Iron sulfide coated polydopamine nano-material capable of being used for toxicity reduction and efficacy enhancement of cervical cancer radiotherapy as well as preparation method and application of iron sulfide coated polydopamine nano-material

Abstract

The invention belongs to the technical field of biological medicines, and discloses an iron sulfide coated polydopamine nano material for toxicity reduction and efficacy enhancement of cervical cancer radiotherapy, and a preparation method and application of the iron sulfide coated polydopamine nano material. The method comprises the following steps: dissolving ammonium ferrous sulfate and trisodium citrate in ethylene glycol to obtain a solution A, dissolving polyethyleneimine in ethylene glycol to obtain a solution B, adding the solution B into the solution A, and stirring and reacting at normal temperature to obtain a mixed solution; adding a thioacetamide solution into the mixed solution, then dropwise adding triethanolamine, carrying out stirring reaction at normal temperature, carrying out solvothermal reaction after stirring is ended, and centrifuging and washing a product after the reaction is ended, so as to obtain a two-dimensional iron sulfide nanosheet; and adding dopamine hydrochloride, and stirring in absolute ethyl alcohol at room temperature to obtain the iron sulfide coated polydopamine nano material. The synthesis method is simple, convenient and high in operability; the synthesized product is stable and has repeatability; the traditional Chinese medicine composition can be used for sensitizing radiation therapy of cervical cancer, can relieve radiation colitis and reduce side effects of radiation therapy, and is beneficial to clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an iron sulfide-coated polydopamine nanomaterial that can be used to reduce toxicity and enhance the efficacy of radiotherapy for cervical cancer, its preparation method, and its application. Background Technology

[0002] Cervical cancer is the fourth most common cancer among women worldwide, with its incidence and mortality rates rising, posing a serious threat to women's health. Radiotherapy (RT) is a routine clinical treatment for cervical cancer, especially suitable for advanced or locally advanced cervical cancer. However, its clinical efficacy faces two major challenges: (1) the inherent radioresistance of tumor cells greatly reduces the treatment effect and increases the risk of recurrence and metastasis; (2) clinical statistics show that up to 90% of patients undergoing pelvic RT experience acute gastrointestinal reactions, including severe symptoms such as nausea, diarrhea, and rectal bleeding. Because the intestinal tissue is anatomically close to the target area and has high radiation sensitivity, it is particularly prone to radiation-induced enteritis, which may even trigger systemic inflammatory response syndrome, seriously affecting the treatment effect and the patient's quality of life. During RT-induced intestinal inflammation, macrophages polarize from the M2 phenotype to the M1 phenotype. This immune reprogramming leads to the upregulation of pro-inflammatory factors (such as IL-6 and TNF-α) and the decrease of anti-inflammatory factor levels, thereby forming a persistent pro-inflammatory microenvironment. It is noteworthy that this pathological process is closely related to the imbalance of reactive oxygen species (ROS) metabolism. In the complex inflammatory response network, key ROS components such as superoxide anion (·O2-), hydroxyl radical (·OH), and hydrogen peroxide (H2O2) are crucial signaling molecules reflecting the severity of inflammation. Their tissue content is significantly positively correlated with the severity of radiation enteritis, providing a theoretical basis for alleviating intestinal inflammation by eliminating ROS. In clinical practice of tumor radiotherapy (RT), ROS-dependent radiosensitization is a key mechanism for ensuring tumor-killing efficacy. However, this differs from the therapeutic requirement of eliminating ROS in radiation enteritis regions, highlighting the necessity of developing novel nanomedicine delivery systems with spatiotemporally specific ROS self-regulation capabilities.

[0003] Iron-based nanomaterials have shown great potential in the field of ROS regulation due to their unique redox properties. In the acidic microenvironment of tumors, Fe... 2+ The Fenton-mediated reaction can catalyze the formation of highly toxic ·OH from H₂O₂. Notably, ·OH possesses extremely strong oxidizing power and can induce ferroptosis through a lipid peroxidation chain reaction, thereby enhancing the radiosensitivity of tumors. Furthermore, based on the self-regulating properties of ROS in iron-based nanomaterials, it holds promise for enhancing ROS generation in tumor regions while simultaneously exerting ROS scavenging functions in normal intestinal tissues. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a method for preparing and applying iron sulfide-coated polydopamine nanomaterials that can be used to reduce toxicity and enhance the efficacy of radiotherapy for cervical cancer. The synthesis method of the iron sulfide-coated polydopamine nanomaterials prepared by this invention is simple and highly operable; the synthesized product is stable and reproducible; the iron sulfide-coated polydopamine nanomaterials synthesized by this invention can be used to sensitize cervical cancer radiotherapy, while also alleviating radiation enteritis and reducing radiotherapy side effects, which is beneficial for clinical application.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing iron sulfide-coated polydopamine nanomaterials that can be used to reduce toxicity and enhance the efficacy of radiotherapy for cervical cancer, comprising the following steps: (1) Dissolve ferrous ammonium sulfate and trisodium citrate in ethylene glycol to obtain solution A, dissolve polyethyleneimine in ethylene glycol to obtain solution B, add solution B to solution A, and stir the reaction at room temperature to obtain a mixture; (2) Thioacetamide solution was added to the mixture, and then triethanolamine was added dropwise. The mixture was stirred at room temperature. After stirring, a solvothermal reaction was carried out. After the reaction was completed, the product was centrifuged and washed to obtain two-dimensional iron sulfide (FeS) nanosheets.

[0006] (3) The iron sulfide nanosheets obtained in step (2) are mixed with dopamine hydrochloride in anhydrous ethanol and stirred at room temperature. After the reaction is completed, the product is centrifuged and washed to obtain iron sulfide-coated polydopamine (FeS@PDA, FP) nanomaterials.

[0007] Furthermore, in step (1), the ratio of ferrous ammonium sulfate, trisodium citrate, and ethylene glycol in solution A is 0.6 mmol: 0.2 mmol: 15 mL; the ratio of polyethyleneimine and ethylene glycol in solution B is 500 mg: 5 mL; and the ratio of the volume of ethylene glycol in solution A to the volume of ethylene glycol in solution B is 3:1.

[0008] Furthermore, in step (1), the stirring is magnetic stirring with a rotation speed of 800 rpm; the stirring reaction time is 2 h.

[0009] Furthermore, in step (2), the concentration of the thioacetamide solution is 0.05M; the ratio of the amount of ferrous ammonium sulfate added to the thioacetamide solution and triethanolamine is 0.6 mmol: 15 mL: 2 mL; the stirring is magnetic stirring at a speed of 800 rpm, and the stirring reaction time is 1~10 min.

[0010] Furthermore, in step (2), the temperature of the solvothermal reaction is 200℃ and the time is 24 h; the centrifugation speed is 14000 rpm and the time is 10 min; the washing is three times with anhydrous ethanol.

[0011] Furthermore, in step (3), the ratio of the amount of iron sulfide nanosheets, dopamine hydrochloride and anhydrous ethanol added is 1 mg: 1 mg: 2 mL.

[0012] Furthermore, in step (3), the stirring is magnetic stirring at a speed of 400 rpm; the stirring reaction time is 10 h; the centrifugation speed is 14000 rpm and the time is 10 min; and the washing is washing twice with deionized water.

[0013] In a second aspect, the present invention provides an iron sulfide-coated polydopamine nanomaterial prepared by the preparation method described above.

[0014] A third aspect of the present invention provides the application of the iron sulfide-coated polydopamine nanomaterial in enhancing the radiosensitization of cervical cancer and alleviating radiation enteritis.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) The method for preparing iron sulfide-coated polydopamine nanomaterials in this invention is simple and highly operable; the synthesized materials are stable and reproducible.

[0016] (2) The iron sulfide-coated polydopamine nanomaterial synthesized in this invention has excellent acid response to the tumor microenvironment and can induce ferroptosis in tumor cells, which is more conducive to clinical application.

[0017] (3) The iron sulfide-coated polydopamine nanomaterial synthesized in this invention has the ability to resist inflammation in normal tissues, can relieve radiation colitis, and can reduce the side effects of radiotherapy. Attached Figure Description

[0018] Figure 1 Transmission electron microscopy image of iron sulfide nanosheets (FeS); Figure 2 Transmission electron microscopy image of iron sulfide-coated polydopamine nanomaterial (FeS@PDA, FP); Figure 3 X-ray diffraction pattern of FeS nanomaterials; Figure 4 The hydration particle size of FeS and FP nanomaterials; Figure 5 The image shows the cell-killing effect of FP material on HeLa cells with and without RT. Figure 6The image shows the effect of FP material on the migration ability of HeLa cells with and without RT. Figure 7 To detect the inhibitory effect of different concentrations of FP nanomaterials on GPX4 protein expression using Western blot (WB): Figure 7 a represents the WB developing band; Figure 7 b is a quantitative diagram of GPX4 protein expression; Figure 8 Fluorescence images of ROS removal in FP materials at different time points; Figure 9 Images showing the colon length in mice from different treatment groups; Figure 9 Image a is a mouse colon; Figure 9 b is the length quantization diagram; Figure 10 Images showing the effects of hematoxylin-eosin (H&E) staining on the colon of mice in different treatment groups. Detailed Implementation

[0019] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] As introduced in the background section, cervical cancer currently exhibits a certain degree of radiation resistance, leading to poor radiotherapy outcomes. Furthermore, radiotherapy patients inevitably experience intestinal damage, resulting in significant side effects.

[0022] Based on this, the purpose of this invention is to provide a method for preparing and applying iron sulfide-coated polydopamine nanomaterials that can be used to reduce toxicity and enhance the efficacy of radiotherapy for cervical cancer. This invention uses ferrous ammonium sulfate and thioacetamide as raw materials, obtaining a precursor solution through a simple stirring reaction, followed by a solvothermal reaction to obtain two-dimensional iron sulfide nanosheets (FeS), which are then combined with polydopamine to obtain iron sulfide-coated polydopamine nanomaterials (FP). The method for synthesizing the iron sulfide-coated polydopamine nanomaterials for reducing toxicity and enhancing the efficacy of radiotherapy for cervical cancer prepared by this invention is simple and highly operable, exhibits good ferroptosis-sensitizing radiotherapy capabilities, can protect the intestines during radiotherapy of tumor cells, and effectively alleviate radiation colitis.

[0023] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0024] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0025] Example 1: Preparation of two-dimensional iron sulfide nanosheets Solution A was prepared by dissolving 0.6 mmol of ferrous ammonium sulfate and 0.2 mmol of trisodium citrate in 15 mL of ethylene glycol. Solution B was prepared by dissolving 500 mg of polyethyleneimine (PEI) in 5 mL of ethylene glycol. Solution B was then added to solution A, and the mixture was reacted at room temperature with magnetic stirring at 800 rpm for 2 h to obtain a final mixture. Subsequently, 15 mL of 0.05 M thioacetamide solution was added to the mixture, followed by 2 mL of triethanolamine. The mixture was then reacted at room temperature with magnetic stirring at 800 rpm for 5 min. After stirring, the entire mixture was transferred to an autoclave and heated at 200 °C for 24 h. Finally, the mixture was collected by centrifugation at 14000 rpm for 10 min and washed three times with anhydrous ethanol to obtain two-dimensional iron sulfide nanosheets (FeS).

[0026] Example 2: Preparation of iron sulfide-coated polydopamine material 1 mg of iron sulfide nanosheets and 1 mg of dopamine hydrochloride were dissolved in 2 mL of anhydrous ethanol and stirred at 400 rpm for 10 h at room temperature. The resulting product was centrifuged at 14000 rpm for 10 min, the supernatant was discarded and the precipitate was collected and washed twice with deionized water to obtain the final iron sulfide-coated polydopamine material (FP).

[0027] Experimental Example: Material Property Analysis Transmission electron microscopy observation Dissolve an appropriate amount of two-dimensional iron sulfide nanosheets in anhydrous ethanol to prepare a 1 mg / mL solution. Use a pipette to add 20 μL of the solution to a copper grid. Dry the solution to prepare a transmission electron microscope (TEM) sample and observe it using a TEM.

[0028] The results are as follows Figure 1 The iron sulfide prepared as shown is in the shape of two-dimensional thin sheets with a particle size of about 100 nm.

[0029] Dissolve an appropriate amount of FP material in anhydrous ethanol to prepare a 1 mg / mL solution. Use a pipette to add 20 μL of the solution to a copper grid. Dry the sample to prepare it for transmission electron microscopy (TEM) detection and observe it using a TEM.

[0030] The results are as follows Figure 2 The iron sulfide-coated polydopamine prepared as shown is in the shape of a two-dimensional sheet with a particle size of about 100-120 nm.

[0031] X-ray diffraction (XRD) test: Take 20 mg of iron sulfide powder, fill it tightly into the groove of the glass sample cell and scrape it flat to form a smooth surface. Place the prepared sample cell on the sample stage of the X-ray diffractometer for testing.

[0032] The results are as follows Figure 3 The X-ray diffraction peaks of the prepared iron sulfide shown are consistent with the crystal structure of FeS.

[0033] Dynamic Light Scattering Analyzer (DLS) Test: Take ethanol dispersions containing 30 μg of FeS and FP respectively, centrifuge to remove the supernatant, and resuspend in 1 mL of pure water to prepare dispersions of 30 μg / mL. Measure the hydrated particle size of FeS and FP using a dynamic light scattering analyzer (DLS).

[0034] The results are as follows Figure 4 As shown, the results indicate that the hydrated particle size of FeS nanomaterials is approximately 125 nm, and the hydrated particle size of FP nanomaterials is approximately 169 nm.

[0035] Cytotoxicity assay: Prepare a 6-well plate, add 1×10 to each well. 5Personal cervical cancer cells (HeLa cells) were cultured in a 37°C constant temperature incubator containing 5% CO2 for 24 h. The cells were then treated in two groups: (1) FP group: the cells were co-incubated with FP materials prepared in Example 2 at different concentrations of 0, 10, 20, 40 and 80 μg / mL for 6 h; (2) FP+RT group: the cells were co-incubated with FP materials prepared in Example 2 at different concentrations of 0, 10, 20, 40 and 80 μg / mL for 6 h, and then irradiated with X-rays at a dose of 6 Gy. After that, the medium was changed and cultured for another 24 h. MTT medium was added to each well and cultured for 3.5 h. The MTT medium was removed, and 70 µL DMSO was added to each well. The cells were shaken on a shaker for 10-15 min to dissolve the purple solid completely. The absorbance value (OD value) at 490 nm was read using an ELISA reader. The number of viable cells was determined based on the measured OD value, and the results were plotted.

[0036] The results are as follows Figure 5 As shown, the survival rate of HeLa cells gradually decreased with the increase of FP concentration, and the survival rate of the FP+RT group was even lower, indicating that FP material has a significant cytotoxic effect on tumor cells and a significant radiosensitizing effect.

[0037] Cell scratch assay: Prepare two-well cell scratching accessories, add 5×10 to each well. 4 HeLa cells were cultured for 24 h in a 37°C constant temperature incubator containing 5% CO2. The cells were then treated in four groups: (1) FP group: cells were co-incubated with FP material at a concentration of 40 μg / mL for 6 h, and then the scratched accessories were removed; (2) FP+RT group: cells were co-incubated with FP material at a concentration of 40 μg / mL for 6 h, and then the scratched accessories were removed and the cells were irradiated with X-rays at a dose of 6 Gy; (3) Control group: no drug was administered and no X-ray irradiation was performed; (4) RT group: no drug was administered and X-ray irradiation was performed with a radiation dose of 6 Gy. After treatment, the cells were photographed under a microscope, and then the medium was changed and the cells were cultured for another 24 h before being photographed again.

[0038] The results are as follows Figure 6 As shown, the migration rate of the RT group alone was higher, while the addition of FP material significantly inhibited the migration ability of tumor cells and had a stronger effect than either material alone.

[0039] Western Blot experiment: HeLa cells were loaded at a rate of 2 × 10 5Cells were seeded per well in a six-well plate and treated in four groups: (1) Control (FP concentration of 0 μg / mL); (2) FP of 20 μg / mL; (3) FP of 80 μg / mL; (4) FP of 80 μg / mL and ferroptosis inhibitor. After incubation for 24 h, cells from each group were collected, and total cell protein was extracted by adding protein lysis buffer. Protein quantification was performed according to the BCA reagent instructions. After electrophoresis, the separated proteins were transferred to a PVDF membrane. After membrane transfer, the membrane was blocked with skim milk powder for 1 h. Using GAPDH as an internal control, diluted anti-GPX4 primary antibody solution (diluted according to the recommended concentration in the instructions for each antibody) was added, and the membrane was incubated overnight at 4°C. After washing with PBST, secondary antibody was added and shaken slowly on a shaker for 1 h. After washing with PBST, the membrane was developed and photographed to analyze the Western blot results.

[0040] like Figure 7 As shown, the results indicate that FP can downregulate the expression of the key ferroptosis protein GPX4 in HeLa cells, and upregulate it after the addition of ferroptosis inhibitors, indicating that FP material can effectively induce ferroptosis in cells.

[0041] ROS cleanup experiment: Prepare confocal cells, adding 2×10⁻⁶ ppm to each well. 5 Mouse embryonic fibroblasts (3T3 cells) were cultured in a 37°C incubator containing 5% CO2 for 24 h. After treatment with reactive oxygen species positive control reagent for 30 min, the FP material prepared in the previous step was incubated with the cells at a concentration of 40 μg / mL for 0 h, 2 h, 6 h and 12 h respectively. After removing the supernatant culture medium, DCFH-DA working solution (1 mL / well) was added for staining for 20 min. The intracellular DCF fluorescence was observed using CLSM with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0042] like Figure 8 As shown, the results indicate that the green fluorescence gradually weakens over time, suggesting that the FP material continuously exerts its ROS scavenging effect in normal cells.

[0043] Mouse intestinal length measurement experiment: Six-week-old female C57 mice were divided into four groups: PBS, RT, FP, and FP+RT, with three mice in each group. The PBS group received 100 μL of PBS via tail vein injection as a control. The RT group received 13 Gy of X-ray irradiation in the lower abdomen. The FP group received 100 μL of FP material via tail vein injection at a concentration of 4 mg / kg. The FP+RT group received 100 μL of FP material via tail vein injection at a concentration of 4 mg / kg and 13 Gy of X-ray irradiation in the lower abdomen. The colons of the mice were dissected and photographed three days later.

[0044] like Figure 9 As shown, the results indicate that the RT group had the shortest colon length and the most severe damage, while there was no significant difference between the PBS group and the FP group. The FP+RT group showed a significant alleviating effect.

[0045] Mouse HE staining experiment: Six-week-old female C57 mice were divided into four groups: PBS, RT, FP, and FP+RT, with three mice in each group. The PBS group received 100 μL of PBS via tail vein injection as a control. The RT group received 13 Gy of X-ray irradiation in the lower abdomen. The FP group received 100 μL of FP material via tail vein injection at a concentration of 4 mg / kg. The FP+RT group received 100 μL of FP material via tail vein injection at a concentration of 4 mg / kg and 13 Gy of X-ray irradiation in the lower abdomen. Three days later, the mouse colons were dissected, and the tissues were fixed, embedded, dewaxed, dehydrated in a gradient, and sectioned. Finally, the tissues were stained with HE according to standard methods and observed using a slide scanner.

[0046] like Figure 10 As shown, the results indicate that the RT group suffered the most severe damage to the colon due to crypt destruction and mucosal thinning. There was no significant difference between the PBS group and the FP group, while the FP+RT group showed a significant effect in alleviating damage.

[0047] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing iron sulfide-coated polydopamine nanomaterials that can be used to reduce toxicity and enhance the efficacy of radiotherapy for cervical cancer, characterized in that, The preparation method of the iron sulfide-coated polydopamine nanomaterial includes the following steps: (1) Dissolve ferrous ammonium sulfate and trisodium citrate in ethylene glycol to obtain solution A, dissolve polyethyleneimine in ethylene glycol to obtain solution B, add solution B to solution A, and stir the reaction at room temperature to obtain a mixture; (2) Thioacetamide solution was added to the mixture, and then triethanolamine was added dropwise. The mixture was stirred at room temperature. After stirring, a solvothermal reaction was carried out. After the reaction was completed, the product was centrifuged and washed to obtain two-dimensional iron sulfide nanosheets. (3) The iron sulfide nanosheets obtained in step (2) are mixed with dopamine hydrochloride in anhydrous ethanol and stirred at room temperature. After the reaction is completed, the product is centrifuged and washed to obtain iron sulfide-coated polydopamine nanomaterials.

2. The preparation method according to claim 1, characterized in that, In step (1), the ratio of ferrous ammonium sulfate, trisodium citrate and ethylene glycol in solution A is 0.6 mmol: 0.2 mmol: 15 mL; the ratio of polyethyleneimine and ethylene glycol in solution B is 500 mg: 5 mL; and the ratio of the volume of ethylene glycol in solution A to the volume of ethylene glycol in solution B is 3:

1.

3. The preparation method according to claim 2, characterized in that, In step (1), the stirring is magnetic stirring with a rotation speed of 800 rpm; the stirring reaction time is 2 h.

4. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the thioacetamide solution is 0.05M; the ratio of the amount of ferrous ammonium sulfate added to the thioacetamide solution and triethanolamine is 0.6 mmol: 15 mL: 2 mL; the stirring is magnetic stirring at a speed of 800 rpm, and the stirring reaction time is 1~10 min.

5. The preparation method according to claim 4, characterized in that, In step (2), the temperature of the solvothermal reaction is 200℃ and the time is 24 h; the centrifugation speed is 14000 rpm and the time is 10 min; the washing is three times with anhydrous ethanol.

6. The preparation method according to claim 1, characterized in that, In step (3), the ratio of the amount of iron sulfide nanosheets, dopamine hydrochloride and anhydrous ethanol added is 1 mg: 1 mg: 2 mL.

7. The preparation method according to claim 6, characterized in that, In step (3), the stirring is magnetic stirring at a speed of 400 rpm for 10 h; the centrifugation is performed at a speed of 14000 rpm for 10 min; and the washing is performed by washing twice with deionized water.

8. The iron sulfide-coated polydopamine nanomaterial prepared by the preparation method according to any one of claims 1-7.

9. The application of the iron sulfide-coated polydopamine nanomaterial prepared by the preparation method according to any one of claims 1-7 or the iron sulfide-coated polydopamine nanomaterial according to claim 8 in sensitizing cervical cancer radiotherapy and alleviating radiation enteritis.

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