that the nano-particle Fe (III) -Qu/CJ NPs are prepared.
6. The preparation method of the nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment is characterized by comprising the following preparation steps:
synthesis of Cro-Jul: adding a certain amount of julolidine and croconic acid into a system of toluene and n-butanol, stirring at room temperature for a certain time, heating and refluxing for reaction, and separating and extracting to obtain a compound Cro-Jul after the reaction is finished;
synthesis of Fe (III) -Qu: feCl is added under continuous stirring 3 Dripping ethanol solution into quercetin ethanol solution, stirring, adjusting pH to alkaline, reacting for a certain time, standing, cooling, filtering, centrifuging, washing with solvent, and oven drying to obtain the final product.
Synthesis of Fe (III) -Qu/CJ NPs:
taking a certain amount of Cro-Jul, fe (III) -Qu and PEG respectively, dissolving the Cro-Jul in DMSO, dissolving the Fe (III) -Qu and the PEG in PBS, dropwise adding the Cro-Jul and the Fe (III) -Qu into the PEG solution respectively, and continuously stirring; the solvent is removed from the prepared solution, and the particle size is reduced by ultrasonic treatment, so that the nano-particle Fe (III) -Qu/CJ NPs are prepared.
7. As claimed inThe preparation method of the nanometer photodiagnosis and treatment reagent for PTT/iron death/CDT cooperative treatment is characterized in that the mass ratio of the julolidine to the croconic acid is more than or equal to 2:1; the FeCl 3 And quercetin in a mass ratio of 1:1; the mass ratio of Cro-Jul, fe (III) -Qu and PEG is 1:1:2.
8. A composition for integration of bioimaging, tumor treatment and tumor diagnosis comprising the nanophotodiagnosis reagent according to any one of claims 1 to 5 or the nanophotodiagnosis reagent prepared by the preparation method according to any one of claims 6 to 7.
9. Use of a nano-photodiagnosis and treat reagent according to any one of claims 1 to 5 or prepared by a preparation method according to any one of claims 6 to 7 in biological imaging, tumor treatment, and tumor diagnosis and treat integration.
10. Use of a nanophotodiagnosis and treat reagent according to any one of claims 1 to 5 or a nanophotodiagnosis and treat reagent prepared by the preparation method according to any one of claims 6 to 7 in a medicament for treating tumor.
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